INTRODUCTION

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NF-B belongs to the Rel family of transcription factors which regulate genes involved in immune and inflammatory responses (3, 5, 70). In mammals, the Rel family is composed of RelA/p65, c-Rel, RelB, p50 (NF-B1), and p52 (NF-B2), which have sequence similarity over approximately 300 amino acids in the amino-terminal half of the protein. NF-B subunits are able to homo- or heterodimerize to form transcription factor complexes with a range of DNA-binding and activation potentials. Although all Rel members bind DNA, only RelA/p65 (hereafter referred to as p65), c-Rel, and RelB have extended carboxy termini harboring transactivation function (70). The most widely studied form of NF-B is a heterodimer of the p50 and p65 subunits and is a potent activator of gene transcription (56).

In most cells, NF-B is found sequestered in the cytoplasm bound in an inactive complex with its natural biological inhibitor IB (3, 70). The IB family members include IB, IB, p105/IB (precursor of p50), p100 (precursor of p52), and IB (41, 74). Each has in common a series of ankyrin repeats which interact with the DNA-binding domain and the nuclear localization signal of NF-B, thus maintaining the transcription factor as an inactive complex. Activation of NF-B is induced by a variety of diverse stimuli including inflammatory cytokines, phorbol esters, bacterial toxins (such as lipopolysaccharide) viruses, UV light, and a variety of mitogens (4, 5). Treatment of cells with these stimuli activate the recently discovered IB kinase complex, leading to the phosphorylation of serines 32 and 36 of IB or serines 19 and 23 of IB (19, 46, 52, 77). This phosphorylation event targets IB for ubiquitin-dependent degradation through the 26S proteasome complex, resulting in the release and nuclear translocation of NF-B (22, 68).

In addition to its well-established role in activating the transcription of genes involved in immunological responses, studies indicate that NF-B also functions in promoting cell growth. For instance, lymphocytes from mice lacking p50, p65, or c-Rel are defective in mitogenic responses (20, 38, 58, 65), and p50/p52 double-knockout animals fail to generate mature osteoclasts and B cells (25, 35). Recent reports also demonstrate the expression of NF-B/Rel proteins in the proliferative zone of the developing avian limb bud and the requirement of NF-B for the proper growth of this tissue (15, 36). In addition, deregulated NF-B activity has been associated with oncogenesis, since reports show elevated NF-B/Rel levels in primary breast cancers (18, 66). NF-B is activated by oncogenic Ras and is required by Ras to induce foci in NIH 3T3 cells (23). Similarly, the chimeric oncoprotein Bcr-Abl, implicated in acute lymphoblastic and chronic myelogenous leukemias, also requires NF-B to induce cellular transformation (54). Consistent with this latter study, Hodgkin's lymphoma cells depleted of NF-B activity revealed strongly impaired tumor growth in mice (7). The ability of NF-B to protect cells against chemotherapeutic drugs or TNF-mediated apoptosis function (9, 69, 72, 75), suggests that NF-B-regulated growth control may be related to its cell survival properties. In fact, inhibition of NF-B led to apoptosis in cells expressing oncogenic forms of Ras (45). Finally, recent demonstrations that cellular proliferation defects, attributed to the absence of NF-B, are associated with a delay in cell cycle progression in G₁ (7, 28), in addition to the previously described physical association with NF-B and CBP/p300 (26, 50), establishes a link between NF-B and regulators of the cell cycle. Although the above cited reports strongly suggest a role for NF-B in cell growth control, the molecular mechanism(s) underlying this regulation remains unclear.

To gain insight into the role of NF-B in regulating cell growth, we first used a well-established skeletal myogenesis model, which is characterized by the maturation of precursor myoblasts into differentiated contractile myotubes. This cellular process is dependent on the activation or induction of the myogenic basic helix-loop-helix (bHLH) and MEF2 families of transcription factors that stimulate tissue-specific gene expression, as well as changes in cell cycle regulators that cause myoblasts to undergo irreversible growth arrest (39, 48, 71). The latter process is regulated by a balance in activities of cyclin-cyclin-dependent kinase (cdk) complexes and their respective known kinase inhibitors. The signal to induce differentiation, achieved most commonly in tissue culture by removing growth factor-rich medium, causes the downregulated expression of cyclins A and D1 and kinases cdk2 and cdc2, with an induced synthesis of the cdk inhibitors p18 and p21 and stabilization of the p27 protein (71). This regulated switch in activities leads to the dephosphorylation of the product of the retinoblastoma susceptibility gene (pRb), which maintains cells in a G₁-arrested state by inhibiting the E2F-DP1 transcription factor complex. Exit from the cell cycle, therefore, is critical for myogenic transcription and completion of the differentiation program.

