XinDe Song

Abstract

Transforming growth factor β1 (TGFβ1) is a pluripotent cytokine that works in conjunction with other cytokines to modulate their effects on cell function. Our previous work demonstrated that the combination of TGFβ1 and 1α,25-dihydroxyvitamin D3 had a synergistic effect on alkaline phosphatase induction (500 to 700%) in human osteoblastic osteosarcoma cells (MG63). The current studies focused on the molecular basis of alkaline phosphatase induction, including the regulation of the signal transduction and modification of 1α,25-dihydroxyvitamin D3 receptors (VDR). We demonstrated that alkaline phosphatase activity was regulated at the transcription level. TGFβ1 at 1 ng/ml alone had little effect on alkaline phosphatase mRNA induction and 1α,25-dihydroxy vitamin D3 at 10-8 M increased alkaline phosphatase mRNA to about 200% over the control value. The combination of TGFβ1 and 1α,25-dihydroxyvitamin D3 significantly increased alkaline phosphatase mRNA about 800% over the control. The peak response was at 24 hours. Studies of the signal transduction mechanism provided evidence that the protein kinase C pathway was involved in the alkaline phosphatase stimulation by the combination of TGFβ1 and 1α,25-dihydroxyvitamin D3; MG63 cells express at least seven protein kinase C isoforms and PKCy was activated by TGFβ1 or 1α,25-dihydroxyvitamin D3 after 30 minute treatment. Studies on the VDR demonstrated that TGFβ1 or its combination with 1α,25-dihydroxyvitamin D3 significantly increased VDR mRNA about 300% and 500% respectively over the control by 3 hours. 1α,25-dihydroxyvitamin D3 increased VDR mRNA about 200% by 24 hour. The VDR protein level was also significantly elevated by the combination of TGFβ1 with 1α,25-dihydroxyvitamin D3. The peak response at 6 hours was 300% above the control level. Our studies further showed that a 2 hour treatment with TGFβ1 and 1α,25-dihydroxy vitamin D3 significantly stimulated VDR phosphorylation to 700% and 300% above the control, respectively. When cells were treated with the combination of TGFβ1 and 1α,25-dihydroxyvitamin D3, there was an additive effect on VDR phosphorylation to about 1000% over the control value. Conclusions: (1) PKCy is involved in the TGFβ and 1α, 25-dihydroxyvitamin D3 signal transduction pathway. (2) TGFβ potentiates the action of 1α,25-dihydroxyvitamin D3 by stimulation of VDR mRNA, VDR protein and VDR phosphorylation. These changes further enhance the effect of 1α,25-dihydroxy vitamin D3 on the cellular levels of alkaline phosphatase mRNA and alkaline phosphatase activity.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

Jon E. Wergedal

Second Advisor

Thomas A. Linkhart

Third Advisor

Lora M. Green

Fourth Advisor

E. Clifford Herrmann

Fifth Advisor

Kelvin A. W. Hill

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1996

Date (Title Page)

3-1996

Language

English

Library of Congress/MESH Subject Headings

Osteosarcoma; Transforming Growth Factor beta; Calcitriol

Type

Dissertation

Page Count

xiv; 176

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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Included in

Biochemistry Commons

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