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Peptide Elongation Factor eEF1A-2/S1 Expression in Cultured Differentiated Myotubes and Its Protective Effect against Caspase- 3-mediated Apoptosis*

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1α, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1α to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1α in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1α, whereas cells transfected with antisense eEF1A-1/EF-1αare protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1α and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.

Apoptosis is an active process enabling normal metazoan development and tissue homeostasis by permitting the deletion of unwanted or damaged cells (1,2). In mammalian cells, the control of cell death or survival is governed by the interplay between the Bcl-2 family members and the activation of caspase proteases (for review, see Refs. [3][4][5][6][7]. Once activated, caspases can cleave several proteins to inactivate their functions, which are essential to survival. Upon differentiation induction of skeletal muscle cell lines, undifferentiated myoblasts are susceptible to apoptosis but become resistant as they differentiate into myotubes. It is suggested that this resistance may be dependent upon the induction of two repressors of cell cycle traverse: p21Cip1 and Rb (8,9). However, no study has yet discussed the apoptotic induction mechanism in differentiated myotubes.
During skeletal muscle development, a switch occurs in the expression of the two homologous peptide elongation factor 1As. In mouse and rat, eEF1A-1 1 /EF-1␣ is expressed during embryonic and early postnatal development and is replaced by eEF1A-2/S1 2 weeks after birth; the latter is the only homolog expressed in adult muscle (10,11). In fact, eEF1A-2/S1 is expressed in brain, heart, and skeletal muscle, all tissues largely composed of long lived terminally differentiated cells (12)(13)(14). eEF1A-2/S1 has been shown by in vitro translation assay to support elongation activity similarly to eEF1A-1/EF-1␣; it binds aminoacyl-tRNA and delivers it to the ribosomal aminoacyl site during the elongation phase of protein biosynthesis (15)(16)(17). Deletion of the promoter and first noncoding exon of the eEF1A-2/S1 gene is associated with the wasted mouse phenotype, characterized by muscle atrophy, neurological impairment, immunological deficiency, and death after fewer than 30 days postpartum (18). In skeletal muscle, absence of either form of eEF1A homologs is observed 3 weeks after birth in wasted mutant mice, thus impairing protein translation (11). No mechanism has yet been identified to explain the switch in peptide eEF1As seen in skeletal muscle, heart, and brain during development. Furthermore, it remains unclear why eEF1A-2/S1 is only expressed in long lasting terminally differentiated cells such as neurons, cardiomyocytes, and myofibers. Because both elongation factors, eEF1A-1/ EF-1␣ and eEF1A-2/S1, have similar canonical function in protein translation, we hypothesized that the developmental switch could be explained by the noncanonical functions of the proteins.

MATERIALS AND METHODS
Cell Culture-The mouse-derived skeletal myoblast C2C12 cell line (American Type Culture Center, ATCC) and rat-derived cultured myoblast L6A line (a generous gift from Dr. Dan Goldman, University of Michigan, Ann Arbor) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 g/ml streptomycin and kept at 37°C in a 10% CO 2 humidified atmosphere incubator. After confluent cultures were rinsed twice with serum-free DMEM, myoblast differentiation was induced by replacing the 10% fetal bovine serum-containing culture medium by DMEM supplemented with 2% horse serum. The yield of myotubes achieved was visually estimated to be between 60 and 80%. For serum deprivation, myotubes were rinsed twice with serumfree DMEM to remove all trace of serum and then cultured after the third change in the same medium. Cultured C2C12 cells were harvested for analysis at 3, 6, 9, 12, and 15 days of differentiation and at 3, 6, and 9 days of serum deprivation. Cultured L6 cells were collected daily after induction of either differentiation or apoptosis.
DNA Fragmentation Assays-Cultured C2C12 and L6 myotubes were processed in the same manner. The medium of serum-deprived cells was collected and added to the scraped cells to allow centrifugation of both adhering and floating cells. Cell pellets were resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 100 mM NaCl, 0.5% SDS, and 0.5 mg/ml proteinase K) and incubated overnight at 56°C. Afterward, samples were treated with 50 g/ml RNase A for 2 h followed by a phenol/chloroform extraction. The DNA was precipitated using 0.3 M final concentration of sodium acetate, pH 5.2, and isopropyl alcohol. The DNA pellet was then rinsed with 75% cold ethanol and resuspended in water. After being spun for 20 min in a microcentrifuge, samples were loaded onto a 1.8% agarose and Tris-boric acid-EDTA buffer gel containing ethidium bromide to allow electrophoretic separation of the fragmented DNA. The separated fragments were visualized on the agarose gel using a UV transluminator, and profiles of oligonucleotides were taken with a digital camera.
In Situ DNA Degradation Assay by Terminal Nucleotidyl Transferase-mediated UTP Nick End Labeling (TUNEL) Staining Reaction-DNA fragmentation analysis of individual cell was performed by DNA nicked-end labeling using terminal-d-transferase, as described by Gavrieli et al. (23). Differentiated L6 myotubes were treated with trypsin and then grown on coverslips; one set was serum deprived 2 days after seeding. Differentiated cells were fixed using cold methanol/acetone (1:1) at Ϫ20°C for 10 min. Fixed cells were rehydrated with PBS for 30 min at room temperature and then rinsed twice with double-distilled water. Afterward, coverslips were covered with terminal transferase reaction buffer (30 mM Tris-HCl, pH 6.8, 100 mM sodium cacodylate, 5 mM cobalt chloride, 0.5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 10 M biotin-16-dUTP (Roche), and terminal transferase (Promega) at 0.3 unit/l); the reaction was performed at 37°C for 1 h in a humidified chamber and then terminated by transferring the coverslip samples to a TB buffer containing 300 mM sodium chloride and 30 mM sodium citrate for 15 min at room temperature. The specimens were then rinsed in double distilled water and incubated in 2% bovine serum albumin for 10 min. After the addition of serum albumin, the specimens were incubated with 20 g/ml streptavidin and fluorescein isothiocyanate (Roche) in PBS for 30 min at room temperature and subsequently washed twice in PBS along with 0.4 g/ml propidium iodide in the second washing. The propidium iodide was removed by rinsing the samples twice in PBS. Coverslips were mounted in PBS containing 50% glycerol and examined with a Nikon fluorescence microscope.
Protein Extraction, Gel Electrophoresis, and Western Blotting-Myotubes cultures were rinsed with PBS and then gently scraped into 1 ml of PBS. Collected cells were centrifuged at 4,000 rpm for 3 min. The pellets were resuspended into RIPA buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA) containing fresh protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 2 g/ml of both leupeptin and pepstatin). Afterward, cell extracts were sonicated for 1 min to break cell membranes; proteins were quantified by a modified Bradford method, using the Bio-Rad protein assay according to the manufacturer's instructions, and compared with a ␥-globulin standard curve. Equal amounts of total protein for each sample were separated on a SDS-polyacrylamide gel and then transferred onto a nitrocellulose membrane by the standard transfer method. Membranes were blocked in Tris-buffered saline solution containing 0.25% Tween 20 and 5% fat-free powdered milk. Before incubation with primary antibodies, membranes were rinsed in Tris-buffered saline-Tween 20 solution. Primary antibodies used were HT7 for eEF1A-1/EF-1␣, CB5 for eEF1A-2/S1 (22), MF-20 monoclonal antibody for myosin heavy chain (24) (the clone was a generous gift from Dr. L. Chalifour, Lady Davis Institute, Montreal, Canada); Caspase-3 (H-277) and poly(ADP-ribose) polymerase (PARP) (H-250) antibodies were purchased from Santa Cruz, and monoclonal ␤-actin antibody from Amersham Biosciences, Inc. After incubation overnight at 4°C or 1 h at room temperature, the membranes were washed four times, 10 min each, in Tris-buffered saline-Tween 20. Blots were subjected to a second hybridization with either horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (Cappel) or goat anti-mouse antibodies (Cappel) and then washed five times with Tris-buffered saline-Tween 20. Bands were detected using a chemiluminescent ECL procedure, according to the manufacturer's protocol (Amersham Biosciences, Inc.). For ␤-actin detection, previously used membranes were soaked in stripping buffer (70 mM Tris-HCl, pH 6.8, 2% SDS, and 0.1% ␤-mercaptoethanol) at 60°C for 30 min and hybridized following the procedures described above. Films were quantified by densitometry (Molecular Dynamics) and expressed as the ratio to the maximal level of detection.
