**Figure 2.** Mechanical Response Threshold: Spinal Nerve Ligation (SNL) Chung Model (Chung), Contralateral (Contra) Paw Withdrawal. Contralateral paw withdrawal thresholds following surgery and administration of AQU-118. Data are presented as mean ± SEM.

#### *2.2. Transcript Expression Changes in the DRG 20 Days after SNL-Surgery (D5) between Vehicle and Sham Groups*

Comparisons of MMP-2, MMP-9, IL-1β and IL-6 mRNA expression levels in the left (ipsilateral) DRG between vehicle and sham groups were measured and all were found to be significantly elevated except for MMP-9 (Figures 3 and 4). An apparent correlation was observed between the mRNA expression levels of MMP-2 and IL-1β (Figure 5). However, no such correlation was observed between MMP-2 and IL-6 mRNA levels (Figure 5).

**Figure 3.** MMP2 (**A**) and MMP9 (**B**) mRNA expression levels in ipsilateral DRG for sham and SNL vehicle groups. MMP-2 mRNA expression levels were found to be significantly elevated after SNL-surgery while MMP-9 mRNA was found to be not significant. Data presented as the average from *n* = 10 per group ± SEM and analyzed with a two-tailed unpaired t-test (ns, not significant; \*\*\*\*, *p* < 0.0001).

**Figure 4.** IL-1β (**A**) and IL-6 (**B**) mRNA expression levels in ipsilateral DRG for sham and SNL vehicle groups. Both IL-1β and IL-6 mRNA expression levels were found to be significantly elevated after SNL-surgery in vehicle group compared to sham. Data presented as the average from *n* = 10 per group ± SEM and analyzed a with two-tailed unpaired t-test. (\*\*\*, *p* < 0.001; \*\*\*\*, *p* < 0.0001).

**Figure 5.** Correlation between MMP2 and (**A**) IL-1β mRNA and (**B**) IL-6 mRNA expression levels in left DRG of SNL & sham Vehicle groups. Pearson correlation coefficients (with 95% confidence) were calculated for sham vehicle (*r* = 0.7372; two-tailed *p* value = 0.015) and SNL vehicle (*r* = 0.8135; two-tailed *p* value = 0.0042) for MMP2 vs IL-1β mRNA. Similar analysis of MMP2 vs. IL-6 mRNA showed no clear correlation with IL-6 transcript level: sham vehicle (*r* = 0.4912; two tailed *p* value = 0.1494) and SNL vehicle (*r* = 0.1622; two tailed *p* value = 0.6543).

#### *2.3. Protein Level Changes in the DRG 20 Days after SNL-Surgery (D5) between Vehicle and Sham Groups*

IL-1β (Pro and cleaved form), MBP, IL-6 and caspase-3 protein levels were measured via western blotting of tissue obtained from the left (ipsilateral) DRG and compared between sham and vehicle groups. All protein targets were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. The results show that cleaved IL-1β was found to significantly decrease in the ipsilateral DRG of the vehicle group as compared to sham while no significant changes were observed in contralateral DRG (Figure 6). Caspase-3 was found to significantly increase in the ipsilateral DRG while both the higher and lower molecular weight bands of MBP were found to significantly decrease in the vehicle group as compared to the sham (Figure 7). The pro forms of IL-1β and IL-6 were not detected in the DRG by western blotting.

**Figure 6.** Comparison of cleaved IL-1β protein level in ipsilateral and contralateral DRG for sham and vehicle groups. Cleaved, active IL-1β protein levels in left (ipsilateral) DRG (**A**) significantly decreased in the vehicle group (*n* = 10) compared to the sham (*n* = 10). (**B**) No difference between the sham (*n* = 10) and vehicle group (*n* = 10) in right (contralateral) DRG. Data presented as the average ± SEM. An unpaired t-test was performed: ns, not significant; \*\*, *p* < 0.01.

**Figure 7.** Comparison of Caspase-3 and MBP protein levels in in the ipsilateral DRG for sham and vehicle groups. (**A**) Caspase-3 protein levels (via western blot) in the left (ipsilateral) DRG significantly increased in the vehicle group compared to the sham. (**B**) MBP levels (via western blot) significantly decreased for both the upper band (20 kDa) and (**C**) lower band (15 kDa) in the vehicle group of the left (ipsilateral) DRG as compared to the sham. Protein level is presented normalized to GAPDH and relative to the sham group. Data presented as the average ± SEM. An unpaired t-test was performed: \*\*\*, *p* < 0.001; \*\*\*\*, *p* < 0.0001.

#### *2.4. Protein Level Changes in the DRG 20 Days after SNL-Surgery (D5) between AQU-118 and Vehicle Groups*

MBP (both higher and lower molecular weight bands) and caspase-3 protein levels were measured via western blotting of tissue obtained from the left (ipsilateral) DRG of the AQU-118 group (*n* = 20) and compared with the vehicle (*n* = 20). All protein targets were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. In the samples from AQU-118 treated animals, caspase-3 levels were significantly decreased in the ipsilateral DRG but there were no significant changes observed in the levels of MBP as compared to samples obtained from vehicle-treated animals (Figure 8). The DRG samples were tested for MMP-2 mRNA expression levels and retested for caspase-3 protein levels in the AQU-118 (*n* = 10) and vehicle (*n* = 10) groups. (Figure 9). Comparison of these samples showed that the AQU-118 (*n* = 10) group exhibited a statistically significant decrease in the caspase-3 protein level as compared to the vehicle group (*n* = 10), while still exhibiting elevated MMP-2 mRNA expression levels (Figure 9).

**Figure 8.** Comparison of caspase-3 and MBP in the ipsilateral DRG for vehicle and AQU-118 groups. (**A**) Caspase-3 protein levels in the left (ipsilateral) DRG significantly decreased in the AQU-118 group (*n* = 20) compared to the vehicle (*n* = 20). (**B**) There were no significant changes in MBP levels in both the upper band (20 kDa) and (**C**) lower band (15 kDa) in AQU-118 group (*n* = 20) of the left (ipsilateral) DRG as compared to the vehicle group (*n* = 20). Protein level is presented normalized to GAPDH and relative to the vehicle group. Data presented as the average ± SEM. An unpaired t-test was performed: ns, not significant; \*, *p* < 0.05.

**Figure 9.** Comparison of MMP2 mRNA expression levels versus caspase-3 in a subset of ipsilateral DRG tissue for both vehicle and AQU-118 groups. Caspase 3 protein and MMP-2 mRNA expression levels were performed in the ipsilateral DRG tissue (via western blot) for both the AQU-118 (*n* = 10) and vehicle (*n* = 10) groups and compared to each other. The protein level is presented normalized to GAPDH and relative to the vehicle group (no samples were excluded in analysis). Data presented as the average ± SEM. An unpaired t-test was performed: \*\*, *p* < 0.01.

#### **3. Discussion**

We have shown, for the first time, that oral dosing with AQU-118 attenuates mechanical allodynia and reduces caspase-3 protein levels in the DRG, likely via inhibition of MMP-2/-9. These findings are significant because this is the first analysis of the molecular and biochemical factors that mediate the behavioral effects seen with oral dosing with AQU-118, including the significantly reduced mechanical allodynia in both the rat SNL and CCI-IoN models of neuropathic pain as well as in the naloxone precipitated morphine withdrawal rodent model [14,15]. In this study we specifically looked at changes in biomarker levels after SNL surgery and then after dosing with AQU-118. We confirmed that after SNL surgery there is an increase in mechanical allodynia (Figure 1), which corresponds to an elevation in both the mRNA MMP-2 expression levels (Figure 3A) and caspase-3 protein levels (Figure 7A) in the DRG as compared to the sham. Once per day (SID) oral dosing with AQU-118 produces a statistically significant decrease in mechanical allodynia (Figure 1), which corresponds to a decrease in the caspase-3 protein levels (Figures 8 and 9) as compared to the vehicle group.

The significant increase in MMP-2 but not MMP-9 mRNA expression levels, 25 days after SNL surgery, is consistent with the work of Ji and coworkers. They found that MMP-9 was upregulated in the DRG soon after SNL but declined several days later; this decline in MMP-9 was soon followed by a sharp increase in MMP-2 that remained elevated up to 21 days after SNL-surgery [12]. Our observation of an apparent linear correlation between the MMP-2 mRNA expression levels and that of the expression level of IL-1 β but not IL-6 (Figure 5A,B) is also consistent with the hypothesis that MMP-2 induces neuropathic pain symptoms through activation of IL-1 β and astrocyte activation [12]. However, one would also expect a concomitant elevation in the level of cleaved IL-1 β protein in the DRG which we did not observe. Instead, a significant decrease in the level of cleaved IL-1 β was observed in the ipsilateral DRG of the vehicle group as compared to the sham. (Figure 6A,B). Also, while there was an up-regulation in mRNA IL-6 in the DRG (Figure 4B), no IL-6 protein was

detected [19]. Repeated attempts were made to measure IL-6 using multiple kits and reagents; while IL-6 protein was easily detected using commercially available protein standards, no IL-6 protein was measured in the DRG samples [19]. Although protein levels cannot reliably be predicted from mRNA levels, the absence of detectable IL-6 protein in the rat DRG samples in this study was surprising [20]. Several explanations could account for this result. The levels of IL-6 in the tested samples may have been below the limits of detection of the assays due to low levels of translation. Alternatively, cytokine-producing cells (i.e., glial, astrocytes & Schwann cell) are not evenly distributed throughout the DRG, which would produce variable results in quantification or detection of proteins depending upon the specific sampling location [21].

Another protein that decreased in the DRG after SNL surgery was MBP. This is consistent with the work of Shubayev and coworkers who observed a decrease in both the higher and lower molecular weight bands of MBP after L5 spinal nerve crush (SNC) [13]. They also observed that the decrease in MBP coincided with an initial increase in both MMP-9 mRNA expression and MMP-9 activity in the DRG [13]. However, while Shubayev and coworkers found that daily intraperitoneal (ip) injection of a potent broad spectrum MMP inhibitor (GM6001) was able to reverse the decrease in MBP, we saw no such reversal with oral dosing with AQU-118. Potential explanations for this disparity include the possibility that the amount of MMP inhibitor required for reversal of MBP breakdown may be higher than what was used in our current study, or that there is a significant time delay before a reversal in MBP levels occur.

While we did not observe a reversal in MBP decline after oral dosing with AQU-118, we did observe a decrease in caspase-3 protein levels in the DRG that coincided with a decrease in mechanical allodynia. This is similar to what was observed by Shubayev and coworkers, who measured a significant decrease in the level of caspase-3 in the DRG at the same time as a decrease in mechanical allodynia was noted following daily injections of GM6001 [13]. Given the fact that we observe similar decreases in both mechanical allodynia and caspase-3 protein levels, but no reversal in the levels of MBP, it is possible that either there is a significant delay between the reduction of caspase-3/apoptosis and remyelination, or that MBP breakdown is not directly connected to a caspase-3 dependent apoptotic pathway.

Previous studies have observed a strong correlation between mechanical allodynia and neuronal apoptosis [18,22–25]. For example, Sekiguki and co-workers reported that the level of mechanical allodynia observed within various surgically induced rodent models (nerve root compression versus spinal nerve compression) of neuropathic pain correlated with the level of DRG neuronal apoptosis [18]. It is well known that caspase-3 functions as the key executioner of apoptosis in neuronal cells [26–28]. Inhibition of caspase 3 has been found to reduce pain behaviors in rodent models of neuropathic pain [23–25]. Additionally, hypoxia-reoxygenation, which induces apoptosis in brain endothelial cells, also results in an increase in the expression of MMP-2, MMP-9, and caspase-3, and when these cells are exposed to a non-selective MMP inhibitor the levels of both caspase-3 and apoptosis significantly decrease [29]. This phenomenon has also been observed more recently in an in-vitro and in-vivo rat model of traumatic brain injury (TBI), supporting the idea that inhibition of MMP-2 reduces neuronal apoptosis, at least in part, through decreasing caspase-3 levels or activity [30]. These studies reinforce our findings that inhibition of MMP -2/-9 via AQU-118 reduces caspase 3 protein levels and nerve damage-induced mechanical allodynia. Clearly more studies are required to determine what intermediaries are involved in AQU-118's ability to attenuate mechanical allodynia via caspase-3. The objective of this research is to identify biomarkers that can be used for monitoring the effects of AQU-118 in the clinic as well as to potentially screen patients with neuropathic pain who would be most likely to benefit from treatment with AQU-118.

#### **4. Materials and Methods**

#### *4.1. Testing of Orally Administered AQU-118 in the SNL-Rat Model of Neuropathic Pain*

#### 4.1.1. Animals

The use of animals was approved by the Institutional Animal Care and Use Committee (IACUC) at PsychoGenics, Inc. (IACUC 243\_0214, Pain in rats, approved 9 May 2014 and IACUC 234\_0314 Surgical procedures addendum for rats, approved 24 March 2014). A total of 90 male Sprague Dawley rats (200–225 g) were obtained from Envigo (Indianapolis, IN, USA). Upon receipt, rats were assigned unique identification numbers and were group housed with 3 rats per cage in polycarbonate cages with micro-isolator filter tops. All rats were examined, handled, and weighed prior to initiation of the study to assure adequate health and suitability. During the course of the study, 12/12 light/dark cycles were maintained. Rodent chow and water were provided *ad libitum* for the duration of the study.

Spinal Nerve Ligation (SNL) Chung Model [31,32]: Under general anesthesia with continuous inhalation of isoflurane, surgery was performed with aseptic procedures in a surgery unit. Sterile ophthalmic ointment was used to lubricate the eyes. Animals were observed continuously for the level of anesthesia, testing for the animal's reflex response to a tail or paw pinch and closely monitoring the animal's breathing. A heating pad was used to maintain body temperature at 37 ◦C while the animals recovered from anesthesia. The skin at the area of the lower lumber and sacral level of the rat was shaved and disinfected with betadine and alcohol. A left longitudinal incision at the level next to the vertebral column was made, and the left paraspinal muscles were separated. The transverse process of L6 was removed and nearby connective tissue cleaned to expose L5 and L6 spinal nerves [31,32]. After the nerves were isolated and clearly visualized, 4-0 silk threads were used to ligate the left L5. The muscles were sutured with 4-0 silk threads and the wound closed by staples. All rats received an analgesic (Carprofen, 5 mg/kg, subcutaneous, s.c.) immediately before and the next day after surgery. Each rat was monitored until awake and moving freely around the recovery chamber. Animals were then single-housed for the duration of the study.

