Molecular Mechanism of the Stimulation of Alkaline Phosphatase Activity in Human Bone Cells by 1,25(OH)2 D3

<p>To facilitate this study an <em>in vitro</em> 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.</p> <p>This study also indicated that 1,25(OH)₂D₃ stimulated the <em>de novo</em> 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.</p> <p>Evaluations of the mechanistic action of 1,25(OH)₂D₃showed 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 <em>de novo</em> synthesis of a 1,25(OH)₂D₃-inducible protein, which has been tentatively called the "ALP mRNA stabilizing factor".</p> <p>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.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Year

1991

Author dc:creator

• Kyeyune-Nyombi, Eru

Contributors dc:contributor

• Donna Dee Strong
• K-H William Lau
• Thomas A. Linkhart
• John R. Farley
• R. Bruce Wilcox
• E Clifford Herrmann

• Biochemical Phenomena, Metabolism, and Nutrition
• Biochemistry
• Genetic Phenomena
• Laboratory and Basic Science Research
• Osteopathic Medicine and Osteopathy
• Calcitriol -- analysis; Alkaline Phosphatase -- analysis; Osteoblasts -- metabolism; Bone Development.

Statement dc: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.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/1490

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-2266