s2fieldsofstudy: Biology Chemistry

text:
A New Mouse Liver-specific Gene, Encoding a Protein Homologous to Human Antimicrobial Peptide Hepcidin, Is Overexpressed during Iron Overload*

Considering that the development of hepatic lesions related to iron overload diseases might be a result of abnormally expressed hepatic genes, we searched for new genes up-regulated under the condition of iron excess. By suppressive subtractive hybridization performed between livers from carbonyl iron-overloaded and control mice, we isolated a 225-base pair cDNA. By Northern blot analysis, the corresponding mRNA was confirmed to be overexpressed in livers of experimentally (carbonyl iron and iron-dextran-treated mice) and spontaneously (β2-microglobulin knockout mice) iron-overloaded mice. In addition, β2-microglobulin knockout mice fed with a low iron content diet exhibited a decrease of hepatic mRNA expression. The murine full-length cDNA was isolated and was found to encode an 83-amino acid protein presenting a strong homology in its C-terminal region to the human antimicrobial peptide hepcidin. In addition, we cloned the corresponding rat and human orthologue cDNAs. Both mouse and human genes named HEPC are constituted of 3 exons and 2 introns and are located on chromosome 7 and 19, respectively, in close proximity toUSF2 gene. In mouse and human, HEPC mRNA was predominantly expressed in the liver. During both in vivo and in vitro studies, HEPC mRNA expression was enhanced in mouse hepatocytes under the effect of lipopolysaccharide. Finally, to analyze the intracellular localization of the predicted protein, we used the green fluorescent protein chimera expression vectors. The murine green fluorescent protein-prohepcidin protein was exclusively localized in the nucleus. When the putative nuclear localization signal was deleted, the resulting protein was addressed to the cytoplasm. Taken together, our data strongly suggest that the product of the new liver-specific gene HEPCmight play a specific role during iron overload and exhibit additional functions distinct from its antimicrobial activity.

The understanding of iron metabolism in mammals especially of the mechanisms involved in cellular iron transport and in the regulation of intracellular iron amount is of a great importance since diseases associated with iron overload are common in humans and can be responsible for the shortening of life expectancy. Genetic hemochromatosis is characterized by digestive hyperabsorption of iron. In 1996, Feder et al. (1) reported that the C282Y mutation of HFE gene was responsible for this disease. Other iron overload diseases include ␤-thalassemias, genetic diseases characterized by hemolytic anemia, in which iron overload results both from digestive hyperabsorption of iron and blood transfusions (2,3). In addition, it has been shown that iron excess may occur in chronic liver diseases such as alcoholic liver diseases (4) or hepatitis B and C infections (5,6). For these various diseases, clinical data suggest that iron excess favors the development of hepatic lesions likely due to the preponderant role of the liver in iron storage. This is particularly true in untreated genetic hemochromatosis in which many authors demonstrated a relationship between the increase in liver iron concentration and the risk of cirrhosis and hepatocellular carcinoma (7)(8)(9)(10)(11)(12).
Recently, additional elements involved in iron metabolism have been characterized as follows: (i) HFE potentially modulating iron absorption by its interaction with transferrin receptor (13,14); (ii) Nramp2 (also named DCT1 or DMT1) involved in iron uptake at the apex of the enterocyte (15,16); (iii) transferrin receptor 2 involved in hepatic iron uptake (17); (iv) hephaestin, implicated in iron transfer from enterocyte to the plasma (18), and frataxin which play a role in the mitochondrial iron metabolism (19).
Despite these recent advances, molecular mechanisms leading to hepatic iron overload remain poorly understood. In addition, molecular mechanisms involved in the development of iron-related lesions of the liver are not well known. However, it is clear that modulation of hepatic gene expression, related directly or indirectly to iron excess, takes place during the development of hepatic lesions. Several specific mRNAs are known to be modulated by the iron amount of the liver. Thus, transferrin receptor mRNA, presenting an iron-responsive element (IRE), 1 is repressed during iron overload. Moreover, even if not yet fully demonstrated, genes containing an IRE in their 3Ј-untranslated region may be good candidates to be 1 The abbreviations used are: IRE, iron-responsive element; aa, amino acid; DFO, desferrioxamine; EST, expressed sequence tag; FCS, fetal calf serum; GFP, green fluorescent protein; LIC, liver iron concentration; LPS, lipopolysaccharide; PCR, polymerase chain reaction; RT, reverse transcription; SSH, suppressive subtractive hybridization; FGF-2, fibroblast growth factor 2; bp, base pair; NLS, nuclear localization signal.
repressed by iron excess in the liver. This is especially the case for Nramp2 (15) and glycolate oxidase (20). For genes devoid of IRE, it is known that collagen ␣1(I) chain (21)(22)(23)(24) and transforming growth factor-␤1 mRNAs (23,25,26), encoding two proteins potentially involved in the development of liver fibrosis, are both induced by hepatic iron excess. Other mRNAs, such as ␥-glutamyltransferase (27) and heme oxygenase 1 (28), which are involved in oxidative stress, were also found to be up-regulated in the liver by high amounts of iron.
Since only a few genes have been reported to be abnormally expressed in the liver when iron is in excess, it became evident that identification of new hepatic genes up-regulated by iron would be helpful for a better understanding of mechanisms leading to iron overload and/or liver injury. The aim of our work was to identify new genes positively regulated by iron. For this purpose, we used the previously described carbonyl iron overload mouse model (29). We performed a suppressive subtractive hybridization between cDNAs obtained from the liver of 3% carbonyl iron-overloaded and control mice. This methodology allows for the analysis of differential gene expression in two different conditions (30). It has already been shown to be useful for identifying the up-regulation of complement C4 in hepatic stellate cells issued from rats overloaded by carbonyl iron (31). More recently, this technology enabled the characterization and the identification of IREG1, an iron transporter for which expression was up-regulated under the conditions of increased iron absorption in the duodenum (32). By using this method, we were able to isolate and characterize a novel gene strongly induced in the liver of iron overloaded mice.
All these animals were maintained in accordance to French law and regulations in a temperature-and light-controlled environment. They were given free access to tap water and food.

Modulation of Liver Iron Concentration in Vivo
Carbonyl Iron Overload-Iron overload was performed as described previously (29). Briefly, mice were iron-overloaded by 0.5, 1.5, and 3% carbonyl iron supplemented in the diet (AO3, UAR, France) for 8 months. Control mice have a carbonyl iron-free diet. For 3% carbonyl iron-overloaded mice, liver iron concentration was on average 10-fold higher than the control value (115 mol of iron/g of dry weight liver versus 10 mol, respectively). Liver iron concentration increased 4-and 2-fold, respectively, over the control values for 1.5 and 0.5% carbonyl iron-overloaded mice after 8 months. To study mRNA expression during early stages of iron overload, 3% carbonyl iron was given for only 2 months. Results were compared with mice overloaded by 3% carbonyl iron for 8 months.
Iron-Dextran Overload-A single subcutaneous injection of iron-dextran was performed at the dose of 1 g of iron/kg of body weight (Sigma, France) according to Carthew et al. (35). Control mice were injected with a mixture containing dextran and phenol at the same concentration as in iron-dextran solution. Animals, fed with an AO3 diet (UAR, France), were sacrificed 2 months later. In another experiment, a timedependent iron overload was performed, and animals were sacrificed 1-4 and 8 weeks after the iron-dextran injection.
Spontaneous Iron Overload-␤ 2 -Microglobulin-deficient and the corresponding control mice, 8 weeks old, were fed with 113 diet (UAR, France) for 12 months and then sacrificed. Ten ␤ 2 -microglobulin-deficient mice, 11 weeks old, were used to study the effect of iron depletion on HEPC mRNA expression. They were divided into two groups. The first group was given a low iron diet (UAR, France; 13 mg/kg). The control group was given a similar food containing 230 mg of iron/kg. Mice were sacrificed 2 months later.

Liver Iron Concentration
Liver iron concentration (LIC) was evaluated biochemically according to the method of Barry and Sherlock (36) on liver specimens that have been previously fixed in 4% formalin.

Total RNAs and Poly(A) ϩ RNA Preparations
Total RNAs were extracted from liquid nitrogen-frozen tissue specimens using SV total RNA isolation system (Promega, Madison, WI). For suppressive subtractive hybridization, poly(A) ϩ RNAs were isolated separately from livers of 4 mice overloaded with 3% of carbonyl iron for 8 months and from 4 control mice using Invitrogen's FastTract kit (Invitrogen, Groningen, The Netherlands). mRNAs were then pooled per each group of mice.

Suppressive Subtractive Hybridization
Suppressive subtractive hybridization (SSH) was performed between control and iron-overloaded group mice according to the manufacturer's instructions using "PCR-Select cDNA Subtraction kit" (CLONTECH, Palo Alto, CA) (30). The subtractive product, corresponding to overexpressed genes in condition of iron excess, was recovered by PCR. Ironinduced cDNAs were cloned into pCRII vector using a T/A cloning kit (Invitrogen). Each cDNA was sequenced using Thermosequenase kit (Amersham Pharmacia Biotech) with ␣-33 P-radiolabeled dideoxynucleotides (1500 Ci/mmol). Samples were analyzed on a 5% urea-polyacrylamide gel. Nucleic and amino acid homology searches were performed using the BLAST program (37).

Cloning of the Full-length cDNA
A 5Ј stretch cDNA library (CLONTECH) derived from BALB/cJ liver was screened with the [␣-32 P]dCTP-radiolabeled 225-bp cDNA obtained by SSH to isolate the full-length murine cDNA. The final isolated 410-bp cDNA was sequenced in both strands. Rat and human orthologue cDNAs were generated, respectively, by RT-PCR from rat liver and human hepatoma HepG2 mRNAs using the following primers: 5Ј-CACGAGGGCAGGACAGAAGGCAAG-3Ј and 5Ј-CAAGGTCATTGC-TGGGGTAGGACAG-3Ј for rat cDNA and 5Ј-GACTGTCACTCGGTCC-CAGACACCAG-3Ј and 5Ј-GGGGCAGGAATAAATAAGGAAGGG-3Ј for the human cDNA. After an initial denaturation at 94°C for 5 min, PCRs were set out as follow: 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 30 cycles. All cDNAs were then cloned into a pCRII-TOPO vector (Invitrogen) and were sequenced.

Identification of Genomic Structure of the Mouse and Human Genes
The mouse genomic clone CT7-8N15 and the human genomic clone R30879 (GenBank TM accession numbers AC020841 and AD000684, respectively) were used to elucidate the genomic organization of the mouse and human HEPC genes by sequence comparison with the mouse and human cDNAs. Search for transcription binding sites was performed using MatInspector (38).