Viability Assay and Caspase-3 Inhibitor-Viability tests were performed using the trypan blue exclusion assay, as described by Duttaroy et al. (25). Viability percentages were calculated by dividing the number of dead blue cells by the total cell number. Each experiment was repeated three times. A cell-permeable, irreversible peptide inhibitor of caspase-3, DEVD-FMK (Calbiochem), was dissolved in dimethyl sulfoxide and added to the culture medium at a final concentration of 75 M. The culture medium was changed every day, with the addition of fresh inhibitor-containing medium, and control cells were plated with the same concentration of dimethyl sulfoxide without inhibitor. Cell viability was measured as described above.
Caspase Assays-Serum-deprived and control cell cultures were rinsed once in cold PBS and then collected in cold PBS by scraping the cells off the plate. After centrifugation and removal of PBS, cell pellets were kept at Ϫ80°C until caspase assays were performed. The frozen pellets were resuspended in caspase lysis buffer (10 mM HEPES, pH 7.4, 2 mM EDTA, and 0.1% CHAPS) supplemented with protease inhibitors (5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml pepstatin A, 10 g/ml aprotinin, and 20 g/ml leupeptin). Freezethaw cell lysis cycles were performed by alternatively transferring the samples from an ethanol/dry ice bath to a 37°C water bath five times. The supernatant was collected after 20 min of centrifugation at full speed (Х 12,000 rpm) in a cold microcentrifuge. Assays were performed in caspase buffer (10 mM PIPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, and 5 mM dithiothreitol, final concentration), to which 50 M final concentration of substrate and 5 l of protein extract were added to yield a final volume of 100 l. Caspase-3 DEVD-AFC and caspase-1 YVAD-AFC peptide substrates (Biomol) were dissolved in dimethyl sulfoxide. The assays were performed in black wall, clear bottom plates using a Bio-Rad fluorometer and read at 598 nm after excitation at 390 nm wavelengths. The results were compared against an AFC curve generated during the reaction. To measure specific activity, proteins were quantified according to the method described above.
Adenovirus Construction, Cell Infection, and ␤-Galactosidase Assay-Recombinant adenovirus expressing eEF1A-2/S1 was prepared with ADENO-QUEST (Quantum Biotechnologies, Montreal). Mouse eEF1A-2/S1 cDNA (26) was subcloned into the pQBI-AdCMV5 transfer vector. Cotransfection of QBI-293A cells with viral DNA and the transfer vector was performed by the precipitated calcium phosphate procedure, according to the manufacturer's protocol. Plaques were picked up and amplified in QBI-293A cells. Screening for positive recombinants was performed by Western blotting using CB5 antibody, which specifically recognizes eEF1A-2/S1 protein (22). Among the positive clones, one named A5.4 was used to generate the experimental virus. Control virus for mock infection was generated by transfection of QBI-293A cells with nondigested viral DNA. The ␤-galactosidase gene was already included in the viral DNA by the manufacturer; insertion of a new gene into the vector does not affect its transcription and thus can be used as a marker of infection. After amplification, cells were broken by freeze/ thaw cycles; and after centrifugation, the supernatant culture media containing viruses were kept frozen. Virus titration was done according to the manufacturer's instructions. Growing cultures of L6 cells were infected with mock or recombinant adenovirus supernatant diluted to 200 multiplicity of infection, enough to cover the cell monolayer at 37°C for 1.5 h. After incubation, the medium was changed to fresh DMEM supplemented with 10% fetal bovine serum. Infection efficiency was measured after 2 days and again after 6 days (corresponding to 4 days of differentiation) by ␤-galactosidase assay, using the following protocol. Cells were fixed for 10 min with 0.2% glutaraldehyde and 2% formaldehyde in PBS and then washed three times for 10 min with PBS. Afterward, an enzymatic reaction was performed in ␤-galactosidase buffer (5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 in PBS) with 1 mM 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (Sigma) as substrate. After 3-5 h at 37°C, ␤-galactosidase-positive blue cells were counted, and the results were expressed as percentage of the total number of cells.
eEF1A-1/EF-1␣ Transfection and Viability Assay-Cultured L6 myoblasts were grown and differentiated in six-well plates, as described above. After 3 days of differentiation, cells were cotransfected with 5 g of constitutively expressing Rous sarcoma virus ␤-galactosidase vector, along with 10 g of pBK CMV-eEF1A-1/EF-1␣ sense or antisense (full-length), or empty pBK CMV vector used as a control (19,20) using LipofectAMINE 2000 (Invitrogen), with the exception that the cells were not subcultured the previous day. Efficiency of transfection was assessed by ␤-galactosidase staining, which revealed that myotubes were transfected. Cells were serum deprived 24 h after transfection and collected on a daily basis as described previously into cold PBS. After centrifugation, pellets were kept frozen at Ϫ80°C until protein extraction was performed. For the extraction, the pellets were resuspended in 100 l of 0.25 M Tris-HCl, pH 7.8, and then submitted to three freezethaw cycles by transferring the samples between an ethanol-dry ice bath and a 37°C water bath. Afterward, the extracts were centrifuged at 12,000 rpm for 8 min in a refrigerated bench top centrifuge, and the supernatants were retained for the assay. Viability was quantified by measuring ␤-galactosidase activity, as described by Chen et al. (19) and Oral et al. (27). The activity was assessed in 300 l of reaction solution containing 3.33 l of 100 ϫ magnesium buffer (0.1 M MgCl 2 , 4.5 M ␤-mercaptoethanol), 66 l of 4 mg/ml o-nitrophenyl-␤-D-galactopyranoside, 230.67 l of 0.1 M sodium phosphate buffer, pH 7.5 (41 ml of 0.2 M Na 2 HPO 4 , 9 ml of 0.2 M NaH 2 PO 4 , and 50 ml of water), to which 20 l of protein extract was added. After a 10-min incubation at 37°C in a water bath, the reaction was terminated by the addition of 500 l of 1 M Na 2 CO 3 , and the optical density of the samples was measured by spectrophotometry at 420 nm. Blanks were generated at the same time, and proteins from a positive sample were added after reaction termination with sodium carbonate. Efficiency of transfection was determined by ␤-galactosidase staining of the cells, as described above. For Western blotting, cells were selected with 250 g/ml of Geneticin for 4 days before collection.

Differentiation and Serum Deprivation of Mouse C2C12 and
Rat L6A Myoblast Cell Lines-To initiate our study on the functions of eEF1A-1/EF-1␣ and eEF1A-2/S1, mouse C2C12 and rat L6 myoblasts were first cultured to confluence and then induced to differentiate into multinucleated myotubes by changing the culture medium from a high (10% fetal bovine serum) to a low (2% horse serum) serum concentration; both cell lines gave rise to large myotubes upon differentiation. After 4 days of differentiation for L6 cells, and 6 days for C2C12, myotube cultures were switched from medium containing 2% horse serum to serum-free medium, to induce apoptotic cell death in differentiated myotubes by serum deprivation.
Western Blot Analysis of eEF1A-1/EF-1␣, eEF1A-2/S1, and Myosin Heavy Chain in L6 and C2C12 Differentiation and Serum Deprivation Assays-Protein samples of L6 and C2C12 cultures were collected at different time points during differentiation and processed for Western blotting with specific antibodies against eEF1A-2/S1, eEF1A-1/EF-1␣, and myosin heavy chain, used as a marker of differentiation. After 3 days of differentiation, L6 cells start expressing both eEF1A-2/S1 pro-tein and the differentiation marker myosin heavy chain (Fig.  1A), whereas eEF1A-1/EF-1␣ protein decreases to around 20% of its initial expression after differentiation. In C2C12 cultures, eEF1A-2/S1 and myosin heavy chain also appear after 3 days of differentiation (Fig. 1B), accompanied by a 3-day gradual loss in protein level of eEF1A-1/EF-1␣, which later remains stable at a low level in C2C12 myotube cultures. The kinetic of eEF1A-2/S1 protein appearance correlates with myosin heavy chain, a late marker of differentiation, suggesting that eEF1A-2/S1 is a protein that appears late in the differentiation process.
