GSBS Student Publications

Title

Control of 1,25-dihydroxyvitamin D3 receptor-mediated enhancement of osteocalcin gene transcription: effects of perturbing phosphorylation pathways by okadaic acid and staurosporine

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Cell Biology; Department of Endocrinology

Date

12-1-1995

Document Type

Article

Medical Subject Headings

Alkaloids; Animals; Enzyme Inhibitors; Ethers, Cyclic; Okadaic Acid; Osteocalcin; Phosphoprotein Phosphatases; Phosphorylation; Promoter Regions (Genetics); Protein Kinase C; Rats; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoid X Receptors; Staurosporine; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

The 1,25-dihydroxyvitamin D3 (vitamin D) receptor (VDR) is a key trans-activating protein that mediates calcium regulation as well as cellular proliferation and differentiation. Phosphorylation of the VDR contributes significantly to its functional activity, but the specific mechanisms that mediate this regulation are not well understood. Phosphorylation may influence DNA binding, ligand binding, and protein-protein interactions, including heterodimerization and/or transactivation functions. We used a protein kinase C inhibitor, staurosporine (ST), and an inhibitor of serine-threonine phosphatases, okadaic acid (OA), to elucidate the contribution of VDR phosphorylation to vitamin D-mediated transcription of the osteocalcin (OC) gene. Vitamin D-induced transcription was assayed in transfected ROS 17/2.8 osteosarcoma cells using chloraminphenicol acetyltransferase constructs containing the vitamin D-responsive element (VDRE) at its native locus in the rat OC promoter as well as fused to a heterologous promoter. Both ST and OA inhibit VDRE-mediated and vitamin D-dependent enhancement of OC gene transcription as well as OC biosynthesis, as assessed by RIAs. Results from gel mobility shift and Western blot analyses using nuclear proteins from ROS 17/2.8 cells show that binding of the VDR-retinoid-X receptor heterodimer complex to the OC VDRE is not inhibited in the presence of ST. In contrast, OA does inhibit the formation of complexes interacting with both the OC and osteopontin VDREs; immunoprecipitation studies using 32P-labeled ROS 17/2.8 cells reveal that OA treatment result in ligand-independent hyperphosphorylation of the VDR. Our results suggest that two distinct phosphorylation events modulate rat VDR function. One event is related to transactivation, and the other is also critical to the VDRE-binding activity of VDR-retinoid X receptor-DNA complexes with consequential effects on transactivation.

Rights and Permissions

Citation: Endocrinology. 1995 Dec;136(12):5685-93.

Related Resources

Link to article in PubMed

Journal Title

Endocrinology

PubMed ID

7588324