Tissue-specific Expression
Total mRNA was isolated from the following 8-week old BALB/cJ mice tissues: liver, stomach, duodenum, intestine, ileum, colon, lungs, kidneys, brain, heart, testis, ovaries, thymus, pancreas, bladder, muscle, uterus, and bone marrow, and subjected to Northern blot analysis using the murine 225-bp cDNA as a probe. The expression pattern of the human mRNA was analyzed by RNA blot assay using multiple tissue expression array (CLONTECH), a membrane to which poly(A) ϩ RNAs from different fetal and adult human tissues and cell lines have been immobilized in separate dots. Hybridization was carried out in the ExpressHyb hybridization solution (CLONTECH) in the presence of 32 P-labeled human-specific cDNA probe according to the manufacturer's instructions.

Cell Isolation
Hepatocytes were isolated by enzymatic dissociation from the liver of normal BALB/cJ mice or Sprague-Dawley rat according to Guguen et al. (39). Briefly, after cannulation of the portal vein, the liver was perfused with calcium-free Hepes buffer (0.33 mM, pH 7.6) for 5 min for the mouse and 15 min for the rat. Then the liver was perfused with Hepes buffer (0.33 mM, pH 7.6) containing collagenase 0.025% and calcium chloride (0.075%) for 8 min and delivered at 10 ml/min for the mouse and 30 ml/min for the rat. After enzymatic digestion, hepatocytes were obtained by sedimentation (20 min) and then by centrifugation (700 rpm; 1 min) in a Leibovitz medium (Eurobio, France). The supernatant corresponding to the sedimentation step of rat or mouse hepatocytes was centrifuged (1400 rpm; 4 min) to obtain enriched hepatic nonparenchymal cell fraction as adapted from Doolittle and Richter (40). The presence of contaminating hepatocytes evaluated by light microscopy (hepatocytes are bigger cells than non-parenchymal cells) was lower than 5%. Freshly isolated non-parenchymal cells and hepatocytes were frozen immediately in liquid nitrogen before total RNA extraction.

Cell Cultures
Mouse hepatocytes have been seeded in a mixture of minimum essential medium (75%) and M199 (25%) (Eurobio, France) supplemented with 100 IU/ml penicillin, 30 g/ml streptomycin sulfate, 1 mg/ml bovine albumin serum, 5 g/ml bovine insulin, and 10% fetal calf serum (FCS). Four hours later, medium was renewed by an identical medium containing hydrocortisone hemisuccinate, 5 ϫ 10 Ϫ5 or 1 ϫ 10 Ϫ6 M. The day after, hepatocytes have been maintained in the same medium supplemented or not with 10% FCS. Thereafter, hepatocytes were maintained in culture for 2 or 4 days. The medium was renewed every day. In addition, we used human HepG2 (41) rat FAO (42) hepatocarcinoma-derived cell lines that were maintained in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 30 g/ml streptomycin, and 1 ϫ 10 Ϫ6 M hydrocortisone hemisuccinate sulfate or a mixture of Ham's F-12 and NCTC 135 medium (v/v) supplemented with 10% FCS, 100 IU/ml penicillin, and 30 g/ml streptomycin sulfate, respectively. Mouse hepatic cell line (43) derived from immortalized adult mouse hepatocytes was maintained accordingly to Paul et al. (43).

In Vitro Iron Exposure and Iron Depletion of Hepatocytes
During the first 24 h, hepatocytes were maintained under conditions identical to those described previously. The next day, the culture medium was changed by a similar medium devoid of FCS and supplemented with 1 ϫ 10 Ϫ6 M hydrocortisone hemisuccinate. Iron exposure was performed by the addition of citrate-iron (5 and 10 M of iron) in the medium according to the procedure of Azari and Baugh (44). We chose iron-citrate for performing iron exposure because it is a physiologic form of iron that is found in the serum of hemochromatic patients (45) and because the internalization of iron-citrate into hepatocytes is a well described process (46,47). The ratio was 1/10 for iron as compared with citrate. Control cultures were incubated in the absence or in the presence of citrate alone (50 and 100 M). Iron depletion was performed by addition of desferrioxamine (DFO, Ciba Geigy) at the concentration of 10 M (48, 49). In addition, a culture was carried out in presence of both DFO 10 M and citrate-iron 10 M. All the cultures were maintained for 2 days. A similar experiment was performed except that the culture medium was supplemented with 10% FCS.

LPS Treatments
For in vivo experiments, 5-week-old mice BALB/cJ mice were used. LPS (Westphal preparation from Escherichia coli 055:B5; Sigma) was given intraperitoneally in 0.2 ml of sterile, pyrogen-free saline (0.9% NaCl) at the dose of 0.1 mg/kg to mice fasted overnight (50). Animals were sacrificed 90 min later, and their livers were frozen in liquid nitrogen. For in vitro experiments, the primary cultures of mouse hepatocytes, maintained in absence of FCS, were exposed to LPS (20 g/ml diluted in pyrogen-free saline, 0.9% NaCl) for 24 h according to Griffon et al. (51).

Construction of GFP-Prohepcidin Protein Chimeras
The cDNA containing the entire open reading frame of prohepcidin was generated by PCR using the following primers 5Ј-CGGGATC-CGATGATGGCACTCAGCACT-3Ј and 5Ј-CCCAAGCTTCTATGTTTT-GCAACAGAT-3Ј and full-length mouse prohepcidin cDNA as template.
The nuclear localization signal (NLS) deletion mutants was constructed by two-step PCR method as described by Le Seyec et al. (52). Briefly, in a first step, two parallel reactions were performed using the following two sets of primers: P1, 5Ј-CGGGATCCGATGATGGCACTCAGCACT-3Ј, and P2, 5Ј-GATGGGGAAGTTGGTGTCTCTCTGCATTGGTATCGC-AATGT-3Ј, for the first reaction and P3, 5Ј-ACATTGCGATACCAATG-CAGAGAGACACCAACTTCCCCATC-3Ј, and P4, 5Ј-CCCAAGCTTCT-ATGTTTTGCAACAGAT-3Ј, for the second reaction. In a second step, the two PCR products were purified, mixed, and used as template for PCR amplification using P1 and P4 primers. These two PCR products corresponding to wild type and NLS-deleted mutant forms of prohepcidin were digested with BamHI and HindIII and subcloned into with BglII-HindIII digested pEGFP-C3 plasmid (CLONTECH, Palo Alto, CA).

Cell Transfections
The human osteosarcoma cell line U-2OS (53) was obtained from ATCC (Manassas, VA) and grown in Dulbecco's modified Eagle's medium containing 10% FCS. Then these cells were transiently transfected using Lipofectin (Life Technologies, Inc.) according to the manufacturer's instructions. Analysis of protein localization was performed by fluorescence microscopy.

Statistical Analysis
Results were expressed as mean Ϯ S.D. Mann-Whitney test was used for estimation of statistical significance when appropriate. A p value less than 0.05 was considered statistically significant.

