Adipocyte differentiation is regulated both positively and negatively by external growth factors such as insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). A key component of the adipocyte differentiation process is PPAR␥, peroxisomal proliferator-activated receptor ␥. To determine the relationship between PPAR␥ activation and growth factor stimulation in adipogenesis, we investigated the effects of PDGF and EGF on PPAR␥1 activity. PDGF treatment decreased ligand-activated PPAR␥1 transcriptional activity in a transient reporter assay. In vivo [ 32 P]orthophosphate labeling experiments demonstrated that PPAR␥1 is a phosphoprotein that undergoes EGF-stimulated MEK/mitogen-activated protein (MAP) kinase-dependent phosphorylation. Purified PPAR␥1 protein was phosphorylated in vitro by recombinant activated MAP kinase. Examination of the PPAR␥1 sequence revealed a single MAP kinase consensus recognition site at Ser 82 . Mutation of Ser 82 to Ala inhibited both in vitro and in vivo phosphorylation and growth factor-mediated transcriptional repression. Therefore, phosphorylation of PPAR␥1 by MAP kinase contributes to the reduction of PPAR␥1 transcriptional activity by growth factor treatment.Peroxisome proliferator-activated receptors (PPARs) 1 are members of the nuclear hormone receptor superfamily (1). These receptors heterodimerize with retinoic acid-like receptor, RXR, and become transcriptionally active when bound to ligand. The three PPAR isoforms (␣, ␦, and ␥) differ in their C-terminal ligand binding domains, and each appears to bind and respond to a specific subset of agents including hypolipidemic drugs, long chain fatty acids, aracadonic acid metabolites, and antidiabetic thiazolidinediones (2-4). PPAR␥ is expressed predominantly in mouse white and brown fat, with lower levels in liver, whereas PPAR␣ is present in heart, kidney, and liver (5, 6). PPAR␦ expression is ubiquitous (7,8).Ectopic expression of either PPAR␣ or PPAR␥ in NIH-3T3 cells is sufficient to induce adipocyte differentiation in the presence of PPAR␥ activators (9, 10). The rapid induction of PPAR␥ during adipocyte differentiation and its enriched expression in adipose tissues suggest that PPAR␥ is responsible for the initiation and maintenance of the adipocyte phenotype in vivo (9). Previously two isotypes of PPAR␥ (PPAR␥1 and PPAR␥2) have been identified in 3T3-L1 adipocytes (11). Zhu et al. (12) have demonstrated that these two isotypes are derived from a single PPAR␥ gene by alternative promoter usage and RNA splicing. However, thus far, no functional difference has been found between the two isotypes.Adipogenesis is a complex process; multiple hormones and factors regulate the conversion of progenitor cells to adipocytes. Insulin and/or insulin-like growth factor enhance the ability of PPAR ligand to induce differentiation of both 3T3-L1-and PPAR␥-overexpressing cell lines (9, 13). In contrast, growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast gro...

The cold shock response in Escherichia coli follows an abrupt shift in growth temperature from 37 to 10°C (8). Cold shock causes the cessation of growth for 4 to 5 h, concomitant with a severe reduction in the number of proteins synthesized. During this lag period, the relative synthesis of several cold shock proteins increases. These proteins include CS7.4, NusA, RecA, H-NS, polynucleotide phosphorylase, translation initiation factors 2p and 2a, pyruvate dehydrogenase (lipoamide), and the dihydrolipoamide acetyltransferase of pyruvate dehydrogenase (5,8,9). The cold shock proteins demonstrate a 2-to 10-fold increase in synthetic rate towards the end of the lag period, with the exception of CS7.4, which shows a rapid 10-fold increase in synthesis (< 1 h) and a > 100-fold increase by the time growth resumes (5, 8).The molecular mechanisms responsible for the cold shock response have not been defined. However, it has been suggested that CS7.4 is a positive transcriptional regulator of cold shock protein synthesis (9). CS7.4 has a remarkable similarity to the DNA binding domain of a family of eukaryotic nucleic acid-binding proteins, known as the Y-box transcription factors (3,5,18,19,23,24). In this report, we provide evidence consistent with CS7.4 functioning as a transcriptional activator of another newly identified cold shock protein: the A subunit of DNA gyrase. We suggest that a number of genes encoding cold shock proteins may be coordinately regulated by CS7.4. MATERIALS AND METHODSBacterial strains and plasmids. The host E. coli strains for this work were derivatives of E. coli K-12: MG1655 and W3110 (2,16

PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma are a group of transcription factors that are involved in numerous processes, including lipid metabolism and adipogenesis. By comparing liver mRNAs of wild-type and PPARalpha-null mice using microarrays, a novel putative target gene of PPARalpha, G0S2 (G0/G1 switch gene 2), was identified. Hepatic expression of G0S2 was up-regulated by fasting and by the PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. Surprisingly, the G0S2 mRNA level was highest in brown and white adipose tissue and was greatly up-regulated during mouse 3T3-L1 and human SGBS (Simpson-Golabi-Behmel syndrome) adipogenesis. Transactivation, gel shift and chromatin immunoprecipitation assays indicated that G0S2 is a direct PPARgamma and probable PPARalpha target gene with a functional PPRE (PPAR-responsive element) in its promoter. Up-regulation of G0S2 mRNA seemed to be specific for adipogenesis, and was not observed during osteogenesis or myogenesis. In 3T3-L1 fibroblasts, expression of G0S2 was associated with growth arrest, which is required for 3T3-L1 adipogenesis. Together, these data indicate that G0S2 is a novel target gene of PPARs that may be involved in adipocyte differentiation.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

We describe the cloning and characterization of two cDNAs from Xenopus laevis that encode sequencespecific DNA binding proteins called FRG Y1 and FRG Y2 (frog Y-box proteins 1 and 2). During oogenesis and embryogenesis, the genes encoding these proteins are differentially expressed. FRG Y1 mRNA is present in oocytes, embryos, and all adult tissues examined, whereas FRG Y2 mRNA is found only in testis and immature oocytes. The FRG Y1 and FRG Y2 proteins are shown to stimulate transcription from a promoter containing a Y box (CTGATTGGCCAA). This promoter element is found in both mammalian major histocompatibility complex class II and Xenopus germ-cell-specific genes. FRG Y1, FRG Y2, and a human Y-box binding protein are homologous and represent a distinct family of sequence-specific DNA binding proteins. We identify protamine-like regions that are present within this family of transcription factors, suggesting that they use unusual means of binding to DNA.