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Parathyroid Hormone Activation of Matrix Metalloproteinase 13 Transcription in Osteoblasts: cooperation of p300, PCAF, and Runx2

by
Minnkyong Lee
Bachelor of Science, 2005
Ewha Womans University
Seoul, Korea

Thesis Advisor: Nicola C. Partridge, Ph.D.

Graduate Program of Physiology & Integrative Biology

School of Public Health/Research Building
Room 258

Friday, March 26, 2010
2:00 pm


Abstract

Parathyroid hormone (PTH) is a peptide hormone secreted from the parathyroid glands in response to low serum calcium levels. It plays a central role in regulating serum ion concentrations through its actions at its primary target sites, kidney and bone. In bone, PTH regulates the transcription of many genes involved in bone remodeling in osteoblasts through mechanisms that are still poorly understood. One of these genes is matrix metalloproteinase-13 (MMP-13), which is involved in bone remodeling and early stages of endochondral bone formation. Previous reports from our laboratory have shown that p300 and Runx2 are key players in the PTH-dependent transcriptional activation of MMP-13. Here, we identify PCAF as another factor downstream of PTH involved in MMP-13 transcription. PCAF was found to coimmunopreciptate with p300 in the osteoblastic cell line, UMR 106-01. While there was no change in the net amount of PCAF associated with p300, we were able to detect an increase in the acetylation of PCAF after 1 h PTH treatment. ChIP assays showed that PCAF is recruited to the MMP-13 proximal promoter region in increasing amounts with longer PTH treatment which is associated with an increase in histone acetylation at the promoter region, and increased MMP-13 transcription. Knockdown of PCAF, p300, or Runx2 by siRNA decreased MMP-13 mRNA expression after PTH treatment in both UMR 106-01 cells and primary osteoblasts. ChIP assays showed that all three factors were required for recruitment of RNA polymerase II and histone acetylation at the MMP-13 proximal promoter after PTH treatment. We found that there is a mutual dependency between p300 and PCAF in order to be recruited to the MMP-13 promoter after PTH treatment. In promoter-reporter assays, p300 and PCAF had an additive effect on PTH stimulation of MMP-13 promoter activity, and this required both of their HAT activities. Our findings demonstrate that PCAF acts downstream of PTH signaling as a transcriptional coactivator that is necessary for PTH stimulation of MMP-13 transcription. PCAF cooperates with p300 and Runx2 to mediate PTH activation of MMP-13 transcription, which requires the HAT activity of both p300 and PCAF.


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