Role of accessory factors and steroid receptor coactivator 1 in the regulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

In the liver, glucocorticoids induce a 10-15-fold increase in the rate of transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene, which encodes a key gluconeogenic enzyme. This induction requires a multicomponent glucocorticoid response unit (GRU) comprised of four glucocorticoid accessory factor (AF) elements and two glucocorticoid receptor binding sites. We show that the AFs that bind the gAF1, gAF2, and gAF3 elements (hepatocyte nuclear factor [HNF]4/chicken ovalbumin upstream promoter transcription factor 1 and HNF3beta) all interact with steroid receptor coactivator 1 (SRC1). This suggests that the AFs function in part by recruiting coactivators to the GRU. The binding of a GAL4-SRC1 chimeric protein completely restores the glucocorticoid induction that is lost when any one of these elements is replaced with a GAL4 binding site. Thus, when SRC1 is recruited directly to gAF1, gAF2, or gAF3, the requirement for the corresponding AF is bypassed. Surprisingly, glucocorticoid receptor is still required when SRC1 is recruited directly to the GAL4 site, suggesting a role for the receptor in activating SRC1 in the context of the GRU. Structural variants of GAL4-SRC1 were used to identify requirements for the basic-helix-loop-helix and histone acetyltransferase domains of SRC1, and these are specific to the region of the promoter to which the coactivator is recruited.

Characterization of the human liver fructose-1,6-bisphosphatase gene promoter.

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1, 6-bisphosphate to fructose 6-phosphate and P(i). Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2, 6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor kappaB (NF-kappaB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25 bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-kappaB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.

Salicylate-induced growth arrest is associated with inhibition of p70s6k and down-regulation of c-myc, cyclin D1, cyclin A, and proliferating cell nuclear antigen.

Salicylate and its pro-drug form aspirin are widely used medicinally for their analgesic and anti-inflammatory properties, and more recently for their ability to protect against colon cancer and cardiovascular disease. Despite the wide use of salicylate, the mechanisms underlying its biological activities are largely unknown. Recent reports suggest that salicylate may produce some of its effects by modulating the activities of protein kinases. Since we have previously shown that the farnesyltransferase inhibitor l-744, 832 inhibits cell proliferation and p70(s6k) activity, and salicylate inhibits cell proliferation, we examined whether salicylate affects p70(s6k) activity. We find that salicylate potently inhibits p70(s6k) activation and phosphorylation in a p38 MAPK-independent manner. Interestingly, low salicylate concentrations (/=5 mm) are required to block p70(s6k) activation by epidermal growth factor + insulin-like growth factor-1. These data suggest that salicylate may selectively inhibit p70(s6k) activation in response to specific stimuli. Inhibition of p70(s6k) by salicylate occurs within 5 min, is independent of the phosphatidylinositol 3-kinase pathway, and is associated with dephosphorylation of p70(s6k) on its major rapamycin-sensitive site, Thr(389). A rapamycin-resistant mutant of p70(s6k) is resistant to salicylate-induced Thr(389) dephosphorylation.

NF-kappa B inhibits glucocorticoid and cAMP-mediated expression of the phosphoenolpyruvate carboxykinase gene.

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated by a variety of agents. Glucocorticoids, retinoic acid, and glucagon (via its second messenger, cAMP) stimulate PEPCK gene transcription, whereas insulin, phorbol esters, cytokines, and oxidative stress have an opposing effect. Stimulation of PEPCK gene expression has been extensively studied, and a number of important DNA elements and binding proteins that regulate the transcription of this gene have been identified. However, the mechanisms utilized to turn off expression of this gene are not well-defined. Many of the negative regulators of PEPCK gene transcription also stimulate the nuclear localization and activation of the transcription factor NF-kappaB, so we hypothesized that this factor could be involved in the repression of PEPCK gene expression. We find that the p65 subunit of NF-kappaB represses the increase of PEPCK gene transcription mediated by glucocorticoids and cAMP in a concentration-dependent manner. The mutation of an NF-kappaB binding element identified in the PEPCK gene promoter fails to abrogate this repression. Further analysis suggests that p65 represses PEPCK gene transcription through a protein.protein interaction with the coactivator, CREB binding protein.

Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin. The role of winged helix/forkhead proteins.

Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provided compelling evidence that HNF3 proteins, per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein.

Transducin-like enhancer of split proteins, the human homologs of Drosophila groucho, interact with hepatic nuclear factor 3beta.

Members of the hepatic nuclear factor 3 (HNF3) family, including HNF3alpha, HNF3beta, and HNF3gamma, play important roles in embryonic development, the establishment of tissue-specific gene expression, and the regulation of gene expression in differentiated tissues. We found, using the glutathione S-transferase pull-down method, that the transducin-like Enhancer of split (TLE) proteins, which are the human homologs of Drosophila Groucho, directly associate with HNF3beta. Conserved region II of HNF3beta (amino acids 361-388) is responsible for the interaction with TLE1. A mammalian two-hybrid assay was used to confirm that this interaction occurs in vivo. Overexpression of TLE1 in HepG2 and HeLa cells decreases transactivation mediated through the C-terminal domain of HNF3beta, and Grg5, a naturally occurring dominant negative form of Groucho/TLE, also increases the transcriptional activity of this region of HNF3. These results lead us to suggest that TLE proteins could influence the expression of mammalian genes regulated by HNF3.

Activation of the ras mitogen-activated protein kinase-ribosomal protein kinase pathway is not required for the repression of phosphoenolpyruvate carboxykinase gene transcription by insulin.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the first committed step in hepatic gluconeogenesis. Glucagon and glucocorticoids stimulate PEPCK gene transcription, whereas insulin has a dominant inhibitory effect. We have shown that inhibitors of 1-phosphatidylinositol 3-kinase (PI 3-kinase) block this action of insulin. In contrast, three distinct agents, all of which prevent activation of p42/p44 mitogen-activated protein (MAP) kinase, have no effect on the regulation of PEPCK transcription by insulin. However, a subsequent report has suggested that this pathway is involved in the inhibition of cAMP-induced PEPCK gene transcription by insulin. To address these conflicting data, we re-examined the Ras MAP kinase pathway, not only with respect to regulation of PEPCK gene transcription, but also for regulation of PI 3-kinase and p42/p44 MAP kinase. Overexpression of constitutively active Ras (V61) (or Raf-1 (RafCAAX)) partially represses PEPCK transcription in hepatoma cells. However, an inhibitor of MAP kinase kinase blocks this action of RafCAAX but has no effect on regulation of PEPCK gene transcription by insulin. Second, the action of a dominant negative Ras (N17Ras) on PEPCK gene transcription correlates more closely with the inhibition of PI 3-kinase than with the inhibition of p42/p44 MAP kinase. Third, insulin cannot activate p42/p44 MAP kinase in the presence of cAMP even though cAMP-induced PEPCK gene transcription is inhibited by insulin. This data confirms that the Ras MAP kinase pathway is not required for the regulation of PEPCK gene transcription by insulin and demonstrates the importance of employing multiple techniques when investigating the function of signaling pathways.