Using the murine C2C12 skeletal muscle cell line, we demonstrate that NF-B functions in proliferating myoblasts to inhibit their differentiation process. This was determined by showing that C2C12 myoblasts contain NF-B in their nuclei and that NF-B DNA-binding activity and transactivation function are reduced during myogenesis. In addition, myoblasts generated to lack NF-B activity are greatly accelerated in their differentiation program. Transfections in 10T1/2 cells showed that NF-B strongly blocks the ability of the myogenic transcription factor, MyoD, to induce myogenesis. Furthermore, this latter regulation is specific to the transactivation-competent p65 subunit of NF-B, arguing that NF-B inhibits myogenic differentiation through its activation of gene expression. The observation that C2C12 cells lacking NF-B display a reduction in their proliferation rate and exit the cell cycle faster than do control cells suggests that inhibition of myogenesis by NF-B is in part related to its growth-promoting activity. Importantly, these cells also exhibit a marked reduction in cyclin D1 protein and mRNA levels. The results of experiments performed with 10T1/2 fibroblasts indicate that NF-B regulation of cyclin D1 is one mechanism by which this transcription factor inhibits myogenic differentiation. From this differentiation model, we expanded our study to identify the level at which NF-B regulated cyclin D1. The results show that this regulation occurs at the transcriptional level and is mediated by several authentic NF-B DNA-binding sites in the cyclin D1 promoter. Furthermore, by using diploid fibroblasts, we addressed the potential relevance of NF-B regulation of cyclin D1 with respect to the cell cycle. Our data show that in cells stimulated to reenter the cell cycle, NF-B activity is required for cyclin D1 transcriptional initiation and hyperphosphorylation of pRb, leading to progression into S phase. Similar to what was observed in C2C12 cells, embryonic fibroblasts lacking NF-B activity also exhibit a reduction in proliferation, in conjunction with lower levels of cyclin D1. Taken together, these data establish that the ability of NF-B to control cellular proliferation and differentiation are processes tightly coupled to its ability to transcriptionally regulate cyclin D1.

	MATERIALS AND METHODS

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Cell culture. Murine C2C12 myoblast cells obtained from the American Type Culture Collection and primary murine myoblasts (a generous gift from J. Samulski) were cultured at 37°C in Dulbecco's modified Eagle's medium with high glucose (DMEM-H), supplemented with 20% fetal bovine serum (FBS) and antibiotics (Life Technologies). The cells were grown at subconfluency and passaged every 2 to 3 days. To induce differentiation, the cells were grown overnight to 60 to 70% confluency in growth medium (GM), washed once with phosphate-buffered saline (PBS), and then switched to DMEM-H supplemented with 2% horse serum and 10 µg of insulin per ml plus antibiotics (DM). C3H10T1/2 clone 8 mouse embryo fibroblasts (10T1/2), also obtained from American Type Culture Collection, were cultured in DMEM-H containing 15% FBS plus antibiotics and passaged every 2 to 3 days. HeLa cells were cultured in DMEM-H with 5% FBS and 5% calf serum, NIH 3T3 cells were grown in DMEM-H with 10% Colorado calf serum, and for mouse embryo fibroblasts (MEFs), cells were grown in DMEM-H plus 10% FBS.

Plasmids. For reporter plasmids, 3xB-Luc or 3xBmut-Luc contain three tandem repeats of the wild-type or mutated B site, from the major histocompatibility complex (MHC) class I enhancer, respectively, fused to the luciferase reporter gene (obtained from B. Sugden, University of Wisconsin, Madison, Wis.). TnI-Luc contains a muscle-specific enhancer in the troponin I gene fused to luciferase, and 4RTK-Luc contains four E boxes from the muscle creatine kinase enhancer (gifts of S. Konieczny, Purdue University). Cyclin D1 promoter reporter constructs were described earlier (1). For expression plasmids, p50 and p65 subunits of NF-B were expressed from a cytomegalovirus (CMV)-driven promoter as previously described (10). The mutant IB plasmid, designated IBSR, was a gift of D. Ballard (Vanderbilt University). A cyclin D1 expression plasmid was generated by removing a 1,300-bp EcoRI fragment containing the mouse cyclin D1 cDNA from the pBSSK plasmid and inserting it into the EcoRI site of pCMV5. pCDNA3-D3 plasmid expressing human cyclin D3 was a gift of C. Sherr (St. Jude Children's Research Hospital). pEMC11s plasmid expressing MyoD was obtained from the H. Weintraub laboratory (University of Washington). Oncogenic ras was expressed from the H-ras (V-12) plasmid as previously described (45).