#### 4.1.2. Von Frey Test for Mechanical Allodynia

Withdrawal from a mechanical stimulus was measured by applying von Frey (VF) filaments (Stoelting, Wood Dale, IL, USA) of ascending bending force to the plantar surface of the hind paws, ipsilateral and contralateral to the surgical manipulation. Filaments ranged from 0.69 to 26 g (0.692, 1.202, 1.479, 2.041, 3.63, 6, 8, 10, 15 and 26). Each filament was applied 3 times to determine withdrawal. A positive response was defined as withdrawal from the von Frey filament. Confirmation of the paw withdrawal threshold (PWT) was tested by assessing the response to the filament above and below the withdrawal response. Rats were brought to the experimental room and allowed to habituate in the room for one hour prior to testing, and acclimated to the observation chambers for 15 minutes prior to taking PWT measurements.

#### *4.2. Pre-Operative Baseline Testing*

Prior to surgery, all rats were tested using the VF test. Rats that had an ipsilateral PWT of less than 12 g were excluded from the study.

#### *4.3. Post-Operative Testing*

Two weeks following surgery, baseline VF responses were taken and animals were balanced and assigned to treatment groups based on their post-operative PWT values. Animals with a VF score over 4.5 g were excluded from the study. The animals were divided into four groups. One group (*n* = 20) which only had sham surgery was assigned as the sham group (Table 1). The next three groups consisted of those rodents that had undergone SNL surgery and were assigned as the vehicle group (*n* = 40), AQU-118 group (*n* = 20) and positive control (i.e., gabapentin) group (*n* = 10) (Table 1). Rats were dosed with either AQU-118 of vehicle (via oral gavage once per day for 5 days. Gabapentin was administered (via oral gavage) only on von Frey test days (Day 1, 3, and 5). Von Frey PWT values were measured at 60 min following compound administration (Table 1).

#### 4.3.1. Compound Formulation/Administration

AQU-118 (160 mg/kg, 0.34 mmoles/kg, once per day dosing, SID) was dissolved in 0.5% methylcellulose (400 centipoise, cps) to give a fine suspension and administered orally (P.O.). for 5 days, 60 min prior to VF testing, at a dose volume of 5 mL/kg. AQU-118 was prepared fresh daily. The vehicle (0.5% methylcellulose,400 cps) was administered at a dose volume equivalent to the test compound (5 mL/kg). Gabapentin (100 mg/kg, 0.58 mmoles/kg, SID; Toronto Research Chemicals, North York, ON, Canada) was dissolved in saline and administered P.O. on Days 1, 3, and 5 of the treatment periods, 60 min prior to VF testing, at a dose volume of 1 mL/kg. Gabapentin was prepared daily.

#### 4.3.2. Tissue Collection & Preparation

After VF test on Day 5, rats were anesthetized by CO2. An incision was made in the right atrium and an automated pump was used to perfuse the animal with ice cold PBS. The spinal cord (L4–L6) and DRGs (L4, L5, and L6) from both sides (into separate left and right tubes) were collected, flash frozen, and stored in a −80 ◦C freezer until analysis. Frozen DRG tissue samples were homogenized in RIPA buffer (75 μL per sample) using a handheld pestle homogenizer, 30 s on ice. Samples were cleared by centrifugation at 14,000 rpm for 15 min at 4 ◦C. Supernatants were collected and protein amount was determined using Bio-Rad DC protein kit (Biorad Laboratories, Hercules, CA, USA). For DRG samples that were analyzed by both qPCR and Western, frozen DRG samples were crushed in mini liquid nitrogen cooled mortar. Ten percent of the tissue powder was used in RNA/transcript analysis. The remaining tissue was lysed in RIPA buffer and processed as indicated above.

#### *4.4. RNA and cDNA Preparation for qPCR*

Sample preparation was performed via a modification of a published literature procedure [33]. DRG tissue samples were homogenized 2 × 1 min at 25 Hz in 750 μL of QIAzol Lysis Reagent with Tissue Lyser (Qiagen, Valencia, CA, USA) and 5 mm stainless steel beads (Qiagen). Disrupted samples were incubated at room temperate for 5 min. For RNA extraction, manufacturer protocol for RNeasy 96 Universal Tissue Kit (Qiagen) for RNA isolation was followed. Briefly, 150 μL of Chloroform was added and samples were shaken vigorously for 15 s followed by 3 min incubation at room temperature. The aqueous phase was separated from the organic phase by centrifugation at 6000× *g* at 4 ◦C for 15 min. The aqueous phase was then transferred to a new 96-well block and total RNA was precipitated with equal volume of 70% ethanol and then transferred to a RNeasy 96-well plate followed by centrifugation at 6000× *g* at room temperate for 4 min. Total RNA bound to column membranes was treated with RNase-Free DNase set (Qiagen) for 30 min, followed by 3 washing steps with RW1 and RPE buffers (provided with RNeasy 96 Universal Tissue Kit). RNA was eluted with 20 μL RNase-Free water.

RNA was quantified using Nanodrop 8000. Total RNA (0.5 μg of RNA) was reverse transcribed into cDNA with 3.2 μg random hexamers (Roche Applied Science, Indianapolis, IN, USA), 1 mM each dNTP (Roche Applied Science), 20U Protector RNase Inhibitor (Roche Applied Science), 1xTranscriptor Reverse Transcription reaction buffer and 10U Transcriptor Reverse Transcriptase (Roche Applied Science) in 20 μL total volume. The reactions were allowed to proceed at room temperature for 10 min, 55 ◦C for 30 min and were then inactivated at 85 ◦C for 5 min in a GeneAmp PCR Systems 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). cDNA samples were diluted 10-fold with RNase-Free water for qPCR analysis.

#### *4.5. qPCR Analysis*

All qPCR reagents and TaqMan Expression Assays were purchased from ThermoFisher Scientific (Waltham, MA, USA). Briefly, 5 μL of diluted cDNA was amplified with qPCR primer and probe sets in 1x TaqMan Fast Advanced Master Mix in 20 μL final reaction volume. Reactions were run on Applied Biosystems 7900HT Fast Block System with the following parameters: 95 ◦C for 20 s; 40 cycles of (95 ◦C/3 s; 60 ◦C/30 s). Each cDNA sample was run in triplicate. The Ct value for serially diluted pooled (SNL) cDNA was plotted against the log value of dilution factor and the slope of the linear regression was determined. Only assays with r2 values greater than 0.95 were used in this study. PCR efficiency was calculated as follows:

PCR Efficiency = 10 − 1/slope

The relative mRNA expression for each target was calculated following the method of Pfaffl [34]:

ΔCtsample for Target = Ct Target control − Ct Target sample

ΔCtsample for GAPDH = Ct GAPDHcontrol − Ct GAPDH sample

Normalizing Target to GAPDH for each sample = PCR Efficiency Target (<sup>Δ</sup>Ctsample for Target)/PCR Efficiency GAPDH (ΔCtsample for GAPDH)

Normalized target expression was expressed relative to the average of the sham group.

#### *4.6. Western Blotting*

The procedure was performed following a published literature procedure [35]. IL-1β antibodies were purchased from three different vendors and validated using known standards of which only the antibody purchased from EMD Millipore passed and was used in the study. IL-6 antibodies were purchased from three different vendors of which two passed and were used in the study (R & D Systems and ThemoFisher) [19]. Caspase-3 antibody was purchased from Santa Cruz Biotechnology and MBP was purchased from Santa Cruz Biotechnology, both of which were validated and were used in the study. GAPDH was purchased from Cell Signalling Technology. Protein samples were denatured in Laemmli buffer/2-mercaptoethanol for 5 min at 95 ◦C. Denatured protein samples were separated by SDS-PAGE. After electrophoresis, proteins were transferred from gel to LFP-PVDF membranes by electroblotting. Non-specific binding of antibodies was blocked with 5% *w*/*v* dried milk in 1× TBST for one hour. After a brief rinse in TBST, the blots were probed with primary antibody (each diluted at 1:1000 in 1% *w*/*v* milk with 1× TBST) at 4 ◦C overnight. Protein-PVDF blots were washed once for 15 min followed by 3 more 5-min washes with TBST. Protein-PVDF blots were then incubated with the appropriate secondary antibody (diluted in 1:1000 prepared with 1% milk in TBST) for 1 h at room temperature. Protein-PVDF blots were washed once for 15 minutes followed by one more wash for 5 min. Antibody binding was detected using the ECL Plus Western Chemifluorescence Detection Kit (ThermoFisher Scientific). The detection solution was made fresh according to manufacturer's directions and dispensed onto membranes. After 5 min incubation, the protein-PDVF membranes were scanned using Typhoon 9410 scanner (GE Healthcare Bioscience, Pittsburgh, PA, USA) using 457 nm blue laser for excitation and 520 nm emission filter at 400 V. The scanned images from the Typhoon were analyzed with ImageQuant TL software version 7.0 (GE Healthcare Bioscience, Piscataway, NJ, USA). Band intensities were determined using the Rolling Ball method. Each protein target was first normalized to the in-lane housekeeping protein GAPDH (from the same gel). The normalized protein target for each sample was presented as a ratio relative to the sham vehicle group.

#### *4.7. Statistical Analyses*

Data at all time points post-surgery were analyzed by two-way repeated measures analysis of variance (RM ANOVA) with time as the within-subjects factor and treatment as the between-subjects factor. This was followed by Fisher PLSD post-hoc comparisons where appropriate (Appendix A). Pre-surgery baseline paw withdrawal data were analyzed by one-way ANOVA. An effect was considered significant if *p* < 0.05. Data are presented as the mean ± standard error of the mean (SEM) {\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.0001}.

#### **5. Conclusions**

The results of this study demonstrate for the first time that attenuation of mechanical allodynia via the oral dosing of the dual active MMP-2/MMP-9 inhibitor, AQU-118, in the SNL rat model of neuropathic pain is likely due to the regulation of apoptotic pathways, and specifically through reduction of caspase-3. We have confirmed previous reports that 25 days after SNL surgery, mRNA levels of MMP-2 but not of MMP-9 are elevated in the DRG. We have also been able to confirm previous reports that MBP declines and caspase-3 protein levels increase 25 days after SNL surgery. This study observed a linear relationship between levels of mRNA MMP-2 and that of IL-1β but not of IL-6. While increases in MBP or that of IL-1 β were not observed after daily oral dosing with AQU-118, their participation in attenuating mechanical allodynia cannot be ruled out at this point. While identifying a protein (caspase-3) that changes at the site of nerve damage that coincides with a reduction of mechanical allodynia is a good first step in helping elucidate a mechanism of action for AQU-118, more research will need to be performed to determine if this observation is applicable to non-rodent animal models of neuropathic pain. Additionally, while our initial focus has been on AQU-118's effect on attenuating neuropathic pain, it would be of interest to see if other neurological disorders that implicate a caspase-3 mediated apoptotic pathway would benefit from the intervention of an orally active MMP-2/-9 inhibitor.

**Author Contributions:** A.C., A.B. and A.H. performed the surgery and/or behavioral portions of the SNL rat model. M.Y.K. performed the biomarker portion of the SNL-rat model and edited the manuscript. J.B. and J.S. performed the biomarker portion of the SNL-rat model. A.G. supervised the biomarker portion of the SNL-rat model. T.H. supervised the surgical and behavioral portions of the SNL rat model. M.F. assisted in the design of the biomarker portion of the SNL-rat model and wrote and edited the manuscript. V.R.N. chemically synthesized and purified AQU-118. I.S. conceived and chemically synthesized AQU-118, designed the behavioral and biomarker portions of the SNL-rat model and wrote the manuscript. All authors read and approved the final manuscript.

**Funding:** This work was supported by a Small Business Innovation Research (SBIR) grant (#1R44DE022207-02) from the National Institutes of Health (NIH)/National Institute of Dental & Craniofacial Research (NIDCR).

**Conflicts of Interest:** I.S. owns stock in Aquilus Pharmaceuticals, Inc. All other authors declare they have no competing interests.

#### **Appendix A**

**Figure A1.** Statistical analysis of post-operative von-Frey (VF) baseline paw withdrawal threshold (PWT) of the ipsilateral hind paws. All Chung surgical groups showed a significant decrease in the PWT compared to the sham surgical group. No significant difference in post-operative baseline PWT was detected between the individual Chung surgical groups.

**Figure A2.** Fisher's protected least significant difference (PLSD) test of post-operative VF baseline PWT of the ipsilateral hind paws.

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**Figure A3.** Ipsilateral Two-Way RM ANOVA statistical analysis for behavioral portion (presented in Figure 1).

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**Figure A4.** Contralateral Two-Way RM ANOVA statistical analysis for behavioral portion (presented in Figure 2).

#### **References and Note**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Matrix Metalloproteinase-3 is Key E**ff**ector of TNF-**α**-Induced Collagen Degradation in Skin**

**Ursula Mirastschijski 1, Blaž Lupše 1, Kathrin Maedler 1, Bhavishya Sarma 1, Arlo Radtke 2, Gazanfer Belge 2, Martina Dorsch 3, Dirk Wedekind 3, Lisa J. McCawley 4, Gabriele Boehm 5, Ulrich Zier 1, Kazuhiro Yamamoto 6, Sørge Kelm <sup>1</sup> and Magnus S. Ågren 7,\***

Received: 10 October 2019; Accepted: 17 October 2019; Published: 22 October 2019

**Abstract:** Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (*p* < 0.001) in KO skin explants compared to WT control skin but did not differ (*p* = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (*p* = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.

**Keywords:** extracellular matrix; inflammation; cytokines; proteinases; interstitial collagens

#### **1. Introduction**

More than half a century ago, Gross and Lapière discovered collagenase-1, the first matrix metalloproteinase (MMP) [1]. Since then, research on MMP has yielded vast information on these zinc-dependent endopeptidases [2,3]. Expanding knowledge on MMP function and its impact on physiological and pathological processes has increased the interest in MMP substrates and has fueled further research [4]. Stromelysin-1 (MMP-3) belongs to the MMP family and is induced during development [5], wound repair [6], inflammation [7,8] and cancer [9]. Apart from cleaving extracellular matrix proteins, e.g., E-cadherin, laminins and type IV collagen, MMP-3 activates cytokines, growth factors [3,4] and other MMP members, e.g., the collagenases MMP-1, MMP-8 and MMP-13 and gelatinase B (MMP-9) [3]. In contrast to the collagenases MMP-1, MMP-8 and MMP-13, which are the main effectors of type I collagen degradation during wound repair, MMP-3 is incapable of cleaving native type I collagen. MMP-3 has been implicated in many inflammatory diseases, e.g., ultraviolet-B irradiation and photoaging [10], arthritis [11], after lung injury [8], vascular disease [12] and intestinal inflammation [13]. MMP-3-deficient mice show diminished inflammatory responses to various stimuli [14,15] and reduced cutaneous wound contraction [16]. Pro-inflammatory tumor necrosis factor-α (TNF-α) is an important mediator of inflammatory processes. TNF-α induces a variety of MMPs, e.g., MMP-1, MMP-3, MMP-8, MMP-9 and MMP-13 [4]. Apart from TNF-α converting enzyme (TACE) [17], MMP-3 was shown to activate TNF-α [18,19], yielding a 17 kDa peptide [20]. Activated TNF-α induces the downstream expression and secretion of MMP-3 [21,22]. In our previous study, human skin explant cultures challenged with TNF-α reacted with a concomitant and increased MMP-1, MMP-3 and collagen degradation. In this human model, we hypothesized that the increased collagen degradation was due to the activation of the collagenase MMP-1 by MMP-3 [23].

