The overall objective of this project is the determination of the role of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] on energy metabolism in the monocyte-macrophage system in particular and in bone and intestine in general. Studies suggest that 1,25(OH)2D3 has a paracrine and/or autocrine role in monocytic cells. During differentiation to macrophages, monocytes develop the capability to express and metabolize 1,25(OH)2D3. This is associated with the specific and significant induction of creatine kinase-B (CK-B) expression to meet the increased energy demands of phagocytes. U937D cells, a human monocytic cell line, can be induced to differentiate to macrophage-like cells by 1,25(OH)2D3. Our preliminary data indicate that CK-B expression in U937D cells is stringently regulated, predominantly by translational repression which involves the interaction of a cis-acting element in the highly-conserved 3' untranslated region of the CK-B mRNA with a trans-acting repressor (Ch'ng et.al., Science 1990,248,1003-1006). This repression is relieved following 1,25(OH)2D3-induced differentiation.
Our specific aims are to determine the mechanisms by which 1,25(OH)2D3 modulate the expression of CK-B in U937D cells and their role in the changes during differentiation in this and other 1,25(OH)2D3-modulated systems. Studies will be directed at defining the cis-acting element and purifying the repressor protein by affinity chromatography. RNA mobility-shift assays and SDS- PAGE will be used to monitor the purification process. The repressor cDNA will be cloned by screening a lambda gt11 library constructed from U937D cells with degenerate oligonucleotide probes whose sequences are derived from partial amino acid sequencing of the purified protein. An alternative would be to screen the library with polyclonal antibodies to the repressor protein. Detailed studies can then be conducted on the effect of 1,25(OH)2D3 on CK-B mRNA abundance, CK-B mRNA-repressor interaction, and repressor mRNA and protein abundance in the monocyte/macrophage system in particular and in bone and intestine in general. The significance of the 1,25(OH)2D3-modulated energy enhancement on changes during differentiation will be studied by inhibiting CK-B induction, with antisense RNA (Ch'ng et.al. Proc. Natl. Acad. Sci. U.S.A. 1989; 86:10006-10010). This project will provide further knowledge on the physiological role of 1,25(OH)2D3 in the immune system, bone and intestine.
| Ch'ng, J L; Ibrahim, B (1994) Transcriptional and posttranscriptional mechanisms modulate creatine kinase expression during differentiation of osteoblastic cells. J Biol Chem 269:2336-41 |
