Abstract

To facilitate this study an in vitro human model system was established that exhibited many aspects of normal osteoblasts. The human osteosarcoma cell line (TE85 cells) expressed a skeletal alkaline phosphatase activity (an accepted bone cell differentiation marker) which was stimulated by 1,25(OH)2D3 (a potent differentiating agent), under serum-free conditions in a dose-dependent, time-dependent, and cell density-dependent manner. Cytochemical analysis of the stimulation of ALP activity by 1,25(OH)2D3 showed that 1,25(OH)2D3 increased the number of TE85 cells that expressed detectable ALP activity, suggesting that 1,25(OH)2D3 promoted the process of osteoblast differentiation and maturation.

This study also indicated that 1,25(OH)2D3 stimulated the de novo synthesis of ALP activity in TE85 cells by the following evidence: 1) Treatment of cellular membranes with 1,25(OH)2D3 did not directly activate pre-existing ALP activity, 2) a brief pre-treatment with 1,25(OH)2D3 (1 hour) was sufficient to stimulate ALP activity 47 hours later, 3) 5,6-Dicloro-1-β-D-ribofuranosylbenzimidazole (DRB) and cycloheximide, inhibitors of transcription and protein translation, respectively, each blocked the stimulation of ALP activity by 1,25(OH)2D3, and 4) the stimulation of ALP activity by 1,25(OH)2D3 was accompanied by a corresponding increase in steady state level of ALP mRNA, showing a temporal cause-and-effect relationship between the two parameters.

Evaluations of the mechanistic action of 1,25(OH)2D3 showed that the increase in the steady state level of ALP mRNA was a result of both an increased rate of ALP gene transcription (measured by a nuclear run-off assay) and by a posttranscriptional increase in ALP mRNA stability. The increase in ALP mRNA stability was shown to depend not on a continuous presence of 1,25(OH)2D3 on the cells, but rather on nascent protein synthesis (i.e., cycloheximide blocked the increase in ALP mRNA stability). This finding suggested that increased ALP mRNA stability was mediated through a de novo synthesis of a 1,25(OH)2D3-inducible protein, which has been tentatively called the "ALP mRNA stabilizing factor".

Based on the findings in this study, it is proposed that 1,25(OH)2D3 stimulates human ALP activity through a complex set of mechanisms, involving both transcriptional and posttranscriptional events. Although there is a precedence for the regulation of gene expression by steroid hormones through modulation of both gene transcription and mRNA stability, this is the first time that 1,25(OH)2D3 has been shown to regulate gene expression, or affect the state of osteoblast differentiation, through a combination of mechanisms.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

Donna Dee Strong

Second Advisor

K-H William Lau

Third Advisor

Thomas A. Linkhart

Fourth Advisor

John R. Farley

Fifth Advisor

R. Bruce Wilcox

Sixth Advisor

E Clifford Herrmann

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1991

Date (Title Page)

3-1991

Language

English

Library of Congress/MESH Subject Headings

Calcitriol -- analysis; Alkaline Phosphatase -- analysis; Osteoblasts -- metabolism; Bone Development.

Type

Dissertation

Page Count

xi; 157

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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