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Transforming growth factor beta and epidermal growth factor alter calcium influx and phosphatidylinositol turnover in
L L Muldoon1, K D Rodland, B E Magun
1Oregon Health Sciences University, Portland 97201.
Abstract:
Transforming growth factor type beta (TGF beta) 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 TGF beta and the modulation of EGF-stimulated processes by TGF beta, we have examined second messenger levels in response to growth factor treatment. The addition of EGF or prolonged treatment with TGF beta increased the rate of 45Ca influx in serum-deprived, confluent Rat-1 cells, while the addition of EGF to TGF beta-pretreated cells produced an additive increase in Ca2+ influx. The stimulation of Ca2+ influx by TGF beta 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 TGF beta. Both EGF and TGF beta 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 TGF beta 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 TGF beta-stimulated increases 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 TGF beta.
Insights
Transforming growth factor beta (TGF-β) and epidermal growth factor (EGF) both increase calcium (Ca2+) influx and inositol trisphosphate (IP3) levels in Rat-1 cells. These processes are coupled, with extracellular Ca2+ influx driving IP3 accumulation.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Calcium Homeostasis
Background:
- Transforming growth factor beta (TGF-β) modulates cellular responses to epidermal growth factor (EGF).
- Signal transduction mechanisms are hypothesized to mediate TGF-β's transforming effects and its modulation of EGF-stimulated processes.
- Investigating second messenger levels provides insight into these growth factor-mediated cellular changes.
Purpose of the Study:
- To test the hypothesis that altered signal transduction mechanisms mediate TGF-β's effects and its modulation of EGF-stimulated processes.
- To examine second messenger levels, specifically calcium (Ca2+) influx and inositol trisphosphate (IP3) accumulation, in response to EGF and TGF-β treatment in Rat-1 cells.
Main Methods:
- Serum-deprived, confluent Rat-1 cells were treated with EGF and/or TGF-β.
- Calcium-45 (45Ca2+) influx rates were measured.
- Inositol trisphosphate (IP3) accumulation was quantified.
- The effects of actinomycin D, cobalt, cadmium, and specific calcium channel blockers (nifedipine, verapamil) on Ca2+ influx and IP3 release were assessed.
Main Results:
- Both EGF and TGF-β individually increased 45Ca2+ influx and IP3 accumulation in Rat-1 cells.
- TGF-β-induced Ca2+ influx required prolonged incubation (>1 h) and was inhibited by actinomycin D, suggesting a requirement for new gene transcription.
- EGF and TGF-β exhibited similar time and concentration dependencies for stimulating Ca2+ influx and IP3 accumulation.
- Extracellular Ca2+ was required for IP3 accumulation in response to both growth factors.
- Inhibition of Ca2+ influx by blockers (cobalt, cadmium, EDTA) also inhibited IP3 release, while specific calcium channel blockers like nifedipine and verapamil did not.
- These findings suggest that Ca2+ influx and IP3 release are coupled processes in Rat-1 cells.
Conclusions:
- Both EGF and TGF-β stimulate Ca2+ influx and IP3 release through mechanisms that appear to be coupled in Rat-1 cells.
- Extracellular Ca2+ influx is a key factor responsible for the observed increase in IP3 accumulation induced by both EGF and TGF-β.
- The data suggest that these growth factors do not primarily act via L-type voltage-sensitive calcium channels but rather through a distinct calcium influx pathway coupled to inositol phosphate metabolism.