The pleiotropic effects of insulin and insulin-like growth factor-I (IGF-I) are mediated via the intrinsic tyrosine kinase activity of their receptors. The potential role of insulin and IGF-I receptor tyrosine kinases in the bovine luteal cell was investigated in terms of autophosphorylation of the receptor and phosphorylation of endogenous and exogenous substrates. Insulin and IGF-I receptors were isolated by wheat germ agglutinin-agarose chromatography and immunoprecipitation with antiphosphotyrosine antibodies (alpha PY20). Insulin and IGF-I treatment of purified receptors or luteal cells in culture resulted in phosphorylation of a protein of 95,000 mol wt. This phosphoprotein was further identified as the autophosphorylated beta-subunit of the insulin/IGF-I receptor by immunoprecipitation with an anti-beta-subunit receptor antibody. The protein tyrosine kinase activity of the receptors was also stimulated after insulin or IGF-I treatment, resulting in a 3- to 4-fold increase in phosphorylation of a synthetic substrate poly(Glu4:Tyr1). Insulin and IGF-I treatment also increased (4.5-fold) phosphatidylinositol-3-kinase (PI-3-kinase) activity in alpha PY20 immunoprecipitates from whole cell lysates, suggesting that this enzyme was regulated by a receptor tyrosine kinase-mediated mechanism. The presence of PI-3-kinase in alpha PY20 immunoprecipitates was further confirmed by the sensitivity of the enzyme to detergents and by immunoblot analysis using a PI-3-kinase antibody. The actions of insulin and IGF-I on tyrosine kinase and PI-3-kinase activity were associated with increased protein and DNA synthesis. Insulin and IGF-I treatment of cultured luteal cells for 18 h increased [3H]thymidine incorporation (6- to 7-fold). The effects of insulin and IGF-I were optimal at 10 micrograms/ml and 50 ng/ml, respectively, and were not additive. The stimulatory effects of insulin and IGF-I on thymidine incorporation were blocked by genistein, a specific inhibitor of protein tyrosine kinase. These findings demonstrate a cascade of insulin and IGF-I receptor tyrosine kinase-mediated pathways in the bovine luteal cell, manifested as increased activity of signal transduction enzymes and increased DNA synthesis.
The rapid growth of the human fetal adrenal gland, which is primarily a reflection of the growth of the unique fetal zone, is regulated by ACTH acting indirectly to stimulate the expression of locally produced growth factors, of which IGF-II and bFGF appear to play key roles. Through most of gestation, the outer definitive zone appears to function as a reservoir of progenitor cells which move centripetally to populate the rest of the gland. At the end of pregnancy, the fetal zone undergoes senescence through an apoptotic process. Activin and TGF-beta are capable of inducing apoptosis in the fetal zone. Corticotropin-releasing hormone, which is produced by the placenta in markedly increased amounts at the end of gestation, may orchestrate a variety of processes, including direct stimulation of fetal adrenal steroidogenesis, culminating in the initiation of parturition.
CRH directly stimulates dehydroepiandrosterone sulfate (DHEAS) production in human fetal adrenal cells. In the human fetal and adult pituitary, CRH acts via protein kinase A (PKA). We determined the CRH signal transduction pathway in fetal adrenal cells, i.e. whether CRH modulates human fetal adrenal steroidogenesis via PKA and/or protein kinase C (PKC). In primary cultures, CRH increased inositol trisphosphate. After CRH treatment, inositol tris-, bis-, and monophosphates increased within 1 min, reaching maximal levels at 5 min. In contrast, PGF2alpha, known to act via PKC, induced a sustained response for up to 20 min. The response to CRH was dose dependent, maximal at 1 micromol/L at both 1 and 5 min. CRH increased DHEAS production, with a much lesser effect on cortisol. CRH did not stimulate inositol phospholipid in adult adrenal glands, suggesting that this pathway is unique to the fetal adrenal. CRH increased messenger ribonucleic acid encoding 17alpha-hydroxylase/17,20 lyase (P450c17), but not 3beta-hydroxysteroid dehydrogenase/delta(4-5) isomerase. However, 3betaHSD expression was stimulated by ACTH. PKC, but not PKA, inhibitors blocked CRH-stimulated P450c17 induction, whereas PKA inhibitors blocked ACTH-stimulated cortisol. Thus, CRH is coupled to the phospholipase C-inositol phosphate second messenger system and preferentially induces the expression of P450c17 and DHEAS, suggesting a unique role of CRH regulating human fetal adrenal function via PKC.
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