Insulin-like growth factor-I activates extracellularly regulated kinase to regulate the p450 side-chain cleavage insulin-like response element in granulosa cells.

IGF regulates steroidogenesis in granulosa cells through expression of the cytochrome P450 side-chain cleavage enzyme (P450scc) (CYP11A1), the rate-limiting enzyme in this biosynthetic process. We showed previously that the polypyrimidine tract-binding protein-associated splicing factor (PSF) acts as a repressor, whereas Sp1 is an activator, of P450 gene expression. The aim of the present study was to investigate IGF-stimulated ERK signaling regulating P450scc gene expression in the immortalized porcine granulosa cell line JC-410. We used a reporter gene under control of the IGF response element from the P450scc promoter. Inhibition of ERK phosphorylation with U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] blocked IGF-I induction of IGF response element reporter gene activity. Western blotting revealed that IGF-I treatment resulted in phosphorylation of ERK that was specifically inhibited by U0126. ERK activation led to phosphorylation of T739 (an ERK site) on Sp1 that was diminished by U0126 or overexpression of PSF. Coimmunoprecipitation and Western blotting of nuclear extracts showed that phosphorylated ERK (pERK) bound PSF under basal conditions. IGF-I caused dissociation of pERK from PSF. Finally, chromatin immunoprecipitation analysis showed that PSF and Sp1 constitutively occupy the P450scc promoter independent of IGF-I treatment. These events provide a potential molecular mechanism for release of PSF repression of P450scc expression by dissociation of pERK and subsequent pERK-mediated phosphorylation of Sp1 to drive transcriptional induction of the P450scc gene in the absence of altered binding of PSF or Sp1 to the promoter. Understanding IGF-I regulation of these critical ovarian signaling pathways is the first step to delineating ovarian hyperstimulation syndromes such as polycystic ovarian syndrome.

Identification of ERK and JNK as signaling mediators on protein kinase C activation in cultured granulosa cells.

PKC signaling is critical for follicular development and the induction of ovulatory genes including Pgr, Prkg2, and Cyp11a1 (SCC). We investigated PKC signaling mechanisms in the JC-410 porcine granulosa cell line stably expressing an SCC-luciferase reporter gene containing 2kb of the porcine SCC promoter. Addition of phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, induced the promoter approximately 6-fold over the basal levels in 4h. This effect was predominantly mediated by the PKC beta and delta isoforms. PMA-mediated induction of the SCC promoter was sensitive to inhibition of ERK1/2 or JNK. Inhibition of p38 MAP kinase or Src tyrosine kinase did not alter the PMA-mediated inducibility of the promoter. SCC promoter induction in response to PMA treatment required basal EGF-receptor activity, but did not involve ectodomain shedding. Western blot analyses using phospho-specific antibodies showed that PMA treatment of JC-410 cells induced phosphorylation of MEK1/2, ERK1/2, and its downstream target p90 RSK at 15min. We also documented the rapid phosphorylation of JNK1/2 in response to PMA treatment. Phosphorylation of ERK and JNK was robust and sustained in contrast to activation of PKA and EGF-receptor signaling in these cells. Pretreatment of JC-410 granulosa cells with IGF-1 had a synergistic effect on PMA-mediated induction of the SCC promoter. We demonstrated the importance of PMA activation of ERK signaling and the synergism with IGF-1 by showing similar responses for Prkg2 expression in primary granulosa cells. In conclusion, our studies demonstrated PMA activation of ERK and JNK signaling which is relevant in the regulation of gene expression during follicular development, ovulation, and luteinization.

IGF-1 controls GLUT3 expression in muscle via the transcriptional factor Sp1.

Glucose transporter 3 (GLUT3), while first found in human fetal muscle, is predominantly expressed in brain and neural tissue. By several independent techniques we have previously shown that GLUT3 is expressed in human skeletal muscle cells. The structure of the human GLUT3 gene has not been previously reported nor has there been any evaluation of the 5'-untranslated region (UTR). To this end, we have cloned and sequenced the human GLUT3 gene. Insulin-like growth factor-1 (IGF-1) increased endogenous Glut3 protein in cultured L6 myotubes, and similarly stimulated luciferase activity in a construct of the human GLUT3 5'-UTR linked to a luciferase reporter gene. Actinomycin D, an inhibitor of mRNA synthesis, prevented IGF-1 stimulation of Glut3 protein. Transfection of L6 cells with Sp1 increased Glut3 and augmented IGF-1 stimulation of Glut3 expression. Knockdown of Glut3 expression in cultured L6 muscle cells using small interference RNA (siRNA) specific for Glut3 significantly reduced myocyte glucose uptake. DNAse footprinting and gel shift assays showed Sp1 specifically bound to the human GLUT3 5'-UTR. Substitution mutants of the human GLUT3 5'-UTR luciferase construct indicated that only one of three Sp1 site clusters was involved in IGF-1 action. These data, using both a human GLUT3 5'-UTR construct and L6 cells' endogenous promoter, suggest that IGF-1 plays a role in maintaining muscle GLUT3 expression and basal glucose uptake via the transcriptional factor Sp1.