The primary aim of this project is to study the lack of MMP-3 and the effect of excessive MMP-3 on collagen degradation in skin. For this purpose, we incubate skin explants from MMP-3 knock-out [24] and MMP-3 transgenic mice [25]. Under the applied culture conditions, collagen catabolism dominates and the synthesis of new interstitial collagens is minimal [23]. MMP-1 is not expressed in mice but MMP-13 is thought to be the main collagenase and functional homologue of human MMP-1 [26]. Therefore, we study the interplay between MMP-3 and MMP-13. To enhance the extracellular detection of MMP-13, its endocytosis is blocked by adding the receptor-associated protein (RAP). Finally, the responsiveness of MMP-3-deficient or MMP-3-overexpressing murine skin to an inflammatory stimulus provided by TNF-α is studied. The broad-spectrum MMP inhibitor GM6001 is included in some experiments to elucidate the origin of collagen-degrading enzymes.

#### **2. Results**

#### *2.1. MMP-3 Expression in the Skin of the Four Murine Genotypes*

First, we quantified the MMP-3 mRNA (Figure 1A) and protein (Figure 1B) in the native skin of the four groups of mice. MMP-3 mRNA and protein were undetectable in the skin of MMP-3-deficient mice (KO). MMP-3 mRNA (*p* = 0.016) and protein levels were higher in the skin of transgenic (TG) animals compared with their wild-type (WTT) controls. WT and WTT contained similar MMP-3 mRNA and protein (approximately 0.2–0.3 ng) levels.

**Figure 1.** MMP-3 expression in native mouse skin. (**A**) MMP-3 mRNA levels determined by qPCR analysis. Boxes represent the 25th–75th percentile, whiskers the 5th–95th percentile and the horizontal lines within the boxes indicate the median values. (**B**) Representative western blot of six independent experiments of MMP-3 protein expression. Loading was normalized to the total protein (30 μg/lane) of the tissue extracts. GAPDH indicates the loading/transfer of proteins to the PVDF membrane. The arrow indicates the latent MMP-3 at 56 kDa. The position of the 50 kDa molecular weight marker is indicated to the right. KO (BL10), *n* = 5; WT (BL10), *n* = 5; WTT (FVB), *n* = 3; TG (FVB), *n* = 4.

#### *2.2. Collagen Degradation in Incubated Skin Explants of the Four Murine Genotypes*

We measured hydroxyproline in the media as an indicator of cleaved collagen molecules in the skin. MMP-3-deficient (KO) skin explants (BL10 genetic background) released a total amount of 90 μg hydroxyproline into the media over the eight-day incubation period, WT (BL10) skin 188 μg, WTT (FVB) skin 304 μg and MMP-3-overexpressing TG (FVB) skin 285 μg hydroxyproline (Table 1). Notably, the accumulated hydroxyproline release was higher in FVB WTT (304 μg) versus BL10 WT (188 μg) mice. Regression analysis was carried out to assess the rate of collagen degradation over the eight-day culture period and showed significant differences in the total hydroxyproline released between KO and WT (*p* < 0.001) skin (Table 1 and Figure 2A) but not between WTT and TG (*p* = 0.47) skin (Table 1 and Figure 2B).

**Table 1.** Hydroxyproline (μg) in conditioned media over eight days of culture (mean ± SEM).

**Figure 2.** The effect of the MMP-3 genotype (control), TNF-α treatment and GM6001 treatment on collagen degradation in skin measured by the release of hydroxyproline-containing peptides into media from KO and WT (**A**), and WTT and TG (**B**) skin explants incubated over eight days. The timely progression of hydroxyproline release was assessed by regression analysis of the slopes. Mean ± SEM (bars). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. The number of animals (*n*) used for theses analyses is given in Table 2. TNF-α (10 ng/mL). GM6001, broad-spectrum MMP inhibitor (10 μM). KO, MMP-3 knock-out; WT, wild-type control to KO; WTT, wild-type to transgenic mice (TG); TG, MMP-3 overexpression in skin. Light green bars, day 2; blue bars, day 4; yellow bars, day 6; dark green bars, day 8.

Functional MMP-3 activity was demonstrated by casein zymography in a conditioned medium from skin explants of WT mice but not from skin explants of KO mice (Figure 3A). Tissue extracts of incubated skin explants produced the two characteristic and specific <sup>3</sup> <sup>4</sup> and <sup>1</sup> <sup>4</sup> type I collagen fragments by the action of tissue collagenases. TIMP-1 blocked this endogenous collagenolytic activity. Furthermore, incubation with the organomercurial APMA partly compensated for the lack of MMP-3 (Figure 3B). Consequently, we next wanted to identify the collagenase(s) responsible for the observed collagenolytic activity.

**Figure 3.** MMP-3 (**A**) and collagenase (**B**) activities. (**A**) Casein zymography of conditioned media collected after eight days of incubation of KO and WT skin explants. Addition of the selective MMP-3 inhibitor UK370106 (1 μM) during incubation of the casein containing SDS-PAGE gels abolished the lysis bands at the arrowheads observed in the control WT media. Open arrowhead, latent MMP-3; solid arrowhead, active MMP-3. (**B**) Type I collagenolytic activity of tissue extracts of skin explants incubated for eight days. Collagenase activity of the extracts was determined in the absence (buffer) or in the presence of rhTIMP-1 (200 nM) or APMA (1 mM). KO, MMP-3 knock-out; WT, wild-type control to KO.

#### *2.3. MMP-13 in MMP-3-Deficient and MMP-3-Overexpressing Conditions*

Only true tissue collagenases are capable of cleaving native type I collagen. In rodents, these are MMP-1a [27], MMP-8 and MMP-13 [26]. MMP-3 indirectly contributes to collagenolysis via the activation of collagenases [28]. Here, neither MMP-1a nor MMP-8 was detected by western blot analysis. The main collagenase responsible for tissue homeostasis in rodents is also claimed to be MMP-13 [26]. Therefore, we focused on MMP-13 by determining the mRNA and protein levels in native and incubated skin.

In native skin, MMP-13 mRNA levels were similar in the KO, WT and WTT groups, while MMP-13 transcripts were undetectable in TG skin (Figure 4A). After eight days of incubation, MMP-13 mRNA levels differed among the groups with more (*p* = 0.03) MMP-13 mRNA present in KO skin compared with WTT skin (Figure 4B).

The MMP-13 protein in conditioned media was analyzed by western blot analysis (Figure 4C). After four days of culture, bands corresponding to latent (57 kDa) and active (48 kDa) MMP-13 were fainter in KO compared with WT. The catalytic domain of MMP-13, represented by the band at approximately 29 kDa, was prominent in WT but not in KO. Overall, the expression of MMP-13 was higher in WT compared to KO. In WTT, only faint bands of latent MMP-13 and of the catalytic domain fragment at 29 kDa were found. In contrast, TG-conditioned media presented prominent bands at the positions of latent and active MMP-13. Because MMP-13 detection can be jeopardized by rapid intracellular uptake via the LRP-1 receptor [29,30], the LRP-1 receptor antagonist RAP was used to inhibit MMP-13 endocytosis [31]. The presence of RAP during incubation yielded similar MMP-13 bands but fewer intermediate MMP-13 forms and more of the active MMP-13 compared to the controls (no RAP). After six and eight days of culture, no or minimal amounts of MMP-13 were detected in conditioned media.

**Figure 4.** MMP-13 mRNA (**A**,**B**) and protein expression (**C**) in MMP-3 deficiency and overexpression. (**A**,**B**) MMP-13 mRNA expression in (**A**) native skin and (**B**) in incubated skin after eight days of culture. Boxes represent the 25th–75th percentile, whiskers the 5th–95th percentile and the horizontal lines within the boxes indicate the median values. (**C**) MMP-13 protein in conditioned media after four days of the incubation of skin explants analyzed by western blot. Molecular weights in kDa are indicated to the left. The black arrow to the right indicates the position of latent MMP-13 (57 kDa), the white arrow indicates the active MMP-13 (48 kDa) and the grey arrow to the right indicates the fragment containing the catalytic domain (29 kDa). The band below the 29 kDa band represents the C-terminal domain of MMP-13 [30]. The displayed western blot is representative of four animals in each group. The loading of samples was calculated relative to a pool of day four-conditioned media from all control samples (50 μL of each sample were mixed). The reference pool was run together with the samples on 10% SDS-PAGE gels that were stained with Coomassie blue. The density of the common band at approximately 70 kDa was used for normalization (Supplementary Figure S1). KO, MMP-3 knock-out; WT, wild-type control to KO; WTT, wild-type to transgenic mice (TG); TG, MMP-3 overexpression in the skin. +RAP, addition of receptor-associated protein (RAP).

*2.4. E*ff*ect of Pro-Inflammatory TNF-*α *on MMP-3 Tissue Levels, MMP-13 Secretion and Collagen Degradation*

We next sought to investigate MMP-3 expression under an inflammatory stimulus provided by TNF-α. Expectedly, TNF-α addition increased the presence of latent and active MMP-3 in WT skin but not in KO skin (Figure 5).

**Figure 5.** Effect of TNF-α on MMP-3 tissue levels in KO and WT skin after eight days of incubation in the absence or presence of TNF-α (+TNF) analyzed by western blot. Loading was normalized to the total protein (10 μg/lane) of the tissue extracts. Molecular weights in kDa are indicated to the right. Positions of protein markers in kDa are to the right. KO (*n* = 4), MMP-3 knock-out skin; WT (*n* = 4), wild-type control to KO skin.

Moreover, TNF-α increased the secretion and activation of latent MMP-13 in the conditioned media of WT and KO after four days of culture with more fragments and less latent MMP-13 in WT and more latent MMP-13 in KO (Figure 6A). In contrast to the effect of TNF-α on skin from these animals with BL10 background, only mild effects were observed in the mice with FVB background (WTT and TG). TNF-α treatment increased the fragmentation of MMP-13 from incubated WTT skin explants and active MMP-13 at 48 kDa from incubated TG skin explants (Figure 6B).

**Figure 6.** Effect of TNF-α on MMP-13 levels in conditioned media of KO and WT (**A**), and WTT and TG (**B**) skin explants incubated without or with 10 ng/mL TNF-α (+TNF) for four days and analyzed by western blot. The black arrow indicates the position of latent MMP-13 (57 kDa), the white arrow indicates the active MMP-13 (48 kDa) and the grey arrow the fragment containing the catalytic domain (29 kDa). Loading of samples was calculated relative to a pool of day four-conditioned media from all (*n* = 17) control samples (50 μl of each sample were mixed). The reference pool was run together with the samples on 10% SDS-PAGE gels that were stained with Coomassie blue. The density of the common band at approximately 70 kDa was used for normalization (Supplementary Figure S1). The positions of protein markers in kDa are indicated to the left. KO (*n* = 5), MMP-3 knock-out; WT (*n* = 5), wild-type control to KO; WTT (*n* = 3), wild-type to transgenic (TG); TG (*n* = 4), MMP-3 overexpression in skin.

The incubation of WT skin explants with TNF-α was translated into increased (*p* = 0.0001) collagen degradation compared with the control-treated WT skin explants over the eight-day experimental period (Figure 2A). In contrast, the TNF-α addition to KO skin explants did not increase (*p* = 0.06) collagen degradation further compared with the control. No significant differences in collagen degradation were found between TNF-α-treated WTT and TG skin explants (Figure 2B).

#### *2.5. E*ff*ect of Pro-Inflammatory TNF-*α *on MMP-2 Secretion in MMP-3-Deficient and -Overexpressing Conditions*

Apart from MMP-3 [32], MMP-2 plays an auxiliary role in activating latent MMP-13 with the conversion of the intermediate form into the fully activated form of MMP-13 [33]. In the day eight-conditioned media, no significant differences were found among the four groups with or without TNF-α addition, although the FVB (WTT and TG) mice tended to secrete less MMP-2 compared with the BL10 (KO and WT) mice.

#### **3. Discussion**

Pro-inflammatory MMP-3 and TNF-α are associated with many diseases that are characterized by massive tissue destruction and collagen degradation. Previously, we showed that TNF-α promoted collagenolysis in human skin via the activation of MMP-1, possibly mediated by MMP-3 [23]. Furthermore, earlier studies indicated that MMP-3 increases collagenase activity in cultured murine bone and cartilage [34,35]. To investigate if MMP-3 per se has a role in collagen catabolism, we incubated skin from MMP-3-deficient (KO) and MMP-3-overexpressing mice (TG) and their respective wild-type counterparts (WT and WTT). We found massively reduced collagen degradation and fragmentation in MMP-3-deficient skin, which could possibly be ascribed to the reduced activation of the collagenase MMP-13.

The observed effect on collagen degradation could entirely be ascribed to the action of MMP-3 because addition of the non-selective MMP inhibitor GM6001 to the WT skin cultures did not decrease collagen loss more than that of the KO group. It should be emphasized that our sample size was small and therefore the risk of making a type II error is apparent.

We isolated proteinases from the incubated murine KO skin that produced the two <sup>1</sup> <sup>4</sup> and <sup>3</sup> 4 fragments of native type I collagen triple helix. The extracted proteinases could be activated by APMA and completely blocked by TIMP-1. This implied that one or more collagenases degraded the interstitial collagens of the skin and that the lack of collagenase activation appeared to be an underlying mechanism for reduced collagenolysis in MMP-3 deficiency. Corroborating these data are the findings by van Meurs et al. [35], who observed the specific single cleavage site in type II collagen in cultured cartilage from wild-type mice but not from MMP-3-deficient mice.

MMP-13 is thought to be the most powerful collagenase under normal and pathologic conditions [36]. The two other rodent collagenases, MMP-1a and MMP-8, were not expressed in significant amounts in our skin explant model. Consequently, we focused on the presence and activation state of MMP-13. We could show that MMP-3-deficient skin expressed similar amounts of MMP-13 mRNA compared to controls. The detection of the secreted MMP-13 protein is difficult due to its high affinity binding to LRP [29,30]. Hence, we added the LRP-1 receptor agonist RAP [31] to block MMP-13 endocytosis. Despite this manipulation, similar amounts of MMP-13 were present in the conditioned media of KO and WT samples treated with RAP. Nevertheless, differences in the MMP-13 levels were unlikely the cause for differential collagenolysis seen in MMP-3-deficient conditions. On the other hand, activated MMP-13 molecular species were reduced in KO skin in comparison with the wild-type counterpart. Apart from MMP-3, trypsin, plasmin, cathepsin B, MMP-2, MMP-14 and possibly MMP-8 can activate latent MMP-13 [32,33]. Here, MMP-2 was most likely not involved. Despite of multiple possibilities for MMP-13 activation, MMP-3 seemed to be the main activator of MMP-13 in our ex vivo skin model and the absence of the activation function of MMP-3 was not compensated for by other proteinases.

