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Induction of differentiation in v-Ha-ras-transformed MDCK cells by prostaglandin E2 and 8-bromo-cyclic AMP is
1Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Abstract:
We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells.
Insights
Prostaglandin E2 (PGE2) and 8-bromo-cyclic AMP (8-Br-cAMP) induce differentiation in Ha-ras-transformed MDCK cells by decreasing inositol 1,4,5-trisphosphate (IP3) levels, suggesting a role for the inositol phosphate signaling pathway.
Area of Science:
- Cell Biology
- Signal Transduction
- Molecular Endocrinology
Background:
- Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells exhibit altered signaling pathways compared to parental cells.
- Understanding the mechanisms of differentiation induction is crucial for cellular research.
Purpose of the Study:
- To investigate the signal transduction mechanisms involved in glucagon responsiveness induction by PGE2 and 8-Br-cAMP in Ha-ras-transformed MDCK cells.
- To elucidate the role of inositol 1,4,5-trisphosphate (IP3) and cyclic AMP (cAMP) in this differentiation process.
Main Methods:
- Utilized Ha-ras-transformed MDCK cells as a model system.
- Measured steady-state levels of intracellular cAMP and IP3.
- Administered PGE2 and 8-Br-cAMP and monitored changes over time.
- Assessed the effects of phorbol ester and serum on differentiation induction.
Main Results:
- Transformed MDCK cells showed higher basal IP3 levels but similar cAMP levels compared to parental cells.
- PGE2 and 8-Br-cAMP treatment led to increased cAMP and decreased IP3 levels in a dose- and time-dependent manner.
- A lag period of 8-16 hours was observed between cAMP elevation and IP3 reduction, followed by a 2-day lag before differentiation induction.
- Inhibition of IP3 reduction and differentiation induction by phorbol ester or serum suggested a causal link.
Conclusions:
- A decrease in IP3 levels is likely causally involved in the induction of differentiation in transformed MDCK cells.
- Cyclic AMP may directly regulate the phospholipid signaling pathway.
- Perturbation of the inositol phosphate signaling pathway is implicated in differentiation induction by PGE2 and 8-Br-cAMP.