Erding Hu

Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor

Peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) is an adipocyte-specific nuclear hormone receptor that has recently been identified as a key regulator of two fat cell enhancers. Transcriptional activation by PPAR gamma 2 is potentiated by a variety of lipids and lipid-like compounds, including naturally occurring polyunsaturated fatty acids. We demonstrate here that retroviral expression of PPAR gamma 2 stimulates adipose differentiation of cultured fibroblasts. PPAR activators promote the differentiation of PPAR gamma 2-expressing cells in a dose-dependent manner. C/EBP alpha, a second transcription factor induced during adipocyte differentiation, can cooperate with PPAR gamma 2 to stimulate the adipocyte program dramatically. Our results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.

Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARγ

Adipocyte differentiation is an important component of obesity and other metabolic diseases. This process is strongly inhibited by many mitogens and oncogenes. Several growth factors that inhibit fat cell differentiation caused mitogen-activated protein (MAP) kinase-mediated phosphorylation of the dominant adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and reduction of its transcriptional activity. Expression of PPARγ with a nonphosphorylatable mutation at this site (serine-112) yielded cells with increased sensitivity to ligand-induced adipogenesis and resistance to inhibition of differentiation by mitogens. These results indicate that covalent modification of PPARγ by serum and growth factors is a major regulator of the balance between cell growth and differentiation in the adipose cell lineage.

Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.

The orphan nuclear receptor, peroxisome proliferator-activated receptor (PPAR) gamma, is implicated in mediating expression of fat-specific genes and in activating the program of adipocyte differentiation. The potential for regulation of PPAR gamma gene expression in vivo is unknown. We cloned a partial mouse PPAR gamma cDNA and developed an RNase protection assay that permits simultaneous quantitation of mRNAs for both gamma l and gamma 2 isoforms encoded by the PPAR gamma gene. Probes for detection of adipocyte P2, the obese gene product, leptin, and 18S mRNAs were also employed. Both gamma l and gamma 2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma 1 expression was also detected at lower levels in liver, spleen, and heart; whereas, gamma l and gamma 2 mRNA were expressed at low levels in skeletal muscle. Adipose tissue levels of gamma l and gamma 2 were not altered in two murine models of obesity (gold thioglucose and ob/ob), but were modestly increased in mice with toxigene-induced brown fat ablation uncoupling protein diphtheria toxin A mice. Fasting (12-48 h) was associated with an 80% fall in PPAR gamma 2 and a 50% fall in PPAR gamma mRNA levels in adipose tissue. Western blot analysis demonstrated a marked effect of fasting to reduce PPAR gamma protein levels in adipose tissue. Similar effects of fasting on PPAR gamma mRNAs were noted in all three models of obesity. Insulin-deficient (streptozotocin) diabetes suppressed adipose tissue gamma l and gamma 2 expression by 75% in normal mice with partial restoration during insulin treatment. Levels of adipose tissue PPAR gamma 2 mRNA were increased by 50% in normal mice exposed to a high fat diet. In obese uncoupling protein diphtheria toxin A mice, high fat feeding resulted in de novo induction of PPAR gamma 2 expression in liver. We conclude (a) PPAR gamma 2 mRNA expression is most abundant in adipocytes in normal mice, but lower level expression is seen in skeletal muscle; (b) expression of adipose tissue gamma1 or gamma2 mRNAs is increased in only one of the three models of obesity; (c) PPAR gamma 1 and gamma 2 expression is downregulated by fasting and insulin-deficient diabetes; and (d) exposure of mice to a high fat diet increases adipose tissue expression of PPAR gamma (in normal mice) and induces PPAR gamma 2 mRNA expression in liver (in obese mice). These findings demonstrate in vivo modulation of PPAR gamma mRNA levels over a fourfold range and provide an additional level of regulation for the control of adipocyte development and function.

Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor γ

Peroxisome proliferator activated receptor (PPAR)gamma is an orphan member of the nuclear hormone receptor superfamily and is expressed at high levels specifically in adipose tissue. Recent data suggest that this factor is a central regulator of adipocyte gene expression and differentiation. Fibroblastic cell lines that express PPARgamma ectopically can be induced to differentiate into fat cells by a variety of lipids and lipid-like activators of PPARs, suggesting that this protein may function to link adipogenesis with systemic lipid metabolism.

ADIPOCYTE DIFFERENTIATION : A TRANSCRIPTIONAL REGULATORY CASCADE

The adipose cell is now known to play a complex role in energy homeostasis, energy storage and signaling to other tissues concerning the state of energy balance. The past few years have seen an explosive increase in our knowledge of the transcriptional basis of adipocyte differentiation. Factors such as peroxisome proliferator-activated receptor gamma, the CCAAT/enhancer binding protein family members, and adipocyte determination- and differentiation-dependent factor 1 play important regulatory roles in this process. Furthermore, these factors provide a focus for beginning to understand how various hormones and metabolites influence the development of adipose tissue in vivo.