EMSAs. Nuclear extracts for electrophoretic mobility shift assays (EMSAs) were prepared as previously described (16), except that 0.25% Nonidet P-40 was used to extract nuclei. A 5-µg portion of extract were preincubated with 1 mM phenylmethylsulfonyl fluoride and 1 µg of poly(dI-dC)-poly(dI-dC) in a volume of 12 µl for 10 min. This mixture was subsequently incubated in a total volume of 20 µl at room temperature for 20 min with 2 × 10⁴ cpm of a ³²P-labeled oligonucleotide probe containing a B site (underlined) from the class I MHC promoter (5'-CAG GGC TGG GGA TTC CCC ATC TCC ACA GTT TCA CTT C-3'). The buffer consisted of 10 mM Tris-HCl (pH 7.7), 50 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol. Complexes were resolved on a 5% polyacrylamide gel in Tris-glycine buffer (25 mM Tris, 190 mM glycine, 1 mM EDTA) at 25 mA for 2 to 3 h at room temperature. The gels were dried and exposed on film for approximately 1 to 3 days. For supershift EMSAs, antibodies against specific NF-B were added to the nuclear extract and incubated for 10 min prior to the addition of phenylmethylsulfonyl fluoride and poly(dI-dC)-poly(dI-dC). The antibodies used for this portion of the study were p65 (Rockland), p50 (NLS; Santa Cruz Biotechnology), c-Rel (C; Santa Cruz Biotechnology), and RelB (C-19; Santa Cruz Biotechnology). NF-B-binding sites in the cyclin D1 promoter were determined by generating a series of oligonucleotides corresponding to both wild-type and mutant (M) putative NF-B sites within the human cyclin D1 promoter (47). The oligonucleotides have the following sequences (NF-B wild-type and mutated sites are underlined): 858, 5'-GTG CAG TTG GGG ACC CCC GCA AGG ACC GAC TGG TCA A-3'; 858(M), 5'-GTG CAG TTC CCG ACC CCC GCA AGG ACC GAC TGG TCA A-3'; 749, 5'-ACC ATC TTG GGC TGC TGC TGG AAT TTT CGG GCA TTT A-3'; 749(M), 5'-ACC ATC TTG GGC TGC TGC TCC CCT TTT CGG GCA TTT A-3'; 39, 5'-GGA CTA CAG GGG AGT TTT GTT GAA GTT GCA AAG TCC T-3'; and 39(M), 5'-GGA CTA CAC CCC AGT TTT GTT GAA GTT GCA AAG TCC T-3'.

Transfections and viral infections. To generate C2C12 cells with stably integrated luciferase reporter plasmids, cells were seeded at a density of 2 × 10⁵ cells in 6-cm dishes 24 h prior to transfection. Cotransfections with 4 µg of reporter plasmid 3xB-Luc or 3xBmut-Luc and 1 µg of pcDNA3 (Invitrogen) containing the neomycin resistance marker were performed with Superfect reagent as recommended by the manufacturer (Qiagen). At 48 h posttransfection, the cells were trypsinized and cultured at 1/30 their density in 1 mg of Geneticin (G418; Life Technologies) per ml. Mixed populations containing either wild-type or mutant versions of the B sites were allowed to expand under selection, and from the wild-type population an individual clone expressing similar basal promoter activity was selected. Cell extracts were prepared and luciferase assays were performed as previously described (16).

Western blot analysis. Cells were harvested in PBS, and whole-cell lysates were prepared by resuspending cell pellets in ice-cold RIPA buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40), and incubating the suspension on ice for 20 to 30 min. For pRb Western blots, lysis buffer included phosphatase inhibitors (50 mM sodium fluoride, 1 mM orthovanadate, 50 mM -glycerophosphate, 80 µM cantharidin). Supernatant lysates were collected following high-speed centrifugation for 20 min at 4°C. Equal amounts of extract were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Schleicher & Schuell). Blocking was performed in 5% nonfat dry milk-1× TBST (25 mM Tris-HCl [pH 8.0], 125 mM NaCl, 0.1% Tween 20). Primary and secondary antibodies were diluted in 0.5% nonfat dry milk-1× TBST, and the incubations proceeded for 30 min at room temperature, with the exception of pRb blots, where the primary antibody was incubated for 1 h. Washes were performed in 1× TBST for 5 to 10 min and repeated five times. Specific proteins were visualized by enhanced chemiluminescence (Amersham Life Science). Antibodies to IB (C-21), myogenin (M-225), cyclin D1 (R-124), cyclin A (C-19), MyoD (M-318), and p21 (C-19) were obtained from Santa Cruz Biotechnology, pRb antibody was obtained from Pharmingen, cyclin D3, cdk4, and p27 antibodies were generous gifts of Y. Xiong (University of North Carolina).

