Glucose starvation reduces IGF-I mRNA in tumor cells: evidence for an effect on mRNA stability

The purpose of this study was to characterize the mechanisms by which glucose regulates IGF-I gene expression in rat C6 glioma cells and in rat GH3 pituitary adenoma cells. Glucose starvation for periods of 12 to 48 h decreased IGF-I mRNA levels. In contrast, there was no stimulation of IGF-I mRNA by medium glucose between 1 and 25 mM over a 24-h period. Studies with hexoses and glycolytic metabolites suggested that glucose metabolism was required to maintain IGF-I mRNA. Glucose starvation lowered IGF-I mRNA half-life in both C6 and GH3 cells. Protein synthesis inhibition lowered IGF-I mRNA by about 20% in glucose-fed C6 and GH3 cells, while potently increasing IGF-I mRNA in glucose-starved C6 cells and not altering IGF-I mRNA in glucose-starved GH3 cells. Our results suggest that in these tumor cells, IGF-I mRNA stability is reduced by glucose starvation, secondary to a deficiency in intracellular glucose metabolism. Ongoing protein synthesis is not required for this mRNA de-stabilizing effect in GH3 cells. Rather, in glucose-starved C6 cells, decreased IGF-I mRNA stability may result from the action of a labile protein.     

Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels.

Fibroblasts represent one of the in vivo sites of insulin-like growth factor-I (IGF-I) production. In this study rat dermal fibroblasts in culture were used as a model system to assess the effect of activation of protein kinase-C on the levels of the mRNAs encoding IGF-I and another growth factor, basic fibroblast growth factor (bFGF). IGF-I and bFGF mRNA levels were determined using a solution hybridization/RNase protection assay. Treatment of cells in serum-free medium containing 0.25% BSA (MEM + BSA) with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) decreased IGF-I and increased bFGF mRNA levels in a time- and dose-dependent fashion. The peak effect of 100 nM PMA on IGF-I mRNA levels occurred at 9 h, whereas the peak effect on bFGF mRNA levels occurred after 3 h of incubation. In dose-response studies, half-maximal inhibition of IGF-I mRNA levels was achieved with approximately 0.08 nM PMA, while half-maximal stimulation of bFGF mRNA levels was achieved with approximately 3 nM PMA. Inhibition of protein synthesis with cycloheximide abrogated the effect of PMA on bFGF mRNA levels, but only partially inhibited the effect of PMA on IGF-I mRNA levels. Studies employing sphingosine or staurosporine to inhibit protein kinase-C or preincubation in high doses of PMA to down-regulate protein kinase-C suggested that the effect of PMA on IGF-I and bFGF mRNA levels was mediated by activation of protein kinase-C, although both staurosporine and sphingosine had independent effects on the levels of these mRNAs and down-regulation of protein kinase-C had a sustained effect on IGF-I mRNA levels. Ligands known to activate protein kinase-C were then tested. Treatment of cells with 100 micrograms/ml of the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol decreased IGF-I mRNA levels to 25% and increased bFGF mRNA levels to 520% of the level present in cells maintained in MEM + BSA. Treatment of cells with thrombin or bradykinin also decreased IGF-I mRNA levels and increased bFGF mRNA levels, but whereas the effect of thrombin on IGF-I mRNA levels was marked, the effect of bradykinin was minimal, and whereas the effect of thrombin on bFGF mRNA levels was sustained, the effect of bradykinin was transient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulated expression of sterol carrier protein 2 in the ovary: a key role for cyclic AMP.

Sterol carrier protein 2 (SCP2) is believed to play an important role in the intracellular movement of cholesterol in steroidogenic cells. We examined the distribution of SCP2 gene expression in the rat ovary and the role of gonadotropins and cyclic AMP in the regulation of SCP2 mRNA levels. In situ hybridization revealed that the most steroidogenically active ovarian compartments (e.g., corpora lutea and theca cells) contain significant amounts of SCP2 mRNA whereas granulosa cells have modest levels. Gonadotropins, which promote follicular growth and luteinization, increased the ovarian content of SCP2 mRNA as assessed by Northern blotting along with increases in cytochrome P450scc mRNA. Using steroidogenic transformed rat granulosa cells (Grs-21), a cyclic AMP analogue (8-Br-cAMP) was found to increase SCP2 mRNA and protein levels within 24 h of treatment. P450scc mRNA was also induced whereas actin mRNA levels were not affected. The 8-Br-cAMP stimulation of SCP2 mRNA accumulation was completely inhibited by actinomycin D and cycloheximide. The cyclic AMP analogue also increased SCP2 mRNA levels in a non-steroid hormone producing transformed rat granulosa cell line Gs-8. We conclude that SCP2 gene expression in the ovary is correlated with the state of differentiation of granulosa cells. Gonadotropic hormones which stimulate luteinization of the cells increase SCP2 gene expression. These actions of gonadotropins appear to be mediated at least in part by cyclic AMP through a mechanism requiring ongoing RNA and protein synthesis. However, SCP2 gene expression is not obligatorily coupled to steroidogenic activity, as cyclic AMP analogues can increase SCP2 mRNA in a line of transformed ovarian granulosa cells incapable of synthesizing hormones.     

IGF-I mRNA levels in bovine satellite cell cultures: effects of fusion and anabolic steroid treatment

Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17beta-estradiol (E(2)) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-alpha, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E(2) or trenbolone ranging from 0.001 to 10 nM. IGF-I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9-times control values, P < 0.02) and at 0.1, 1, and 10 nM E(2) (2.9-, 3.5-, and 3.5-times control values, respectively, P < 0.0001). Additionally both 1 and 10 nM trenbolone increased IGF-I mRNA levels to 1.7-times control values (P < 0.02). ER-alpha mRNA was detectable in BSC cultures, and levels were increased (2.3-times control levels, P < 0.001) in cultures treated with 0.001 nM E(2) but not in cultures treated with higher concentrations of E(2). Androgen receptor mRNA levels also were increased (1.5-times control levels, P < 0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone. Levels of IGFBP-3 were increased (1.4-times control values, P < 0.02) by treatment with 0.001 nM E(2) but not by treatment with high concentrations of E(2). Myostatin mRNA levels were not affected by any concentration of either of the steroids. Although, levels of IGF-I mRNA were 10-times greater (P < 0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 h with 10 nM E(2) increased IGF-I mRNA levels (2.5-times control levels, P < 0.02). Both E(2) and trenbolone increased (3)H-thymidine incorporation rate (1.5-times control levels, P < 0.001) in BSC cultures in media containing serum from which IGFBP-3 had been removed by anti-IGFBP-3 affinity chromatography. In summary, treatment of BSC cultures with either E(2) or trenbolone increased IGF-I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture.     

Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.