Besides being capable of activating proMMP-13, MMP-3 can activate growth factors and cytokines, notably proIL-1β. IL-1β in turn influences MMP-3 and MMP-13 expression and secretion [37,38].

Having established that collagen loss is severely reduced under MMP-3-deficient conditions and that the activation, rather than the concentration, of MMP-13 decreases collagen in skin, we next investigated the effect of pro-inflammatory TNF-α on MMP-13 expression and collagen loss. TNF-α is known to induce the expression of MMP-3 [23,39], MMP-9 [18,40] and MMP-13 [41]. TNF-α induced MMP-3 in WT but not in KO skin explants. Moreover, TNF-α augmented the activation of MMP-13 in the WT skin explants, presumably via MMP-3 induction. The increased MMP-13 activation by TNF-α would explain the increased collagen degradation and fragmentation in WT skin explants not found in the KO group. One can speculate about the impact of immune cells under in vivo conditions. Lauridsen et al. [42] reported higher levels of MMP-3 when pericytes were activated with TNF-α and co-cultured with neutrophils. Our results may well explain the pronounced collagen degradation and tissue destruction found in highly inflammatory and chronic progressing diseases.

The dependence of collagen degradation on the genetic background of the mice (BL10 versus FVB) was intriguing. Collagenolysis was not increased in the skin of the FVB mice overexpressing MMP-3. If we assume that the MMP-13 activity is the rate-limiting factor for collagenolysis in our model, then the content of MMP-13 is critical. MMP-13 levels were seemingly higher in MMP-3-overexpressed skin. This may indicate that the activity of MMP-3 was not increased in the MMP-3 transgenic group. Many proteinases activate proMMP-3 [28], for example, mast cell-derived proteinases [43].

Wild-type skin from BL10 mice responded with a massive increase of collagen degradation, MMP secretion and activation when TNF-α was added to cultures, whereas FVB murine skin did not respond to the TNF-α challenge. A similar finding was reported by Martin et al. [44] using a breast cancer model in C57Bl/6 and FVB/N mice. MMP-mediated effects on tumor angiogenesis were only found in C57BL/6 mice, with no effect in the FVB/N strain. Therefore, the choice of experimental animals with the appropriate genetic background is pivotal with respect to the interpretation of results and translation to the human situation.

#### **4. Materials and Methods**

#### *4.1. Animals*

MMP-3-deficient mice and the corresponding wild-type mice were donated by Dr. Gary Rosenberg, University of New Mexico, Health Science Center, Albuquerque, NM, USA [24]. The animals were crossed back to a homogeneous background of BL10 (C57BL10.RIIIH2r-Mmp3tm1Mol/Ztm) mice for 10 generations. Skin from four-month-old or six-month-old homozygous BL10 MMP-3 knock-out (KO) and BL10 wild-type (WT) mice was used in this study.

Mice overexpressing MMP-3 under the keratin-5 promoter were first described by Dr. Lisa McCawley [25]. This overexpression is confined mainly to the keratinocytes of the skin. The sperm of MMP-3-overexpressing mice was used to inseminate mice of the FVB genotype. The offspring were mated to mice with a homozygous background. Backcrossing was performed for 10 generations. Skin from three-month-old FVB MMP-3-overexpressing, transgenic (TG) and FVB wild-type (WTT) mice were used.

The animals were kept in Makrolon® Type 2L cages at 21 ◦C, with a relative humidity of 55% ± 5% and a light cycle of 10 h/14 h. Sterilized commercial softwood granulate bedding was used (Lignocel, Altromin, Lage, Germany). Autoclaved commercial pellets (Altromin 1314, Altromin, Lage, Germany) and autoclaved water were provided ad libitum. The microbiological status was examined as recommended by FELASA (Federation of European Laboratory Animal Science Associations) and the absence of listed microorganisms was confirmed [45]. We did not test for the presence of *Rodentibacter sp., Helicobacter spp., Staphylococcus aureus* or *Klebsiella oxytoca*.

The experiments were approved by the local Institutional Animal Care and Research Advisory Committee (522-27-11/02-00/118, 10 September 2013).

#### *4.2. Experimental Model and Treatment Groups*

After decapitation, the dorsal skin from the male mice of all genotypes was shaved, disinfected and excised under sterile conditions. The skin samples (approximately 5 cm × 5 cm) were prepared within 36 h after harvesting. The skin explants were excised with a sterile 8 mm or a sterile 4 mm trephine and incubated submerged in a serum-free culture medium in 24-well culture plates (Nunc, Roskilde, Denmark) at 37 ◦C in a humidified atmosphere of 5% CO2/air in a keratinocyte growth medium (KGM)-2 containing 6 mmol/L glucose, 50 ng/mL amphotericin-B, 100 μg/mL penicillin, 100 U/mL streptomycin and supplemented with 1.4 mM CaCl2 [23].

For each animal genotype, i.e., KO, WT, WTT or TG (Table 2), 8 mm skin explants were incubated in 1.0 mL medium in the absence (control) or presence of rmTNF-α (Sigma-Aldrich, St. Louis, MO, USA) or the broad-spectrum hydroxamate MMP inhibitor GM6001 (Merck-Millipore, Molsheim, France).

**Table 2.** Experimental groups.

Because MMP-13 is rapidly endocytosed intracellularly, extracellular detection of MMP-13 can be difficult. The internalization of MMP-13 is mediated by LRP-1 [29,30]. RAP is a ligand-binding antagonist for LRP-1 and inhibits proteinase endocytosis [31]. Hence, we used RAP in a second series of experiments to facilitate extracellular MMP-13 detection by blocking rapid internalization. Skin explants (4 mm) were incubated in 0.25 mL medium in the absence (control) or presence of the indicated treatments in Table 3.

**Table 3.** Experimental setting for the RAP (receptor-associated protein) experiment.

<sup>1</sup> 10 ng/mL. <sup>2</sup> 10 μM. <sup>3</sup> 250 nM.

Conditioned media were harvested and replaced by a fresh medium every second day. After eight days of incubation, the experiments were terminated, media were harvested, and skin explants were kept at −80 ◦C until analysis for MMP-3 and MMP-13 mRNA, and MMP-3 protein. Conditioned media were stored at −20 ◦C until analysis for hydroxyproline and MMP-13 protein.

#### *4.3. Analyses*

#### 4.3.1. Hydroxyproline Assay

Hydroxyproline levels in the conditioned media from days two, four, six and eight were measured colorimetrically [23].

#### 4.3.2. MMP-3 and MMP-13 mRNA Determined by qPCR

RNA was isolated from skin explants (approximately 30 mg) using TRIzol reagent (Thermo Fisher Scientific, Schwerte, Germany). Total RNA (approximately 100 ng) were reverse-transcribed into cDNA and subsequently analyzed for 40 cycles in qPCR. The qPCR was conducted on a 7500 Fast Real Time PCR system (Thermo Fisher Scientific, Schwerte, Germany) with TaqMan probes for murine MMP-3 (Mm00440295\_m1) and murine MMP-13 (Mm00439491\_m1). Murine β-actin (Mm02619580\_g1) and murine peptidylprolyl isomerase A (Mm02342430\_g1) were used as housekeeping genes. Each reaction was run in triplicate and the relative quantity of MMP-3 or MMP-13 was determined using the ΔΔCT method [46].

#### 4.3.3. Casein and Gelatin Zymography

Casein zymography (β-casein, Sigma-Aldrich, St. Louis, MO, USA) was performed as described elsewhere [23]. Mark12™ unstained protein standard (LC5677, Thermo Fisher Scientific, Schwerte, Germany) was run in a parallel lane. After electrophoresis, the gels were incubated in 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl2, 1 μM ZnCl2 and 0.1% Triton X-100 with or without the selective MMP-3 inhibitor UK370106 (R&D Systems, Minneapolis, MN, USA) for 72 h at 37 ◦C.

Gelatin zymography was used to analyze the MMP-2 content in 4× concentrated (Amicon Ultra-0.5 filter, Merck-Millipore, Darmstadt, Germany) conditioned media [23]. rmMMP-2 (924-MP, R&D Systems, Minneapolis, MN, USA) was run at 2 ng in parallel to estimate the MMP-2 content of the samples by the image analysis of digitized gels [47].

#### 4.3.4. Collagenase Activity

Tissue extracts of skin explants that had been incubated for eight days were prepared together with 1 mM Pefabloc SC (Roche Diagnostics GmbH, Mannheim, Germany) and concentrated 4x by Amicon Ultra-0.5 [23]. The collagenolytic activity was determined by incubating the extracts with native (trypsin-resistant) type I collagen from bovine skin for 240 h at 22 ◦C, as described elsewhere [23]. rhTIMP-1 (R&D Systems, Minneapolis, MN, USA) was added to one set of reaction vials to a final concentration of 200 nM and APMA (Sigma-Aldrich, St. Louis, MO, USA) to another set of reaction vials to a final concentration of 1 mM. TIMP-1 or APMA were present during the entire 240 h incubation period.

#### 4.3.5. Western Blot Analyses

Native and incubated skin explants were homogenized (Ultra-Turrax® T25 Basic, IKA Werke GmbH, Staufen, Germany) in RIPA (radioimmunoprecipitation assay) buffer or modified RIPA buffer at pH 7.4 (5 μL/mg tissue) containing 0.1 M Tris-HCl, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS (sodium dodecyl sulfate) [48] and EDTA (ethylendiamintetraacetate)-free proteinase inhibitor cocktail and 1 μM pepstatin A. Homogenates were then centrifuged at 12,000× g for 10 min, and the supernatants were stored at −20 ◦C until analysis. Protein concentration was determined using the Pierce BCA (bicinchoninic acid) Protein Assay kit (Thermo Fisher Scientific, Schwerte, Germany).

Tissue extracts, conditioned media, molecular weight markers (RPN800E, Sigma-Aldrich, St. Louis, MO, USA) and rmMMP-3 (RPA101Mu01, Cloud-Clone Corporation, Katy, TX, USA) were electrophoresed on 10% SDS-PAGE gel under reducing conditions and electrotransferred for seven min using iBlot apparatus (Invitrogen, Carlsbad, CA, USA) onto a polyvinylidene fluoride (PVDF) membrane (Immobilon®, Merck-Millipore, Darmstadt, Germany). Membranes were blocked in 5% fat-free milk (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h at room temperature and incubated with primary antibodies for 18 h at 4 ◦C (rabbit polyclonal anti-mouse MMP-1a antibody (1:200 dilution; 250750, Abbiotec, San Diego, CA, USA), anti-mouse MMP-3 peptide antibody [49,50], rabbit polyclonal anti-MMP-8 (1:1000 dilution; ABT38, Merck-Millipore, Darmstadt, Germany), rabbit polyclonal anti-MMP-13 antibody (1:250 dilution; ab39012, Abcam, Cambridge, UK), mouse monoclonal anti-β-actin antibody (1:40,000 dilution; A5441, Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-β-actin antibody (1:1000 dilution; #4967, Cell Signaling Technology, Leiden, The Netherlands) or rabbit monoclonal anti-GAPDH antibody (1:1000 dilution; #2118, Cell Signaling Technology, Leiden, The Netherlands)). Then, membranes were incubated for 1 h at room temperature with IgG-horseradish

peroxidase secondary antibodies (Jackson ImmunoResearch, Ely, UK), and a specifically bound antibody was detected using an immunodetection kit (Amersham ECL Prime Western Blotting Detection Reagent, GE Healthcare Life Sciences, Amersham, UK), and then exposed to X-ray film (Amersham Hyperfilm ECL, GE Healthcare Life Sciences, Amersham, UK).

For the analysis of MMP-13 in conditioned media by western blot, the loading of samples was normalized from Coomassie blue-stained 10% SDS-PAGE gels because the BCA assay was not sensitive enough for these samples. On each gel, the samples were run with a reference sample composed of conditioned media from all control-treated samples (50 μL of each sample were mixed) in each specific experiment. The density of a common band at approximately 70 kDa was determined by ImageJ™ and used for the normalization of protein loading (Supplementary Figure S1).

#### *4.4. Statistics*

The Shapiro–Wilk test was used to assess for normality. A Student's t-test was used for the statistical analysis of results with normal distribution, and otherwise the Wilcoxon Rank Sum test and the Mann–Whitney U test using GraphPad Prism 8.0.2 software (GraphPad Software Inc., San Diego, CA, USA) were applied. For the comparison of hydroxyproline release over time, the slopes for two independent samples were compared after regression analysis. The Bonferroni correction was used in the cases of multiple comparisons. *p* < 0.05 was considered significant.

#### **5. Conclusions**

Collagen degradation in murine skin is MMP-3 and MMP-13 dependent, with MMP-3 being a potent activator of MMP-13. The pro-inflammatory cytokine TNF-α enhanced collagenolysis via the up-regulation of MMP-3 and increased the activation of MMP-13. Co-targeting MMP-3 in inflammatory diseases seems an appropriate measure to enhance the anti-inflammatory impact of clinically used drugs.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/20/ 5234/s1.

**Author Contributions:** Conceptualization, U.M., S.K. and M.S.Å.; methodology, B.L., K.M., B.S., A.R. and G.B.; software, U.Z.; validation, U.M., S.K. and M.S.Å.; formal analysis, U.M. and U.Z.; investigation, B.L., K.M., B.S., G.B., A.R., D.W. and M.D.; resources, D.W., M.D., K.Y. and S.K.; data curation, U.M., U.Z. and G.B.; writing—original draft preparation, U.M., B.L., K.M., B.S., G.B., D.W., A.R., G.B. and M.S.Å.; writing—review and editing, U.M., K.M., A.R., G.B., L.J.M. and M.S.Å.; visualization, U.M. and U.Z.; supervision, U.M., K.M., S.K., G.B. and M.S.Å.; project administration, U.M.; funding acquisition, U.M., K.M. and K.Y.

**Funding:** The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement NO. 243195 (to U.M.). K.Y. is supported by Versus Arthritis Career Development Fellowship (21447). B.L.'s work was supported by the DFG grant MA 4172/13-1 (to K.M.).