When C2C12 and L6 myotubes are serum deprived to induce apoptosis, eEF1A-1/EF-1␣ and eEF1A-2/S1 expression patterns are reversed from that seen during differentiation. In cultured L6 myotubes, eEF1A-2/S1 protein disappears after 2 days of serum deprivation, whereas eEF1A-1/EF-1␣ protein level increases simultaneously, to almost double the level seen in control myotubes ( Fig. 2A). A similar pattern is observed in cultured C2C12 myotubes; eEF1A-1/EF-1␣ protein expression level remains low for the first 3 days of serum deprivation but then increases and reaches a higher protein expression level than in the differentiated state (Fig. 2B). Unlike eEF1A-1/EF-1␣, eEF1A-2/S1 protein disappears after 9 days of serum deprivation. Myosin heavy chain was used to confirm that cells were differentiated during the serum deprivation experiment; ␤-actin was used as a loading control.
Cell Viability and DNA Fragmentation Assay-Cell viability of serum-deprived C2C12 and L6 myotubes was measured using the trypan blue exclusion assay, where blue cells are counted as dead. As seen in Fig. 3A, only 15% of C2C12 myotube cultures remain alive after 8 days of serum deprivation, whereas more than 60% of the control cells cultured with 2% FIG. 1. eEF1A-2/S1 and eEF1A-1/EF-1␣ protein expression during muscle differentiation in L6 and C2C12 myoblasts. eEF1A-2/S1 and eEF1A-1/EF-1␣ proteins were detected using CB5 and HT7 specific antibodies, respectively. Panel A, L6 cells were differentiated under low serum conditions and collected on a daily basis for protein analysis. Lane 0 corresponds to undifferentiated cells, and lanes 1-4 represent 1-4 days of differentiation, respectively. MF-20 monoclonal antibody recognizing myosin heavy chain (MHC) was used as a marker of differentiation. Relative expressions of eEF1A-2/S1 and eEF1A-1/ EF-1␣ during differentiation are shown in the graph, and the error bars represent that S.D. Panel B, as in panel A, but for mouse C2C12 myoblasts collected at 0, 3, 6, and 9 days of differentiation, respectively. In both panels A and B, ␤-actin was used as a loading control. horse serum remain viable at the same time point. In L6 myotube cultures, less than 10% of serum-deprived cells survive past 3 days, whereas about 40% of the control cells survive. To characterize myotube death further, DNA fragmentation assays were performed. As shown in Fig. 3B, oligonucleosome fragments appeared in C2C12 myotube cultures after 3 days of serum deprivation and increased steadily up to 9 days of serum deprivation, whereas control myotube cultures do not display any detectable DNA fragmentation. The earliest dying L6 cells start to show fragmented DNA after 2 days of serum deprivation. Thus, serum deprivation induces apoptotic cell death in differentiated cultures of myotube. TUNEL staining was performed to confirm apoptosis as the mechanism for cell death (Fig. 4). The left part of the composite panel (Fig. 4, A, C, and E) represents the control differentiated myotube cultures maintained in DMEM supplemented with 2% horse serum; the right part (Fig. 4, B, D, and F) represents serum-deprived cultured myotubes. Bright TUNEL-positive stained nuclei, a hallmark of apoptosis, are observed in serum-deprived myotube cultures as indicated by full arrows in Fig. 4F, whereas the control counterpart remains negative for TUNEL staining (Fig. 4E,  open arrows). Propidium iodide staining was used as a coun-terstain for all nuclei (Fig. 4, C and D), and phase contrast pictures (Fig. 4, A and B) reveal that the cells were indeed differentiated myotube syncytia.
Caspase-3 and PARP Western Blots, Caspase Activity, and Caspase-3 Inhibitor Effects on Cell Viability-We have assessed the apoptotic biochemical program by measuring caspase-3 activity with the cleavage of DEVD-AFC fluorescent peptide substrate. High caspase-3 activity is observed after 2 days of serum deprivation, whereas the activity in control cells remains close to the base-line level for the same period of time (Fig. 5A). As expected for caspase-mediated cell death, caspase-3 activity is seen when the cells are dying as shown by the kinetic of viability measurements (Fig. 5C). Caspase-1 fluorescent YVAD-AFC peptide was used as a negative control; caspase-1 activity remains constant in both serum-deprived and control myotube cultures. To verify that caspase-3 activity indeed occurs in apoptotic myotube cultures, proteins from control and serum-deprived cells were processed for Western blotting with caspase-3 and PARP rabbit polyclonal antibodies. As shown in Fig. 5B, the p17 catalytic fragment of active caspase-3 protein, obtained after activation by cleavage of p32 zymogen form of caspase-3, is seen on the Western blot at both 2 and 3 days of serum deprivation. These time points correspond to the peak of activity level in caspase-3 fluorogenic peptide cleavage assays (Fig. 5A). During the same period of time, a p85-cleaved PARP fragment, a marker of cellular caspase-3 protein activity (28), is detected in the same samples, demonstrating that caspase-3 is indeed activated in differentiated myotube cultures after serum deprivation. To confirm further that a caspase-3-dependent death mechanism indeed occurs, DEVD-FMK caspase-3 peptide inhibitor was used in differentiated cell cultures. As seen in Fig. 5C, after 3 days of serum deprivation in the presence of the inhibitor, around 60% of the differentiated cells are still alive, whereas less than 10% FIG. 2. eEF1A-2/S1 and eEF1A-1/EF-1␣ protein expression in serum deprivation-induced apoptosis of L6 and C2C12 myotube cultures. After 4 days of L6 differentiation and 6 days of C2C12 differentiation, myotube cultures were serum deprived to induce apoptosis. ϩ1 and ϩ2 represent the number of days of serum deprivation after differentiation, indicated by the first number (4 or 6). Panel A, eEF1A-2/S1 and eEF1A-1/EF-1␣ protein expression in L6 myotube cultures during serum deprivation. Control cells (C) were maintained in DMEM supplemented with 2% horse serum, and serum-deprived cells (S) were kept in serum-free medium. The lane marked M indicates 3-month-old rat skeletal muscle protein extract used as a control. Myosin heavy chain (MHC) was used as a marker of differentiation and ␤-actin as a loading control. Graphs represent the relative expression of both elongation factors in control and serum-deprived samples, and the error bars represent S.D. Panel B, serum-deprived C2C12 myotube cultures. As above, the graph represents the relative expression of eEF1A-2/S1 and eEF1A-1/EF-1␣ in C2C12 cells. The asterisk marks a contaminant band.  Fig. 2, the ϩday system is used to label the samples from the day of serum deprivation. In C2C12 cells, control cells at 6 (initial day of serum deprivation), 9, 12, and 15 days of differentiation and serum-deprived cells for 3, 6, and 9 days were used. of the serum-deprived control cells remain viable. The inhibitor was potent to protect differentiated cells against serum deprivation-induced myotube cell death. This protection from cell death by the use of caspase-3 inhibitor confirms that upon serum deprivation of differentiated myotubes, the cell death mechanism is caspase-3-dependent, as suggested by Western blotting.
Western Blots and Viability of eEF1A-2/S1-infected Cells-Previous figures have shown that eEF1A-2/S1 was lost in cultures of dying myotubes but present before induction of apoptosis (see Fig. 2). Adenovirus clone A5.4 expressing eEF1A-2/S1 was used to infect undifferentiated L6 cells to see whether eEF1A-2/S1 protects or rescues cells from serum deprivationinduced apoptosis. In general, infection efficiency, determined by ␤-galactosidase assay, is on average about 85%, whereas infection efficiency for C2C12 cells is too low (around 40%); therefore only L6 cultures were used for this study. Undifferentiated L6 cells were infected and allowed to differentiate into myotubes. After differentiation, cells were serum deprived, and their viability was measured by trypan blue exclusion assay. As shown in Fig. 6A, about 45% of eEF1A-2/S1-infected myotube cultures remain alive after 3 days of serum deprivation, whereas only about 15% of the control (uninfected) and mockinfected (␤-galactosidase) cells remain viable. After 2 days of serum deprivation, mock-infected cultures seem protected by the adenoviral infection, but that effect was not seen after 3 days of serum deprivation, revealing the true protective effect of eEF1A-2/S1. To confirm further the protective effect of eEF1A-2/S1, caspase-3 activity was measured in serum-deprived eEF1A-2/S1-infected differentiated L6 cells. After 2 days of serum deprivation, A5.4-infected cultures exhibit between 50 and 60% of the caspase-3 activity of control cultures (Fig. 6B). As mentioned above, the adenoviral infection seems to protect myotubes cultures after 2 days of serum deprivation, but this protection is not seen when caspase-3 activity is measured (no significant difference), thus confirming that the adenovirus does not protect myotubes cultures, as seen after 3 days of serum deprivation viability. The reduced caspase-3 activity and low eEF1A-1/EF-1␣ protein expression seen in eEF1A-2/S1-infected cells confirm the role of eEF1A-2/S1 as a rescuer from cell death in differentiated skeletal myocyte cultures. As shown in Fig. 6C, eEF1A-1/EF-1␣ and eEF1A-2/S1 Western blots were performed to verify the efficiency of eEF1A-2/S1 adenoviral expression as well as the corresponding change in eEF1A-1/EF-1␣ expression. As expected, only A5.4-infected cells express eEF1A-2/S1 protein before differentiation, revealing the efficiency of transfection. The absence of myosin heavy chain expression was used as a marker for the undifferentiated state in these samples. After differentiation, all samples express eEF1A-2/S1 protein, but after 3 days of serum deprivation, only the A5.4-infected myotubes still express eEF1A-2/S1 protein as shown by Western blotting. Interestingly, A5.4-in- FIG. 4. Detection of apoptotic myotubes by TUNEL staining. Differentiated L6 myotube cultures were grown on coverslips and then serum deprived to induce apoptosis. Control cells were maintained in DMEM supplemented with 2% horse serum during the duration of the experiment (panels A, C, and E). After 3 days of serum deprivation, L6 myotube cultures were processed for TUNEL staining (panels B, D, and F). Phase contrast pictures show control (panel A) and apoptotic (panel B) myotubes. Propidium iodide was used as a counterstain to reveal the presence of multinucleated myofibers (panels C and D). TUNEL staining revealed the absence of positive apoptotic myonuclei in the control myotube (panel E) and bright, condensed positive myonuclei in apoptotic myotube (panel F). Open arrows indicate the presence of nonapoptotic nuclei (panels C and E), and full arrows indicate the presence of apoptotic nuclei (panels D and F). All pictures were taken at the same magnification (ϫ400). fected L6 cultures express less eEF1A-1/EF-1␣ protein than their control counterparts, noninfected and mock-infected cultures.