RESULTS
Isolation of an Iron-induced Gene-SSH was performed between livers of mice overloaded by 3% of carbonyl iron during 8 months and control mice. Among clones obtained, we isolated a 225-bp cDNA presumably overexpressed under the condition of iron overload. By using this cDNA as a probe, we studied the expression of the corresponding mRNA in the liver of mice overloaded with carbonyl iron during 8 months and in the liver of control mice. As presented in Fig. 1, the size of mRNA hybridizing with the cDNA probe was about 500 bp. The amount of this mRNA was increased in a dose-dependent manner by the carbonyl iron treatment. Its expression was 9.8-fold higher (p Ͻ 0.01) in the liver of 3% carbonyl iron-overloaded mice compared with the control mice. This overexpression was confirmed in a second independent experiment where mice have been overloaded with 3% carbonyl iron for 8 months (8.6 times; p Ͻ 0.05) (data not shown).
Cloning of Full-length cDNA and Analysis of HEPC Gene Organization-To obtain a full-length clone, we screened a mouse liver cDNA library with the 225-bp probe obtained by SSH. The isolated cDNA fragment was 410 bp in length that is similar to previously estimated mRNA size. Thus, this clone likely represents the full-length cDNA ( Fig. 2A). By using the sequence of this mouse cDNA, we searched for homologous sequences in EST GenBank TM data base and found at least two apparently full-length mouse EST clones W12913 (419 bp) and AI255961 (429 bp) showing 100 and 93%, respectively, identity in the overlapping region with our cDNA. Further data base screening led us to identify highly related 412-bp rat and 430-bp human ESTs (GenBank TM accession numbers AW534367 and AI937227, respectively). The last two cDNAs were isolated by RT-PCR and verified by sequencing.
Sequence analysis of the cloned mouse cDNA corresponding to the EST W12913 revealed the presence of consensus Kozak start site (54) at position 35 and open reading frame for a predictive protein of 83 amino acids (aa) (Fig. 2A). A protein data base search allowed us to identify a significant homology (76% of identity) between the C-terminal region of predictive mouse protein and the 25-aa mature chain of human hepcidin (GenBank TM accession number P81172), a secreted peptide purified from human urine. Thus, it is likely that we cloned mouse orthologue of human hepcidin. Because the isolated cDNA encodes a proform of this peptide, it was named prohep-cidin1. Consequently, the second mouse EST clone AI255961 encoding an 83-aa protein that exhibits 90% identity with prohepcidin1 was referred to as prohepcidin2. The corresponding mouse genes were named Hepc1 and Hepc2. Analysis of the deducted amino acid sequences of human and rat cDNA clones revealed that both encode 84-aa proteins and showed 54 and 77%, respectively, identity with mouse prohepcidin1. Alignment of the predicted mouse, rat, and human amino acid sequences is presented in Fig. 2B. We noted the presence of repeated 6 leucines and the conserved locations of 8 cysteines. Moreover, these proteins are rich in positively charged residues (10 from 83) represented by arginine and lysine.
The GenBank TM search revealed the presence of both mouse Hepc1 and Hepc2 genes on mouse genomic clone CT7-8N15 (GenBank TM accession number AC020841). Comparison of mouse prohepcidin1 cDNA sequence to that of CT7-8N15 clone enabled us to determine the exon/intron organization of the mouse gene. We deduced that this gene was small (1654 bp) and composed of 3 exons and 2 introns (Fig. 2C). Just on the 5Ј upstream flanking region of this gene, we found the gene Usf2 (upstream stimulatory factor 2) that was located in tail-to-head orientation. The 1240-bp region separating the end of Usf2 gene from the start of exon 1 of Hepc1 gene was analyzed for predictive transcription factor binding sites using MatInspector. This investigation pointed out the presence of putative HNF3␤, C/EBP␤, and NF-B regulatory element binding sites and a TATA box located about 70 bp upstream of the first start codon. Interestingly, nucleotide sequence and organization of Hepc2 gene showed a very high degree of similarity to that of Hepc1 suggesting that mouse Hepc1 and Hepc2 genes arose from relatively recent duplication of ancestral gene. As Usf2 is located on chromosome 7 in mouse (55), we deduced that HEPC genes were also on chromosome 7. In addition, we determined the genomic organization of the human gene taking advantage of the human genomic clone R30879 from chromosome 19 that matched 100% with the human cDNA (Fig. 2D). Similar to the mouse counterpart, human HEPC gene includes 3 exons, lies in close proximity to the USF2 gene, and contains potential binding sites for HNF3␤, C/EBP␤, and NF-B in the upstream regulatory region.
Tissue-specific Expression-After having identified an mRNA encoding a protein belonging to the antimicrobial peptide family, regulated by iron in the liver, we looked for its tissue-specific expression in mice. By Northern blot, using the cDNA of 225 bp obtained by SSH as a probe, we observed that HEPC mRNA was predominantly expressed in the liver and very weakly in stomach, intestine, colon, lungs, heart, and FIG. 2. A, sequence of the full-length (410 bp) murine HEPC cDNA. B, alignment of putative mouse prohepcidin1 with its rat and human counterparts using ClustalW 1.7 program. Black color corresponds to identical residues and gray color to similar residues. C and D, schematic representation of the genomic organization of (C) mouse Hepc1 gene and (D) human HEPC gene. Boxes represent exons. Gray and white colors correspond to coding and to untranslated regions, respectively. thymus (Fig. 3A). By using multiple tissue expression array membrane and human cDNA as a probe, we studied mRNA expression in human adult and fetal organs and found that it was very strongly expressed in the adult and fetal liver and to a lesser extent in adult left atrium of heart, fetal heart, and adult spinal cord (Fig. 3B). Non-hepatic cell lines which were investigated did not express HEPC mRNA (Fig. 3B). Since HEPC mRNA was specifically expressed in the liver of both humans and mice, we sought the hepatic cellular types expressing HEPC mRNA. By using the mouse fragment of cDNA as a probe, we found that HEPC mRNA was expressed in freshly isolated hepatocytes both in mouse and in rat (Fig. 4, A and B). In enriched non-parenchymal cells isolated from mouse and rat livers, this mRNA expression was much weaker (Fig. 4, A and B) as compared with hepatocytic expression. As expected, albumin mRNA was strongly expressed in hepatocytes and was undetectable in nonparenchymal cells. The amount of HEPC mRNA rapidly and dramatically decreased in mouse hepatocytes maintained under serum-free conditions, and addition of FCS partially stabilized expression of HEPC transcripts (Fig.  4C). Furthermore, HEPC transcripts were undetectable in human, rat, and mouse hepatic cell lines that we studied by Northern blot (data not shown). However, we evidenced a PCR product generated from reverse-transcribed HepG2 mRNA using human HEPC-specific primers. These data suggest that expression of HEPC mRNA is likely related to hepatocyte differentiation status.
Iron Regulation of HEPC mRNA Expression-To confirm that HEPC mRNA expression was linked to the amount of iron in the liver, two other types of in vivo iron overload were studied as follows: experimental iron overload using iron-dextran, and spontaneous iron overload observed in ␤ 2 -microglobulin (Ϫ/Ϫ) mice. In addition, to ascertain that a decrease of liver iron concentration leads to a drop of HEPC mRNA expression, ␤ 2 -microglobulin-deficient mice were fed with an iron-poor diet. In the liver of mice injected by iron-dextran and sacrificed 2 months later, the amount of HEPC mRNA was 4 times higher compared with the control mice (p Ͻ 0.01) (Fig. 5A). In this experiment, LIC was 170.6 Ϯ 29 mol of iron/g of dry liver for iron-overloaded mice and 7 Ϯ 0.8 mol of iron/g of dry liver of control mice. After 4 months, mRNA expression was induced 6 times (p Ͻ 0.05) in iron-overloaded mice compared with controls (data not shown). In 12-month-old ␤ 2 -microglobulin (Ϫ/Ϫ) mice (developing spontaneous iron overload), hepatic expression of HEPC mRNA was also found to be overexpressed (2 times, p Ͻ 0.05) compared with the control wild type mice (Fig. 5B). In these mice, the LIC reached 23 mol of iron/g of dry liver for ␤ 2 -microglobulin knockout mice versus 2.5 mol for control mice.
In the liver of ␤ 2 -microglobulin (Ϫ/Ϫ) mice fed a low iron diet, the liver iron concentration was 4 times lower in comparison with ␤ 2 -microglobulin (Ϫ/Ϫ) mice fed a normal diet. By Northern blot analysis, this decrease of LIC was accompanied by a 5.5 times (p Ͻ 0.03) reduction of HEPC mRNA in the liver of iron-deprived mice compared with spontaneous iron-overloaded mice (Fig. 5C).
Then, to determine whether overexpression was observed in earlier steps of iron overload, we looked for hepatic mRNA expression in mice overloaded with 3% carbonyl iron for 2 months and compared it with that observed after 8 months of treatment. Under these conditions, HEPC mRNA expression was induced as soon as 2 months of carbonyl iron treatment (10 times, p Ͻ 0.03). Its amount was comparable with that observed at 8 months of 3% carbonyl iron administration. Likewise, in the iron-dextran model, overexpression was observed in an independent experiment, as soon as 1 week after iron injection (2.5 times) and was similar to that observed after 2 months (2-fold increase) (data not shown).
Next, to evaluate the direct effect of iron on HEPC mRNA expression, we studied mRNA expression in vitro in mouse hepatocytes either exposed to iron or depleted of iron. Whatever the presence or absence of FCS, the addition of iron citrate or iron chelator (DFO) to the culture medium did not induce significant changes in HEPC mRNA expression (data not shown).
Induction of HEPC mRNA Expression by LPS-Based on the similarity of prohepcidin to antimicrobial peptides and considering that some of them are induced by LPS, we tested the effect of LPS on HEPC mRNA expression in vivo in mouse liver and in vitro using hepatocytes cultures. As shown in Fig. 6, the steady state level of mRNA was 4.3 times higher (p Ͻ 0.05) in the liver of mice injected with LPS. This result was confirmed by in vitro experiments that showed a 7-fold increase of HEPC mRNA in mouse hepatocytes treated with LPS compared with control culture (Fig. 6).
Localization of the GFP-Prohepcidin Chimera-To determine the cellular localization of prohepcidin protein, it was expressed as a fusion with GFP. In contrast to GFP that exhibited a homogeneous intracellular pattern of fluorescence (Fig. 7A), the fluorescence of cells transfected with expression vector encoding the GFP-prohepcidin chimera was specifically localized in nuclei (Fig. 7B). Predictive studies using PSORT II program (56) enabled us to identify a putative nuclear localization signal (KRRK) in prohepcidin protein. To verify the functional role of this sequence, these four amino acids were deleted from prohepcidin protein, and the resulting mutant was fused to GFP. In this configuration, in most of the transfected cells, fluorescence was predominantly found in the cytosol, and the nuclei appeared devoid of fluorescence (Fig. 7C). DISCUSSION To identify novel hepatic iron up-regulated genes, we took advantage of the carbonyl iron overload model that we previ-ously developed in mice (29). We performed an SSH between cDNAs obtained from livers of control and iron-overloaded mice, and we isolated a 225-bp cDNA corresponding to a gene that was overexpressed in the liver of carbonyl iron-overloaded mice.
To confirm that the observed mRNA overexpression was directly related to iron overload itself and not to other parameters related to the carbonyl iron model, we investigated hepatic mRNA expression in other in vivo models of iron overload.
The fact that the HEPC mRNA was induced in all these different models led us to conclude that liver iron excess itself is responsible for the up-regulation of mRNA expression. In addition, the amount of mRNA was directly linked to liver iron concentration as evidenced by the dose dependence of mRNA induction in carbonyl iron-overloaded mice and the decrease of HEPC mRNA expression in ␤ 2 -microglobulin (Ϫ/Ϫ) mice fed with a low iron content diet. Thus, iron overload triggers HEPC mRNA overexpression, and iron depletion leads to a decrease of its expression. However, it seems that mRNA expression was not dependent on the duration of iron overload. Indeed, HEPC mRNA was strongly up-regulated as early as 2 months and 1 week in carbonyl iron and iron-dextran models, respectively, and similar levels were maintained thereafter.
In contrast to in vivo situation where HEPC mRNA was strongly overexpressed during iron overload, the amount of transcript was not modified by in vitro exposure of primary mouse hepatocytes (hepatic cells that predominantly expressed HEPC mRNA) to iron citrate. Cultured hepatocytes are widely used as a tool to perform metabolic and pharmacologic studies as an alternative to animal models (57). However, it is likely that the absence of HEPC mRNA induction by iron was related to hepatocyte phenotypic changes that occur with time in culture. Indeed, when maintained for a few days in primary cultures without differentiation promoting factors, hepatocytes lost the expression of several liver-specific genes (58). In particular, hepatocytes in conventional culture failed to maintain expression of genes encoding members of the CYP2B subfamily of the cytochromes P450 and lost their inducibility by phenobarbital (59). The fact that HEPC mRNA expression (i) is higher in human adult liver compared with fetal liver, (ii) decreases spontaneously in conventional mouse hepatocyte culture, and (iii) is not detected in hepatic cell lines could support the hypothesis that expression of HEPC mRNA is dependent of hepatocyte differentiation status.
Full-length mouse cDNA as well as human and rat orthologues were cloned, and genomic organization of mouse and human genes was established. In addition, we determined that mouse genome contains two highly related Hepc genes. Both Hepc genes, which exhibit similar genomic organization, are located in tandem on chromosome 7 and likely arose from duplication of an ancestral gene. In humans, HEPC gene was found on chromosome 19 in a locus that is syntenic to the region containing Hepc genes on chromosome 7 in mouse (60).
The translation of both murine cDNAs leads to an 83-aa protein that shows strong homology in its C-terminal region to mature forms of a human antimicrobial peptide, the hepcidin, suggesting that we have identified the murine proforms of hepcidin. As shown in the accompanying paper by Ganz and co-workers (86), peptides of different length (20, 22, and 25 aa) corresponding to mature forms of hepcidin were purified from human urine. It should be noticed that Krause et al. (61) also reported in a recent paper, published after submission of our manuscript, a 25-aa peptide that is similar to the 25-aa hepcidin form described by Park et al. (86). The human peptides of 20 and 25 aa, resulting from the processing of the 84-aa proform, exhibited antimicrobial activities against bacteria and fungi. The high number of cationic residues in the mouse protein and the conserved position of cysteines between human and mouse proteins argue in favor of an antimicrobial role of mature forms of hepcidin in mice.
Interestingly, both in mice and in humans, HEPC transcripts were found exclusively in the liver. The presence of potential binding sites for liver-enriched transcription factors C/EBP␤ (62,63) and HNF3␤ (64), known to regulate expression of several hepatic genes, indicates that these transcription regulators could confer liver specificity to hepcidin. To our knowledge, it is the first demonstration of a liver-specific mammalian antimicrobial peptide that is reminiscent of insect antimicrobial peptides in the fat body, the functional equivalent of mammalian liver (65,66). However, we did not find any structural homologues of mammalian prohepcidin in the Drosophila genome.
Considering that mature forms of human hepcidin were purified from urine, it was unexpected to find that mouse GFPprohepcidin chimera was localized in the nuclei of mammalian cells. We demonstrated that nuclear localization of this protein was related to the presence of an NLS because its deletion led to a cytoplasmic expression of prohepcidin mutant form. This confirmed the functional role of this sequence. However, the mechanism involved in the transport of the mature form of prohepcidin to the extracellular zone is not fully understood. The presence of hepcidin in biological fluids is unlikely resulting from necrosis of hepatocytes when considering that normal liver is characterized by a very low level of renewal and that this peptide was isolated from the urine of healthy persons (see accompanying article (86)). At least two mechanisms could be taken into consideration. The prohepcidin could be transitorily addressed to the nucleus before relocation to the cytoplasm and secretion. This process has been described for at least one secreted protein, the fibroblast growth factor 2 (FGF-2). It has been shown that the GFP low molecular weight FGF-2 form chimera was rapidly translocated to the nucleus after exposure of cells to the expression vector and then retro-transported to the cytoplasm, after which this molecule was secreted (67). Alternatively, the GFP-prohepcidin might be processed in the cytoplasm releasing the secreted mature C-terminal hepcidin, and the N-terminal part of prohepcidin containing NLS could be addressed to the nucleus. Indeed, the prohepcidin potential NLS sequence also matches the signal sequence cleavage site (RX(R/K)R2X) for furin and related mammalian subtilisin/ Kex2p-like propeptide convertases that could process prohepcidin to the secreted forms (68). It is tempting to speculate that under normal physiological conditions most of prohepcidin would be cleaved in the liver by furin-like convertases to produce mature chains of hepcidin. Indeed, at least three enzymes of this family, namely furin, PACE4, and PC8, are expressed in liver (69). However, considering our data, we cannot exclude that, due to the presence of functional NLS, at least some amount of prohepcidin could be addressed to the nucleus. In addition, in pathological situations in which expression or activity of processing enzymes would be reduced or inhibited, the localization and the fate of prohepcidin could be modified.
Patients developing iron overload have been reported to be more sensitive to bacterial infections, suggesting a relationship between iron and infection (70 -72). In addition, it has been demonstrated that lactoferrin, a protein that binds iron in milk, has antimicrobial activity after cleavage (73). However, why an mRNA encoding the antimicrobial peptide hepcidin is induced during iron overload remains an open question. It is well known that expression of several antimicrobial peptides in mammals can be enhanced by exposure to bacterial cells or components such as LPS (74 -76). In our work, we also found a strong positive effect of LPS both in vivo and in vitro on HEPC mRNA expression. Moreover, we found that, in vitro, HEPC mRNA was maintained only in the presence of FCS suggesting that this effect could be linked to the presence of traces of endotoxin in the serum (77). All these data suggest that iron induction of HEPC mRNA probably resulted from inflammation related to chronic iron overload. In our previous report (29), histological studies did not detect important inflammation in the liver of iron-overloaded mice. However, a subtle inflammatory process has been noticed in genetic hemochromatic patients with heavy iron overload (78). In addition, heavy iron deposits that are observed during iron overload in the Kupffer cells of long term carbonyl iron-overloaded mice (29) and irondextran mice very soon after iron injection, but also during heavy iron overload in human diseases, could modify cytokine expression in the liver. Alternatively, it cannot be excluded that the LPS effect on HEPC mRNA expression could be at least partially iron-mediated, since some data have reported that endotoxin and endotoxin-induced cytokines tumor necrosis factor-␣, interleukin-1␤, and interleukin-6 (79 -81) enhanced liver cell uptake of transferrin-bound iron.
Finally, our data do not exclude the possibility that murine prohepcidin could play a distinct role in addition to its antimicrobial activity. Indeed, antimicrobial peptides are also known to prevent the oxidative stress (82), to exert antitumor activity (83,84), and to be involved in regulation of angiogenesis (85).
In conclusion, we isolated a novel liver-specific murine mRNA that is overexpressed during iron overload in mice and that encodes an 83-aa protein homologous to the human antimicrobial peptide hepcidin. Further studies are required to determine the physiological role of prohepcidin and its mature forms during iron overload.


