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 muM 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. In protein kinase-C-down-regulated cells, treatment with A23187 or thapsigargin decreased IGF-I mRNA levels to approximately 30% of levels in protein kinase-C-down-regulated cells reexposed to MEM + BSA. These data demonstrate that increased [Ca2+]i decreases IGF-I mRNA levels in cultured fibroblasts independently of the activation of protein kinase-C. In summary, alterations in [Ca2+]i may be important in vivo in the regulation of IGF-I production.