**Acknowledgments:** Antonio Jimenez-Caliani, Cristina Barria, Petra Berger, Katrischa Hennekens and Nilima Dinesh at the University of Bremen are acknowledged for technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Stimulation of Peritoneal Mesothelial Cells to Secrete Matrix Metalloproteinase-9 (MMP-9) by TNF-**α**: A Role in the Invasion of Gastric Carcinoma Cells**

**Teruaki Oku 1,†, Kentaro Shimada 1,†, Hiroki Kenmotsu 1, Yusuke Ando 1, Chisato Kurisaka 1, Rikio Sano 1, Makoto Tsuiji 1, Shinya Hasegawa 2, Tetsuya Fukui <sup>2</sup> and Tsutomu Tsuji 1,\***

Received: 12 October 2018; Accepted: 6 December 2018; Published: 9 December 2018

**Abstract:** It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.

**Keywords:** TNF-α; matrix metalloproteinase; peritoneal mesothelial cell; gastric cancer; metastatic dissemination

#### **1. Introduction**

Metastatic peritoneal dissemination causes a poor prognosis for patients with advanced gastric cancer. Cancer metastasis depends on multiple interactions between cancer cells and various factors derived from the host microenvironment, including cytokines, motility factors, cell adhesion molecules, and matrix-degrading enzymes. On the other hand, many recent studies have suggested the relevance of inflammation to cancer progression. In particular, tumor necrosis factor-α (TNF-α) and other inflammatory cytokines have been shown to participate in the initiation and progression of cancer [1–5]. TNF-α is a multifunctional cytokine that modulates various aspects of cancer cell phenotypes, such as cell proliferation, migration, invasion, and metastatic potential, in addition to causing the death of cancer cells. TNF-α, either alone or in combination with transforming growth factor-β, has also been shown to induce so-called epithelial-mesenchymal transition (EMT), which includes changes in both morphological and invasive phenotypes [6,7]. It has been well established that TNF-α induces the production of matrix metalloproteinases (MMPs) by carcinoma cells originating from various tissues,

and these MMPs facilitate cancer cell invasion and metastasis [8–13]. A recent clinical study of patients with gastric cancer indicated that TNF-α expression was significantly different between patients with and those without peritoneal metastasis and was one of the risk factors for peritoneal metastasis of gastric cancer [14].

We have studied the interaction of gastric carcinoma cells with the extracellular matrix (ECM) deposited by peritoneal mesothelial cells and found that MKN1 human gastric carcinoma cells secreted MMP-9 accompanied by enhanced invasion through Matrigel-reconstituted basement membranes when these cells adhered to ECM containing laminin-332, a major component of submesothelial basement membranes [15]. Moreover, the phenotype of MKN1 cells was found to be more invasive in the presence of TNF-α derived from tumor-associated macrophage-like cells in association with enhanced MMP-9 secretion [16]. In the present study, we co-cultured MKN1 cells with mesothelial cells isolated from murine peritoneum and examined the effects of treatment with TNF-α on MMP-9 production by these cells. The TNF-α treatment of these cells resulted in the induction of MMP-9 secretion not only from MKN1 carcinoma cells but also from mesothelial cells. This result prompted us to characterize the enhanced MMP-9 production by mesothelial cells upon TNF-α treatment. We also present evidence showing that MMP-9 secreted from mesothelial cells plays a significant role in the invasion of gastric carcinoma cells. For this purpose, we used a reconstituted mesothelium consisting of peritoneal mesothelial cells and Matrigel basement membranes in a Boyden chamber system.

#### **2. Results**

#### *2.1. Mesothelial Cells Secreted MMP-9 in Response to Stimulation with TNF-α*

We first examined the effects of TNF-α on MMP-9 secretion in the co-cultures of MKN1 and peritoneal mesothelial cells. Zymographic analysis of the conditioned medium of the co-culture of these cells revealed that MMP-9 secretions from both MKN1 and murine peritoneal mesothelial cells were markedly increased in the presence of TNF-α (10 ng/mL) (Figure 1). Since murine MMP-9 has a slightly higher molecular mass (105 kDa for proMMP-9) than its human counterpart (92 kDa) due to the presence of an extra amino acid sequence in murine MMP-9 [17], the two molecules can be distinguished from each other by zymography. By contrast, the MMP-2 level was not significantly changed in the conditioned medium after TNF-α treatment. The upregulation of MMP-9 secretion from both types of cells by TNF-α treatment (10 ng/mL) was confirmed when TNF-α was added to the separate cultures of MKN1 and mesothelial cells (Figure 2A). We observed low-level secretion of MMP-2 from both cell types in the absence of TNF-α, and the secretion was not substantially influenced by TNF-α treatment in either case. We then examined the dose-response of the potentiation of MMP-9 secretion by TNF-α. In the zymographic analysis, we detected MMP-9 activity in the conditioned medium of mesothelial cell culture in the presence of TNF-α (1 ng/mL) for 24 or 48 h, and the MMP-9 secretion was dose-dependently increased (up to 100 ng/mL) (Figure 2B). In the kinetic analysis, we detected MMP-9 activity after the culture with TNF-α for 12 h, and MMP-9 secretion was increased at 24 and 48 h (Figure 2C). These results clearly indicated that TNF-α treatment induced MMP-9 secretion from mesothelial cells, in addition to enhancing MMP-9 secretion from MKN1 gastric carcinoma cells.

We next conducted RT-qPCR to measure mRNA levels for MMP-9 in mesothelial cells after TNF-α treatment. The results shown in Figure 3A indicated that the mRNA level in mesothelial cells was elevated at 6 h after the stimulation by TNF-α (10 ng/mL) and reached the maximum level at 12 h, where it remained until 24 h after the stimulation. The increase in MMP-9 mRNA expression was found to be dose-dependent within the range of 1–100 ng/mL of TNF-α (Figure 3B), which was in good agreement with the results of the zymographic analysis (Figure 2B). However, the mRNA for MMP-2 exhibited no significant change after TNF-α treatment.

**Figure 1.** Zymographic analysis of MMPs in the conditioned medium of the co-culture of MKN1 and mesothelial cells after TNF-<sup>α</sup> treatment. A suspension of MKN1 cells (5 <sup>×</sup> 104 cells/0.2 mL) in ASF104 medium was added to a monolayer of murine peritoneal mesothelial cells in a 96-well culture plate and the cells were cultured with/without TNF-α (10 ng/mL) for 48 h. The culture supernatant was analyzed by gelatin zymography.

**Figure 2.** Effects of TNF-α on the secretion of MMP-9 from peritoneal mesothelial cells as assessed by zymography. (**A**) Monolayers of peritoneal mesothelial cells and MKN1 cells were separately cultured in ASF104 serum-free medium with/without TNF-α (10 ng/mL) for 48 h. (**B**) Mesothelial cells were cultured with TNF-α (0, 1, 10 or 100 ng/mL) for 24 or 48 h. (**C**) Mesothelial cells were cultured with TNF-α (10 or 100 ng/mL) for 6, 12, 24 or 48 h.

**Figure 3.** RT-qPCR analysis of mRNA for MMP-2 and MMP-9 in mesothelial cells after treatment with TNF-α. (**A**) Mesothelial cells were cultured in ASF104 serum-free medium containing TNF-α (10 ng/mL) at 37 ◦C for 0, 6, 12 or 24 h. Total RNA was then isolated, and RT-qPCR for the genes for MMP-2 (*open bar*) and MMP-9 (*closed bar*) was carried out as described in Section 4. (**B**) Mesothelial cells were cultured in ASF104 medium containing TNF-α (0, 1, 10 or 100 ng/mL) for 6 h. The gene expressions of MMP-2 (*open bar*) and MMP-9 (*closed bar*) were analyzed by the relative standard curve method using Gapdh as an internal control. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student's *t*-test. \*\*\* *p* < 0.005 vs. control.

#### *2.2. TNF-α Potentiates MKN1 Cell Invasion through the Reconstituted Mesothelium*

Because the above experiments indicated that mesothelial cells secreted MMP-9 in response to TNF-α treatment, we designed an artificial, reconstituted mesothelium where a monolayer of mesothelial cells was cultured on a Matrigel layer in a Boyden chamber system (Figure 4A) and examined the effects of TNF-α on carcinoma cell invasion. Mesothelial cells isolated from the murine peritoneum grew as a monolayer with polygonal morphology after 4–5 days (Figure 4B). The transmigration of MKN1 cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner (Figure 4C).

We previously found that the interaction between α3β1 integrin on cancer cells and laminin in the mesothelium played an important role in the cancer cell adhesion and invasion [15,18]. Next, we examined the effects of the anti-α3 integrin antibody on the transmigration of MKN1 cells through the reconstituted mesothelium. The cell invasion potentiated by TNF-α was significantly inhibited by the anti-α3 integrin antibody (Figure 5A), suggesting the importance of an α3β1 integrin-dependent process in the invasion. The adhesion of MKN1 cells to a monolayer of mesothelial cells was also increased after the TNF-α treatment of mesothelial cells and was partially inhibited by the anti-α3 integrin antibody (Figure 5B). Mochizuki et al. [19] reported that the treatment of mesothelial cells with TNF-α induced their morphological change followed by an increase in the areas of intercellular gaps. This process may cause exposure of the submesothelial extracellular matrix (ECM) in the intercellular gaps. Because laminin-332, a counter-ligand for α3β1 integrin, is a major component of submesothelial ECM, TNF-α treatment might facilitate the adhesion of MKN1 cells to the mesothelium via α3β1

integrin/laminin-332 interaction. In RT-qPCR analysis, we observed a slight increase in expression of the γ2 subunit of laminin-332 after TNF-α treatment of mesothelial cells (Figure S1), and this might also have caused the increased adhesion of MKN1 cells.

**Figure 4.** Cell invasion assay using a reconstituted artificial mesothelium in a Boyden chamber (Transwell) system. (**A**) The inner chamber with a membrane (8.0 μm pore) was composed of a monolayer of peritoneal mesothelial cells on a Matrigel layer and was utilized to examine the migration of MKN1 cells. The outer chamber was filled with ASF104 medium supplemented with HT1080 serum-free conditioned medium as a chemoattractant. (**B**) Microscopic observation of a monolayer of mesothelial cells (scale bar = 20 μm). (**C**) After mesothelial cells were treated with TNF-α (1, 10 or 100 ng/mL) and washed with ASF104 medium, MKN1 cells (1 <sup>×</sup> 105 cells/0.2 mL) were placed in the inner chamber and incubated at 37 ◦C for 16 h. The cells migrating into the outer chamber through the membrane were counted under a microscope after staining with Diff-Quik. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student's *t*-test. \*\*\* *p* < 0.005 vs. the control.

#### *2.3. The Exogenous Addition of MMP-9 Promotes MKN1 Cell Invasion*

Because our experiments demonstrated that TNF-α potentiated MMP-9 secretion from both mesothelial cells and cancer cells, we next addressed the effect of the exogenous addition of MMP-9 on the invasive behavior of cancer cells. When MMP-9 purified from human THP-1 leukemic cells (Figure S2) was added to the culture of MKN1 cells, their invasion into Matrigel basement membranes was increased by approximately 29% (Figure 6A). To exclude the contribution of secretion of MMP-9 and MMP-2 from cancer cells, we established MMP-knockdown cells by the RNA interference procedure (Figure S3). The silencing of MMP-9 expression in MKN1 cells resulted in marked impairment of the invasion (~90% inhibition), whereas MMP-2 silencing exhibited only a moderate effect (~30% inhibition) (Figure 6A,B). The addition of MMP-9 to the culture potentiated the invasion of MMP-9-knockdown cells in a dose-dependent manner (Figure 6B). These results suggest that the invasion of cancer cells is influenced by MMP-9 secreted from TNF-α-stimulated mesothelial cells.

**Figure 5.** Invasion and adhesion of MKN1 cells and effects of the anti-α3 integrin antibody. A monolayer of mesothelial cells was stimulated with TNF-α (10 ng/mL) for 6 h. (**A**) MKN1 cells (1 <sup>×</sup> 105 cells/0.2 mL) in ASF104 serum-free medium were added to the inner chamber of the reconstituted mesothelium and incubated at 37 ◦C for 16 h. The cells that had migrated into the outer chamber through the membrane were counted under a microscope. (**B**) Fluorescently labeled MKN1 cells were added to the monolayer of mesothelial cells in a 96-well culture plate, and incubated at 37 ◦C for 40 min. After non-adherent cells were removed by washing, adherent cells were lysed with 1% Nonidet P-40 and measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). For the inhibition experiments, MKN1 cells were treated with the anti-α3 integrin antibody (SM-T1, 10 μg/mL) at 0 ◦C for 30 min. Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student's *t*-test. \*\* *p* < 0.01, \*\*\* *p* < 0.005.

**Figure 6.** Potentiation of the invasion of MMP-9-knockdown MKN1 cells by exogenous MMP-9. MMP-2- or MMP-9-knockdown MKN1 cells were prepared by RNA interference as described in the Materials and Methods section. (**A**) The MMP-9-knockdown MKN1 cells (*right*) and parent cells (*left*) were assayed for in vitro invasion using Matrigel basement membranes in the presence or absence of MMP-9 purified from THP-1 cells. After culturing for 16 h, the cells that had migrated into the lower chamber were microscopically observed (40×). (**B**) MMP-2- or MMP-9-knockdown MKN1 cells were subjected to an invasion assay, and the migrated cells were counted under a microscope (*Left*). The invasion assay was performed in the presence of purified MMP-9 (2.5–10 μg/mL) (*right*). Experiments were performed in triplicate, and the data are presented as the mean ± SEM. Statistical data analysis was conducted using the Student's *t*-test. \*\*\* *p* < 0.005.

#### **3. Discussion**

It has recently been recognized that inflammatory cytokines such as TNF-α play crucial roles in metastatic dissemination. For example, TNF-α treatment of human gastric cancer cells was shown to increase MMP-9 mRNA and protein expression levels [20]. In the present study, we demonstrated that peritoneal mesothelial cells also secreted MMP-9 in response to TNF-α (Figures 1 and 2) and that the enhanced secretion of MMP-9 was associated with upregulation of MMP-9 transcription (Figure 3). In addition, we suggested the importance of the MMP-9 secreted by host mesothelial cells in gastric carcinoma invasion by utilizing a reconstituted mesothelium consisting of a monolayer of peritoneal mesothelial cells layered on a Matrigel basement membrane (Figure 4). The increase in cell invasion was shown to partly involve an integrin-dependent process (Figure 5). In addition to matrix-degrading enzymes secreted by the cancer cells themselves, MMP-9 derived from the host microenvironments, including peritoneal mesothelial cells, appeared to contribute to the cancer cell invasion into submesothelial matrices (Figure 6). Although mesothelial cells were thought to play a structural role in maintaining the smooth surface of the peritoneum and possess rather static properties, recent studies have shown that mesothelial cells produce various growth factors (e.g., vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, and transforming growth factor-β) and ECM components (e.g., cytokeratin, vimentin and type I and III collagen) [21]. Therefore, peritoneal mesothelial cells are considered to be potentially active cells that dynamically respond to environmental changes. Chen and co-workers reported that TNF-α induced MMP-9 synthesis in human pleural mesothelial cells associated with *Mycobacterium tuberculosis* infection and that this process might be implicated in the pathogenesis of tuberculous pleuritis [22]. TNF-α is a strong activator for NF-κB, and MMP-9 expression is known to be upregulated via activation of NF-κB signaling in various types of cells [23,24]. The signaling pathway involved in the enhanced MMP-9 expression in mesothelial cells should be clarified in future studies.

