Understanding 1,25(OH)2D3's role in controlling osteoclastic bone resorption will aid in developing clinical approaches to osteoporosis. This proposal will study 1,25(OH)2D3's modulation of two genes critical to osteoclast differentiation and function. We have previously shown that 1,25(OH)2D3 decreases expression of the macrophage colony stimulating factor receptor (MCSFR) while increasing expression of osteoclast characteristics in the HL-60 cell, which serves as a model for the early osteoclast precursor. We postulate that 1,25(OH)2D3's potentiation of osteoclast differentiation involves downregulating the ability of the precursor cell to respond to MCSF: precursor cells with decreased MCSFR would have an increased probability of entering the osteoclast pathway and a decreased probability of MCSF induced entry into the macrophage pathway. We will first show that 1,25(OH)2D3 decreases the expression of MCSFR in marrow cells as they enter the osteoclast pathway by measuring (125)I-MCSF binding and in situ hybridization of MCSFR mRNA in individual osteoclasts and monocytes in culture. The molecular mechanism by which 1,25(OH)2D3 decreases expression of the MCSFR gene will be defined by nuclear run on and study of MCSFR transcript elongation in HL-60 cells. 1,25(OH)2D3 regulation of osteoclast function will be investigated in the second specific aim: the applicant has previously shown in HL-60 cells that 1,25(OH)2D3 and protein kinase C synergistically increase the expression of carbonic anhydrase Il. Regulation of CA II may be a critical means of controlling bone resorption as it is an enzyme necessary for osteoclast function. We propose to study the ability of 1,25(OH)2D3 and PKC to regulate CA Il in mature cultured osteoclasts using reverse transcription polymerase chain reactions to quantify changes in CA Il mRNA. Finally, the interaction of 1,25(OH)2D3 and PKC to enhance CA Il gene transcription will be studied in the HL-60 cell line by nuclear run on. These studies should help eluciate the mechanisms of 1,25(OH)2D3 action in osteoclasts.
| Biskobing, D M; Fan, D (2000) Acid pH increases carbonic anhydrase II and calcitonin receptor mRNA expression in mature osteoclasts. Calcif Tissue Int 67:178-83 |
| Biskobing, D M; Fan, D; Fan, X et al. (1997) Induction of carbonic anhydrase II expression in osteoclast progenitors requires physical contact with stromal cells. Endocrinology 138:4852-7 |
| Biskobing, D M; Fan, D; Rubin, J (1997) c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells. Calcif Tissue Int 61:205-9 |
| Fan, X; Biskobing, D M; Bain, S et al. (1996) Ketoconazole and phorbol myristate acetate regulate osteoclast precursor fusion in primary murine marrow culture. J Bone Miner Res 11:1274-80 |