extfieldsofstudy: Medicine Biology

text:
Demonstration of Polysaccharide Capsule in Campylobacter jejuni Using Electron Microscopy

ABSTRACT Recently, we reported that Campylobacter jejuni, an important gastrointestinal pathogen, has the genetic determinants to produce a capsular polysaccharide (Karlyshev et al., Mol. Microbiol. 35:529–541, 2000). Despite these data, the presence of a capsule in these bacteria has remained controversial. In this study we stain C. jejuni cells with the cationic dye Alcian blue and demonstrate for the first time by electron microscopy thatC. jejuni cells produce a polysaccharide capsule that is retained in the coccoid form but is absent in a kpsMmutant.

Campylobacter jejuni is currently a major cause of bacterial gastrointestinal disease in humans in many countries (13). Despite deciphering the genomic sequence of one isolate, NCTC 11168 (12), many questions relating to the biochemistry, physiology, and pathogenicity of the microorganism remain unanswered (20). Capsules frequently play key roles in the survival of bacteria and in immune evasion of several bacterial pathogens (16). Recently, it has been suggested that the high-molecular-weight fractions of C. jejuni lipopolysaccharide may be capsular polysaccharide (CPS) (3). Using the NCTC 11168 genome sequence data, we reported the genetic and biochemical characterization of these molecules and found that they are related to group II and III CPS found in other pathogenic bacteria (8). Despite detection of CPS in C. jejuni, to date there have been no reports on the direct visualization of capsules in this species. In this study, using electron microscopy (EM), we demonstrate for the first time that this important pathogen does produce a polysaccharide capsule surrounding the surface of the cell. Capsule detection was facilitated by our recent discovery that CPS of C. jejuni has a strong affinity to Alcian blue dye (9).
Bacterial polysaccharide capsules are usually heavily hydrated polymers loosely attached to the bacterial cell surface (16) cluding treatment with antibodies, different dyes, and chemical cross-linking (2). Alcian blue has been used for staining capsules in several bacterial species, including Klebsiella pneumoniae (2), Neisseria gonorrhoeae (6,14), Bordetella bronchiseptica (10), Pasteurella multocida (5), and Haemophilus pleuropneumoniae (7). Both whole bacteria (10) and thin sections (2,5,6,7,14) were used successfully. To avoid disturbing fragile capsular material, bacteria are often fixed and stained directly on agar plates, followed by thin-section analyses (7,14). Ruthenium red dye has also been used for staining bacterial CPS, for example, in Vibrio parahaemolyticus (4,5). Alcian blue may stabilize the capsule, since treatment of cells only during the initial fixation step but not at subsequent stages facilitated capsule detection (10). Although Alcian blue is known to have a high affinity for acidic polysaccharides (1), the mechanism by which the dye binds is unclear.
Fixation and processing steps were performed within a fume cupboard at room temperature in vials on a rotary mixer. Agar pieces with bacteria were put into fixative containing 1% Alcian blue 8GX (or 0.1% ruthenium red) and 3% glutaraldehyde prepared in 0.075 M sodium cacodylate buffer (pH 7.4) and incubated for 2. crose solution overnight in a refrigerator at 4°C, washed in ultrapure MilliQ water, and dehydrated through a graded series of 30 to 100% ethanol for 10 min per step, transferred sequentially into 3:1, 1:1, and 1:3 ethanol-TAAB resin (TAAB Laboratories, Reading, United Kingdom) mixtures for 1 h per step, and finally into 100% TAAB resin overnight. The samples were incubated in fresh 100% TAAB resin for 3 h, embedded in silicone flat molds, and placed into a 60°C oven to polymerize for 2 days. The blocks were removed from the molds and trimmed and 100-nm ultrathin sections were cut using a glass knife and a Leica Ultracut R ultramicrotome, (Leica Microsystems, Milton Keynes, England). The sections were floated onto MilliQ water and then placed onto copper grids coated with a thin Pioloform support film. The grid-mounted sections were then stained with 2% alcoholic uranyl acetate for 10 min in the dark, thoroughly washed in MilliQ water, then stained for a further 10 min on a drop of Reynolds' lead citrate (15) in a CO 2 -free atmosphere, washed in MilliQ water, and allowed to air dry before examination and photography on a Jeol 1200EX transmission electron microscope at 80 kV (Jeol Ltd, Welwyn Garden City, England). Micrographs were taken on Agfa Scienta EM film, and the resulting images were printed on Agfa multicontrast resin-coated paper using an Agfa Rapidoprint processor and Agfa chemistry.
Ruthenium red dye was unable to stain C. jejuni cells (Fig.  1). By contrast, clearly defined capsules were detected after cells on agar blocks were fixed in the presence of Alcian blue ( Fig. 2A and 2B). The capsule was presented as a thick amorphous structure covering bacterial cells. The thickness of the capsule usually varied between 70 and 100 nm, whereas the diameter of cross-cut cells was in the range of 200 to 250 nm. Some capsules were extended by a distinct massive fibrous layer (F) containing a characteristic bright internal area (Fig.  2C). As F structures have been reported during Alcian blue EM visualization of capsules in other microorganisms (2, 5), we investigated their nature. Surprisingly, such F structures could also be observed after treatment of blank agar blocks with Alcian blue. We suggest that F structures visible in EM are aggregates of Alcian blue dye. Such aggregates, which can often be observed on EM photographs between the cells, stick to encapsulated cells (Fig. 2C) and can easily be differentiated from amorphous capsule layer with irregular structure ( Fig. 2A  and B).
To investigate whether the capsule in the G1 strain consists of CPS, Alcian blue staining and EM were performed on defined insertional G1 mutants. One of the mutations, pldA, does not affect CPS production (9), and this mutant was used as a positive control. By contrast, the kpsM mutation was found to abolish production of CPS molecules (8). A capsule was seen in the pldA mutant, but it was somewhat thinner than the capsule observed in the wild-type strain (Fig. 3A). The reason for this difference remains unclear, although one cannot exclude that capsule formation can be modulated by the outer membrane phospholipase product of the pldA gene. As expected, no capsule was detected in the kpsM mutant by Alcian blue staining (Fig. 3B), confirming that the structure detectable in wild-type G1 cells is CPS.
The results presented in Fig. 1 to 3 were from a 2-day-old culture. It is known that C. jejuni cells are prone to undergo transformation from a spiral to a coccoid morphology after prolonged incubation on a growth medium (19). We investigated whether this transformation has any effect on capsule staining. Four-day agar cultures of strain G1 were analyzed in EM after staining with either ruthenium red or Alcian blue dye. Most cells after 4 days of growth had become oval, with an average size of about 900 nm, i.e., about three times the crosssection of the spiral forms ( Fig. 4A and B). As with 2-day-old cultures, no capsule could be detected with ruthenium red (Fig. 4A). A dark internal area presumably consisting of a nucleoid could be observed in the older culture. Interestingly, in contrast to 2-day-old cultures, the space between the nucleoid and cell wall is nonstainable. This probably indicates dramatic reduction in biochemical (transcriptional) activity in the coccoid cells. Similar observations were noticed when the same culture was treated with Alcian blue. A difference in this case, however, was the presence of a capsule (Fig. 4B). As in strain G1, a capsule was detected in wild-type strain X but not in the X kpsM::kan mutant (data not shown). The use of kpsM mutants in both cases was essential in eliminating the possibility of false-positive results due to a potential of Alcian blue to form aggregates and interact with other molecules on the cell surface (14).
We also attempted to detect a capsule in the sequenced strain NCTC 11168 (12). A gene cluster related to CPS production is present in this strain (8). However, attempts to detect capsule in this strain have not been successful despite the observation that CPS of this strain is stainable with Alcian blue (9). One possibility is that the CPS of NCTC 11168 has lower affinity for Alcian blue and staining conditions on a gel (acid pH) are different from those used for staining-fixation for EM. This correlated with the lower intensity of CPS in the NCTC 11168 strain compared to that of strain G1 (Fig. 5). It is possible that despite CPS production in NCTC 11168, the mechanism of capsule formation in this strain is impaired or specific environmental signals are required for capsule formation. Reversible loss of capsule has been reported in some bacteria (4). As the kpsM gene is present in all nine C. jejuni strains tested (data not shown) and this gene is involved in both CPS biosynthesis and capsule formation in strain G1, we suggest that the majority of C. jejuni strains make or have the potential to make a polysaccharide capsule.
Through a combination of Alcian blue staining and EM, we have shown for the first time visualization of a C. jejuni capsule. This finding has confirmed the prediction from sequence analysis that C. jejuni cells may produce CPS and paves the way for the full genetic, biochemical, and structural analysis of this subcellular organelle. We have also demonstrated that despite apparent significant structural and biochemical changes, the coccoid form of C. jejuni retains the capsule. By similarity to the polysaccharide capsules of other microorganisms, C. jejuni capsules may be involved in enhancing survival of the microorganism in the environment by affording resistance to desiccation and increased adherence to various surfaces via biofilm formation, as well as in colonization and resistance to nonspecific and specific host immunity (reviewed in reference 16).
C. jejuni remains a major conundrum in microbiology. One paradox is how a microorganism that is difficult to culture and that has reduced viability when cultivated in vitro is able to survive in the environment to be the most frequently isolated bacterial food-borne pathogen (13,18). The polysaccharide capsule described in this study might be an important new factor essential for the survival and pathogenicity of C. jejuni. Its discovery should facilitate future investigations on the survival, transmission, and pathogenesis of this problematic foodborne pathogen and may assist in the development of improved intervention strategies to reduce the burden of C. jejuni infection.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
The Ca2+-binding Proteins S100A8 and S100A9 Are Encoded by Novel Injury-regulated Genes*