The origin of TNF-α involved in the stimulation of mesothelial cells is unclear at present. Considering that micrometastasis in the early stage of peritoneal dissemination takes place mainly in the omentum, where many small lymphoid tissues called milky spots are present [25,26], macrophages and lymphoid cells accumulated in milky spots are likely to secrete TNF-α. Another candidate would be the macrophages infiltrating into tumors, which are collectively called tumor-associated macrophages (TAMs) and are known to produce various cytokines depending on the stage and types of tumors [16,27,28].

A recent clinical study on patients with gastric cancer indicated a positive correlation between TNF-α expression and peritoneal metastasis [14]. The results showed that the TNF-α expressions were significantly different between the metastatic group and non-metastatic group, and the logistic regression analysis indicated that TNF-α expression was a risk factor for peritoneal metastasis of gastric cancer. TNF-α has been shown to deteriorate malignant phenotypes of cancer cells, including cell proliferation, migration, epithelial-mesenchymal transition, and productivity of cytokines and growth factors. In addition to these effects on cancer cells, it seems important that the phenotypic changes of mesothelial cells induced by TNF-α, especially the enhancement of MMP-9 secretion, may cause peritoneal metastasis, and thus a poor prognosis for patients with gastric cancer. The relationship between the level of MMP-9 in the gastric carcinoma environment and the malignant invasive phenotypes of gastric carcinoma, including lymph node metastasis, has been extensively studied, and the data suggest that MMP-9 plays important roles in the progression of gastric cancer [29–31]. Therefore, it would be feasible that MMP-9 and its endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs) are preventive and therapeutic targets for cancer metastasis.

We have previously studied the interaction of gastric carcinoma cells with the mesothelium and found that the integrin-dependent adhesion of carcinoma cells to the submesothelial matrix promotes MMP-9 secretion from cancer cells [15]. TNF-α treatment of mesothelial cells has been shown to induce morphological changes characterized by cell rounding and disruption of the cell–cell contacts, leading to the expansion of intercellular gaps [19]. It is likely that the adhesion of cancer cells to the exposed submesothelial matrix is stabilized, which might promote further upregulation of MMP-9 secretion and successive invasion of cancer cells. In conclusion, the present study strongly suggests that TNF-α-induced MMP-9 secretions from mesothelial cells play an important role in the metastatic dissemination of gastric cancer.

#### **4. Materials and Methods**

#### *4.1. Reagents*

RPMI1640 medium, trypsin, Triton X-100, Brij 35, TriReagent and hexadimethrine bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan). Bovine serum albumin (BSA), gelatin, dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA) and polyethylene glycol (PEG) 6000 were purchased from Wako Pure Chemical Industries (Osaka, Japan). Polyethylenimine "Max" was purchased from Polysciences Inc. (Warrington, PA, USA). Fetal calf serum (FCS) was obtained from Biosera (Boussens, France). ASF104 serum-free medium was supplied by Ajinomoto (Tokyo, Japan). Matrigel was purchased from BD Biosciences (San Diego, CA, USA). The PrimeScript RT Reagent Kit and KAPA SYBR FAST qPCR Kit Master Mix (2×) ABI Prism were obtained from Takara Bio Inc. (Shiga, Japan) and KAPA Biosystems (Boston, MA, USA), respectively. ViraPower Lentiviral Packaging Mix and puromycin dihydrochloride were supplied by Life Technologies (Carlsbad, CA, USA). Oligonucleotides were supplied by FASMAC (Kanagawa, Japan). Recombinant murine TNF-α was a product of Peprotech (Rocky Hill, NJ, USA). A fluorescent dye, 3 -*O*-acetyl-2 , 7 -*bis*(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethyl ester (BCECF-AM), was obtained from Dojindo Laboratories (Kumamoto, Japan). Gelatin Sepharose 4B was a product of GE Healthcare (Piscataway, NJ, USA).

#### *4.2. Cells*

The MKN1 human gastric carcinoma, HT1080 human fibrosarcoma, and THP-1 human acute monocytic leukemia cell lines were supplied by the RIKEN Cell Bank (Tsukuba, Japan) and cultured in RPMI 1640 medium supplemented with 10% FCS at 37 ◦C under 5% CO2. Mesothelial cells were isolated under sterile conditions essentially as described by Nakashio et al. [32]. Briefly, the parietal peritoneum and diaphragm excised from ddY mice were washed with phosphate-buffered saline (PBS) and incubated with 0.25% trypsin in PBS at 37 ◦C for 30 min with gentle mixing every 5 min. After an equal volume of RPMI 1640/10% FCS was added to the cell suspension, the mixture was centrifuged at 2000 rpm for 10 min. The pelleted cells were suspended and incubated with ammonium-chloride-potassium (ACK) lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) to remove red blood cells at room temperature for 1 min. After 10 volumes of RPMI 1640 medium/10% FCS was added, the mesothelial cells were recovered by centrifugation at 2000 rpm for 10 min. The pelleted cells were resuspended in RPMI 1640 medium/10% FCS and passed through nylon mesh (40 <sup>μ</sup>m), and an aliquot (2 × 105 cells/0.1 mL) was placed in a 96-well culture plate that had been coated with 10% Matrigel at 4 ◦C for 16 h. The cells were cultured at 37 ◦C for 4–5 days until the growth of a monolayer of polygonal mesothelial cells. This procedure was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Hoshi University School of Pharmacy and Pharmaceutical Sciences (accredited by the Ministry of Education, Culture, Sports, Science and Technology, Japan).

#### *4.3. Gelatin Zymography*

MMP activity was assayed by gelatin zymography essentially as described previously [33]. Briefly, MKN1 and/or mesothelial cells were treated with TNF-α (0, 1, 10, 100 ng/mL) in ASF104 serum-free medium for 0–48 h, and the culture supernatants were mixed with sample buffer for SDS-polyacrylamide gel electrophoresis (Laemmli's buffer without reducing agent). Specimens were electrophoresed on a polyacrylamide gel (7.5%) containing 0.15% gelatin. The gel was washed three times with 1% Triton X-100 for 20 min each, and three times with water for 20 min each. Then gelatinolytic activity was detected by incubating the gel for 16 h at 37 ◦C in 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 1 μM ZnCl2 followed by staining with Coomassie brilliant blue (CBB) R-250 (Merck, Darmstadt, Germany).

#### *4.4. Quantitative RT-PCR (RT-qPCR)*

Total RNA was isolated from mesothelial cells using TriReagent, and cDNA was synthesized with a PrimeScript RT reagent kit using the total RNA as a template according to the manufacturer's protocols. The qPCR reactions were conducted with an Applied Biosystems StepOne system (Life Technologies) using a KAPA SYBR FAST qPCR Kit Master Mix (2×) ABI Prism under the following conditions: Denaturation at 95 ◦C for 20 s followed by 40 cycles of 95 ◦C for 5 s, 55 ◦C for 30 s and 72 ◦C for 30 s. Melting curve analysis and agarose gel electrophoresis of the PCR products were performed to verify the specific amplification. All samples were analyzed in triplicate and quantified by the relative standard curve method using the *Gapdh* housekeeping gene as an internal control. The sequences of the primers used were as follows: *Mmp2*, 5 -ATC GCT CAG ATC CGT GGT G-3 (forward) and 5 -GGA GCT CAG GCC AGA ATG T-3 (reverse); *Mmp9*, 5 -ATG TAC CCG CTG TAT AGC TAC C-3 (forward) and 5 -ATA GTG GGA CAC ATA GTG GGA G-3 (reverse); *Gapdh*, 5 -TGA AGC AGG CAT CTG AGG G-3 (forward) and 5 -CGA AGG TGG AAG AGT GGG AG-3 (reverse).

#### *4.5. Cell Invasion Assay Using Reconstituted Mesothelium*

Reconstituted artificial mesothelium consisting of a monolayer of mesothelial cells on a Matrigel layer in a Boyden chamber (Transwell) system was utilized for cell invasion assay. Murine mesothelial cells were isolated as described above and cultured in a 10% Matrigel-coated inner chamber with a polyethylene terephthalate (PET) membrane (pore size, 8 μm; Falcon 353097; BD Biosciences) combined with an outer chamber (Falcon 353504) for 4–5 days until the cells grew as a confluent monolayer. An MKN1 gastric carcinoma cell suspension (1 × 105 cells/0.2 mL) was placed in the inner (upper) chamber, and the outer (lower) chamber was filled with ASF104 serum-free medium supplemented with HT1080 serum-free conditioned medium. The addition of HT1080 conditioned medium (20%) increased the chemotactic activity, although the conditioned medium contained a moderate level of MMP-9 secreted by HT1080 cells. After the chambers were incubated at 37 ◦C for 16 h, the cells that had migrated into the lower chamber through the membrane were stained with Diff-Quik (International Reagents, Kobe, Japan) and counted under a microscope.

#### *4.6. Cell Adhesion Assay*

MKN1 cells were labeled with the fluorescent dye BCECF-AM (3 μM) at 37 ◦C for 40 min [18]. The labeled cells (1 × 105 cells/0.1 mL in 1% BSA containing RPMI 1640 medium) were added to the monolayer of mesothelial cells in a 96-well culture plate as described above. After the plate was incubated at 37 ◦C for 40 min, non-adherent cells were removed by gentle washing three times with PBS. Adherent cells were lysed with 0.1 mL of 1% Nonidet P-40, and the lysates were diluted with 9 volumes of PBS. The fluorescence intensities were measured with a fluorescence spectrophotometer (Ex = 490 nm, Em = 520 nm). The percent adhesion was calculated as follows: (The fluorescence intensity of adherent cells/fluorescence intensity of total cells) × 100.

#### *4.7. Flow Cytometry*

The expression of α3 integrin in MKN1 cells was measured by a flow cytometer (BD FACSVerse; BD Biosciences, San Diego, CA, USA), using monoclonal antibodies against human integrin α3 subunit (SM-T1) [34] and FITC-labeled anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA).

#### *4.8. MMP-Knockdown MKN1 Cells*

Knockdown of MMP-2 and MMP-9 was conducted by using lentivirus-mediated RNA interference. The short hairpin RNAs (shRNAs) of target sequences against MMP-2 (185–203: AGG AGA GCT GCA TCC TGT T) [35] and MMP-9 (343–361: AAG TGG TAC CAC CAT AAC A) [36] were cloned into the pSIH-H1 vector (System Biosciences, Palo Alto, CA, USA) according to the manufacturer's protocol. Lentiviral particles were produced in HEK293FT cells after the co-transfection of pSIH-H1 vector and ViraPower (three packaging plasmids) using polyethylenimine "MAX" [37] for 48 h and concentrated by the PEG precipitation method. Briefly, the culture medium containing viral particles was mixed with a one-third volume of 4 × PEG solution (32% PEG, 400 mM NaCl, 40 mM HEPES-NaOH, pH 7.4) and incubated at 4 ◦C overnight. After centrifugation (3000× *g*, 30 min), the obtained precipitate was dissolved in serum-free RPMI 1640 medium. The virus solution was mixed with hexadimethrine bromide (8 μg/mL at final concentration), and the mixture was added to pre-cultured MKN1 cells (1 × <sup>10</sup><sup>5</sup> cells/2 mL in a 6-well plate). The plate was then centrifuged (1800× *<sup>g</sup>*, 90 min) at 32 ◦<sup>C</sup> and incubated at 37 ◦C for 48 h. The cells were successively cultured for 24 h after the medium was replaced with RPMI 1640 medium/10% FCS, followed by selection with puromycin (2.5 μg/mL).

#### *4.9. Purification of MMP-9*

MMP-9 was purified from the conditioned medium of human monocytic leukemia THP-1 cells based on the method of Morodomi et al. [38]. THP-1 cells were cultured in ASF104 medium containing PMA (5 nM) at 37 ◦C for 3–4 days. The conditioned medium was mixed with gelatin Sepharose 4B which had been equilibrated with TNC buffer (50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij 35, pH 7.5) and incubated at 4 ◦C for 16 h with gentle agitation. After gelatin Sepharose 4B was washed three times with TNC buffer, MMP-9 was eluted with DMSO (5%)-containing TNC buffer. The eluate was then applied to a PD-10 desalting column (GE Healthcare, Piscataway, NJ, USA) to replace the buffer with ASF104 medium. The purity of MMP-9 was estimated to be >95%, as analyzed by SDS-polyacrylamide gel electrophoresis followed by CBB and silver staining. The amino acid sequence of the purified protein was analyzed by peptide mass fingerprinting. The tryptic fragments obtained from the specimen were applied to MALDI/TOFMS (AXIMA-QIT; Shimadzu/Kratos, Kyoto, Japan) as described previously [39]. Major mass peaks detected by the MS analysis and the corresponding amino acid sequences to human MMP-9 were as follows: *m*/*z* 1345.74 for 25QSTLVLFPGDLR36, *m*/*z* 1184.62 for 43QLAEEYLYR51, *m*/*z* 1701.90 for 77QLSLPETGELDSATLK92, *m*/*z* 1084.55 for 107FQTFEGDLK115, *m*/*z* 1680.91 for 144AFALWSAVTPLTFTR158, *m*/*z* 2350.10 for 163DADIVIQFGVAEHGDGYPFDGK184, *m*/*z* 3175.37 for 185DGLLAHAFPPGPGIQGDAHFDDDELWSLGK214, *m*/*z* 2012.89 for 222FGNADGAACHFPFIFEGR239, *m*/*z* 2073.87 for 250SDGLPWCSTTANYDTDDR267, *m*/*z* 999.47 for 268FGFCPSER275, *m*/*z* 997.50 for <sup>325</sup> LFGFCPTR332, *m*/*z* 1385.79 for 547GSRPQGPFLIADK559, *m*/*z* 739.30 for 560WPALPR565, *m*/*z* 873.45 for 579LFFFSGR585, *m*/*z* 1532.83 for 586QVWVYTGASVLGPR599, *m*/*z* 1440.80 for 604LGLGADVAQVTGALR618, *m*/*z* 823.45 for 624MLLFSGR630 and *m*/*z* 1921.91 for 653MFPGVPLDTHDVFQYR668 (UniProt P14780). Based on these results, the purified protein was confirmed to be MMP-9.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/12/ 3961/s1. Figure S1: RT-qPCR analysis of mRNA for the γ2 subunit of laminin-332 in mesothelial cells; Figure S2: Purification of MMP-9 from THP-1 cells; Figure S3: Zymographic analysis of MMPs in the conditioned medium of MMP-2- or MMP-9-knockdown MKN1 cells.

