E2F/p107 and E2F/p130 complexes are regulated by C/EBPalpha in 3T3-L1 adipocytes.

We have previously found that loss of C/EBPalpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein levels and to an induction of S phase-specific E2F/p107 complexes. In this paper, we investigated C/EBPalpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that conditionally express C/EBPalpha. C/EBPalpha and C/EBPbeta proteins are induced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/E2F/p107, E2F/p130 and E2F4. cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells during mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is associated with elevation of C/EBPalpha, but is independent of C/EBPbeta expression. Bacterially expressed, purified His-C/EBPalpha is able to disrupt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differentiation. C/EBPbeta, however, does not disrupt E2F/p107 complexes. A short C/EBPalpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro. Induction of C/EBPalpha in stable 3T3-L1 clones revealed that C/EBPalpha causes disruption of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes are induced in cells expressing C/EBPalpha. Our data suggest that induction of p130/E2F complexes by C/EBPalpha occurs via up-regulation of p21, which, in turn, leads to association with and inhibition of, cdk2 kinase activity. The reduction in cdk2 kinase activity correlates with alterations of p130 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stable clones. Our data suggest two pathways of C/EBPalpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 complexes and induction of E2F/p130 complexes.     

Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition

The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G₁- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be that dephosphorylation of specific amino acids signals apoptosis vs. cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants of Rb to alter serine/threonine to glutamic acid to mimic the phosphorylated state. By calcium phosphate transfection, mutant plasmids were introduced into C33A Rb-null cells, and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. Our results show that only mutation of threonine-821 to glutamic acid (T821E) blocked apoptosis by 50%, whereas other sites tested had little effect. In Rb-null Saos-2 and SKUT-1 cells, the T821E mutation also blocked apoptosis induced by the cdk inhibitor, Roscovitine, by 50%. In addition, we show that endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Thus, our data indicates that dephosphorylation of threonine-821 of Rb is required for cells to undergo apoptosis.     

Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition

The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G₁- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be that dephosphorylation of specific amino acids signals apoptosis vs. cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants of Rb to alter serine/threonine to glutamic acid to mimic the phosphorylated state. By calcium phosphate transfection, mutant plasmids were introduced into C33A Rb-null cells, and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. Our results show that only mutation of threonine-821 to glutamic acid (T821E) blocked apoptosis by 50%, whereas other sites tested had little effect. In Rb-null Saos-2 and SKUT-1 cells, the T821E mutation also blocked apoptosis induced by the cdk inhibitor, Roscovitine, by 50%. In addition, we show that endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Thus, our data indicates that dephosphorylation of threonine-821 of Rb is required for cells to undergo apoptosis.     

Phosphorylation of C/EBPbeta at a consensus extracellular signal-regulated kinase/glycogen synthase kinase 3 site is required for the induction of adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes

Stimulation of adipogenesis in mouse preadipocytes requires C/EBPbeta as well as activation of the MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/glycogen synthase kinase 3 (GSK3) site regulates adiponectin gene expression during the C/EBPbeta-facilitated differentiation of mouse fibroblasts into adipocytes. First, we show that exposure of 3T3-L1 preadipocytes to insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) leads to the phosphorylation of C/EBPbeta at threonine 188. Pretreating the cells with a MEK1-specific inhibitor (U0126) significantly attenuates this activity. Similarly, these effectors activate the phosphorylation of T188 within an ectopic C/EBPbeta overexpressed in Swiss mouse fibroblasts, and this event involves both MEK1 and GSK3 activity. We further show that expression of C/EBPbeta (p34kD LAP isoform) in Swiss mouse fibroblasts exposed to DEX, MIX, and insulin induces expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and some adiponectin but that it does not activate expression of FABP4/aP2. In fact, complete conversion of these fibroblasts into lipid-laden adipocytes, which includes activation of FABP4 and adiponectin expression, requires their exposure to a potent PPARgamma ligand such as troglitazone. Expression of a mutant C/EBPbeta in which threonine 188 has been modified to alanine (C/EBPbeta T188A) can induce PPARgamma production in the mouse fibroblasts, but it is incapable of stimulating adiponectin expression in the absence or presence of troglitazone. Interestingly, replacement of T188 with aspartic acid creates a C/EBPbeta molecule (C/EBPbeta T188D) that possesses adipogenic activity similar to that of the wild-type molecule. The absence of adiponectin expression correlates with a reduced amount of C/EBPalpha in the adipocytes expressing the T188A mutant suggesting that C/EBPalpha is required for expression of adiponectin. In fact, ectopic expression of PPARgamma in C/EBPalpha-deficient fibroblasts (NIH 3T3 cells) produces a modest amount of adiponectin, whereas expression of both PPARgamma and C/EBPalpha in NIH 3T3 cells facilitates production of abundant quantities of adiponectin. These data demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/GSK3 site is required for both C/EBPalpha and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.     

Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27

Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.     

Retinoblastoma protein dephosphorylation induced by D-erythro-sphingosine.

The retinoblastoma gene product (Rb), a nuclear phosphoprotein, functions as a tumor suppressor that is inactivated in retinoblastoma and other malignancies. The hypophosphorylated forms of Rb are observed in the G0/G1 phase of the cell cycle, whereas the hyperphosphorylated forms predominate in S and G2/M phases, suggesting that phosphorylation/dephosphorylation of Rb may regulate progression through the growth cycle. However, little is known about the intracellular signals that regulate phosphorylation/dephosphorylation of Rb. We show that D-erythro-sphingosine potently induces early dephosphorylation of Rb. Initial dephosphorylation was observed as early as 1 h after treatment of hematopoietic cells with sphingosine, whereas complete shift to the dephosphorylated form was seen 4 h after treatment. These effects occurred at concentrations of sphingosine as low as 100-500 nM, with maximal effects observed at 1-2.5 microM. These effects were specific to sphingosine, inasmuch as other lipids, amphiphiles, and long chain amino bases, as well as structural analogs of sphingosine, failed to induce dephosphorylation of Rb. Also, activation of second messenger systems including protein kinase C, cAMP-dependent kinases, and calcium ionophores, as well as inhibition of serine/threonine protein phosphatases, failed to induce dephosphorylation of Rb. Induction of Rb dephosphorylation by sphingosine preceded inhibition of growth and a specific arrest in the G0/G1 phase of the cell cycle. These studies, for the first time, identify an intracellular activator of Rb.