Haluk Avci

Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.

Estrogen receptor-α and Sp1 interact in the induction of the low density lipoprotein-receptor

Both estrogen and selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have been demonstrated to lower plasma low density lipoprotein (LDL)-cholesterol concentrations by stimulation of LDL receptor gene expression. To determine the molecular mechanisms of estradiol- and tamoxifen-induced LDL receptor expression, we performed transient transfection experiments with luciferase reporter gene-constructs under transcriptional control of the human LDL receptor promoter. We demonstrate, that estradiol and tamoxifen stimulate LDL receptor gene expression in human HepG2 hepatoma cells only when estrogen receptor (ER)-α but not when ER-β is cotransfected. Deletion mutants and point mutations of the LDL receptor promoter reveal that estradiol- and tamoxifen-stimulated expression of this gene depends on an intact repeat 3 in the LDL receptor promoter, a cis-element previously shown to interact with Sp1. Gel mobility analyses demonstrated estradiol- and tamoxifen-stimulated binding of nuclear proteins to repeat 3 (bp −56 to bp −36) of the LDL receptor promoter. These data provide an alternative mechanism of LDL receptor gene expression by non-classical estradiol- and tamoxifen-stimulated induction through an ER-α/Sp1 complex.

Phosphorylation of sterol regulatory element-binding protein (SREBP)-1a links growth hormone action to lipid metabolism in hepatocytes

OBJECTIVE Increased lipid accumulation in cells and tissues is a key phenomenon in the development of obesity, insulin resistance, and atherosclerosis. In the regulation of lipid content of cells and tissues the SREBPs play a dominant role as transcription factors. METHODS Since growth hormone (GH) affects lipid metabolism and function of fat as well as liver cells, we have investigated the role of SREBP-1a, SREBP-1c and SREBP-2 in the gene regulatory action of GH in the human liver cell line HepG2 and primary mouse hepatocytes. RESULTS These experiments showed that SREBP-1a couples the stimulatory effect of GH on cholesterol regulated genes, e.g. LDL receptor gene, via sterol sensitive binding cis-element (sre-1). This effect was not depending on RNA expression, but related to phosphorylation of SREBP-1a protein. The result was supported by experiments with the mutant variant SREBP-1a S117A, which is not phosphorylated by Erk-MAP kinases. To analyse a possible role of GH-induced SREBP-1a phosphorylation in cellular physiology we investigated an expression profile of genes coding for central players in lipid transport or lipid metabolism as well as for transcription factors by real time PCR in primary mouse hepatocytes and human hepatoma cell line stably overexpressing the mature form of SREBP-1a or mutated form. CONCLUSION These experiments emphasize the role SREBP-1a and its phosphorylation for gene regulatory effects of GH.

A mutation in the c-Fos gene associated with congenital generalized lipodystrophy

BackgroundCongenital generalized lipodystrophy (CGL) or Berardinelli–Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.Methods and resultsIn a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation (c.–439, T→A) in the patients’ c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.ConclusionThe immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development.

Insulin-regulated transcription factors: molecular link between insulin resistance and cardiovascular risk factors.

Patients with insulin resistance and/or type 2 diabetes have a 5-fold increase in cardiovascular mortality rate. Therefore, it is a current issue of discussion that arterial hypertension, lipid disorders as well as visceral obesity are coronary risk factors, which might belong to a syndrome that is caused by decreased insulin sensitivity. Concerning a possible molecular link between insulin resistance, atherosclerosis and obesity, we focus in our research on questions looking for a molecular link between lipid metabolism, insulin action, and obesity at a gene regulatory level. Alterations in the structure, function and regulation of transcription factors appear to be such signalling steps which might play an essential role in the pathogenesis and therapy of cardiovascular risk factors associated with insulin resistance, eg the so called metabolic syndrome. Recent examples are members of the nuclear hormone receptor superfamily, eg peroxisome proliferator-activated receptor (PPAR) isoforms and sterol regulatory element-binding proteins (SREBPs). Beside their regulation by different metabolites, these transcription factors are also targets of hormones, like insulin and leptin, growth factors, and inflammatory signals. Therefore, they appear to be a point of signalling convergence at a gene regulatory level. Major signalling pathways coupling receptors at the cell surface for hormones, growth factors as well as cytokines to gene regulatory events in the nucleus are the MAP-kinase cascades. We have recently defined different postreceptor defects in these pathways in patients with clinical phenotypes corresponding to congenital lipoatrophy. Therefore, these studies may identify novel pathways which play a role in the control of body weight, insulin sensitivity and cardiovascular risk.