**Author Contributions:** Conceptualization, T.O., K.S. and T.T.; Formal analysis, T.O., K.S., H.K., Y.A., C.K., R.S. and S.H.; Investigation, T.O., K.S., H.K., Y.A., C.K., R.S. and S.H.; Methodology, T.O., M.T., S.H. and T.F.; Project administration, T.T.; Resources, M.T., S.H. and T.F.; Supervision, T.T.; Writing—original draft, T.O.; Writing—review & editing, T.T.

**Funding:** This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (23590092).

**Acknowledgments:** We thank Yoko Sawaki, Ayako Takai, Misaki Ide, and Hiromi Akiyama (Hoshi University School of Pharmacy and Pharmaceutical Sciences) for their technical assistance.

**Conflicts of Interest:** The authors disclose no potential conflicts of interest related to this work.

#### **Abbreviations**

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5**

**Fraser M. Rogerson 1,2,3, Karena Last 1,2, Suzanne B. Golub 1,2, Stephanie J. Gauci 2, Heather Stanton 1,2, Katrina M. Bell <sup>2</sup> and Amanda J. Fosang 1,2,\***

Received: 19 December 2018; Accepted: 24 January 2019; Published: 29 January 2019

**Abstract:** A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5Δcat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5Δcat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-Δcat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

**Keywords:** aggrecan; aggrecanase; ADAMTS; cartilage; arthritis

#### **1. Introduction**

The weight-bearing properties of articular cartilage are conferred by the large and highly charged glycosaminoglycan side chains that are present on the proteoglycan aggrecan. In arthritic disease, the resilience of cartilage is compromised when large aggrecan aggregates are degraded by aggrecanases that are members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of zinc-dependent enzymes within the metzincin family of metalloproteinases. The principal aggrecanases thought to be responsible for aggrecan degradation are ADAMTS-4 [1] and ADAMTS-5 [2].

These enzymes were originally identified by their ability to cleave the aggrecan core protein at the TEGE <sup>373</sup>↓<sup>374</sup> ALGS bond in the aggrecan interglobular domain (IGD) [3–5] and at four additional sites within the chondroitin sulphate (CS)-rich region [6,7] (Figure 1) in response to stimulation with pro-catabolic agents, such as interleukin-1-alpha (IL-1α), IL-1β and retinoic acid. In contrast, ADAMTS-1 [8], -8 [9], -9 [10,11], -16 [11] and -18 [11] were thought to have only weak activity against aggrecan, in vitro and in vivo.

**Figure 1.** Aggrecanase cleavage sites and neoepitopes in the mouse aggrecan core protein. The domain structure of the mouse aggrecan core protein, with globular G1, G2 and G3 domains and intervening interglobular and chondroitin sulphate-rich regions are shown. The amino acid sequences flanking the conserved cleavage sites are shown in boxes with amino acid numbers at the P1 position. Modified from [12].

In the course of our studies, we identified a new aggrecanase activity that was present in cartilage explants from mice deficient in both ADAMTS-4 and ADAMTS-5 catalytic activity (TS-4/5Δcat mice) [13]. We found that retinoic acid treatment of TS-4/5Δcat cartilage explants did not stimulate aggrecanase activity at the classical TEGE <sup>373</sup>↓<sup>374</sup> ALGS or TAQE <sup>1572</sup>↓<sup>1573</sup> AGEG sites, but instead at the FREEE <sup>1467</sup>↓<sup>1468</sup> GLGS and SSELE <sup>1279</sup>↓<sup>1280</sup> GRGTI sites located in the chondroitin sulphate-rich region of the core protein (Figure 1). Thus, retinoic acid stimulates the expression of a novel aggrecanase activity in TS-4/5Δcat cartilage. This unexpected finding reveals that the substrate specificity of this unknown enzyme is distinct from ADAMTS-4 and -5. In addition, and in contrast to ADAMTS-4 and ADAMTS-5, the activity of the novel aggrecanase is stimulated by retinoic acid, but not by interleukin-1α (IL-1α) treatment [13].

In order to identify this novel aggrecanase, we cultured mouse femoral head cartilage explants from wildtype and TS-4/5Δcat mice. We used discrimination between the known effects of IL-1α and retinoic acid on the up-regulation of aggrecanase activity to screen for candidate extracellular proteinases whose mRNA expression levels were specifically increased by retinoic acid, but not by IL-1α treatment. Our results reveal that ADAMTS-9 is a major aggrecanase in TS-4/5Δcat mice, raising the possibility that ADAMTS-9 might be active in cartilage, in vivo, in the presence of inhibitors designed to block ADAMTS-4 and ADAMTS-5 activity.

#### **2. Results**

#### *2.1. Retinoic Acid Regulates the Expression of a Novel Aggrecanase Activity via a Transcriptional Mechanism*

We previously identified a novel aggrecanase activity that cleaves aggrecan in the CS-rich region in cartilage explants from mice deficient in both ADAMTS-4 and ADAMTS-5 [13]. Since this novel activity is stimulated by treatment with retinoic acid, but not IL-1α, we reasoned that the differential in susceptibility to retinoic acid and IL-1α could be used to identify aggrecanase candidates by microarray. We first confirmed that retinoic acid stimulates aggrecanolysis in TS-4/5Δcat cartilage via a transcriptional mechanism by culturing TS-4/5Δcat cartilage explants in the presence or absence of the transcriptional inhibitor, actinomycin D. Western blot analysis of the conditioned media showed that

retinoic acid treatment stimulated the release of the aggrecan FREEE <sup>1467</sup> neoepitope from TS-4/5Δcat cartilage into conditioned medium (Figure 2a, lane 3), compared with the untreated control (Figure 2a, lane 1). However, the retinoic acid-induced increase in FREEE <sup>1467</sup> neoepitope was blocked in the presence of actinomycin D (Figure 2a, lane 4), confirming that the effects of retinoic acid in this system were indeed transcription-dependent. The chondrocytes remained viable in culture for a further 6 days in the presence or absence of retinoic acid, confirming that inhibition of aggrecan cleavage was not due to actinomycin-induced cell death.

**Figure 2.** Identifying candidate aggrecanases in TS4/5Δcat mice. (**a**) Aggrecanase cleavage in TS-4/5Δcat femoral head cartilage explants, cultured untreated (lane 1) or in the presence of actinomycin D (lane 2), retinoic acid (lane 3) or both actinomycin D and retinoic acid together (lane 4) was analysed by FREEE <sup>1467</sup> neo-epitope Western blotting. (**b**) The results of the microarray studies, represented as a volcano plot, identify candidate proteinases for the novel aggrecanase.

#### *2.2. Microarray and qPCR Studies Reveal ADAMTS-9 as a Strong Candidate for the Novel Aggrecanase*

Based on our conclusion that retinoic acid exerts transcriptional control over aggrecanolysis in TS-4/5Δcat chondrocytes, we next used microarrays to shortlist genes encoding aggrecan-degrading proteinases, based on their up-regulation in response to retinoic acid but not IL-1α treatment. We focused on enzyme families known to cleave aggrecan, including the a disintegrin and metalloproteinase (ADAM), ADAMTS, matrix metalloproteinase (MMP), cathepsin and calpain families. The results presented as a volcano plot in Figure 2b show that only three genes matched these criteria: *Adamts-9*, *Mmp-11* and *Calpain-5* (Figure 2b). There was no increase greater than 1.5-fold in the expression of any ADAM or cathepsin gene in IL-1α-treated compared with retinoic acid-treated cultures.

To validate the microarray expression data, we used qPCR to measure the expression of key genes in cultured chondrocytes from TS-4/5Δcat mice. The results shown in Figure 3 confirm

the up-regulation of mRNA expression for *Adamts-5* (Figure 3a), *Mmp-13* (Figure 3b) and *Mmp-11* (Figure 3d) by both IL-1α and retinoic acid, as reported previously, thus excluding these enzymes as the novel aggrecanase. The expression of *Calpain-5* was only weakly increased by IL-1α and retinoic acid (Figure 3c) and was therefore also considered not to be an aggrecanase. Overall, the mRNA data suggested that ADAMTS-9 was the strongest and most likely candidate for the novel aggrecanase. Figure 3e shows that while *Adamts-9* mRNA expression is increased by treatment with retinoic acid, there is no increase in *Adamts-9* gene expression induced by IL-1α. Similarly, retinoic acid, but not IL-1α, increased the expression of ADAMTS-9 protein detected as a band of approximately 200 kDa by Western blot (Figure 3f).

**Figure 3.** q-PCR and Western blot analyses of candidate aggrecanases. Candidate aggrecanases expressed in cultured cartilage explants were analysed by qPCR for genes including *Adamts-5* (**a**), *Mmp-13* (**b**), *Calpain* (**c**), *Mmp-11* (**d**), and *Adamts-9* (**e**). Protein extracts from Wildtype and TS4/TS5Δcat cartilage extracts were also analysed for ADAMTS-9 expression by Western blotting (**f**).

#### *2.3. The Novel Aggrecanase is Inhibited by TIMP-3*

Tissue inhibitor of metalloproteinase (TIMP)-3 is a potent endogenous inhibitor of ADAMTS and MMP family members [14]. We therefore used sensitivity to inhibition with TIMP-3 to further confirm the identity of the novel aggrecanase and found that when femoral head explants from TS-4/5Δcat mice were incubated with retinoic acid in the presence or absence of 500 nM TIMP-3, aggrecan cleavage at the FREEE <sup>1467</sup>↓<sup>1468</sup> GLGSV site was inhibited (Figure 4a, lane 5). An additional FREEE <sup>1467</sup> band migrating at approximately 50 kD and most likely the SSELE 1279\_1467 FREEE fragment previously identified in human cartilage [15] was also generated in the presence of retinoic acid (Figure 4a, lanes 2 and 3) but blocked by high concentrations of TIMP-3 (Figure 4a, lane 5). As a control for the potency of TIMP-3 inhibition, cleavage at the MMP-sensitive DIPEN <sup>342</sup>↓<sup>343</sup> FFGVG site was also examined. TIMP-3 blocked cleavage at DIPEN <sup>342</sup>↓<sup>343</sup> FFGVG at the same high concentration (Figure 4b, lanes 4 and 5).

**Figure 4.** The novel aggrecanase is inhibited by tissue inhibitor of metalloproteinase (TIMP)-3. Western blot analyses show that TIMP-3 inhibits retinoic acid-induced aggrecanolysis at FREEE <sup>1467</sup>↓<sup>1468</sup> GLGSV (**a**) and DIPEN <sup>341</sup>↓<sup>342</sup> FFGVG (**b**) in TS-4/5Δcat cartilage explants.

#### *2.4. ADAMTS9 Expression and Aggrecanase Activity in the Growth Plate*

Finally, we used immunohistochemistry to compare sites of ADAMTS-9 expression and aggrecanase activity in sections of the 3-week mouse growth plate. For both genotypes, FREEE <sup>1467</sup> neoepitope was present in selected regions of the growth plate including the proliferative zone (Figure 5a,b,e,f; P) and a thin layer of cells surrounding the secondary centre of ossification (Figure 5a,b,e,f; right-pointing arrow). The staining surrounding the secondary centre was more punctate in sections from TS-4/5Δcat mice (Figure 5e,f) than from wildtype mice (Figure 5a,b). FREEE <sup>1467</sup> neoepitope was also present at the metaphyseal border in wildtype mice (Figure 5a,b; left-pointing arrow) but was absent at this site in TS-4/5Δcat mice (Figure 5e,f). The different patterns of FREEE <sup>1467</sup> immunostaining at the metaphyseal border in wildtype mice, compared with TS4/5Δcat mice, suggest that FREEE <sup>1467</sup> immunoreactivity in the growth plate is a product of ADAMTS-4 and/or ADAMTS-5 activity. We therefore conclude that ADAMTS-9 is not essential for aggrecanase activity at the metaphyseal border.

**Figure 5.** ADAMTS-9 protein and FREEE <sup>1467</sup> neoepitope are present in the 3-week-old mouse joint. M, metaphysis; P, proliferative zone of the growth plate; left-pointing arrow, metaphyseal border; right-pointing arrow, boundary of the secondary centre of ossification; diagonal dotted arrows, columns of calcified cartilage; H, hypertrophic zone of the growth plate. Panels (**a**,**b**,**e**,**f**) show staining for the FREEE <sup>1467</sup> neoepitope. Panels (**c**,**d**,**g**,**h**) show staining for ADAMTS-9 protein. Panels (**a**–**d**) show cartilage from wildtype mice. Panels (**e**–**h**) show cartilage from TS-4/5Δcat mice.

Unlike the limited staining for the aggrecan FREEE <sup>1467</sup> neoepitope seen in wildtype growth plates (Figure 5 a,b,e,f), ADAMTS-9 immunostaining was strong throughout all zones of the wildtype growth plate, including the metaphysis (M), proliferative zone (P), hypertrophic zones (H) and in bony spicules (*diagonal dotted arrows*) protruding into the calcified region of the hypertrophic zone (Figure 5c,d). In contrast, there was no immunostaining for ADAMTS-9 in the hypertrophic/pre-hypertrophic zones (H) of the growth plate in TS4/5Δcat mice (Figure 5g,h). One interpretation of this unintuitive staining pattern is that ADAMTS-9 is not expressed in the hypertrophic zone (Figure 5g,h). However, a more likely interpretation is that the physical entrapment of residual, undegraded and highly charged aggrecan in the hypertrophic zone blocks diffusion of immunoreagents into this region of the sections, producing a false negative result.

#### **3. Discussion**

ADAMTS-9 is a multi-domain proteinase with key roles in the remodelling of extracellular matrices during growth and development [16–21]. Versican is a major substrate for ADAMTS-9, and in this study we show that the related proteoglycan, aggrecan, which is highly expressed in joint cartilage, is also a substrate for ADAMTS-9. Our results suggest that unlike ADAMTS-4 and ADAMTS-5, which are widely recognised as destructive aggrecanases in joint disease [22,23], the aggrecanolytic activity of ADAMTS-9 might be more important for normal joint development [18] than for pathology.

Previous studies in wildtype mice have reported the mRNA expression of ADAMTS-9 in chondrocytes and cartilage explants [20,21]; however, in the presence of ADAMTS-4 and ADAMTS-5 expression, it has not been possible to show direct evidence of ADAMTS-9 activity. Here, we have used TS-4/5Δcat mice to show that ADAMTS-9 is a bona fide aggrecanase with activity, not at the classical TEGE <sup>373</sup>↓<sup>374</sup> ALGS site in the IGD and TAQE <sup>1572</sup>↓<sup>1573</sup> AGEG site in the C-terminal domains, but instead ADAMTS-9 cleaves at FREEE <sup>1467</sup>↓<sup>1468</sup> GLGSV and SELE <sup>1279</sup>↓<sup>1280</sup> GRGT sites located centrally within the aggrecan core protein. These results extend previous studies, suggesting that ADAMTS-9 might also cleave at TAQE <sup>1771</sup>↓<sup>1772</sup> AGEG near the C-terminal G3 domain [10].