Western Blots and Viability of eEF1A-1/EF-1␣ Sense-and Antisense-transfected L6 Cultures-After 3 days of differentiation, L6 cells were transiently transfected with EF-1␣/EF1A-1 sense or antisense gene cDNA under the control of the CMV promoter (19,20); viability was assessed by measuring ␤-galactosidase activity, as described previously (19,27), providing the advantage that only transfected cells are counted. However, cells must be dead for a certain period of time to become negative for ␤-galactosidase activity. Upon serum deprivation, cells transfected with eEF1A-1/EF-1␣ sense die more rapidly, whereas antisense-transfected cultures are protected from apoptosis and survive better than sense gene-transfected cells (Fig. 7A). After 3 days of serum deprivation, fewer than 25% of the cells transfected with the eEF1A-1/EF-1␣ sense gene remain viable compared with about 55% of the control (mock transfection) and 70% of the eEF1A-1/EF-1␣ gene antisense gene, as measured with this technique. The difference between the antisense and the control is small after 3 days of serum deprivation but significant (p Ͻ 0.05). The efficiency of gene transfer was analyzed by ␤-galactosidase staining, and Western blotting revealed a 50% increase in the abundance of eEF1A-1/EF-1␣ protein presence with sense-transfected cells, whereas antisense-transfected cells show a decrease of 40% in eEF1A-1/EF-1␣ protein abundance (Fig. 7, B and C). Both were compared with cells transfected with the empty pBK CMV vector. The experiments show that: 1) the same effect on enhanced apoptosis of sense eEF1A-1/EF-1␣ is observed in myotubes, as published previously (19 -21) and 2) the effects of eEF1A-1/EF-1␣ and eEF1A-2/S1 in myotube apoptosis are opposite.

DISCUSSION
In this report, we demonstrate that eEF1A-2/S1 and eEF1A-1/EF-1␣ protein expressions are regulated inversely in differentiated myotubule cultures during differentiation and apoptosis. When eEF1A-2/S1 protein is up-regulated during differentiation, eEF1A-1/EF-1␣ protein is down-regulated with the reverse pattern of expression in apoptotic events. Apoptotic cell death is also associated with the activation of caspase-3 protease, which can be inhibited by specific cell-permeable, irreversible inhibitor. The presence of this inhibitor protects differentiated cultures of myotubes from serum deprivation-induced celldeath,thusconfirmingthatthedeathmechanismiscaspase-3dependent in differentiated myotube cultures. DNA fragmentation analysis confirms the apoptotic nature of the death in cultured myotubes and indeed reveals that differentiated myotubes are capable of apoptotic cell death, as TUNEL-positive multinucleated syncytia are observed.
In an attempt to determine eEF1A-2/S1 function in apoptotic cells, the effect of continuous expression of eEF1A-2/S1 was assessed in dying myotubes. eEF1A-2/S1-infected myotubes are more resistant to serum deprivation-induced apoptosis than virus alone infected (␤-galactosidase) or uninfected cells, as observed after 3 days. This result is the first to ascribe a protective or rescuing function to eEF1A-2/S1 protein in apoptosis. Cultures of myotubes bearing the transfected sense eEF1A-1/EF-1␣ gene die more rapidly, whereas cells transfected with the antisense are protected and die more slowly from serum deprivation-induced apoptosis, similar to what was observed previously (19,20). This experiment essentially shows the opposite effects of eEF1A-1/EF-1␣ and eEF1A-2/S1 on myotube survival. In terminally differentiated muscle cells, eEF1A-2/S1 may possibly replace eEF1A-1/EF-1␣, functioning not only in protein synthesis but also preventing apoptosis. This is the first noncanonical function associated with eEF1A-2/S1 and may help unravel the mystery behind the developmental switch between peptide elongation factor 1A observed in brain neurons, heart, and skeletal muscles; the switch may be beneficial to preserve and protect these long lasting cells from inopportune apoptotic cell death. Thus, the absence of eEF1A-2/S1 protein, and its replacement by eEF1A-1/EF-1␣ in dying muscles, as observed in injured muscles (22) and in cultured myotubes, is a mechanism permitting apoptosis to occur.
Both eEF1A-2/S1 and eEF1A-1/EF-1␣ proteins have a similar elongation function in vitro (15,16), and probably in vivo, because only eEF1A-2/S1 protein is expressed in mature brain neurons, heart, and skeletal muscles, where protein translation is maintained. The similarities between eEF1A-2/S1 and eEF1A-1/EF-1␣ may not extend to other noncanonical functions not directly associated with protein biosynthesis. For example, eEF1A-1/EF-1␣ is capable of severing microtubules, whereas no similar function has been shown for eEF1A-2/S1 (29). It is known that microtubule severing or anti-microtubule agents induce apoptosis (30,31). The increase in eEF1A-1/ EF-1␣ protein levels observed during apoptosis may serve to FIG. 6. Effect of continuous expression of eEF1A-2/S1 in serum-deprived L6 myotube cultures. Panel A, a trypan blue exclusion assay was used to measure the viability of differentiated L6 cells after serum deprivation following adenoviral infection. Uninfected cells (black bars) and cells infected with the control ␤-galactosidase adenovirus (white bars) were serum deprived and used as controls. The gray bars represent cells infected with eEF1A-2/S1 A5.4 adenovirus. Panel B, caspase-3 activity was measured in all cells. The same labeling system developed in Fig. 2 was used to label the lanes. Panel C, Western blotting of adenovirus-infected cells before (0), after 4 days of differentiation (4), and after 2 days of serum deprivation (4ϩ2). The uninfected control cells are indicated by C, the ␤-galactosidase control by B, and A5.4-infected cells by S. CB5 and HT7 rabbit polyclonal antibodies were used to detect eEF1A-2/S1 and eEF1A-1/EF-1␣, respectively. MF-20 monoclonal antibody was used to detect myosin heavy chain (MHC), a marker of differentiation. Graphs represent the relative expression of eEF1A-2/S1 and eEF1A-1/EF-1␣ proteins. All error bars represent the S.D. facilitate rapid death of cells, by dismantling their cytoskeleton. In contrast, eEF1A-2/S1, which may not be capable of microtubule severing, may thus indirectly protect cells from apoptosis.