Immunofluorescence. All immunofluorescence experiments were performed directly in 12-well plates. Myoblasts were grown to subconfluency in GM and then switched to DM for 48 h. For 10T1/2 fibroblasts, cells were grown overnight in complete medium following transfections then switched to DM for 72 h. All steps were performed at room temperature. The cells were washed in PBS and fixed in a 2% formaldehyde-1× PBS solution for 30 min. They were permeabilized with 0.5% Nonidet P-40-1× PBS for 5 min and then blocked with horse serum (1:100) in PBS for 30 min. They were washed with PBS, incubated for 1 h with anti-skeletal myosin heavy chain (MY-32; Sigma) diluted 1:500 in 3% bovine serum albumin-1× PBS, washed three times with PBS, and incubated for 1 h in the dark with fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG; Sigma) diluted at 1:250. The cells were washed in PBS and photographed with an Olympus inverted microscope equipped with phase-contrast and UV illumination through an FITC filter. For bromodeoxyuridine (BrdU) staining, MEFs grown on glass coverslips were washed once in PBS, fixed for 5 min at room temperature with 100% cold methanol, washed again in PBS, and permeabilized with 0.25% Triton X-100 in PBS for 5 min at room temperature. Following a PBS wash, the cells were treated for 10 min with 1.5 M HCl, washed four times with PBS, and incubated for 30 min with an FITC-conjugated anti-BrdU antibody (1:10 dilution in 1% bovine serum albumin-PBS [Becton Dickinson]). After several washes in PBS, the cells were counterstained for 3 min with a 1-µg/ml solution of Hoechst dye 33258 (Sigma) in PBS. Following two additional washes in PBS, the coverslips were mounted on glass slides and examined on a Zeiss microscope.

Northern blot analysis. Total RNA was isolated with Trizol reagent as recommended by the manufacturer (Life Technologies). RNA samples were fractionated on an agarose gel and transferred overnight onto a nylon filter. The next day, RNA was cross-linked with a UV cross-linker (Stratagene). For detection of IB, Id-1, Hes-1, cyclin D3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNAs blots were hybridized in QuickHyb buffer supplemented with 100 µg of salmon sperm DNA as recommended by the manufacturer (Stratagene). For detection of cyclin D1 mRNA, blots were hybridized overnight at 42°C in 50% formamide-5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1× PE (50 mM Tris-HCl [pH 7.5], 0.1% sodium pyrophosphate, 1% SDS, 0.25% polyvinylpyrrolidene, 0.25% Ficoll, 5 mM EDTA)-150 µg of salmon sperm DNA. All the probes were generated with a random-primed labeling kit (Life Technologies) in the presence of [-³²P]dCTP (NEN-Dupont). The DNA products were purified over micro-G-50 Sephadex columns (Life Technologies), boiled, and added to the hybridization mixture. Washes were performed twice in 2× SSC-0.1% SDS for 10 min at room temperature and then twice in 0.1× SSC-0.1% SDS for 20 min at 42°C for cyclin D1 detection and 65°C for detection of all other mRNAs.

	RESULTS

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Loss of NF-B activity correlates with myogenic differentiation. To analyze the role of NF-B in cell growth control, we initiated our study by using the C2C12 myogenic differentiation model, since in this cell culture system the gene products involved in regulating terminal cell cycle arrest are largely known (39, 48, 71). First, we analyzed the relative levels and subunit composition of NF-B/Rel complexes found in undifferentiated and differentiated C2C12 cell cultures. EMSAs performed with nuclear extracts prepared from C2C12 cells identified four potential NF-B-containing complexes in proliferating myoblasts (Fig. 1A, lane 1). The levels of complexes I, II, and III decreased as cells became terminally differentiated (Fig. 1A, lanes 1 to 4). The level of complex IV initially decreased but was elevated 72 h following initiation of the differentiation process. EMSA supershifts performed to identify which Rel proteins were contained in these complexes revealed the presence of the p65 subunit in complex III and p50 in complex IV in both undifferentiated and differentiated cells (Fig. 1B, lanes 2 and 3 and lanes 7 and 8). Although the antibody against p50 was not able to supershift complex III, the migration pattern of this complex nevertheless resembled that of the classical p50-p65 heterodimer. None of the antisera specific for NF-B/Rel proteins caused a supershift in complexes I and II, suggesting that NF-B is most probably not a component of these higher-molecular-weight complexes. To address whether these effects were specific to C2C12 cells, EMSA and supershift analyses were repeated with nuclear extracts prepared from primary myoblasts. As seen with the C2C12 cells, loss of p50 and p65 NF-B DNA-binding activity was observed in primary myoblasts undergoing differentiation (Fig. 1C), indicating that a similar reduction of NF-B may occur in naturally developing skeletal muscle.