Our results are consistent with previous studies showing *Adamts-9* mRNA expression in neonatal growth plates [21] and in the proliferative and hypertrophic zones of the 7-week-old mouse growth plate [21]. In addition, we have used immunoreactivity against the FREEE <sup>1467</sup> neoepitope to show that TS-4/5Δcat mice express aggrecanase activity, and we propose that this activity is likely to be the product of ADAMTS-9 (Figure 5).

The distribution of FREEE <sup>1467</sup> neoepitope in the TS-4/5Δcat epiphysis suggests that ADAMTS-9 has a role in the remodelling of the secondary centre of ossification and the proliferative zone of the growth plate. However, whereas the absence of FREEE <sup>1467</sup> neoepitope at the metaphyseal border in TS4/5-Δcat mice suggests that ADAMTS-9 might not degrade aggrecan at this site, a more likely interpretation (and complication) of this staining pattern is that an accumulation of undegraded aggrecan in calcified regions of the hypertrophic zone impedes antibody penetration and compromises immunoreactivity.

Beyond the G2 domain, the aggrecan core protein is heavily decorated with glycosaminoglycan side chains. When ADAMTS-9 cleaves at FREEE <sup>1467</sup>↓<sup>1468</sup> GLGSV, the C-terminal third of aggrecan is lost from the cartilage, while the remaining N-terminal two thirds, immobilised in the cartilage by G1-domain binding to hyaluronan, provide the weight-bearing properties. Accordingly, cleavage at FREEE <sup>1467</sup>↓<sup>1468</sup> GLGSV is less deleterious for weight-bearing than cleavage in the IGD, which releases the entire CS-containing portion of aggrecan.

There are very few studies on the roles of regional aggrecanolysis. In vitro testing of cartilage explants showed that aggrecan loss mediated by IGD cleavage correlates with a loss of mechanical properties, whereas aggrecan loss due to cleavage at sites in the CS-2 domain does not appear to affect biomechanics [24]. In vivo studies to specifically explore the role of C-terminal domain cleavage have not been done, but it is widely held that C-terminal processing of aggrecan is part of normal homeostasis [25,26]. Furthermore, the proportion of C-terminally shortened aggrecan increases with age [27,28], suggesting that loss of the C-terminus is part of normal tissue maturation. Thus, ADAMTS-9 might have a role in maturation and normal cartilage homeostasis.

Alternatively, CS-2 domain cleavage might indeed have a role in cartilage pathology. In vitro evidence suggests that there is a hierarchy for aggrecanase cleavage and that cleavage in the IGD occurs after cleavage in the CS-2 domain [29–31]. If CS-2 domain cleavage is a prerequisite for IGD cleavage, then a CS-2 aggrecanase such as ADAMTS-9 might initiate an aggrecan-degrading cascade. Aggrecan fragments resulting from CS-2 domain cleavage are elevated in human synovial fluids following knee joint injury [32], raising the possibility that ADAMTS-9 contributes to the production of G3-containing fragments from injured cartilage. Furthermore, the G3 domain binds complement factors C1q and C3, thus activating the classical and alternative complement pathways [33], suggesting a possible role for ADAMTS-9 in complement regulation in inflamed joints.

#### **4. Materials and Methods**

#### *4.1. Mice*

The TS-4/5Δcat mice deficient in both ADAMTS-4 and ADAMTS-5 activity have been described previously [13]. The Δ-cat mice were bred from the TS-4 Δ-cat and TS-5 Δ-cat strains [13,22], which have in-frame deletions of exon 4 of the *Adamts-4* gene (TS-4 Δ-cat) and exon 3 of the *Adamts-5* gene (TS-5 Δcat), encoding the respective catalytic domains [22]. Mutant ADAMTS-5 mRNA lacking exon 3 is translated and includes exons encoding the ADAMTS-5 C-terminal domain [34]. The TS-4/5Δcat mice are healthy with no abnormalities in skeletal growth or development. The Murdoch Children's Research Institute Animal Ethics Committee approved all animal procedures under projects A605, A710, A795 and A868. Each experiment was done at least twice with a minimum of two biological replicates, except for the microarray which was done once with three mice per treatment.

#### *4.2. Preparation of Primary Chondrocytes*

Chondrocytes were isolated from the distal femoral and proximal tibial epiphyses of 6-day-old mice. The dissected epiphyses were digested for 1 h with 0.25% trypsin/EDTA to remove loose fibrous tissue, washed with PBS, then incubated overnight at 37 ◦C/5%CO2 with 300 U/mL collagenase II (Worthington Biochemical Corp, Lakewood, NJ, USA) in DMEM and 5% fetal bovine serum (FBS). The isolated cells were washed extensively with medium and seeded at 5 × 105 cells/well in 24-well plates in DMEM, 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM L-glutamine and 20 mM HEPES. After 3 days in culture, the cells were washed, placed in serum-free DMEM and stimulated with either 10 ng/mL IL-1α (Peprotech, Rocky Hill, NJ, USA) or 10 μM retinoic acid (Sigma-Aldrich, St Louis, MO, USA) for a further 3 days. These concentrations of IL-1α and retinoic acid were previously confirmed by dose response to elicit maximal gene expression.

#### *4.3. Culture of Femoral Head Cartilage Explants*

Mouse femoral head cartilage explants were cultured as described previously [12,13]. Explants were incubated with 10 μM retinoic acid ± 10μg/mL actinomycin D (Life Technologies, Carlsbad, CA, USA) for 24 h. Conditioned medium was harvested, and the explants were washed and incubated with fresh retinoic acid for a further 6 days to confirm that actinomycin D had no adverse effect on chondrocyte activity or viability. To examine the inhibition of aggrecanase activity by tissue inhibitor of metalloproteinase-3 (TIMP-3), femoral head explants were co-cultured with 10 μM retinoic acid and 10–500 nM recombinant human TIMP-3 (a gift from Professor Hideaki Nagase, UK) and then analysed for aggrecanase products by Western blotting.

#### *4.4. Western Blotting*

To detect aggrecan neoepitopes, conditioned media from mouse femoral head cultures were dialysed against proteinase inhibitors and deglycosylated as described previously [34]. Deglycosylated samples were resolved by reducing SDS-PAGE on 4–20% gradient 7TGX gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinylidene difluoride membranes. Aggrecan degradation was analysed using polyclonal antibodies [35] that recognise the FREEE <sup>1467</sup> neoepitope [36], created by aggrecanase cleavage in the CS-rich region, and the DIPEN <sup>341</sup> neoepitope [37], created by MMP cleavage in the IGD. To detect ADAMTS-9 protein, conditioned medium from primary chondrocyte cell cultures was blotted and detected with anti-ADAMTS-9 antibody from Abcam (Cambridge, UK), used at 1 μg/mL and detected with anti-rabbit-HRP (1:2000) from Dako (Jena, Germany). The volume of medium loaded was determined empirically for each neoepitope examined and results were normalised to the values for total aggrecan as described previously [12,13]. The anti-DIPEN and anti-FREEE antibodies were used at a concentration of 5 μg/mL.

#### *4.5. Microarray Analysis*

Femoral head cartilage explants harvested from 3-week-old TS-4/5Δcat mice were cultured for 3 days in serum-free DMEM containing 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM L-glutamine and 20 mM HEPES. The explants were either (i) unstimulated, (ii) stimulated with 10 ng/mL recombinant human IL-1 or (iii) stimulated with 10 μM retinoic acid. These concentrations of IL-1α and retinoic acid stimulated maximal aggrecan loss from cartilage explants as described previously [13]. After treatment, the explants were snap frozen and stored at −80 ◦C. Three separate mice (unpooled) were included for each treatment. Total RNA from frozen, crushed cartilage samples was isolated using the RNeasy kit (Qiagen, Hilden, Germany) and the RNA quality was assessed by BioAnalyser. The microarray analyses were done under contract at the Australian Genome Research Facility using the Illumina Sentrix Mouse 1.1 chip. The data were analysed using the R limma package from Bioconductor, utilising the neqc function (quantile normalisation) with non-detected

probes removed and a factor included for batch and biological replicates in the linear mode [38]. The microarray data is available at Gene Expression Omnibus (GSE110754).

#### *4.6. Quantitative Real-Time Polymerase Chain Reaction (qPCR) Analysis of mRNA Expression*

To validate the microarray data identifying the putative aggrecanases, mRNA expression of the candidates was analysed by real-time quantitative PCR (qPCR) with probes and primers from the Universal Probes Library (UPL) (Roche, Basel, Switzerland). The ΔΔCp values derived from the Cp values determined by the Roche Light Cycler 480 were calculated for each gene and normalised against the geometric mean of the genes *Rpl10* and *Rpl26* [39], two reference genes specific for chondrocytes. The primer sequences used for mouse *Mmp13*, *Mmp11*, *Adamts5*, *Adamts9* and *calpain 5* genes are shown in Table 1.

**Table 1.** Primer sequences for qPCR analyses.

#### *4.7. Immunohistochemistry*

Knee joints from 3-week-old mice were fixed in 4% (*w*/*v*) paraformaldehyde for 24 h and then decalcified in 7% EDTA/PBS, pH 7.4, for 3 weeks. Five-micron sections of paraffin-embedded epiphyses were treated with 3% (*v*/*v*) H2O2 to block endogenous peroxidase activity. Antigen retrieval included treatment with 10 mM citrate, pH 6.0, with 0.05% (*v*/*v*) Tween 20 for 30 min at 60 ◦C and then cooling to room temperature prior to incubation with 0.2% (*w*/*v*) hyaluronidase (Sigma) for 1 h at 37 ◦C. Non-specific antibody binding was reduced by treatment with 1.5% (*v*/*v*) normal goat serum. Sections were incubated with either rabbit anti-FREEE <sup>1467</sup> primary antibody (0.5 μg/mL), rabbit anti-ADAMTS-9 antibody (Abcam, Cambridge, UK; cat No 28279, 1:2000 dilution) or no primary antibody overnight at 4 ◦C. The following day, sections were incubated with HRP-labelled anti-rabbit IgG (Dako, Jena, Germany), visualised with diaminobenzidine and counterstained with haematoxylin.

#### *4.8. Declarations Ethics Approval*

The animal procedures used in this study were approved by the Murdoch Children's Research Institute Animal Ethics Committee under ethics project numbers A605 (20 May 2008), A710 (18 April 2012) A795 (16 April 2015), A868 (17 May 2018).

#### *4.9. Availability of Data and Materials*

The data sets generated and analysed during this study are available in the GEO repository at accession number GSE110754.

#### **5. Conclusions**

The results of this study are significant in the context of continuing efforts to identify the physiological aggrecanases and to develop aggrecanase inhibitors for the clinical management of degenerative joint diseases. Overall, we show that in the absence of ADAMTS-4 and -5 activity, ADAMTS-9 is expressed and might therefore have a role in normal aggrecan remodelling during skeletal growth and development. While our findings suggest that ADAMTS-9 has important roles in skeletal growth, it is not clear what role(s), if any, ADAMTS-9 might have in pathological aggrecanolysis. In future work, it will be important to determine whether aggrecanase inhibitors targeting ADAMTS-4 and/or ADAMTS-5 might also affect ADAMTS-9 activity in adult tissues or influence endochondral ossification in developing and juvenile skeletal tissues.

**Author Contributions:** Conceptualisation: A.J.F.; Methodology: F.M.R.; Validation: F.M.R., K.L., S.B.G., S.J.G., A.J.F.; Formal analysis: K.M.B.; Investigation: F.M.R., K.L., S.B.G., S.J.G.; Writing—original draft preparation: F.M.R., A.J.F.; Writing—Review and editing: H.S., A.J.F.; Supervision: A.J.F.; Funding acquisition: F.M.R., A.J.F.

**Funding:** This work was supported by grants from the National Health and Medical research Council Australia (App ID 491203) and by the Victorian Government's Operational Infrastructure Support Program.

**Acknowledgments:** The authors thank Johnson & Johnson Pharmaceutical Research & Development, L.L.C. for supplying the *Adamts-4*- and *Adamts-5*-floxed mice; the Australian Genome Research Facility, Parkville, Victoria, who did the microarrays and Prof. Hideaki Nagase (Kennedy Institute of Rheumatology, UK) for providing TIMP-3.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Di**ff**erences in Shedding of the Interleukin-11 Receptor by the Proteases ADAM9, ADAM10, ADAM17, Meprin** α**, Meprin** β **and MT1-MMP**

**Martin Sammel 1, Florian Peters 1, Juliane Lokau 2, Franka Scharfenberg 1, Ludwig Werny 1, Stefan Linder 3, Christoph Garbers 2, Stefan Rose-John <sup>1</sup> and Christoph Becker-Pauly 1,\***

Received: 28 June 2019; Accepted: 25 July 2019; Published: 26 July 2019

**Abstract:** Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin β, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes.

**Keywords:** interleukin; IL-6; IL-11; trans-signaling; metalloproteases; ADAM; MMP; meprin

#### **1. Introduction**

IL-11 is a crucial factor in several physiological and pathophysiological signaling pathways [1]. It plays an important role in inflammation, osteogenesis, hematopoiesis, and fertility [1–3]. So far, those functions have only been linked to interleukin-11 (IL-11) signaling via the membrane bound IL-11 receptor (IL-11R). In this so-called classic signaling pathway, IL-11 binds to its membrane bound α-receptor, inducing the dimerization of the ubiquitously expressed β-receptor glycoprotein-130 (gp130) on the same cell. This complex then activates intracellular JAK/STAT, MAPK, PI3K signaling pathways [4–6]. The IL-11R can be released from the cell surface [7] by proteolytic ectodomain-shedding, which is analogous to IL-6R solubilization [8]. Soluble receptors such as sIL-6R and sIL-11R can then bind their ligands, IL-6 or IL-11, respectively, before inducing dimerization of gp130 on distinct cells. This pathway is called trans-signaling. sIL-11R can be found in serum of healthy individuals [7], strongly suggesting a physiological function for IL-11 trans-signaling, as known for sIL-6R [9].

Similar to the IL-6R [10,11], the IL-11R consists of three extracellular domains, an immunoglobulin (Ig)-like domain (D1), two fibronectin type III domains (D2 and D3), a stalk region, which is a 54 amino acid long stretch connecting the extracellular part to the transmembrane region (TM), followed by an intracellular domain (ICD) [11,12]. The extracellular domains D2 and D3 form the cytokine binding module, which is essential for signaling [9].

Ectodomain-shedding of the IL-11R and IL-6R requires proteolytic cleavage of the receptors within their stalk regions. IL-6 trans-signaling can be proteolytically induced by a disintegrin and metalloprotease 10 (ADAM10) and ADAM17, as well as by meprin α and meprin β [13]. IL-11R shedding has been attributed to ADAM10 and the neutrophil-derived serine protease neutrophil elastase (NE), thereby inducing trans-signaling [7].