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)

Ligands that activate protein kinase-C differ in their ability to regulate basic fibroblast growth factor and insulin-like growth factor-I messenger ribonucleic acid levels.

In this study, rat dermal fibroblasts were used as a model system to examine the ability of ligands that are known to activate protein kinase-C to regulate the levels of the mRNAs encoding basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I), two growth factors that are thought to be important in processes such as tissue repair and regeneration and wound healing. Treatment of fibroblasts with the phorbol ester phorbol 12-myristate 13-acetate (PMA), thrombin, bradykinin, serotonin, angiotensin-II, or bombesin increased protein kinase-C activity to a similar degree. Treatment of fibroblasts with 1 microM serotonin transiently increased bFGF mRNA levels about 3-fold compared to the level in control cells maintained in serum-free medium with 0.25% BSA and decreased IGF-I mRNA levels by approximately 50% compared to the level in control cells. This is similar to the previously described changes induced by bradykinin in these cells, but different from the more marked and sustained changes induced by thrombin and PMA. In contrast, angiotensin-II and bombesin had no effect on bFGF or IGF-I mRNA levels. The effects of serotonin, bradykinin, and PMA on bFGF and IGF-I mRNA levels were abrogated by preincubation of cells in 250 nM PMA to down-regulate protein kinase-C. In contrast, the effect of thrombin on bFGF mRNA levels was only partially inhibited by down-regulation of protein kinase-C, while its effect on IGF-I mRNA levels was unaffected. The activation of signaling pathways by the different ligands was further investigated to begin to determine the mechanism for the differences in the effects of thrombin vs. serotonin and bradykinin and in the effects of these three ligands vs. angiotensin-II and bombesin. All of the ligands activated phospholipase-D to a similar degree, suggesting that activation of this enzyme was not responsible for the differential effects of the ligands. In contrast, thrombin, serotonin, and bradykinin had marked effects on the hydrolysis of phosphatidylcholine and phosphatidylinositol, whereas bombesin and angiotensin-II had a small effect on phosphatidylcholine hydrolysis and no effect on phosphatidylinositol hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular calcium regulates insulin-like growth factor-I messenger ribonucleic acid levels.

Insulin-like growth factor-I (IGF-I) is involved in repair and regeneration in tissues in which non-GH-mediated regulation of its production has been shown to be important. We have investigated the effects of a second messenger signaling pathway, intracellular calcium, on IGF-I mRNA levels in cultured rat dermal fibroblasts using a RNase protection assay. Intracellular calcium concentrations ([Ca2+]i) were increased using either the calcium ionophore A23187 or thapsigargin. The ability of these agents to increase [Ca2+]i was confirmed by spectrofluorimetry, using fluo-3 as the Ca2+ indicator. Treatment of cells in serum-free medium and 0.25% BSA [Minimum Essential Medium (MEM) + BSA] with 500 nM A23187 or 1 micron thapsigargin decreased IGF-I mRNA levels in a time-responsive manner over 4-8 h. A23187 and thapsigargin also decreased IGF-I mRNA levels to 36% and 47% of control levels, respectively, in a dose-responsive fashion. Basic fibroblast growth factor mRNA levels, which were simultaneously determined, were either unchanged or increased in cells treated with thapsigargin or A23187. Consistent with the change in IGF-I mRNA levels, immunoreactive IGF-I levels in medium conditioned for 48 h by A23187 or thapsigargin decreased to 25% and 14%, respectively, of control levels in cells maintained in MEM + BSA. To determine the role of protein synthesis in the effects of A23187 and thapsigargin, cells were treated with these agents in the presence or absence of cycloheximide. Cycloheximide had no effect on the decrease in IGF-I mRNA levels mediated by thapsigargin, but significantly attenuated the response to A23187. Given these differences in the role of protein synthesis in and the time course of the effects of A23187 and thapsigargin on IGF-I mRNA levels, additivity experiments were performed. Treatment of cells with the combination of A23187 and thapsigargin resulted in IGF-I mRNA levels that were approximately 70% of the levels present in cells treated with either agent alone. These data are consistent with a small additive effect, but suggest that the majority of the effect of A23187 and thapsigargin occurs via the same final pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a rat insulin-like growth factor I gene promoter.

Rat IGF-I mRNAs contain one of two alternative 5'-untranslated regions which are encoded by alternative exons (exons 1 and 2) and whose expression is controlled by alternative promoter elements. We investigated the ability of fragments of DNA which contain exon 1 and its 5'-flanking region to regulate transcription of a luciferase reporter gene in transient transfection assays. Maximal promoter activity was obtained with a construct which contained 412 bp of 5'-flanking region, while constructs which contained 1120 and 1690 bp of 5'-flanking region induced approximately 50% less enzymatic activity. Mapping of transcription start sites by RNase protection assay demonstrated that native start sites were used by these constructs, although the relative use of different start sites was different from start site usage by the endogenous gene. These data demonstrate that the 5'-flanking region of exon 1 is capable of regulating transcription of IGF-I mRNAs.

Inhibition of T4 polynucleotide kinase activity by phosphorothioate and chimeric oligodeoxynucleotides.

Whole and partially modified phosphorothioate oligodeoxynucleotides (ODN) were found to directly inhibit T4 polynucleotide kinase (PNK) activity, while phosphodiester ODN showed no detectable inhibition. This inhibition was found to be length dependent, as demonstrated by a 28-mer phosphorothioate ODN with an IC50 of 12 nM, and an 8-mer phosphorothioate ODN with an IC50 of 27,000 nM. Inhibition depended on the number and type of modified internucleotide linkages: a 20-mer phosphorothioate ODN had an IC50 of 21 nM, while a chimeric ODN with seven phosphorothioate linkages and an identical sequence showed no inhibition. On the other hand, the same sequence as a chimeric phosphorodithioate ODN (with seven dithioate linkages) had an IC50 of 580 nM. Four different chimeric phosphorodithioate ODN showed markedly different potencies of inhibition, suggesting that inhibition of PNK activity can be sequence specific.