View larger version (80K): [in this window] [in a new window]   FIG. 1.   Loss of NF-B binding activity during myogenic differentiation. (A) Proliferating C2C12 myoblasts (GM) were induced to differentiate (DM), and at the indicated times nuclear extracts were prepared and EMSA was performed with a radiolabeled oligonucleotide containing an NF-B-binding site. (B) C2C12 cells were maintained in GM or switched to DM for 72 h. Supershift EMSA was performed with nuclear extracts preincubated with either no antibody (lanes 1 and 6) or antisera specific for p65 (lanes 2 and 7), p50 (lanes 3 and 8), c-Rel (lanes 4 and 9), or RelB (lanes 5 and 10). NF-B complexes containing p50 and p65 subunits are shown. (C) EMSA and supershift EMSA were performed as described above with nuclear extracts prepared from primary myoblasts undergoing differentiation. (D) Supershift EMSA was performed with nuclear extract prepared from WEHI-231 cells, preincubated either with no antibody or with an antiserum specific to the c-Rel subunit of NF-B.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Myogenic differentiation correlates with loss of NF-B transactivation function. (A) C2C12 myoblasts stably containing a 3xB-Luc reporter plasmid were propagated as either a mixed population or a clonal isolate. Cells were plated in triplicate overnight in 6-cm culture dishes, and on the following day they were maintained in GM or switched to DM for 48 h. At that time, cell extracts were prepared, and relative luciferase units were determined by normalizing to total protein (RLU). Promoter activities were also determined for 3xB-Luc and 3xBmut-Luc populations that were treated or not treated with 10 ng of TNF- per ml for 24 h. (B) C2C12 cells were maintained in GM or differentiated in DM for up to 72 h. At the indicated times, total RNA was prepared and 10 µg of sample was used for Northern blot analysis. The blot was hybridized with an IB-specific probe, and RNA loading was normalized by stripping the blot and reprobing for GAPDH mRNA.

NF-B functions as a negative regulator of myogenesis in the C2C12 model. The results described above suggested a functional role for NF-B in precursor myoblasts. To test this hypothesis, C2C12 cells were generated to express a mutant form of the IB inhibitor for which serines at positions 32 and 36 had been changed to alanines. The resulting protein (referred to as IB superrepressor, or IBSR) is no longer subject to phosphorylation and subsequent proteasome degradation following an NF-B-activating stimulus and therefore functions as a potent and specific inhibitor of NF-B activity (13). The absence of endogenous IB protein in IBSR-containing myoblasts (Fig. 3A), as well as the previously described sensitivity of IBSR-expressing cells or p65^/ cells to TNF-induced killing (9, 69, 72) (Fig. 3B), demonstrated that IBSR was functioning properly to block NF-B activity. The inhibitory activity of the IBSR was confirmed by EMSA, which showed that only IBSR-expressing myoblasts were blocked in their ability to activate NF-B in response to TNF treatment (data not shown). Parental, vector control, or IBSR-expressing myoblasts were examined for the rate at which they underwent differentiation. At 48 h following serum withdrawal, less than 10% of the parental or vector control cells had undergone differentiation, as determined by their myotube phenotype and by their ability to express the late differentiation marker, myosin heavy chain (Fig. 3C). In sharp contrast, nearly all the IBSR-expressing cells had become fully differentiated by this time (Fig. 3C). Similar enhanced rates of differentiation were observed in pooled clones of IBSR-containing cells, demonstrating that these effects were not due to clonal variations (Fig. 3C). An examination of a second temporally regulated myogenic differentiation marker, myogenin, demonstrated greatly accelerated expression of this myogenic transcription factor in C2C12-IBSR cells compared to vector control cells (Fig. 3D). These data suggested that NF-B activity was required in proliferating myoblasts to block myogenic differentiation.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   C2C12 cells lacking NF-B activity have an accelerated rate of differentiation. (A) Whole-cell lysates were prepared from C2C12 parental, vector control, or IBSR proliferating myoblasts, and 50 µg of sample was used for Western blot analysis. IB and IBSR proteins were detected with an IB polyclonal antibody (C-19; Santa Cruz Biotechnology) at a 1:1,500 dilution. The IBSR protein is FLAG tagged and therefore migrates at a slightly higher mobility compared to the endogenous protein. (B) IBSR-expressing myoblasts were seeded in triplicate overnight in 12-well plates, and the following day cells were treated with increasing concentrations of TNF- for 48 h. Cell viability was scored by trypsinization and the trypan blue exclusion method. Cells not treated with TNF- were designated 100% viable. (C) C2C12 parental cells, vector control, an IBSR clone, or five pooled IBSR clones were differentiated in DM for 48 h, at which time the cells were prepared for immunofluorescence to detect for the myosin heavy chain. (D) C2C12 vector control or IBSR cells were induced to differentiate in DM for up to 72 h. At the indicated times, lysates were prepared and Western blot analysis was performed probing for myogenin expression. V (P) and I (P) denote myogenin expression from five pooled vector control or IBSR clones, respectively, that were differentiated for 48 h.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   NF-B inhibits MyoD-induced myogenesis in 10T1/2 fibroblasts. Cells were maintained in growth medium containing 15% FBS and differentiated in DM. The cells were seeded in triplicate overnight in 6-cm dishes, and the following day, cotransfections were performed with Superfect (Qiagen). DNA consisted of 1 µg of a troponin-I-Luc reporter plasmid (TnI-Luc) (A) or 1 µg of the 4RTK-Luc plasmid (B), along with 0.25 µg of an expression plasmid for MyoD alone or in combination with 0.5 µg of expression plasmids for either the activated form of oncogenic ras (H-ras V-12), p65, p50, or 1 µg of IBSR. DNA was standardized to 2.5 µg by the addition of Bluescript plasmid (Stratagene). Cells were maintained in growth medium for 24 h following transfections and then switched to DM for 48 h, at which time cell extracts were prepared and relative light units (RLU) were determined, by normalizing values to total protein. (C) For immunofluorescence analysis, 10T1/2 cells were seeded overnight and the next day similar transfections were performed as described above, except that one-third of the amount of DNA was used. At 24 h following transfections, the cells were switched to DM for 72 h, at which time the cells were fixed and probed for the myosin heavy chain. To score for the number of myotubes formed, cells expressing myosin were counted and averaged from a minimum of 10 randomly selected fields.