Targeted disruption of the c-fos gene demonstrates c-fos-dependent and -independent pathways for gene expression stimulated by growth factors or oncogenes.

The c‐fos proto‐oncogene is believed to play a pivotal role in transducing growth factor‐mediated signals from the extracellular milieu into the nucleus. c‐fos protein dimerizes with c‐jun and related proteins and mediates transcription via AP‐1 sites. Using c‐fos‐deficient mice generated through gene knockout techniques, we derived 3T3‐type cell lines from primary embryonic fibroblasts. The c‐fos‐deficient cells grow normally under optimal culture conditions and show only a slight reduction in growth rate in low serum culture compared with control cells. They also express mRNA for most of the Fos and Jun family members at normal levels. The overall levels of AP‐1 DNA binding activity are normal and several genes (c‐jun, MCP1, metallothionein) known to contain functional AP‐1 sites are expressed normally in the c‐fos‐deficient and control cells. In contrast, mRNA for the metalloproteases stromelysin (MMP‐3) and type I collagenase (MMP‐1), which are often induced by oncogenes and growth factors and have been implicated in tumor invasiveness, cannot be induced by epidermal growth factor or platelet‐derived growth factor in c‐fos‐deficient cells. Transformation of mutant cells with polyoma middle T oncogene essentially restores wild‐type levels of stromelysin expression, while transformation with v‐src leads to only a weak induction of the metalloprotease. These results clearly demonstrate that some AP‐1‐dependent genes require c‐fos for full expression while others do not; oncogenes may activate expression of metalloproteases via either fos‐dependent or fos‐independent mechanisms. These results also imply that c‐fos may play an important regulatory role in the invasive behavior of malignant tumors, independent of any role this proto‐oncogene might play in cell growth per se.

PPARγ and the control of adipogenesis

We recently cloned PPARγ as a factor that binds to an enhancer which has specificity for adipose cells. When expressed ectopically, PPARγ converts fibroblasts into bona fide preadipose cells. Upon application of activators or PPARγ ligands, these cells differentiate into fat cells. Most recently, we have been trying to understand the nature of natural ligands that activate PPARγ and the protein domains that control adipogenesis. With regards to ligands, we have shown that an unusual prostanoid, 15-deoxyΔ12,14PG 12, can bind to PPARγ and activate it. A second transcription factor that is induced early in differentiation, ADD1/SREBP1, appears to promote the formation of PPARγ ligands. Transfection of this molecule, a member of the bHLH family, causes the secretion of molecules that can serve as ligands for PPARγ. This ligand-like activity is specific for the γ isoform of PPAR. Current studies are attempting to identify these potentially novel ligands. With regard to structure-function of PPARγ, we first analyzed the adipogenic activity of the three isoforms of PPAR: α, γ and δ. Using appropriate activators of each it is clear that PPARγ has the most adipogenic action. PPARα can be adipogenic with high levels of the strongest activators and PPARδ does not stimulate fat cell differentiation. To identify the domain(s) of PPARγ responsible for differentiation, chimeras between PPARγ and PPARδ were created and transfected into fibroblasts. This has allowed the isolation of relatively small regions of this molecule that are responsible for differentiation.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA AND THE CONTROL OF ADIPOGENESIS

The adipose cell is now known to play a complex role in energy homeostasis, storing energy and signaling to other tissues concerning the state of energy balance. The past several years have seen an explosive increase in our knowledge of the transcriptional basis of adipocyte differentiation. This review describes the role of peroxisome proliferator-activated receptor gamma in this process, and describes how other transcription factors may affect adipogenesis by modulating the amount or activity of peroxisome proliferator-activated receptor gamma. Furthermore, peroxisome proliferator-activated receptor gamma and other adipogenic transcription factors provide a focus for beginning to understand how various hormones and metabolites influence the development of this tissue in vivo.

DEF-1, a Novel Src SH3 Binding Protein That Promotes Adipogenesis in Fibroblastic Cell Lines

ABSTRACT The Src homology 3 (SH3) motif is found in numerous signal transduction proteins involved in cellular growth and differentiation. We have purified and cloned a novel protein, DEF-1 (differentiation-enhancing factor), from bovine brain by using a Src SH3 affinity column. Ectopic expression of DEF-1 in fibroblasts resulted in the differentiation of a significant fraction of the culture into adipocytes. This phenotype appears to be related to the induction of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ), since DEF-1 NIH 3T3 cells demonstrated augmented levels of PPARγ mRNA and, when treated with activating PPARγ ligands, efficient induction of differentiation. Further evidence for a role for DEF-1 in adipogenesis was provided by heightened expression of DEF-1 mRNA in adipose tissue isolated fromobese and diabetes mice compared to that in tissue isolated from wild-type mice. However, DEF-1 mRNA was detected in multiple tissues, suggesting that the signal transduction pathway(s) in which DEF-1 is involved is not limited to adipogenesis. These results suggest that DEF-1 is an important component of a signal transduction process that is involved in the differentiation of fibroblasts and possibly of other types of cells.