To gain insight into the molecular mechanisms underlying cutaneous wound repair, we performed a large scale screen to identify novel injury-regulated genes. Here we show a strong up-regulation of the RNA and protein levels of the two Ca2+-binding proteins S100A8 and S100A9 in the hyperthickened epidermis of acute murine and human wounds and of human ulcers. Furthermore, both genes were expressed by inflammatory cells in the wound. The increased expression of S100A8 and S100A9 in wound keratinocytes is most likely related to the activated state of the keratinocytes and not secondary to the inflammation of the skin, since we also found up-regulation of S100A8 and S100A9 in the epidermis of activin-overexpressing mice, which develop a hyperproliferative and abnormally differentiated epidermis in the absence of inflammation. Furthermore, S100A8 and S100A9 expression was found to be associated with partially differentiated keratinocytes in vitro. Using confocal microscopy, both proteins were shown to be at least partially associated with the keratin cytoskeleton. In addition, cultured keratinocytes efficiently secreted the S100A8/A9 dimer. These results together with previously published data suggest that S100A8 and S100A9 are novel players in wound repair, where they might be involved in the reorganization of the keratin cytoskeleton in the wounded epidermis, in the chemoattraction of inflammatory cells, and/or in the defense against microorganisms.

After cutaneous injury, a series of biological events takes place that aims at the reconstruction of the damaged skin. Among them are the migration, proliferation, and differentiation of inflammatory, epithelial, and mesenchymal cells. These cells exert specific functions in a temporally and spatially coordinated manner such as the removal of irreversibly destructed tissue, the deposition of new extracellular matrix, and the reestablishment of the cutaneous barrier (1,2). These proc-esses are well described at the histological level, but little is known about their molecular basis.
To gain insight into the molecular mechanisms that underlie the repair process, we performed a large scale subtractive hybridization screen to systematically identify genes that are differentially expressed in injured compared with normal skin. To minimize the risk of detecting differences in gene expression levels due to changes in cellular composition rather than to transcriptional regulation, we compared normal skin with early (24 h) wounds, because only minor changes in cell type composition occur during the initial wound healing period.
One of the cDNA clones that we obtained encodes the murine S100A8 protein (also known as calgranulin A, MRP8, leukocyte protein L1, or cytokine CP-10). S100 proteins are intracellular Ca 2ϩ -binding and Ca 2ϩ -modulated proteins that form antiparallel noncovalently linked dimers in solution and play a role in various Ca 2ϩ -mediated cellular functions including cell growth and differentiation, energy metabolism, cytoskeletalmembrane interactions; some of the S100 proteins regulate kinase activities or are secreted and perform extracellular functions (3). S100A8 and its dimerization partner, S100A9 (MRP14), are expressed constitutively in neutrophils and monocytes and, after activation, in macrophages and are secreted by these cells, whereas they are absent from normal skin (4 -11). However, they are up-regulated in the epidermis as well as in other tissues at sites of inflammation in conditions such as psoriasis, rheumatoid arthritis, inflammatory bowel and lung diseases, and allograft rejection (5,(12)(13)(14)(15)(16)(17)(18). Here we demonstrate that S100A8 and S100A9 expression is strongly induced during wound healing. We show that these genes are expressed at high levels in the hyperproliferative wound epithelium during the first week after skin injury as well as in inflammatory neutrophils and macrophages that have infiltrated the wound. Moreover, we detected increased levels of S100A8 and S100A9 in the non-inflamed but hyperproliferative, abnormally differentiated epidermis of activin-overexpressing transgenic mice (19) as well as in activin-overexpressing HaCaT keratinocytes, which differentiate prematurely compared with wild-type cells. We further show that normal and activin-transfected HaCaT cells secrete a substantial fraction of the S100A8/9 proteins they produce into the culture medium. The results presented in this paper indicate that the induction of these genes is not a consequence of the inflammation but rather correlates with an alternative differentiation pathway of the keratinocytes and suggest a novel role for secreted S100A8/9 during wound healing.