NF-B inhibits C2C12 myogenesis through its growth-promoting activity and regulation of cyclin D1. To determine the mechanism by which NF-B regulated myogenesis in vitro, we first examined whether NF-B had any effect on the expression of Id-1 or Hes-1, which are known inhibitors of differentiation (12, 55). Northern analysis performed with mRNA isolated from proliferating C2C12 parental, vector control, or IBSR-expressing myoblasts revealed that the steady-state levels of Hes-1 was only slightly altered whereas Id-1 was more strongly altered in cells lacking NF-B activity (Fig. 5A). This result may indicate that these genes could be involved in NF-B-mediated inhibition of myogenic differentiation. Relevant to the regulatory mechanism of NF-B was that C2C12-IBSR cells maintained in growth medium exhibited a substantial increase in cell doubling time compared to parental or vector control cells (Fig. 5B). Since successful progression of myogenic differentiation is highly dependent on the ability of cells to irreversibly exit the cell cycle in the G₁ state (71), we asked whether the effect on cell growth as a result of the absence of NF-B activity translated to changes in cell cycle progression. Exit from cell cycle was assessed by immunoblot analysis probing for the phosphorylation status of pRb. The results showed that while no obvious differences in pRb states were observed in either vector control or IBSR cells maintained in fresh growth medium (t = 0 h), once cells were induced to differentiate (t = 12 and 24 h) pRb hypophosphorylation occurred much faster in IBSR cells than in control cells (Fig. 5C). These data imply that in immortalized C2C12 cells, inhibition of NF-B accelerates the rate at which cells exit the cell cycle.

View larger version (40K): [in this window] [in a new window]   FIG. 5.   C2C12 cells lacking NF-B exhibit a growth defect and changes in pRb phosphorylation. (A) Total RNA was prepared from C2C12 parental, vector control, or IBSR proliferating myoblasts, and Northern analysis was performed to detect the expression of Hes-1 or Id-1 genes. (B) C2C12 parental, vector control, or IBSR myoblasts were plated in triplicate in 10-cm plates and maintained in GM for 3 days. Every 24 h, the total cell number was determined by trypsinization and trypan blue exclusion. (C) C2C12 vector control (V) or IBSR cells (I) were induced to differentiate for up to 24 h. At the indicated times, cell lysates were prepared with lysis buffer containing phosphatase inhibitors and Western blotting was performed to probe for the hypo- and hyperphosphorylated forms of Rb with a monoclonal antibody (14001A; Pharmigen).

