Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells

Previous work from our laboratory demonstrated that 1,25(OH)(2)D-3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+](i) subsequently increased due to IP3-mediatcd release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)(2)D-3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)(2)D-3 to colonocyte BLM. Recently, we found that 1,25(OH)(2)D-3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+](i) and DAG, two isoforms of protein kinase C (PKC alpha and PKC beta 2), but not other isoforms were activated by 1,25(OH)(2)D-3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta 2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)(2)D-3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKC alpha showed increased 1,25(OH)(2)D-3-stimulated PLD activation, whereas transfectants with antisense PKC alpha had an attenuated response. In addition, 1,25(OH)(2)D-3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/c-src/PKC pathway. (C) 1999 Elsevier Science Inc. All rights reserved.