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

Abstract

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.

FIG. 1.

Phosphorylation of C/EBPβ at a consensus ERK/GSK3 site during the differentiation of 3T3-L1 preadipocytes. (A) Proliferating 3T3-L1 preadipocytes were cultured in growth medium until they reached confluence. At 2 days postconfluence (day 0), the quiescent cells were exposed to DEX, MIX, FBS, and insulin, and total cellular protein was harvested at the indicated times. Equal amounts of protein from each sample were subjected to Western blot analysis using antibodies specific for phospho-ERK1/2 (p-ERK), phospho-C/EBPβ (p-C/EBPβ), ERK, C/EBPβ, PPARγ, C/EBPα, perilipin, and adiponectin. (B) Confluent 3T3-L1 preadipocytes were exposed to various combinations of 1.67 μM insulin (I), 1 nM FGF-2 (F), 20 μM U0126 (U), 50 μM LY294002 (LY), and 10 mM LiCl (Li) in the presence of MIX and DEX for 4 h, and total cellular protein was subjected to Western blot analysis using antibodies specific for phospho-C/EBPβ (pC/EBPβ), C/EBPβ, phospho-ERK1/2 (pERK), and ERK.

FIG. 1.

Phosphorylation of C/EBPβ at a consensus ERK/GSK3 site during the differentiation of 3T3-L1 preadipocytes. (A) Proliferating 3T3-L1 preadipocytes were cultured in growth medium until they reached confluence. At 2 days postconfluence (day 0), the quiescent cells were exposed to DEX, MIX, FBS, and insulin, and total cellular protein was harvested at the indicated times. Equal amounts of protein from each sample were subjected to Western blot analysis using antibodies specific for phospho-ERK1/2 (p-ERK), phospho-C/EBPβ (p-C/EBPβ), ERK, C/EBPβ, PPARγ, C/EBPα, perilipin, and adiponectin. (B) Confluent 3T3-L1 preadipocytes were exposed to various combinations of 1.67 μM insulin (I), 1 nM FGF-2 (F), 20 μM U0126 (U), 50 μM LY294002 (LY), and 10 mM LiCl (Li) in the presence of MIX and DEX for 4 h, and total cellular protein was subjected to Western blot analysis using antibodies specific for phospho-C/EBPβ (pC/EBPβ), C/EBPβ, phospho-ERK1/2 (pERK), and ERK.

FIG. 2.

Phosphorylation of T188 within a consensus ERK/GSK3 site of an ectopic C/EBPβ in response to exposure of Swiss mouse fibroblasts to FGF-2. Swiss mouse fibroblasts expressing either wild-type (Wt), T188A (T-A), or T188D (T-D) forms of C/EBPβ were cultured in growth medium without tetracycline until confluent. Two days later, the cells were exposed to the various effectors in the presence of MIX, DEX, and insulin. (A) The Wt cells were exposed to various combinations of 1 nM FGF-2, 20 μM U0126 (U), 50 μM LY294002 (LY), and 10 mM LiCl (Li) for 30 min and harvested, and the resulting total cell proteins were subjected to Western blot analysis of phospho-C/EBPβ (pC/EBPβ), C/EBPβ, phospho-ERK (pERK), and ERK. (B) Confluent Wt, T-A, and T-D cells were exposed to 1 nM FGF-2 along with DEX, MIX, and insulin for 30 min, and total cell proteins were subjected to Western blot analysis of phospho-C/EBPβ and C/EBPβ.

FIG. 3.

Morphological differentiation of Swiss mouse fibroblasts expressing wild type (Wt), T188A (TA), and T188D (TD) forms of C/EBPβ. The Swiss cell lines corresponding to Wt, TA, and TD forms of C/EBPβ were induced to differentiate for 5 days in the absence or presence of tetracycline (TET), as described in Materials and Methods. The cells were then fixed, stained with Oil Red O, and photographed.

FIG. 4.

Phosphorylation of mouse C/EBPβ at a consensus ERK/GSK3 site (T188) is required for the induction of adiponectin gene expression during the differentiation of mouse fibroblasts. Swiss mouse fibroblast cell lines (wild type [Wt], T188A [T-A], and T188D [T-D]) were grown to confluence in the absence of tetracycline to induce expression of the corresponding C/EBPβ protein. Two days later, the cells were treated with either MIX or troglitazone in the presence of DEX and insulin. The cells were harvested 5 days later and subjected to Western blot analysis of phospho-C/EBPβ (phosC/EBPβ), C/EBPβ, PPARγ, perilipin, adiponectin, aP2, and actin.

FIG. 5.

Phosphorylation of mouse C/EBPβ at a consensus ERK/GSK3 site (T188) is required for C/EBPα expression and for sustaining terminal adipogenesis. (A) Swiss mouse fibroblast cell lines (wild type [WT], T188A [T-A], and T188D [T-D]) were cultured in growth medium with or without tetracycline until confluent. Two days later, the cells were induced to differentiate by exposure to DEX, MIX, FBS, insulin, and troglitazone for 2 days in the absence or presence of tetracycline. At this stage, cells were treated with insulin and troglitazone in the presence (7d+TET) or absence of tetracycline and harvested at the indicated times. (B) The Swiss-LAP cells (WT, T-A, or T-D) were induced to differentiate in the absence of tetracycline for 6 days as outlined for panel A. At this stage, 1 μg of tetracycline (+) or vehicle (−) was added to the culture medium of each cell line to suppress expression of the ectopic C/EBPβ, and cells were harvested for nuclear proteins at the indicated times. Equal amounts of nuclear proteins from each cell sample (A and B) were subjected to Western blot analysis of C/EBPβ, PPARγ, and C/EBPα.

FIG. 6.

Phosphorylation of C/EBPβ at T188 is not required for its ability to bind to a C/EBP regulatory element from the promoter of the C/EBPα gene. Swiss fibroblasts expressing wild-type (WT), T188A (T-A), or T188D (T-D) forms of C/EBPβ were induced to differentiate as described for Fig. 5A, and cells were harvested at either day 7 (A) or the indicated times (B) for preparation of nuclear extracts. Equal amounts of each extract were preincubated for 30 min in the presence or absence of antiserum for either C/EBPβ (Cβa/b) or C/EBPα (Cαa/b) or a 100-fold excess of cold C/EBP-oligonucleotide (C/EBP binding site in the C/EBPα promoter) prior to addition of a ³²P-labeled C/EBP-oligonucleotide. The binding of the oligonucleotide to the different C/EBP complexes was analyzed by EMSA, as described in Materials and Methods. Arrowheads, corresponding supershifted complexes.

FIG. 7.

Induction of adiponectin expression in mouse fibroblasts by PPARγ2 requires expression of C/EBPα. Confluent NIH 3T3 cell lines expressing PPARγ2 with (+C/EBPα) or without (−C/EBPα) were induced to differentiate by exposure to DEX, MIX, FBS, insulin, and troglitazone. At day 2, cells were treated with insulin and troglitazone and harvested at the indicated times for Western blot analysis of PPARγ2, C/EBPα, adiponectin, fatty acid synthase (FAS), aP2, and β-catenin. Noninfected NIH 3T3 fibroblasts were also subjected to the same differentiation protocol for 6 days in order to illustrate the absence of adipogenic proteins.

FIG. 8.

Transcription factors and signaling pathways regulating adiponectin expression during adipogenesis. This model is based on this study and other studies referred to in the text.