Our study of apoptotic cell death in myotube cultures reveals a caspase-3-dependent mechanism. Caspase-3 activation was confirmed by a specific inhibitor, and by Western blot analysis of caspase-3 and PARP cleavage, a cellular marker of caspase-3 activity (28). In differentiated L6 cells, however, caspase-3 activity is lower when eEF1A-2/S1 is present, as seen in A5.4infected cells; after 2 days of serum deprivation, half of the control caspase-3 activity is present in A5.4-infected cells. This may explain why, after 3 days of serum deprivation, about twice as many A5.4-infected cells survive serum deprivationinduced apoptosis. These results suggest that the protection exerted by eEF1A-2/S1 may include a mechanism involved in the regulation of caspase-3 activity. Disruption of the muscle architectural organization triggers the activation of caspase-3, as observed previously in the muscle cell death in muscular dystrophy (32). It will be interesting to analyze the possible analogy between caspase-3 activation in muscular dystrophy and how the expression of both elongation factors eEF1A-1/ EF-1␣ and eEF1A-2/S1 is regulated in this disease state.
The mechanism regulating caspase-3 activation may be related to the efficiency of differential translation of some genes mRNA by eEF1A-1/EF-1␣ or eEF1A-2/S1. Accordingly, in such mechanism, eEF1A-2/S1 may favor the translation of prosurvival genes mRNA, or likewise repress the translation of proapoptotic genes, whereas eEF1A-1/EF-1␣ may work in the opposite manner. Recently, two different studies reveal that the translation of the mRNA of some genes is modified according to the state of the cell, and a change in abundance of a single protein determines the translation speed of some mRNAs (33,34). This differential translation mechanism may explain why eEF1A-1/EF-1␣ protein is less abundant in eEF1A-2/S1-infected cells, which survive serum deprivation-induced apoptosis longer, and also why the eEF1As exert opposite effects on apoptosis.
In conclusion, we demonstrate that a switch between two peptide eEF1As occurs in cultured differentiation, as well as in tissue during development (11). This switch from eEF1A-1/ EF-1␣ to eEF1A-2/S1 may confer to the long lived, terminally differentiated myotube a translation elongation factor, which also prevents accidental cell death from occurring. The eEF1A-2/S1 protective function is the first noncanonical function associated with the protein. More experiments are needed help our understanding of how the protective function of eEF1A-2/S1 in skeletal muscle operates to regulate apoptotic cell death. Furthermore, it will be interesting to see whether this protective function of eEF1A-2/S1 behaves in heart and neurons in a manner that is similar to its behavior in skeletal muscles. It will also be interesting to study in vivo the expression and effect of eEF1A-2/S1 in skeletal muscle during aging and to determine whether the loss of eEF1A-2/S1 may expedite the age-dependent myofiber decline in skeletal muscle.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Delayed Cellular Maturation and Decreased Immunoglobulin κ Light Chain Production In Immature B Lymphocytes Lacking B Cell Linker Protein

B cell linker (BLNK) protein is a component of the B cell receptor (BCR) signaling pathway and BLNK−/− mice have a block in B lymphopoiesis at the pro-B/pre-B cell stage. To study the effect of BLNK mutation at later stages of B cell development, we introduce an innocuous transgenic BCR into BLNK−/− mice and show that two populations of immature B cells distinguishable by their IgMlow (lo) and IgMhigh (hi) phenotypes are found in the bone marrow of these mice in contrast to a single population of IgMhi cells found in control BCR-transgenic BLNK+/+ mice. The mutant IgMlo and IgMhi cells are at an earlier developmental stage compared with the control IgMhi cells as indicated by their differential expression of CD43, B220, and major histocompatibility complex class II antigens and their timing of generation in culture. Thus, in the absence of BLNK the differentiation of immature B cells is delayed. Furthermore, mutant IgMlo cells produce equivalent level of immunoglobulin (Ig) μ but less Ig κ proteins than control and mutant IgMhi cells and this defect is attributed to a decrease in the amount of κ transcripts being generated. Finally, splenic B cells in BCR-transgenic BLNK−/− mice are predominantly of the transitional B cell phenotype and are rapidly lost from the peripheral B cell pool. Taken together, the data suggest a role for BLNK and perhaps BCR signaling, in the regulation of κ light chain expression and continued immature B cell differentiation.

Introduction
B lymphopoiesis occurs through discrete stages, starting first from the pro-B and progressing to the pre-B, immature B and finally, mature B cells. Signal originating from the pre-B cell receptor (preBCR), and possibly the B cell receptor (BCR) * are thought to be required for this highly regulated process to occur (1). The preBCR is made up of the Ig heavy chain and the surrogate light chains while the BCR is composed of the surface Ig, and both forms of the receptors are complexed to the signal-transducing subunits Ig ␣ and Ig ␤ (2). The importance of the preBCR in B cell development is evident in gene knockout studies in mice in which the inactivation of its various components results in the arrest of B cell differentiation at the very early pro-B cell stage (1). Although the role of preBCR signaling in early B cell development is well defined, it is less clear whether BCR signaling plays a role in the intermediate stages of B cell maturation other than the maintenance (3) and activation of mature B cells (2). BCR signaling has been studied extensively. It is known that the engagement of the BCR activates various cytoplasmic protein tyrosine kinases such as Syk, Lyn, Blk, and Bruton's tyrosine kinase (Btk), and this leads ultimately to the activation of transcription factors such as nuclear factor of activated T (NF-AT) and NF-B (4). However, it is not clear how the activation of the proximal protein tyrosine kinases is coupled to the activation of the terminal transcription factors, and this in turn is translated into specific cellular responses such as the activation, proliferation, differentiation, and death of B lymphocytes. Recently, adaptor proteins with modular domains but no intrinsic enzymatic activities have been shown to play a major role in BCR signaling by interfacing tyrosine kinase activation with selective downstream molecules (5). Thus, they may play the critical role of channeling signaling pathways and directing specific cellular responses. One such adaptor proteins in B lymphocytes is the B cell linker protein (BLNK) (6), which is also known as SLP-65 (7) or BASH (8). Upon BCR stimulation, BLNK couples activated Syk to PLC-␥ , Vav, Grb2, and Nck (9). In addition, it binds Btk (10,11) and is required for the activation of the transcription factor NF-B (12).
We and others have generated mice lacking BLNK (13)(14)(15)(16). BLNK Ϫ / Ϫ mice have a major block in B cell development at the pro-B to pre-B cell stage where the preBCR is first expressed. This mutant phenotype is consistent with a role for BLNK in the preBCR signaling of early B lymphopoiesis. As few peripheral B cells are generated in BLNK Ϫ / Ϫ mice (13)(14)(15)(16), this makes the analysis of the role of BLNK in the later stages of B cell differentiation difficult. Although BLNK is known to be involved in the activation of mature B cells as evidenced by the inability of BLNK Ϫ / Ϫ mice to respond to a T cell-independent antigen (13), its role (if any) in regulating some aspects of B cell physiology at the immature B cell stage is less clear. Thus, to further examine the role of BLNK in the intermediate stage of B cell differentiation, we now introduced an innocuous transgenic BCR into BLNK Ϫ / Ϫ mice.
Cell Preparation and Culture. Bone marrow cells were obtained by injecting PBS containing 3% FCS and 0.1% NaN 3 into the femurs and tibia of mice. Splenic cells were obtained by dissociating the tissue with a plastic mesh and rubber-stopper from a 5-ml syringe. All cells were treated with red blood cell lysing solution (0.15 M NH 4 Cl, 1 mM KHCO 3 , and 0.1 mM Na 2 EDTA) to eliminate erythrocytes. For cell culture experiments, IgM ϩ cells were removed from bone marrow samples using anti-IgM mAb-coupled MACS beads (Miltenyi Biotec) in a negative selection protocol. The postselected bone marrow cells were cultured in RPMI 1640 supplemented with 10% FCS. In some experiments, cells were cultured for 48 h in the presence of chemical inhibitors (Calbiochem) of signaling molecules. Flow Cytometry. For FACS ® analyses, cells were stained with optimal amounts of FITC-, PE-, and biotin-conjugated mAbs for 10 min on ice and washed twice with PBS. Biotin-conjugated mAbs were revealed with Streptavidin-CyChrome. For intracellular staining, cells were fixed in 2% PBS-buffered paraformaldehyde for 20 min at 4 Њ C, washed, and stained with either anti-or anti-mAb for 20 min in PBS containing 0.5% saponin (Sigma-Aldrich). FACS ® analyses were performed on a FAC-Scan™ (Becton Dickinson) using CELLQuest™ software. The following mAbs were obtained from BD PharMingen: anti-IgM (R6-60.2), anti-B220 (RA3-6B2), anti-CD21 (7G6), anti-CD24 (HSA), anti-CD43 (S7), anti-(R5-240), anti-(LS136), and anti-MHC class II (2G9). The Ac146 anti-idiotypic antibody was obtained from Dr. K. Rajewsky (Harvard Medical School, Boston, MA).

