The objective of this proposal is to investigate the regulation of bone alkaline phosphatase (AP) by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the osteoblast at the transcriptional, translational, and posttranslational levels in phenotypically- distinct osteoblast-like rat and human osteogenic sarcoma cell lines and in fetal rat calvariae. It is well established that the osteoblast possesses specific, high- affinity receptors for 1,25(OH)2D3 and that the hormone acts to modulate the biosynthetic products of the osteoblast, such as type I collagen, osteocalcin, and alkaline phosphatase, at the level of transcription. Recently, phenotypically-distinct subclones of the osteoblast-like rat osteogenic sarcoma (ROS) 17/2.8 cell line have been isolated. 1,25(OH)2D3 stimulates AP activity and osteocalcin secretion in the G2 subclone (which exhibits low basal AP activity) by a genome-dependent mechanism. In contrast, 1,25(OH)2D3 inhibits alkaline phosphatase activity in the C12 subclone (which exhibits high basal AP activity and undetectable levels of osteocalcin secretion) by genome-independent mechanism. The regulation of AP will be examined systematically in these well-defined ROS clones and selectively in human osteogenic sarcoma (HOS) cells and in fetal rat calvariae by: a) direct enzymatic assay of total AP activity, b) quantitation of bone AP levels by immunoassay, and c) conducting parallel studies of bone AP-mRNA levels. RNA will be isolated from control and 1,25(OH)2D2-treated cultures for use in studies which include: i) in vitro translation in a rabbit reticulocyte lysate, followed by immunoprecipitation of bone AP and characterization of the radiolabelled products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), ii) construction of an expression library in the wavelength gtll vector, followed by screening for AP-producing recombinants and production of AP-specific cDNA probes; and iii) agarose gel electrophoresis, Northern transfer, and hybridization with the 32P-labelled AP-cDNA probe. In addition, posttranslational processing and glycosylation will be examined by pulse-labelling with 35S-methionine, 3H-mannose, or 3H-glucosamine, followed by SDS-PAGE as above. This integrated approach should clarify the mechanisms by which AP is either up- or down-regulated depending on the phenotype of the osteoblast and may contribute to a general understanding of how steroid hormones regulate gene expression in eukaryotic animals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Unknown (R23)
Project #
5R23AR038168-04
Application #
3446436
Study Section
General Medicine B Study Section (GMB)
Project Start
1987-01-01
Project End
1990-12-31
Budget Start
1989-01-01
Budget End
1990-12-31
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095