View larger version (41K): [in this window] [in a new window]   FIG. 6.   C2C12 cells lacking NF-B are downregulated for cyclin D1 protein and mRNA. (A) C2C12 vector control or IBSR cells were differentiated in DM for up to 72 h. At the indicated times, whole-cell lysates were prepared and 50 µg was used in Western blot analyses to probe for various cell cycle proteins. (B) Total RNA was prepared from proliferating parental (P), vector control (V), or IBSR (I) myoblasts, and 10 µg of sample was used in Northern blotting to probe for cyclin D1 or cyclin D3 mRNA.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   NF-B inhibits MyoD transactivation function through the regulation of cyclin D1. (A) 10T1/2 fibroblasts were seeded in triplicate overnight in 6-cm culture dishes, and the next day cotransfection was performed with DNA consisting of 1 µg of the troponin-I reporter plasmid (TnI-Luc) and 0.25 µg of a MyoD expression plasmid, along with either 0.25 µg of an IBSR expression plasmid or the indicated amounts of cyclin D1 or cyclin D3 expression plasmid. DNA was normalized by the addition of Bluescript plasmid (Stratagene). Transfected cells were maintained in growth medium overnight and on the following day were switched to DM for 48 h, at which time extracts were prepared and a luciferase assay was performed. The level of troponin-I activation by MyoD alone was set to a value of 100. (Below) To verify the expression of proteins, parallel transfections were performed and immunoblot analysis was performed to probe for MyoD, cyclin D1, and cyclin D3. (B) Similar transfections were performed in 10T1/2 fibroblasts with the following amounts of expression plasmids: 0.25 µg of MyoD, 1 µg of IBSR, and 2 µg of cyclin D1 or cyclin D3. The cells were differentiated as described above for 72 h, at which point myogenesis was quantitated by counting myotubes from a minimum of 10 fields of cells.

View larger version (109K): [in this window] [in a new window]   FIG. 8.   TNF- inhibits C2C12 myogenesis and stabilizes cyclin D1. C2C12 cells were induced to differentiate in the absence or presence of 20 ng of TNF- per ml. Cytokine addition was repeated at 6 h and every additional 12 h after the induction of differentiation. At 72 h, the cells were washed with PBS, fixed for 10 min at room temperature with 4% paraformaldehyde, and photographed by phase-contrast microscopy. In parallel treatment cultures, cells were harvested at 24 and 48 h and whole-cell lysates were prepared for Western blot analysis. A 50-µg portion of total protein was fractionated by SDS-polyacrylamide gel electrophoresis, and immunoblotting was performed to probe for cyclin D1. The blot was subsequently stripped and reprobed for cdk4, used as an internal control.

NF-B is a direct transcriptional activator of cyclin D1. The use of the C2C12 myogenesis model allowed us to demonstrate that NF-B regulates cyclin D1 expression. Next, we were interested in determining whether this regulation was tissue specific and at what level it was controlled. To address the first of these points, an adenoviral delivery system was used to transiently overexpress IBSR in HeLa cells and MEFs. Northern analysis showed that, similar to what was seen in C2C12 cells stably expressing IBSR, transient inhibition of NF-B activity led to a decrease in steady-state levels of cyclin D1 mRNA (Fig. 9A). This result demonstrated that this regulation was therefore not unique to skeletal muscle cells and, perhaps equally important, did not result from a clonally selectable process. Conversely, transfections in fibroblasts with a plasmid expressing the p65 subunit of NF-B led to increased levels of endogenous cyclin D1 (Fig. 9B), thus underscoring the specificity of the IBSR protein and demonstrating that expression of p65 is sufficient to induce cyclin D1 expression.

View larger version (35K): [in this window] [in a new window]   FIG. 9.   Regulation of cyclin D1 is specific to NF-B occurring in multiple cell types. (A) HeLa cells or MEFs were infected with adenovirus containing either the IBSR or empty vector (AdCMV) at a multiplicity of infection of 50 or 200, respectively. Total RNA was prepared 48 h postinfection, and Northern analysis was performed to detect cyclin D1 mRNA expression. Western blot analysis with an IB-specific antibody is shown to demonstrate the expression of the IBSR in HeLa cells and MEFs by using the adenovirus delivery system. (B) Fibroblasts were transfected with either empty vector or an expression plasmid expressing the p65 subunit of NF-B. The following day, cells were switched to serum-deprived conditions for a 48-h period. Subsequently, whole-cell extracts were prepared and 50 µg was used in immunoblot analysis probing for cyclin D1. The loading efficiency was normalized by reprobing the blot for cdk4 expression.