5-Bromo-2-Deoxyuridine Incorporation Studies.
To study the rate of turnover of peripheral B cells, mice were fed continuously with water containing 1 mg/ml of 5-bromo-2-deoxyuridine (BrdU) over a 1-wk period. Cells were recovered from the animals and stained with anti-B220 and anti-IgM antibodies to identify B cells and later fixed for 30 min in 1 ml ice-cold 70% ethanol and overnight in PBS containing 2% paraformaldehyde and 0.02% Tween-20. Thereafter, cells were washed once in PBS and incubated for 10 min at room temperature in a solution containing 150 mM MgCl 2 , 10 M HCl, and 300 g/ml Dnase 1. Finally, cells were stained intracellularly with anti-BrdU antibody (Becton Dickinson) for 20 min at room temperature, washed twice in PBS, and analyzed on a FACScan™.

Results
Appearance of IgM lo Cells in the Bone Marrow of BLNK Ϫ / Ϫ Mice Expressing an Innocuous Transgenic BCR. Mice lacking the adaptor protein BLNK have a major block in B cell development at the pro-B to pre-B cell transition stage where the preBCR is first expressed (13)(14)(15)(16). This developmental stage arrest is likely due to defective signaling by the preBCR in the absence of BLNK. However, the impairment in B cell differentiation in BLNK Ϫ / Ϫ mice is not absolute as some peripheral B cells are generated (13)(14)(15)(16) and the reason for this leakiness is unknown. To determine if the expression of a transgenic BCR can bypass the early developmental block in BLNK Ϫ / Ϫ mice, we crossed wildtype and BLNK Ϫ / Ϫ mice with mice bearing the V H B1-8 VDJ and V 3-83 VJ gene segments that were inserted by gene-targeting into the physiological Ig heavy (H) and light (L) chain loci, respectively (3,17). The resulting BLNK Ϫ /Ϫ mice bearing the V H B1-8/V 3-83 BCR are designated as TG1 BLNK Ϫ/Ϫ mice. The V H B1-8 gene segment was derived from an antibody directed against the hapten 4-hydroxy-3-nitrophenyl acetyl (18) while the V 3-83 gene segment was derived from an anti-H-2 b antibody (19). However, the V H B1-8/V 3-83 BCR is of unknown but innocuous specificity (20). Mice bearing single alleles of V H B1-8 and V 3-83 are used in all subsequent experiments to ensure that the level of expression of the transgenic H and L chains is physiological and mimics that of the and proteins found in normal mice.
As shown in Fig. 1 A, wild-type mice have a distinct population of B220 ϩ IgM Ϫ pre-B cells in their bone marrow in addition to a population of immature B cells that express a continuum level of IgM on their cell surfaces ( Fig. 1 A, top panel). In contrast, BLNK Ϫ/Ϫ mice have few immature B cells as B cell development in these mice is arrested at the pro-B/pre-B cell stage (13)(14)(15)(16). On the other hand, BLNK ϩ/ϩ B cells that express the transgenic V H B1-8/V 3-83 BCR can bypass the pre-B cell stage of development and directly differentiate into IgM high (hi) immature B cells ( Fig. 1 A, third panel). This is in agreement with previously published data (20) and is not surprising as the pre-B cell stage is where the rearrangement of L chain gene segments takes place but these transgenic B cells already possessed and expressed a functional L chain knock-in V region gene segment and thus exhibited expedited development. The IgM hi phenotype of immature B cells found in these mice probably reflects the development of a monoclonal population of B cells in contrast to the development of a polyclonal B cell population in wild-type mice. Interestingly, BLNK Ϫ/Ϫ mice bearing the V H B1-8/V 3-83 BCR generate a larger fraction of IgM ϩ cells (Ͼ2-3-fold) in their bone marrow compared with BLNK ϩ/ϩ mice bearing the same transgenic BCR. Moreover, two distinct B cell populations are evident in the bone marrow of TG1 BLNK Ϫ/Ϫ mice ( Fig. 1 A, fourth panel). In addition to the IgM hi population that is seen in the TG1 BLNK ϩ/ϩ mice, TG1 BLNK Ϫ/Ϫ mice also have an IgM low (lo) B cell population. The presence of this population of B cells that expresses lower level of BCR on the cell surface is also confirmed by anti-staining ( Fig. 1

B, top panel).
As BLNK is directly involved in BCR signaling (6), it is conceivable that the presence of this IgM lo B cell population in TG1 BLNK Ϫ/Ϫ mice is indicative of the occurrence of some form of receptor editing. Our previous finding that BLNK-deficient B cells maintain intact allelic exclusion of the Ig loci (21) suggests that this is unlikely the case. However, to definitively rule out this possibility, we stained for Ig-expressing cells in TG1 BLNK Ϫ/Ϫ mice. It has been shown previously that as a consequence of receptor editing, there is an increase in the population of Ig-expressing B cells in autoreactive BCR transgenic mice (22). However, FACS ® analyses of bone marrow ( Fig. 1 B, bottom panel) or splenic (data not shown) B cells indicate that only a small fraction of the B cells express the L chain in TG1 BLNK Ϫ/Ϫ mice and this is comparable to the fraction found in TG1 BLNK ϩ/ϩ mice. Furthermore, staining of B cells with the anti-idiotypic mAb Ac146, which recognizes the V H B1-8/V 3-83 combination, indicates that virtually all the B cells in the spleen ( Fig. 1 C) and bone marrow (data not shown) of TG1 BLNK Ϫ/Ϫ mice express the knock-in H and L chains. Taken together, the data suggest that there is no evidence of receptor editing occurring in TG1 BLNK Ϫ/Ϫ mice.
Thus, the data suggest that the introduction of an innocuous transgenic BCR can bypass the early developmental block in BLNK Ϫ/Ϫ mice and leads to the generation of a large population of IgM ϩ cells in their bone marrow. However, a substantial fraction of these cells )%05ف( is of the IgM lo phenotype and appears to represent cells in an as yet undefined stage of differentiation. Delayed Cellular Maturation of IgM ϩ Cells in TG1 BLNK Ϫ/Ϫ Mice. To gain insight into the population of IgM lo cells in TG1 BLNK Ϫ/Ϫ mice, we analyze these cells with respect to a series of maturation markers. As shown in Fig. 2, TG1 BLNK ϩ/ϩ B cells express high levels of the pan-B cell marker B220 and high levels of the heat stable antigen CD24 but low levels of CD43 on their cell surfaces. Thus, they resemble immature B cells found in normal mice. Interestingly, both the IgM hi and IgM lo cells in TG1 BLNK Ϫ/Ϫ B cells express lower levels of B220 and CD24 but higher levels of CD43 on their cell surfaces suggesting that they resemble more of the pro-B/pre-B than the immature B cell phenotype (23). On closer examination, the level of CD43 expression on the mutant IgM lo cells is even higher than that found on the mutant IgM hi cells. Thus, the IgM lo and IgM hi cells in TG1 BLNK Ϫ/Ϫ mice appear to be less "mature" than the IgM ϩ cells found in TG1 BLNK ϩ/ϩ mice.
To confirm this observation, we stained the cells with anti-MHC class II antibodies. The expression of the MHC class II antigens is regulated during B lymphopoiesis such that there is a progressive increase in their expression from the pre-B cell stage onwards with a higher level of expression on immature and mature B cells (24)(25)(26). Indeed, FACS ® analyses indicate that both the IgM lo and IgM hi cells in TG1 BLNK Ϫ/Ϫ mice express significantly lower levels of MHC class II antigens on their cell surfaces compared with the IgM ϩ cells in TG1 BLNK ϩ/ϩ mice (Fig.  2). In addition, the level of MHC class II antigens on mutant IgM lo cells is even lower than that found on mutant IgM hi cells. Thus, the FACS ® analyses suggest that the immature B cells in TG1 BLNK Ϫ/Ϫ mice are at an earlier differentiation stage compared with the immature B cells in TG1 BLNK ϩ/ϩ mice, and furthermore that the mutant IgM lo cells may be even less differentiated than the mutant IgM hi cells.
As the mutant IgM lo and IgM hi cells are relatively immature compared with the control BLNK ϩ/ϩ IgM ϩ cells, we decided to examine the cell cycle status of these cells. However, analysis of the intracellular DNA content of wild-type IgM ϩ and mutant IgM lo and IgM hi cells by 7-AAD staining revealed no significant differences among these cells and that they are all noncycling (data not shown).
