Transforming growth factor β and epidermal growth factor alter calcium influx and phosphatidylinositol turnover in Rat-1 fibroblasts

Transforming growth factor type β (TGFβ) alters the cellular response to epidermal growth factor (EGF) for a variety of processes ranging from early transport activities and gene transcription to mitogenesis. In order to test the hypothesis that altered signal transduction mechanisms may mediate both the transforming effects of TGFβ and the modulation of EGF-stimulated processes by TGFβ, we have examined second messenger levels in response to growth factor treatment. The addition of EGF or prolonged treatment with TGFβ increased the rate of ⁴⁵Ca influx in serum-deprived, confluent Rat-1 cells, while the addition of EGF to TGFβ-pretreated cells produced an additive increase in Ca²⁺ influx. The stimulation of Ca²⁺ influx by TGFβ was only observed at incubation times greater than 1 h and was inhibited by inclusion of actinomycin D, suggesting that a newly transcribed gene product was required for the observed response to TGFβ. Both EGF and TGFβ displayed similar time and concentration dependencies for stimulation of Ca²⁺ influx and for accumulation of inositol trisphosphate (IP₃). The increase in IP₃ accumulation in response to either EGF or TGFβ required the presence of extracellular Ca²⁺, and the observed concentration dependencies were similar for the stimulation of phosphatidylinositol turnover and Ca²⁺ influx. The EGF- and TGFβ-stimulated increased in Ca²⁺ influx could be blocked by cobalt, cadmium, and [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, but not by specific Ca²⁺ channel blockers such as nifedipine or verapamil, suggesting that these growth factors do not act via L-type voltage-sensitive calcium channels. Those calcium blockers which inhibited Ca²⁺ influx also inhibited inositol phosphate release. These data, taken together, indicate that Ca²⁺ influx and inositol phosphate release are coupled in Rat-1 cells and suggest that influx of Ca²⁺ from the extracellular medium is responsible for the changes in IP₃ accumulation observed in response to both EGF and TGFβ.