Protein kinase Ciota enhances the transcriptional activity of the porcine P-450 side-chain cleavage insulin-like response element.

IGF-I enhances steroidogenesis in granulosa cells by stimulating the expression of the rate-limiting steroidogenic enzyme, cytochrome P-450 side-chain cleavage (P-450(scc)). This effect is mediated through an IGF response element (IGFRE) that binds polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) and Sp1. Sp1 is essential for activation of the IGFRE, and PSF functions as a repressor. We investigated mechanisms of modulation of the IGFRE by the atypical protein kinase C (PKC)iota in a porcine stable granulosa cell line, JC-410. PKCiota was found in nuclear extracts, and levels were increased by IGF-I after 24 and 48 h of treatment. Immunoprecipitation experiments demonstrated that PSF and PKCiota associated with each other in nuclear extracts from JC-410 cells. Transient transfection with expression plasmids of kinase-active and kinase-deficient PKCiota isoforms enhanced transcriptional activity of the IGFRE regardless of kinase catalytic activity. Depletion of PKCiota protein by small interfering RNA suppressed basal IGFRE activity but did not prevent IGF-I stimulation of the IGFRE. We conclude that PKCiota enhances transcriptional activity of the porcine P-450(scc) IGFRE independently of kinase activity by a mechanism involving protein-protein interaction with PSF.

PTB-associated splicing factor regulates growth factor-stimulated gene expression in mammalian cells.

An insulin-like growth factor I (IGF-I) response element (IGFRE) in the porcine P-450 cholesterol side-chain cleavage gene (P450scc) binds two transcription factors, Sp1 and polypyrimidine tract-binding protein-associated splicing factor (PSF). In this study, we investigated expression of these transcription factors in mouse Y1 adrenal cells, a cell line that does not increase P450scc expression in response to IGF-I. Western blot analysis showed a greater expression of PSF in Y1 cells when compared with a mouse fibroblast cell line (NWTb3) in which IGF-I stimulates the P450scc IGFRE. The two cell lines expressed Sp1 equally, and IGF-I did not increase expression of either transcription factor. Chromatin immunoprecipitation analysis with Y1 chromatin confirmed that PSF and Sp1 bound to the IGFRE. When increasing amounts of Sp1 were expressed in Y1 cells, the IGFRE became responsive to IGF-I. Moreover, a mutant oligonucleotide IGFRE reporter construct that lacks PSF binding was responsive to IGF-I. In conclusion, Y1 adrenal cells are a physiological example of PSF repression of growth factor-stimulated (IGF-I) gene expression (P450scc). The dynamic nature of this repression is consistent with PSF functioning as a regulator of growth factor-stimulated gene expression in mammalian cells.

NH2 terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element.

An insulin-like growth factor (IGF) I response element (IGFRE) in the porcine P-450 cholesterol side-chain cleavage gene (P450scc) regulates transcription through the binding of two proteins, Sp1 and polypyrimidine tract-binding protein-associated splicing factor (PSF). PSF is a component of spliceosomes and contains RNA-binding domains. In this study, we localized the NH2-terminal amino acid residues necessary for binding of PSF to the IGFRE. Three COOH-terminal truncated proteins (aa 304, 214, and 134) of PSF were designed to empirically partition the NH2-terminal region while excluding the RNA-binding domains. Southwestern analysis showed that only the largest expressed truncated protein, P3, strongly bound the porcine P450scc IGFRE. Truncated PSF protein expression in Y1 adrenal cells showed that P3 repressed transcriptional activity of the IGFRE similar to full-length PSF, whereas P2 (minimal binding to the IGFRE) had no effect. In conclusion, the NH2-terminal region of PSF contains the amino acid residues necessary for binding to the porcine P450scc IGFRE and repressing the transcriptional activity of the element.