EXPERIMENTAL PROCEDURES
Animal Care-BALB/c mice were obtained from Charles River Wiga, Sulzfeld, Germany and from RCC Ltd., Fü llinsdorf, Switzerland. They were housed and fed according to German or Swiss Federal guidelines, and all procedures were approved by the local government of Bavaria or of the Kanton Zü rich.
Wounding, Preparation of Wound Tissue, and RNA Isolation-Several independent wound healing experiments were performed. For each experiment, 24 BALB/c mice (8 -10 weeks of age) were anesthetized with a single intraperitoneal injection of ketamine/xylazine. The hairs on the back of the animals were cut, and the skin was wiped with 70% alcohol. Four full-thickness excisional wounds (5-mm diameter, 8 -10 mm apart) were generated on the back of each animal by excising skin and panniculus carnosus. The excised skin pieces were saved and used as unwounded control samples. The wounds were allowed to dry to form a scab. At different time points after injury (1, 3, 5, 7, and 14 days), animals were sacrificed, and the complete wounds including 2 mm of the wound margins were isolated.
For preparation of RNA, the tissue from four animals was combined, immediately frozen in liquid nitrogen, and stored at Ϫ80°C until used for RNA isolation. Total cellular RNA was prepared as described by Chomczynski and Sacchi (20). For in situ hybridization, immunohistochemistry, and immunofluorescence, wounds were bisected and fixed for 4 h in 4% paraformaldehyde in phosphate-buffered saline (PBS). 1 Half of each wound was transferred to 15% sucrose in PBS before embedding in tissue-freezing medium (Jung, Köln, Germany) and preparation of frozen sections, and the other half was embedded in paraffin wax and processed according to standard procedures.
Human Biopsies-Biopsies were taken by consent from patients presenting ulcera crura (n ϭ 4) of more than 4 months' duration, due to primary or secondary venous insufficiency (size of ulcer Ͼ30 cm 2 ). The biopsies were obtained from the wound edge of chronic wounds and showed no clinical sign of infection. As a control, intact skin from the same patients was taken. For the analysis of physiological wound healing, biopsies were taken from healthy volunteers (n ϭ 4), including day-5 wounds (6-mm diameter) and intact skin. After paraformaldehyde fixation, one biopsy was embedded in tissue freezing medium, and the other biopsy (from the same site and the same patient) was embedded in paraffin wax and processed according to standard procedures. All procedures were approved by the local ethics committee of the Technical University of Munich.
cDNA Synthesis, cDNA Subtraction, Reverse Northern Blot, and Screening-Double-stranded cDNA was synthesized starting from 1 g of total RNA using Superscript II reverse transcriptase (Life Technologies, Inc.) and the SMART PCR cDNA synthesis kit (CLONTECH, Heidelberg, Germany). cDNA subtraction was performed using the PCR-Select cDNA subtraction kit (CLONTECH). cDNA synthesis and subtraction were performed according to the manufacturer's recommendations. 50 ng of subtracted cDNA were cloned directly into 50 ng of the pT-Adv vector (CLONTECH). The ligation mixture was introduced into Escherichia coli SURE (Stratagene, Amsterdam, Netherlands) by electroporation (1.7 kV) using an E. coli pulser (Bio-Rad). The resulting library was separated into aliquots and frozen in 15% glycerol. An aliquot was plated on selective media containing 100 M isopropyl-1thio-␤-D-galactopyranoside and 50 g/ml 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside. A total of 3840 individual recombinant clones were picked and used to inoculate 40 sterile 96-well microtiter plates containing LB medium and ampicillin at 100 g/ml. After PCR amplification of the cDNA insertions according to the manufacturer's instructions (CLONTECH), the products were dotted in an array onto nylon membranes for duplicate screening using a 384 pin replicator (NUNC, Denmark). The cDNAs were denatured by incubating the prepared membranes on polyethylene-coated Whatman paper soaked in 0.4 M NaOH for 20 min followed by washing the membranes with Tris EDTA buffer. Fixation was performed by baking (30 min at 80°C) followed by UV-cross-linking (256 nm, 120 mJ/cm 2 ). The filters were hybridized under stringent conditions (25 mM sodium phosphate buffer pH 7.5, 125 mM NaCl, 7% SDS at 67°C) with equivalent amounts of 32 P-labeled subtracted double-stranded cDNAs of equal specific activity derived from the wound minus skin and the skin minus wound subtractions. Filters were washed under stringent conditions (hybridization buffer diluted 1:10 at 67°C) and analyzed using the PhosphorImager (Storm 860, Molecular Dynamics, Krefeld, Germany). Differentially expressed cDNAs were identified by comparison of the resulting signals.
RNase Protection Assay-RNase protection assays were performed according to (21). 276-and 267-base pair fragments corresponding to the full-length coding sequence of S100A8 and nucleotides 108 -375 of the coding sequence of S100A9 served as templates for the generation of 32 P-labeled antisense riboprobes. The template for the human activin ␤A probe was recently described (19).
Radioactive in Situ Hybridization-Frozen sections (6 m) from the middle of 5-day-old wounds were hybridized as described (22). 246-and 267-base pair fragments corresponding to nucleotides 97-343 of the coding and 3Ј-untranslated sequence of S100A8 and nucleotides 108 -375 of the coding sequence of S100A9 served as templates for the generation of 35 S-labeled antisense riboprobes. After hybridization, sections were coated with Kodak NBT2 nuclear emulsion (Integra Bioscience AG, Wallisellen, Switzerland) and exposed in the dark for 3 weeks. After development, sections were counterstained with Mayer's hemalum and eosin, mounted, and analyzed using a Zeiss Axiophot microscope equipped with a Jenoptik ProgRes 3008 CCD camera.
Immunofluorescence-Paraffin was removed from sections that had been prepared from the middle of 5-day-old wounds and tail skin of activin-overexpressing and wild-type mice. After rehydration, sections were allowed to dry. HaCaT cells were grown to confluence on positively charged microscope slides (Superfrost; Menzel Glä ser, Germany), and some specimens were incubated with 10 M Ca 2ϩ ionophore A23187 (Calbiochem) for 1 h to ensure the activation of Ca 2ϩ -dependent cellular processes before fixation with 4% paraformaldehyde in PBS for 10 min, washing with PBS, and postfixation with ice-cold acetone for 10 min. Specimens were then incubated overnight at 4°C with specific antisera to S100A8 or S100A9 (24,25) and, in some instances, with a mouse monoclonal antibody directed against human cytokeratin 10 (DAKO), diluted in PBS containing 12% bovine serum albumin. After washing with PBS, sections were incubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated secondary antibody directed against rabbit and/or mouse immunoglobulins (Roche Molecular Biochemicals). After washing with PBS, sections were allowed to dry and mounted with Mowiol (Hoechst, Frankfurt, Germany) or n-propyl gallate/Tris/glycerol, and fluorescence was analyzed either by using a Zeiss Axioplan microscope equipped with a Hamamatsu three-color CCD camera or by using a Leica DM IRBE fluorescence microscope equipped with a TCSNT confocal laser system.

SDS-Polyacrylamide Gel Electrophoresis and Western
Blot-Tissues were homogenized in Tris buffer containing 8 M urea, protein concentration was determined by the BCA method, and 30-g aliquots were boiled in sample buffer containing 100 mM dithiothreitol before SDSpolyacrylamide gel electrophoresis and blotting onto nitrocellulose membrane (Protran, Schleicher &Schuell). Membranes were then incubated overnight at 4°C with specific antisera to S100A8 or S100A9 (24,25) followed by alkaline phosphate-conjugated anti-rabbit IgG (Promega, Heidelberg, Germany). Antibody binding was visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega).
Cell Culture and Quantitative Enzyme-linked Immunosorbent Assay of S100 Proteins-Vector-transfected and activin ␤A-transfected HaCaT cells were grown in 100-mm tissue culture dishes in 10 ml of Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (Amimed; Bioconcept, Allschwil, Switzerland) and 200 mg/liter Geneticin (Life Technologies, Inc.). For determination of secreted S100A8/9, the medium was replaced by serum-free DMEM at confluence, and aliquots of 300 l of supernatant were sampled from the dishes immediately and at different time points thereafter. Cells were pelleted from the supernatant, and the supernatant was snap-frozen in liquid nitrogen. In one experiment, the number of dead cells floating in the supernatant was analyzed using trypan blue exclusion and found to be not greater than 0.5% of the total number of cells. At the end of the experiment, the cells on each plate were trypsinized and counted for normalization to cell number. For determination of intracellular S100A8/9, cells were trypsinized at confluence, and an aliquot was counted using a hemocytometer. Cells were then pelleted and snapfrozen in liquid nitrogen. A quantitative sandwich enzyme-linked immunosorbent assay for S100A9 was performed as described previously (26).

