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.

The peroxisome proliferator-activated receptor alpha is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. PPARalpha mediates the effects of fibrates, which are potent hypolipidemic drugs, on gene expression. To better understand the biological effects of fibrates and PPARalpha, we searched for genes regulated by PPARalpha using oligonucleotide microarray and subtractive hybridization. By comparing liver RNA from wild-type and PPARalpha null mice, it was found that PPARalpha decreases the mRNA expression of enzymes involved in the metabolism of amino acids. Further analysis by Northern blot revealed that PPARalpha influences the expression of several genes involved in trans- and deamination of amino acids, and urea synthesis. Direct activation of PPARalpha using the synthetic PPARalpha ligand WY14643 decreased mRNA levels of these genes, suggesting that PPARalpha is directly implicated in the regulation of their expression. Consistent with these data, plasma urea concentrations are modulated by PPARalpha in vivo. It is concluded that in addition to oxidation of fatty acids, PPARalpha also regulates metabolism of amino acids in liver, indicating that PPARalpha is a key controller of intermediary metabolism during fasting.

Glycerol, a product of adipose tissue lipolysis, is an important substrate for hepatic glucose synthesis. However, little is known about the regulation of hepatic glycerol metabolism. Here we show that several genes involved in the hepatic metabolism of glycerol, i.e., cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase (GPDH), glycerol kinase, and glycerol transporters aquaporin 3 and 9, are upregulated by fasting in wildtype mice but not in mice lacking PPARα. Furthermore, expression of these genes was induced by the PPARα agonist Wy14643 in wild-type but not PPARα−null mice. In adipocytes, which express high levels of PPARγ, expression of cytosolic GPDH was enhanced by PPARγ and β/δ agonists, while expression was decreased in PPARγ +/-and PPARβ/δ -/-mice. Transactivation, gel shift, and chromatin immunoprecipitation experiments demonstrated that cytosolic GPDH is a direct PPAR target gene. In line with a stimulating role of PPARα in hepatic glycerol utilization, administration of synthetic PPARα agonists in mice and humans decreased plasma glycerol. Finally, hepatic glucose production was decreased in PPARα-null mice simultaneously fasted and exposed to Wy14643, suggesting that the stimulatory effect of PPARα on gluconeogenic gene expression was translated at the functional level. Overall, these data indicate that PPARα directly governs glycerol metabolism in liver, whereas PPARγ regulates glycerol metabolism in adipose tissue.

Atherosclerosis in inbred mouse strains has been widely studied by using an atherogenic (Ath) diet containing cholesterol, cholic acid, and fat, but the effect of these components on gene expression has not been systematically examined. We employed DNA microarrays to interrogate gene expression levels in liver of C57BL/6J mice fed the following five diets: mouse chow, the Ath diet, or modified versions of the Ath diet in which either cholesterol, cholate, or fat were omitted. Dietary cholesterol and cholate produced discrete gene expression patterns. Cholesterol was required for induction of genes involved in acute inflammation, including three genes of the serum amyloid A family, three major histocompatibility class II antigen genes, and various cytokine-related genes. In contrast, cholate induced expression of genes involved in extracellular matrix deposition in hepatic fibrosis, including five collagen family members, collagen-interacting proteins, and connective tissue growth factor. The gene expression findings were confirmed by biochemical measurements showing that cholesterol was required for elevation of circulating serum amyloid A, and cholate was required for accumulation of collagen in the liver. The possibility that these gene expression changes are relevant to atherogenesis in C57BL/6J mice was supported by the observation that the closely related, yet atherosclerosis-resistant, C57BL/ 6ByJ strain was largely resistant to dietary induction of the inflammatory and fibrotic response genes. These results establish that cholesterol and cholate components of the Ath diet have distinct proatherogenic effects on gene expression and suggest a strategy to study the contribution of acute inflammatory response and fibrogenesis independently through dietary manipulation.The mouse has become established as a key animal model for studies of lipid metabolism and atherosclerosis, due to the development of techniques for genetic manipulation and tools for gene discovery in this species (reviewed in Refs. 1-4). The first studies to demonstrate that the mouse might provide a useful model for characterization of genetic factors affecting atherosclerosis susceptibility appeared more than 30 years ago. These studies surveyed several inbred laboratory mouse strains and demonstrated that some strains develop early atheromatous lesions when fed experimental diets. These diets contained high concentrations of cholesterol (5%) and fat (30%) supplemented either with cholic acid (2%) (5) or fed in combination with irradiation treatments (6). These early diets produced high mortality and were subsequently modified to reduce the concentrations of cholesterol (1.25%), fat (15%), and cholate (0.5%). By using this modified atherogenic (Ath) 1 diet, Paigen et al. (7,8) demonstrated that fatty streak lesion formation is reproducible within a strain, and that strains differ in their susceptibility. The C57BL/6J strain was among the most susceptible and has been extensively used as a model for dietinduced atherosclerosis.Although the At...