Cross talk between genomic and membrane-initiated actions of vitamin D in Caco-2 cells

Kenneth William Hance, Purdue University

Abstract

Vitamin D stimulates intestinal calcium transport through a nuclear vitamin D receptor (nVDR) dependent genomic mechanism. Additional evidence indicates that vitamin D may activate membrane-initiated signal transduction pathways by interacting with a membrane-bound vitamin D receptor (mVDR). We hypothesize that vitamin D-mediated calcium transport and gene expression are dependent upon cross talk between these pathways. We found that pharmacological suppression of protein kinase activity significantly reduced vitamin D-stimulated calcium transport and gene expression in the human intestinal cell line, Caco-2. Conversely, we observed that activation of protein kinase activity with the phorbol ester, PMA, synergistically activated vitamin D-induced gene expression. Metabolic labeling studies in ROS 17/2.8 and Caco-2 cells demonstrated that vitamin D increases nVDR phosphorylation in both cell types. This effect was rapid and could be suppressed by inhibition of protein kinase activity. These studies suggest that rapid, membrane-initiated signaling pathways modulate the genomic actions of vitamin D through effects on nVDR level, receptor function, and phosphorylation status in Caco-2 cells. To identify a specific mechanism that may explain this cross-talk phenomenon, we used confocal microscopy to study effect of vitamin D on nuclear VDR translocation in Bbe cells transiently transfected with a green fluorescent protein-vitamin D receptor (GFP-VDR) chimeric expression vector. In both proliferating and differentiated cells, vitamin D induced a rapid translocation of the GFP-VDR to the nucleus. This suggests that the resistance to vitamin D-mediate gene expression previously observed in proliferating cells is not due to a defect in nuclear translocation. To determine whether cross talk can be explained by an effect of signal transduction on vitamin D-induced nuclear VDR translocation, we measured GFP-VDR redistribution kinetics in response to vitamin D in the presence or absence of pharmacological inhibitors of protein kinase activity. The vehicle used in the inhibitor studies, DMSO, attenuated the rapid translocation of the GFP-VDR rendering definitive conclusions regarding the role of signal transduction in modulating hormone-induced receptor translocation difficult. Additional research designed to elucidate the molecular mechanisms that mediate cross talk will contribute to the development a more comprehensive understanding of the biological effects of vitamin D on its target tissues.

Degree

Ph.D.

Advisors

Fleet, Purdue University.

Subject Area

Nutrition

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