RESULTS
S100A8 and S100A9 Expression Is Strongly Up-regulated after Wounding-Using the PCR-Select kit we constructed a subtractive cDNA library of normal versus wounded murine skin and vice versa in order to identify genes that are either upor down-regulated after skin injury. One of the ESTs we obtained from the screen for up-regulated genes corresponded to the full-length murine S100A8 (mS100A8) cDNA. To confirm the differential expression, we analyzed the mS100A8 mRNA levels during the different stages of wound repair by RNase protection assay. As shown in Fig. 1A, mS100A8 mRNA expression, which was barely detectable in healthy skin, was dramatically up-regulated during the first week after injury and subsequently decreased until it had reached low levels by the end of the second week after wounding, when the wounds were fully healed. Identical results were obtained with probes that were either transcribed from the initial EST or from the full-length sequence (shown is the result obtained with the full-length probe). Moreover, this result was confirmed by analysis of independently prepared RNAs using real time reverse transcriptase-PCR. By this technique, a 40-fold increase in mS100A8 mRNA levels in 1-day wounds compared with intact skin was measured (data not shown).
Because S100A8 is usually co-expressed with its dimerization partner, S100A9, we determined whether mS100A9 mRNA is also up-regulated after injury. Indeed, as shown in Fig. 1B, mS100A9 expression was strongly induced in 1-7-dayold wounds. Moreover, mS100A8 and mS100A9 mRNA levels seemed to be regulated in a strictly coordinated fashion. This result was reproduced twice with different sets of RNAs from independent wounding experiments. To determine the expression levels of mS100A8 and mS100A9 proteins within the wounds, Western blot analysis was performed with specific antisera and an alkaline phosphatase-conjugated secondary antibody. As described previously, the specificity of these antisera has been verified by Western blot analysis of lysates prepared from monocytes and granulocytes as well as by immunoreactivity against recombinant S100A8 and S100A9 and against S100A8 and/or S100A9 transfected fibroblastic cell lines (24,25). Western blot analysis of wound lysates revealed that mS100A8 and mS100A9 proteins were also induced after injury, with kinetics that correlated with those of the mRNAs (Fig. 1, D and E).
The Hyperproliferative Wound Epithelium Is a Novel Site of S100A8 and S100A9 Expression-To identify and localize the mS100A8/A9-expressing cell types within the wound, we performed in situ hybridization on sections of 5-day-old wounds. At this stage after injury, a thick hyperproliferative epithelium as well as extensive granulation tissue containing various inflammatory cells is present in the wound. As shown in Fig. 2, 35 S-labeled antisense probes for mS100A8 (Fig. 2, A and B) and mS100A9 (Fig. 2, C and D) yielded striking hybridization sig-FIG. 1. Expression of S100A8 and S100A9 mRNA and protein is strongly induced during wound healing. Aliquots of 20 g from batches of total RNA that was prepared from murine skin wounds at different times after injury were subjected to RNase protection analysis with antisense probes to the full-length coding sequences for mS100A8 (panel A) or mS100A9 (panel B), or 1-g aliquots were separated on an agarose gel and stained with ethidium bromide (EtBr; panel C). probe, 1000 cpm of the radioactive probes were used as a standard for size and intensity of signal; tRNA, 20 g of tRNA was used as a negative control. The film was exposed overnight without the aid of an intensifier screen. Aliquots of 30 g from batches of total protein lysate that was prepared from murine skin wounds at different times after injury were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with specific antisera to mS100A8 (panel D) and mS100A9 (panel E). Binding of the alkaline phosphate-conjugated secondary antibody was visualized by nitro blue tetrazolium and bromo-chloro-indolyl phosphate. Sizes of marker proteins are indicated on the left of the panels. The bands corresponding to the mS100A8 and mS100A9 monomers are indicated by arrows. A double band for S100A8 is frequently observed and most likely results from partial proteolytic cleavage during tissue preparation. d, day. nals throughout the suprabasal layers of the hyperproliferative epithelium (Fig. 2, A-D), indicating that differentiating wound keratinocytes express high levels of mS100A8 and mS100A9 mRNAs. By contrast, keratinocytes at a distance from the wound did not express these genes (data not shown). Inspection of the granulation tissue at high magnification also revealed a specific population of cells that were decorated with silver grains (Fig. 2, G and H). The shape of their nuclei suggests that these cells are neutrophils and macrophages, which are known producers of S100A8 and S100A9 (4 -11). No signals were obtained with probes transcribed in the sense direction (Fig. 2, E and F, for mS100A9; data not shown for mS100A8).
Subsequently, we performed immunohistochemical stains and immunofluorescence analysis with specific antisera to localize the mS100A8 and mS100A9 proteins. Fig. 3, A-C, shows overviews of sections through 5-day-old wounds that were immunostained using a peroxidase-conjugated secondary antibody. In sections stained for mS100A8 (Fig. 3A) and S100A9 (Fig. 3B), the reaction product was seen within the hyperthickened wound epidermis (arrows in Fig. 3, A and B) as well as in punctate locations throughout the deeper layers of the dermis and the granulation tissue, whereas control sections stained with preimmune sera did not show this specific stain (Fig. 3C). To localize both mS100A8 and mS100A9 in the hyperproliferative epidermis at the wound edge at higher resolution, we performed indirect immunofluorescence on serial sections of several 5-day-old wounds. A typical result is shown in Fig. 3, D-F. Immunofluorescent label decorated the cytoplasm of the suprabasal differentiating wound keratinocytes in the sections that were stained for mS100A8 (Fig. 3D) and mS100A9 (Fig. 3E), whereas no epidermal stain was seen when the first antibody was omitted (Fig. 3F). Furthermore, by staining sections of earlier and later wounds, we determined that in 3-dayold wounds, the suprabasal cells of the emerging hyperproliferative epithelium already were highly immunopositive for mS100A8 and mS100A9, whereas at day 13 after injury, when the epidermis reversed to a non-hyperproliferative state, no immunoreactivity was seen in keratinocytes (data not shown). S100A8 and S100A9 Are Induced in Acute and Chronic Human Wounds-To determine whether the up-regulation of S100A8 and S100A9 also occurs in injured human skin, we prepared digoxigenin-labeled antisense probes for the human homologs, hS100A8 and hS100A9, and hybridized them to sections of human wounds. As seen in Fig. 4, both mRNAs were strongly expressed in suprabasal epithelial cells of acute full-thickness excisional skin wounds that were taken from healthy adult volunteers (n ϭ 4) 5 days after injury (Fig. 4, C-F). Moreover, in sections prepared from chronic venous ulcers (n ϭ 3 for S100A8 and n ϭ 1 for S100A9), we observed a strong hybridization signal in the suprabasal layers of the strongly thickened epithelium around the edge of these chronic wounds (Fig. 4, G and H). Intact human skin was devoid of hybridization signal (Fig. 4, A and B), as were sections that had been hybridized to probes transcribed in the sense direction (data not shown). In confirmation of previous data (13, 15, 16, 18, 28), we also found hS100A8 and hS100A9 expression in lesional but not in the non-affected skin of FIG. 3. Localization of S100A8 and S100A9 proteins in sections of murine wounds. 7-m paraffin sections of 5-day-old murine wounds were stained by the indirect peroxidase method using specific polyclonal antisera directed against mS100A8 (panel A), mS100A9 (panel B), or using pre-immune sera (panel C). Specific reaction product appears in red. Sections were counterstained with Mayer's hemalum and viewed at magnifications of 25ϫ. D, dermis; HE, hyperproliferative epithelium; G, granulation tissue; arrows in panels A and B, immunopositive hyperproliferative epithelium. Serial paraffin sections of 5-day-old murine wounds were stained with specific polyclonal antisera directed against mS100A8 (panel D) or mS100A9 (panel E), followed by Cy3-conjugated anti-rabbit-IgG. In panel F, the specific first antibody was omitted. Shown is a view of the hyperproliferative epithelial tongue at the wound edge at 250ϫ magnification. H, position of a hair shaft; E with an arrow, direction toward normal epidermis; arrows, position of the basal keratinocyte layer. Ctrl, control.
FIG. 2. S100A8 and S100A9 mRNAs are localized to differentiating keratinocytes of the hyperproliferative wound epithelium. 6-m frozen sections of 5-day wounds were hybridized to 35 S-labeled antisense probes, which recognized coding sequences of mS100A8 (panels A, B, and G), mS100A9 (panels C, D, and H), or to a mS100A9 probe transcribed in the sense direction (panels E and F). Sections were exposed for 3 weeks, counterstained with Mayer's hemalum and eosin, and viewed at magnifications of 200ϫ (panels A-F; shown is the area of the epithelial edge to the right of the center of the wound) or 630ϫ (panels G and H). Specific hybridization is indicated by dark grains in the bright field views (panels A, C, E, G, and H) and by bright grains in the dark field views (panels B, D, F). G, granulation tissue; HE, hyperproliferative epithelium; arrows in panels G and H, mS100A8 and mS100A9 expressing inflammatory neutrophils that can be identified by their characteristic segmented nuclei. psoriatic patients (data not shown).
To determine whether hS100A8 and hS100A9 proteins are present in the heterodimeric form in human wounds, we prepared immunohistochemical stains using the monoclonal antibody 27E10, which specifically recognizes the human S100A8/A9 heterodimer (27), and a peroxidase-conjugated secondary antibody. As shown in Fig. 5, the reaction product decorated the suprabasal hyperproliferative keratinocytes of a 5-day-old wound (Fig. 5, B and C), whereas the basal keratinocytes were largely devoid of stain (Fig. 5C, arrow). No antibody stain was detected in intact skin (Fig. 5A). This result shows that hS100A8 and hS100A9 proteins are present in the suprabasal keratinocyte layers of wounded skin, and moreover, they are assembled into the heterodimeric complex. S100A8 and S100A9 Are Up-regulated in the Hyperthickened Epithelium of Activin-overexpressing Transgenic Mice-To determine whether the up-regulation of S100A8 and S100A9 in keratinocytes is related to inflammation or to the hyperproliferative activated state of the epithelium, we analyzed the expression of the two genes in the skin of transgenic mice that overexpress the human transforming growth factor ␤ superfamily member activin A in the basal layer of the epidermis. In the tail skin and to a much lesser extent also in the back skin of these mice, a hyperthickened epithelium develops without obvious signs of inflammation. This epithelium is also characterized by abnormal keratinocyte differentiation (19). In the RNase protection assay shown in Fig. 6, mRNAs from two lines of transgenic mice (lines 30 and 31) expressing high levels of recombinant activin (Fig. 6C) were compared with their wildtype siblings. In both transgenic lines, mRNA levels for mS100A8 (Fig. 6A) and mS100A9 (Fig. 6B) were found to be strongly increased in transgenic compared with wild-type tail skin, whereas expression levels in the back skin of both transgenic and wild-type mice were always lower (Fig. 6, A and B) and slightly variable between individual mice (data not shown). No interleukin 1␤ transcripts were detected by RNase protection analysis of the same batches of tail and back skin RNAs (data not shown). Furthermore, tumor necrosis factor-␣ was not expressed in the skin of these mice, 2 and no inflammatory infiltrate was seen. These findings strongly suggest that upregulation of S100A8 and S100A9 occurs in the absence of inflammation. 2 S. Werner, unpublished data. A and B), acute human wounds at day 5 after injury (panels C-F), or chronic wounds (panels G and H; Ulcus cruris lateralis) were hybridized to digoxigenin-labeled antisense probes for hS100A8 (panels A, C, E, and G) or hS100A9 (panels B, D, F, and H) mRNAs. Reaction product appears in a grayish-purple color. Sections were viewed at magnifications of 25ϫ (panels A-D) and 100ϫ (panels E-H). E, epidermis; D, dermis; G, granulation tissue; HE, hyperproliferative epithelium. Note that some sections (panels B and H) were counterstained with nuclear fast red.

FIG. 4. S100A8 and S100A9 mRNAs are up-regulated in acute and chronic human wounds. Paraffin sections of intact human skin (panels
FIG. 5. The heterodimeric S100A8/9 complex is present in the hyperproliferative epidermis of human wounds. Paraffin sections of intact human skin (panel A) or acute human wounds at day 5 after injury (panels B and C) were stained with a specific monoclonal antibody directed against the hS100A8/9 heterodimer. The reaction product appears in red. Sections were counterstained with Mayer's hemalum and viewed at magnifications of 25ϫ (panels A and B) or 100ϫ (panel C). E, epidermis; D, dermis; G, granulation tissue; HE, hyperproliferative epithelium. Arrow in panel C, basal epithelial layer, exhibiting very little immunoreactivity.
FIG. 6. Increased S100A8 and S100A9 mRNA expression in the tail skin of activin-overexpressing transgenic mice. Aliquots of 20 g from batches of total RNA that was prepared from tail or back skin of two lines (ln 30 or ln 31) of transgenic mice (tg) that overexpress the activin ␤A chain in the epidermis or of their wild-type siblings (wt) were subjected to RNase protection analysis with antisense probes to the full-length coding sequences for mS100A8 (panel A) or mS100A9 (panel B) or with a probe to the human activin ␤A chain (panel C). probe, 1000 cpm of the radioactive probes were used as a standard for size and intensity of signal; tRNA, 20 g of tRNA was used as a negative control. The film was exposed for 2 days (panels A and B) or overnight (panel C) without the aid of an intensifier screen.
To localize mS100A8 and mS100A9 expression in the tail skin, we performed immunofluorescence with serial sections prepared from several transgenic and wild-type mice (Fig. 7). Most sections of wild-type animals stained for mS100A8 and mS100A9 did not contain any labeled epithelial cells. A few patches of immunopositive cells were detected in the normal epidermis of one out of five mice only (Fig. 7, A and C). In transgenic mice, on the other hand, both proteins were coexpressed at high levels by epithelial cells residing within the suprabasal layers of the strongly hyperthickened epidermis (Fig. 7, B and D), although the expression pattern was patchy. No increased numbers of immunopositive inflammatory cells were detected in sections taken from transgenic mice, compared with wild-type mice (Fig. 7, A-D, and data not shown). Sections that had not received specific first antibody were devoid of stain (data not shown). These results demonstrate that up-regulation of mS100A8 and mS100A9 can occur in a hyperproliferative epidermis in the absence of inflammation. S100A8 and S100A9 Are Expressed and Secreted by a Differentiated Subpopulation of HaCaT Keratinocytes-To answer the question of whether the S100A8/9 dimer is secreted by keratinocytes that express it, we used HaCaT human keratinocyte cell lines, which were transfected either with an activin ␤A expression construct, resulting in premature initiation of differentiation in many of these cells (19), or with a vector containing only the neomycin resistance gene. For the detection of the S100A8/S100A9 heterodimer we used a quantitative sandwich enzyme-linked immunosorbent assay for S100A9, since the latter only forms heterodimers with S100A8 but no homodimers. We first determined that HaCaT cells contain intracellular hS100A8/9 heterodimers and, furthermore, that activin-overexpressing cells contain significantly higher levels (37.98 ϩ 2.86 ng/10 6 cells) than neo-transfected control cells (15.5 ϩ 11.6 ng/10 6 cells; the determination was performed twice with cells of slightly different passage number). Next, we analyzed the kinetics of hS100A8/9 secretion into serum-free culture medium. Fig. 8 shows that under these conditions hS100A8/9 is secreted at substantial levels by the HaCaT cells, as the amount accumulating within 24 h in the culture medium corresponds to 35-40% of the intracellular content of an equal number of cells at the beginning of the experiment. Furthermore, the secretion level of activin-overexpressing HaCaT cells was significantly higher than that of wild-type cells (by a factor of 1.5-2.5; the experiment was repeated three times with cells of slightly different passage number, and duplicate plates were prepared for each experiment). By trypan blue staining of the cells floating in the culture supernatant, we verified that the contribution of S100A8/9 proteins released into the culture medium by lysed dead cells was negligible, since less than 0.5% of the total number of cells were dead.
To determine whether every HaCaT cell or only a subpopulation of differentiated cells express S100A8 and S100A9, we performed double immunofluorescence with antibodies directed against hS100A8 or hS100A9 and human cytokeratin 10 (hK10), a marker of early keratinocyte differentiation. Fig. 9, A-C and D-F, show corresponding areas of cultures of activinoverexpressing cells stained with hS100A8 ( Fig. 9A) or hS100A9 (Fig. 9D) and hK10 antibodies (Fig. 9, C and F) and imaged by confocal laser scanning microscopy. As can be seen in the overlays of both signals (yellow color in Fig. 9, B and E), most but not all cells that express either protein also express the other, indicating that, similar to the in vivo situation, hS100A8 and hS100A9 expression in HaCaT cells is associated with the differentiated state. A similar result was also obtained with vector-transfected keratinocytes, although the percentages of K10-, S100A8-, and S100A9-positive cells were lower (data not shown). The subcellular localization of the hS100A8 and hS100A9 proteins in comparison to hK10 was determined by scanning individual optical sections of immunopositive cells at a high magnification (shown for activin-transfected cells in Fig. 9, G-I and K-M). Signals from hS100A8 (Fig. 9G) and hS100A9 (Fig. 9K) immunostainings revealed that both proteins were distributed throughout the cells. However, toward the cell periphery a mesh-like pattern of higher and lower hS100A8/9 protein concentrations was observed. In some peripheral areas of each cell (arrows in Fig. 9, G-M), regions of high S100A8/9 concentration co-localized with the K10 signal (Fig. 9, I and M). This co-localization was also seen in vectortransfected cells and was observed irrespective of treatment with the Ca 2ϩ ionophore A23187 before fixation, presumably because within the immunopositive differentiated cells, Ca 2ϩdependent cellular processes were already activated. These results suggest an association of some of the S100A8/9 proteins with parts of the keratin filament network. FIG. 7. S100A8 and S100A9 proteins are co-expressed within the hyperthickened tail epithelium of activin-overexpressing mice. Serial paraffin sections prepared from the tail skin of activinoverexpressing transgenic mice or their wild-type siblings were stained with specific polyclonal antisera directed against mS100A8 (panels A and B) or mS100A9 (panels C and D) followed by fluorescein-conjugated anti-rabbit-IgG. Shown are a view of an area of wild-type epidermis that contained a few immunopositive cells (panels A and C) and a view of a patch of transgenic epidermis containing several layers of highly immunoreactive suprabasal cells (panels B and D). The arrows in panels A-D, position of the basement membrane separating epidermis and dermis. Sections were viewed at 250ϫ magnification.
FIG. 8. S100A8/9 is secreted by vector-transfected and activinoverexpressing HaCaT human keratinocytes. Aliquots of cell culture supernatant were taken from duplicate confluent plates of activinoverexpressing cells and cells transfected with a vector containing only the neomycin resistance gene (Control) at different time points after switching to serum-free medium. The content of S100A8/9 was measured by a sandwich enzyme-linked immunosorbent assay for S100A9 (26). The absolute values were determined by interpolating a standard curve prepared with defined concentrations of purified human complex of S100A8/A9. Numbers were normalized to 10 6 cells and plotted against time.