View larger version (56K): [in this window] [in a new window]   FIG. 10.   NF-B regulation of cyclin D1 occurs at the transcriptional level. (A) NIH 3T3 cells were plated in triplicate in 12-well plates. Transfections were performed on the following day with Lipofectamine reagent (Life Technologies) mixed with DNA consisting of 0.2 µg of reporter plasmids containing various 5' deletions of the human cyclin D1 promoter, along with 0.15 µg of a p65 expression plasmid. DNA remained on the cells for 3 h in serum-free medium, and the cells were then switched for 48 h to complete medium containing 10% calf serum, at which time the luciferase activity was determined. Values were normalized to basal levels of promoter activity obtained by transfecting cyclin D1 promoter constructs in the absence of p65. (B) EMSAs were performed with nuclear extracts prepared from either NIH 3T3 cells or HeLa cells treated (+) or not treated () with TNF- for 30 min. Putative NF-B-binding sites, located at positions 858, 749, and 39 in the human cyclin D1 promoter, are indicated within the oligonucleotide sequences used for EMSAs. (C) Supershift EMSAs were performed with nuclear extracts prepared from HeLa cells treated with TNF, which were preincubated either with no addition (lanes 1, 6, and 11) or with addition of antisera specific for p50 (lanes 2, 7, and 12), or the p65 subunit (lanes 3, 8, and 13). Arrows denote p65-containing complexes. For competition EMSAs, extracts were preincubated with either a 100-fold molar excess of unlabeled oligonucleotides containing wild-type (xs wt) NF-B-binding sites (lanes 4, 9, and 14), or a 100-fold molar excess of unlabeled oligonucleotides containing mutations in the NF-B sites (xs mut) (lanes 5, 10, and 15). The mutations are shown in the boxed regions above the NF-B-binding sites. (D) The same mutation at position 39 was made in the NF-B-binding site within the 66CD1-Luc reporter plasmid. Both the wild-type and mutant (66CD1-Bmut-Luc) reporter plasmids were separately cotransfected along with a p65 expression plasmid in NIH 3T3 cells under the same transfection conditions as described for panel A.

NF-B is required in early G₁ for cyclin D1 activation and progression into S phase. Having established that cyclin D1 is a novel transcriptional target of NF-B and provided evidence to show how this regulation is important for controlling the differentiation of skeletal muscle cells, we focused the last part of our study on examining the relevance of this regulatory mechanism to cell cycle progression and cell growth. Cyclin D1 was originally identified as a suppressor of G₁ arrest in yeast cells and as a gene that is inducible in the G₁ phase of the cell cycle (44, 76). The expression of cyclin D1 in G₁ is important for cell cycle progression (60, 61), and results demonstrating that acceleration into S phase by cyclin D1 is greater for synchronized cultures emerging from quiescence than for asynchronous cycling cells suggest a specialized role for this D-type cyclin in the G₀/G₁ transition (53). Interestingly, earlier results from our laboratory showed that NF-B was strongly activated when growth-arrested fibroblasts were stimulated by serum to re-enter cell cycle (6). This finding prompted us to investigate whether NF-B was required for cyclin D1 induction in cells reinitiating cell cycle.

View larger version (23K): [in this window] [in a new window]   FIG. 11.   Requirement for NF-B activity in the early G1 phase of the cell cycle. (A) NIH 3T3 cells were made quiescent by being switched for 48 h from complete medium containing 10% serum to medium containing 0.25% calf serum. Serum-deprived cells were cotransfected either with a cyclin D1 promoter reporter plasmid alone (963CD1-Luc) (2.5 µg) or in combination with indicated amounts of IBSR plasmid. The cells were maintained in quiescence or switched to complete medium to induce cyclin D1 transcription and reentry into the cell cycle. Extracts were prepared, and relative luciferase activity was determined by standardizing to total cellular protein. (B) MEFs were made quiescent by culturing cells in 0.1% FBS for 48 h. The cells were then infected under serum-deprived conditions with adenovirus containing either empty vector (CMV) or IBSR at an MOI of 200. At 24 h postinfections, MEFs were either maintained under serum-deprived conditions or induced to reenter the cell cycle by the addition of 10% FBS. Whole-cell lysates were prepared 24 h later, and immunoblot analysis was performed to probe for both pRb and cyclin D1 (under these culture conditions, we determined that pRb hyperphosphorylation in MEFs is maximally detectable between 20 and 24 h following serum stimulation). (C) Subconfluent MEFs were grown on coverslips overnight and were rendered quiescent on the following day. Cells were infected with either control adenovirus (CMV), IBSR, or a combination of IBSR and cyclin D1 viruses (both at a multiplicity of infection of 200). At 24 h following infections, the cells were maintained quiescent or switched for 12 h to growth medium containing 10% serum, at which point the medium was supplemented with 100 µM BrdU (Sigma) for an additional 10 h. The cells were fixed and prepared for immunofluorescence analysis as described in Materials and Methods. The percentage of cells in the S phase was calculated by determining the number of BrdU-positive cells with respect to the total number of cells in a given field.