To further distinguish between the differentiation of IgM lo and IgM hi cells in TG1 BLNK Ϫ/Ϫ mice, we purified and cultured bone marrow B220 Ϫ IgM Ϫ cells for various times. As seen in Fig. 3, B220 Ϫ IgM Ϫ cells from control TG1 BLNK ϩ/ϩ mice give rise mainly to IgM hi cells after 16-24 h of culture and the same population of cells is recovered even after the 72 h time point. In contrast, B220 Ϫ IgM Ϫ cells from TG1 BLNK Ϫ/Ϫ mice generate mainly IgM lo cells after 16-24 h in culture. However, an increasing fraction of IgM hi cells is recovered at the 48-72 h time points. This, together with the in vivo staining data (Fig. 2), suggest that the IgM lo cells likely arise before the IgM hi cells in TG1 BLNK Ϫ/Ϫ mice. Taken together, the overall data indicate that the absence of BLNK appears to delay the maturation of developing B cells in the bone marrow.
Decreased Intracellular Light Chain Expression in IgM lo Cells of TG1 BLNK Ϫ/Ϫ Mice. We next examined the reason for the lower level of BCR expression on IgM lo cells in the bone marrow of TG1 BLNK Ϫ/Ϫ mice. It is possible that these cells produce lesser amount of and/or proteins or alternatively, these IgM lo cells could produce equal amount of cytoplasmic and proteins as the IgM hi cells but they express lower levels of IgM on their cell surfaces. To distinguish between these possibilities, we stain for intracellular and proteins in the IgM ϩ cells of both TG1 BLNK ϩ/ϩ and TG1 BLNK Ϫ/Ϫ mice. As shown in Fig. 4, the IgM hi cells in TG1 BLNK Ϫ/Ϫ mice have equivalent amount of cytoplasmic and compared with the IgM hi cells in TG1 BLNK ϩ/ϩ mice. Interestingly, the IgM lo cells in TG1 BLNK Ϫ/Ϫ mice produce equivalent amounts of but much lesser amounts of compared with the IgM hi The decrease in the production of light chain proteins in the IgM lo cells could be due to a decrease in the generation of the transcripts or in their translation into proteins. If the latter were the reason, then equivalent amount of transcripts should be found in the cells of TG1 BLNK ϩ/ϩ and TG1 BLNK Ϫ/Ϫ mice. To examine these possibilities, we isolated RNA from equal number of purified IgM ϩ cells from TG1 BLNK ϩ/ϩ and IgM lo and IgM hi cells from TG1 BLNK Ϫ/Ϫ mice and subject them to RT-PCR analyses. As shown in Fig. 5, semiquantitative RT-PCR analyses of serially diluted samples indicate that compared with the IgM ϩ cells from TG1 BLNK ϩ/ϩ mice, much less transcripts are found in both IgM hi and IgM lo cells of TG1 BLNK Ϫ/Ϫ mice and with the mutant IgM lo cells having even much lesser transcripts compared with the mutant IgM hi cells. As control and consistent with the intracellular staining data (Fig. 4), equivalent amounts of transcripts is found in all three samples. Hence, the data suggest that the decrease in protein in the mutant IgM lo cells is likely due to a decrease in the amount of transcripts being generated.

Surface IgM Expression Levels on Differentiating B Cells Can Be Modulated by Inhibitors of Signaling Proteins.
As BLNK is an adaptor protein in BCR signal transduction (6) and is known to activate numerous signaling pathways (6-12), we decided to investigate the signaling pathways that might be involved in signaling expression. The small number of B cells in the bone marrow makes it difficult for us to obtain enough cells from either TG1 BLNK ϩ/ϩ or TG1 BLNK Ϫ/Ϫ mice for Western blot analysis of biochemical pathways. Besides, low level basal signaling in the absence of deliberate BCR stimulation may not be detectable using this conventional methodology. Last but not least, it is impossible for us to isolate mutant IgM hi and IgM lo cells from TG1   BLNK ϩ/ϩ mice were purified via positive selection using anti-IgM-coupled magnetic beads while IgM hi and IgM lo cells from TG1 BLNK Ϫ/Ϫ mice were FACS ® sorted. The purity of cells obtained is Ͼ90%. RNA is prepared from equal numbers of purified cells and reverse transcribed into cDNA. The cDNA template is serially diluted threefold and subjected to PCR analyses to detect the expression level of and transcripts. G␣S expression is used as a control for the amount of cDNA template used. BLNK Ϫ/Ϫ mice without stimulating their BCR, as we will have to FACS-sort these cells using fluorochrome-coupled anti-IgM antibodies to identify their phenotype.
We thus employed the alternative method of blocking specific signaling pathways using chemical inhibitors. Bone marrow cells depleted of IgM ϩ cells from TG1 BLNK ϩ/ϩ mice were cultured for 48 h in the presence of varying concentrations of various inhibitors and developing B cells were analyzed for their cell surface expression of IgM. As can be seen in Fig. 6 A, disruption of calcium signaling using calcium chelators such as EGTA at 0.75-500 M or BAPTA/AM at 10-50 M did not affect the level of surface IgM expression on B cells. Similarly, inhibiting the NF-B pathway using SN50 (18-36 M) or the extracellular signal-regulated kinase (ERK) pathway using PD98059 (20-100 M) did not lead to a lower level of surface IgM expression. On the other hand, B cells that differentiated in the presence of LY294002 (2-10 M) that inhibits the activation of PI3K or PP2 (50-200 M) that inhibits src-family tyrosine kinases did have much lower expression of surface IgM. Moreover, the decrease in surface IgM expression on B cells in the presence of LY294002 or PP2 is dosage dependent (data not shown). Finally, as shown in Fig. 6 B, B cells differentiating in the presence of LY294002 or PP2 but not BAPTA/AM and PD98059 also have a lesser amount of intracellular proteins. Thus, it appears that a signaling pathway involving Src-family kinase, PI3K, and BLNK is involved in signaling light chain expression in B cells. Splenic B Lymphocytes in TG1 BLNK Ϫ/Ϫ Mice Are Predominantly Early Stage Transitional B Cells. As seen in Figs.  1 and 7, the introduction of a transgenic BCR leads to a greater than twofold increase in the number of IgM ϩ (IgM lo and IgM hi ) cells in the bone marrow of TG1 BLNK Ϫ/Ϫ mice compared with control animals. This suggests that the expression of a transgenic BCR can bypass the developmental block in BLNK Ϫ/Ϫ mice. However, such an accumulation of B cells is not apparent in the peripheral lymphoid tissues of these mice. Instead, there is a two-to threefold reduction in the number of B cells in the spleen of TG1 BLNK Ϫ/Ϫ mice compared with the control animals (Fig. 7). This suggests that BLNK deficiency may also perturb the further differentiation of peripheral B cells.
Given the finding that the B cells in the bone marrow of TG1 BLNK Ϫ/Ϫ mice are at an earlier differentiation stage compared with those in control mice, we next examined the differentiation of the B cells in the peripheral lymphoid tissues of TG1 BLNK Ϫ/Ϫ mice. Previously, we have shown that the peripheral B cells in BLNK Ϫ/Ϫ mice do not differentiate into the mature IgM lo IgD hi fraction (13). Consistent with that observation, the splenic B cells in TG1 BLNK Ϫ/Ϫ mice are mainly of the IgM hi transitional B cell phenotype (Fig. 8). In addition, we now show that a large fraction of these cells express low level of the MHC class II antigens on their cell surfaces in contrast to the high level expression  of these antigens on splenic B cells found in the control TG1 BLNK ϩ/ϩ mice. Similarly, a large fraction of the splenic B cells in TG1 BLNK Ϫ/Ϫ mice are also CD43 ϩ compared with the cells found in control mice. These CD43 ϩ splenic B cells are not B-1 cells, as the CD5 ϩ B cell subset is not found in BLNK Ϫ/Ϫ mice (13)(14)(15)(16). These data would again suggest that the splenic B cells in TG1 BLNK Ϫ/Ϫ mice are less mature compared with those found in the spleen of control mice.
To determine the precise stage in which BLNK deficiency affects B cell differentiation in the periphery, we examined in detail the transitional B cell population in TG1 BLNK Ϫ/Ϫ mice. Transitional (T) B cells can be further resolved into the earlier T1 and the later T2 cell stages on the basis of CD21 expression (27,28). T1 cells are IgM hi CD2l lo while T2 cells are IgM hi CD21 hi and mature B cells are IgM intermediate (int) CD21 int . As shown in Fig. 8, the splenic B cells in TG1 BLNK Ϫ/Ϫ mice are predominantly T1 cells, suggesting that in the absence of BLNK, the cells failed to develop into the T2 cell stage.