DISCUSSION
To gain insight into the molecular mechanisms that underlie the wound-healing process, we initiated a large scale screen for wound-regulated genes. This strategy was chosen since a series of previous studies from our laboratory had revealed important functional roles of genes that are differentially expressed between wounded and unwounded skin. These genes encode cytokines, growth factors, and their receptors, which play a role in the signaling network that orchestrates skin reconstruction in a temporally and spatially coordinated manner (21, 29 -34), proteins with a protective function, e.g. proteins that limit the amount of inflammation or oxidative stress or control bacterial infection (35)(36)(37), or extracellular matrix molecules, which erect a scaffold into which cells can grow and that can bind growth factors and other essential molecules (38).
In our initial screen, we identified mS100A8 as a gene that was strongly up-regulated after injury and, subsequently, we identified its dimerization partner, mS100A9, which was upregulated in a coordinated fashion. S100A8 and S100A9 have been previously characterized as Ca 2ϩ -binding proteins whose expression is linked to inflammatory processes in myeloid cells and in diseased epidermis, which is characterized by abnormal differentiation (5, 12-17, 28, 39, 40). Here we report the injuryregulated induction of the genes for these two Ca 2ϩ -binding proteins, not only in the inflammatory leukocytes recruited into the wound but also in differentiating suprabasal wound keratinocytes which express them at high levels during the hyperproliferative phase. We further show that human HaCaT keratinocytes express these proteins in cell culture and secrete them at high levels into the culture medium. S100A8 and the S100A8/9 heterodimer are also secreted by other epithelial cells and by activated monocytes (41,42), and there are at least two potential extracellular functions of these proteins that may be of physiological relevance in the context of wound healing. First, the heterodimer has been shown to display striking antimicrobial properties, as it is inhibitory and even toxic toward various strains of pathogenic microorganisms (43)(44)(45). Thus, one possible reason for the up-regulation of the two dimerization partners could be control of microbial infection at the wound site.
Second, murine S100A8 was identified as a potent chemotactic molecule that has the capacity to recruit myeloid cells in vitro and in vivo (46). The moiety performing this function is a non-covalent homodimer of two S100A8 molecules, which is the favored form in low Ca 2ϩ concentrations (3). It was further shown that the non-covalent homodimer is oxidized to a nonfunctional disulfide-linked homodimer by the action of hypochlorite under conditions similar to those prevailing during inflammatory processes. This process may limit leukocyte recruitment to the site of inflammation on one hand and protect cells from excessive oxidative damage by neutralizing reactive oxygen species on the other hand (3). Thus, if the murine S100A8 protein is secreted by some of the cells expressing it within the wound, it may play a role in the regulatory network that controls the inflammatory process. A function of S100A8 in the control of inflammation in the mouse is also suggested by its targeted inactivation phenotype; null embryos are infiltrated with maternal inflammatory cells and resorbed at embryonic day 9.5, whereas in embryos with a functional allele, fetal cells, which infiltrate the deciduum before embryonic day 9.5, express mS100A8 and through this are thought to prevent resorption (47). However, some functional differences may exist between murine and human S100A8. The human protein was neither shown to be chemotactically active nor to form homodimers (3,46,48). Thus, the physiological relevance of these extracellular effects during the process of wound healing is still not clear.
Evidence from activin-overexpressing mice, which do not show inflammation in non-wounded skin (19), suggests that expression of S100A8 and S100A9 by the suprabasal keratinocytes of hyperproliferative epithelia is not a result of the concomitant inflammation. Furthermore, we do not think it likely that up-regulation of S100A8 and S100A9 expression is a straightforward effect of activin signaling, because 1) S100A8 and S100A9 were not up-regulated in the almost normal back skin of the activin-overexpressing mice (Fig. 6), 2) S100A8 and S100A9 were induced in only some of the activin-overexpressing HaCaT cells, which represented a mostly K10-positive differentiated subpopulation (Fig. 9), and 3) we did not obtain evidence of a direct induction of the S100A8 gene in activintreated HaCaT cells (data not shown). Rather, we observed an increase in S100A8 expression in confluent non-transfected or vector-transfected HaCaT cells, which undergo partial differentiation, as reflected by expression of K10 and other markers (49), in comparison to subconfluent cells. 3 This correlates well with previous observations that expression of S100A8 and S100A9 in various epithelial cell lines is linked to their potential to undergo terminal differentiation (28). Thus, we believe that the expression levels of S100A8 and S100A9 in keratinocytes may be linked in a more complex way to the balance between proliferation and differentiation that is altered during wound healing and psoriasis as well as in the activin-overexpressing mice and in cultured keratinocytes.
There are several findings that indicate a role for S100A8 and S100A9 in epithelial differentiation. First, the genes for human S100A8 and S100A9 are co-localized in a cluster, the human epidermal differentiation complex, which also contains 3 I. S. Thorey, S. Durka, and S. Werner, unpublished information.
FIG. 9. Coexpression of S100A8 and S100A9 with the differentiation marker cytokeratin 10 in HaCaT keratinocytes. Activinoverexpressing HaCaT cells were stained with specific polyclonal antisera directed against hS100A8 (panels A and G) or hS100A9 (panels D and K) and with a monoclonal antibody to human cytokeratin 10 (panels C, F, I, M) followed by Cy3-conjugated anti-rabbit-IgG and fluoresceinconjugated anti-mouse-IgG. Cells were viewed by confocal laser microscopy. To detect co-localization, an overlay was prepared of the images from the red and green channels (panels B, E, H, L). Arrows in panels G-M, areas where high level hS100A8 or hS100A9 expression and cytokeratin filaments are co-localized. Cells were scanned at 250ϫ (panels A-F) or 630ϫ (panels G-M) magnification. the genes for other S100 proteins, several cytokeratins, and epidermal differentiation markers such as profilaggrin and involucrin; it has been suggested that expression of these genes could be coordinately regulated (50 -52). Second, in accordance with our observations in HaCaT cells, the S100A8/A9 heterodimer was shown in vitro as well as in intact epithelial cells to bind to keratin filaments in a calcium-dependent manner (25,53), and there is evidence for its association with the epidermal cytoskeleton in inflammatory dermatoses (14). It is not yet known whether S100A8/A9 binding to keratins has any functional consequences. However, the glial-specific Ca 2ϩbinding proteins, S100A1 and S100B, are capable of inhibiting the polymerization of glial intermediate filaments by binding to the subunit glial fibrillary acidic protein, thereby regulating the state of assembly of glial intermediate filaments (54,55). In a similar manner, S100A8/A9 may possibly play a role in cytoskeletal reconstruction processes, which occur in those Ha-CaT cells that have begun to express K10 and that likewise may occur in hyperproliferative keratinocytes during wound healing or skin disease progression.
The detailed molecular processes that lead to up-regulation of S100A8 and S100A9 in epithelia during hyperproliferative processes remain to be elucidated. However, several findings implicate retinoids as negative regulators and the pro-inflammatory and pro-proliferative transcription factor nuclear factor interleukin 6 as a positive upstream regulator of S100A8 expression (56 -58). Repression of S100A8 may thus explain some of the therapeutic effects of retinoids in inflammatory as well as hyperproliferative skin diseases.