As B cells in TG1 BLNK Ϫ/Ϫ mice are arrested at the transitional T1 cell stage, they are likely to be short-lived (27) and not selected into the long-lived mature B cell pool (29). Indeed, one would expect a higher rate of turnover of peripheral B cells in the mutant mice, and this might account for the loss of cells in these animals. To determine if this is the case, we examine the rate of turnover of the peripheral B cells in the mutant mice. TG1 BLNK ϩ/ϩ and TG1 BLNK Ϫ/Ϫ mice were fed with BrdU in drinking water and the fraction of splenic B cells that had incorporated BrdU over a 1-wk period was determined. As shown in Fig. 9, the fraction of IgM ϩ B cells that had incorporated BrdU in the mutant mice is approximately twice that of the wild-type mice, suggesting that there is a greater loss of peripheral B cells in the absence of BLNK.

Discussion
BLNK Ϫ/Ϫ mice have a major block in B cell development at the pro-B/pre-B cell stage and few peripheral B cells are generated (13)(14)(15)(16). This makes the analysis of the role of BLNK in the later stages of B cell development difficult. We show here that the introduction of an innocuous transgenic BCR into BLNK Ϫ/Ϫ mice leads to the generation of a large population of IgM ϩ cells in the bone marrow and thus could circumvent the problem. Our data further indicate that BLNK plays a role in the intermediate stages of B cell differentiation, in particular at the immature and transitional B cell stages. BLNK Ϫ/Ϫ mice bearing an innocuous transgenic BCR develop two populations of immature B cells in their bone marrow that is distinguishable by their surface IgM expression level (Fig. 1). The IgM lo cells in these mutant mice are apparently at an earlier developmental stage compared with the IgM hi cells and both these cell populations are less "mature" than the immature IgM hi cells found in control BLNK ϩ/ϩ mice bearing the same transgenic BCR (Fig. 2). Furthermore, the IgM lo cells in BCR transgenic BLNK Ϫ/Ϫ mice express less Ig light chain proteins compared with the IgM hi cells in both BLNK ϩ/ϩ and BLNK Ϫ/Ϫ mice (Figs. 4 and 5). Thus, in the absence of BLNK the production of light chain is decreased and the cellular maturation of developing B cells is delayed. In addition, BLNK also play a role in the further differentiation of B cells in the periphery (Fig. 8) as almost all the peripheral B cells in the mutant mice are transitional stage 1 (T1) cells.
B cell development occurs in discrete stages and the role of preBCR signaling in driving B lymphopoiesis is well documented as the inactivation of various signaling molecules such as Ig␤ (30), Btk (31,32), and BLNK (13)(14)(15)(16) all lead to an arrest of B cell development at the pro-B cell  Groups of five TG1 BLNK ϩ/ϩ and TG1 BLNK Ϫ/Ϫ mice were continuously fed with BrdU in drinking water for a period of 1 wk and splenic B220 ϩ IgM ϩ cells were stained for their intracellular BrdU content. Statistical significance is determined by a paired two-tailed Student's t test.
stage. In addition, mice with mutations in the cytoplasmic regions of Ig␣ (33) or Ig␤ (34) have impairment in the generation of immature B cells. However, it is less clear whether BCR signaling is required for continued B cell differentiation at the immature B cell stage other than for the maintenance (3) and activation (2) of mature B cells. In particular, it is not known whether the assembly and expression of a functional BCR per se is sufficient for immature B cell development or that its signaling is also required. Our current data from the TG1 BLNK Ϫ/Ϫ mice would suggest the latter since BLNK is an adaptor protein in the BCR signal transduction pathway.
The data presented here suggest that the BCR, upon assembly and presumably initial low level expression on the cell surface, can signal its own increased expression by regulating the transcription of the Ig light chain. Apparently, in the absence of BLNK, basal BCR signaling is perturbed and this leads initially to a decrease in Ig production and subsequently results in the generation of a population of IgM lo cells in the mutant mice. Later, as the transcripts and protein accumulate, the cells then acquire the IgM hi phenotype. In addition, as a consequence of continued impairment in BCR signaling due to the absence of BLNK, the IgM lo mutant cells did not down-regulate CD43 and upregulate B220 and MHC class II antigens. Gradual changes to these cell surface antigens and hence further differentiation occur only after the mutant cells acquire the IgM hi phenotype but the change is still delayed compared with the control IgM hi cells, presumably due to a weaken signal. This would suggest that the strength of the BCR signaling is the determining factor in the further differentiation of immature B cells. A weak signal leads to a delay in the maturation of developing B cells (this paper) while excessive BCR signaling leads to their deletion as in the case of autoreactive B cells (19). It remains to be determined whether BCR signaling directly regulates the expression level of some of the antigens associated with the differentiation of B cells, in particular the MHC class II antigens. This is particularly interesting as the expression level of MHC class II antigens correlates with the expression level of IgM on BCR transgenic BLNK Ϫ/Ϫ cells (Fig. 2) and furthermore, their level is decreased upon the induced deletion of BCR on mature B cells (3).
BLNK is a central molecule in the BCR signaling pathway (6) and is involved in the activation of Rac-JNK (9), Vav and Nck (6), and NF-B (12) signaling. Hence, it remains to be determined if any of these molecules and pathways are also involved in the various aspects of immature B cell differentiation described here, in particular, in the regulation of Ig expression. Our cell culture studies using chemical inhibitors suggest that a Src-family tyrosine kinase and phosphoinositide 3-kinase (PI3K) are involved in the latter aspect (Fig. 6). It is reasonable to assume that a Src family tyrosine kinase is involved as Lyn is proximal to BCR signaling and together with Syk, initiates the whole cascade of BCR signal transduction (2,4). The role of PI3K is far less clear although incidentally, PI3K Ϫ/Ϫ mice (35,36) have similar defects compared with BLNK Ϫ/Ϫ mice (13)(14)(15)(16). PI3K is activated upon BCR stimulation and generates lipid products that recruit Btk and phospholipase C-␥2 (PLC␥2) to the plasma membrane (37,38). As BLNK is part of the B cell "signalosome" that also includes Btk and PLC␥2 (37,38), it will be interesting to determine if Btk Ϫ/Ϫ (31, 32), PLC␥2 Ϫ/Ϫ (39,40), and PI3K Ϫ/Ϫ (35,36) mice bearing transgenic BCR also have similar defects in Ig expression and immature B cell differentiation. These experiments are currently underway. Last but not least, as a note of precaution, our inhibition studies in no way rule out the involvement of other signaling pathways, in particular that of NF-B signaling and Ca 2ϩ fluxes, in the regulation of light chain expression and much work remains to be done in these aspects.
The finding that the majority of the peripheral B cells in TG1 BLNK Ϫ/Ϫ mice are blocked at the transitional B cell stage is also intriguing (Fig. 8). Presumably, BCR signaling involving BLNK may be required for T1 cells to differentiate into T2 cells and later, enter the long-lived mature B cell pool. Many other mutations that affect BCR signaling, for example those of Ig␣, Btk, Syk, and CD45, have also been shown to interfere with transitional B cell maturation (27). However, the exact mechanism that regulates transitional to mature B cell differentiation remains to be determined. Recently, it has been shown that T2 cells can be distinguished from T1 cells by their ability to up-regulate the cell survival Bcl-xL and cell cycle cyclin D2 proteins upon BCR stimulation (28). We have previously shown that BLNK-deficient B cells do not up-regulate these cell survival and cell cycle proteins and have a defect in NF-B signaling (12). Thus, basal or induced NF-B signaling may be required for the differentiation of transitional B cells. Consistent with this argument, mice lacking IB kinase ␣ and therefore unable to activate NF-B signaling do not generate mature B cells (41).
In conclusion, the data presented in this paper indicate that the adaptor protein BLNK plays a critical role in immature and transitional B cell differentiation. Although BLNK may be involved in signaling by other cell surface receptors, the phenotype described here is more consistent with its role in BCR signal transduction. Our data would suggest that once the BCR is assemble in immature B cell, it is capable of signaling and the signals regulate both the increased production of Ig light chains at the transcriptional level as well as the continued cellular maturation of lymphocytes. Elucidation of the signaling pathways associated with BLNK may shed light on how these processes are regulated.