QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 7 April 2009
doi: 10.1242/jcs.040709

This Article Figures Only Full Text Full Text (PDF) All Versions of this Article: jcs.040709v1 122/9/1382    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, M. Articles by Chen, D. Search for Related Content PubMed PubMed Citation Articles by Zhang, M. Articles by Chen, D. Social Bookmarking                         What's this?

PTHrP prevents chondrocyte premature hypertrophy by inducing cyclin-D1-dependent Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation

Ming Zhang^1,*, Rong Xie^2,*, Wei Hou^2,3, Baoli Wang², Run Shen², Xiumei Wang², Qing Wang¹, Tianhui Zhu¹, Jennifer H. Jonason² and Di Chen^2,

¹ Medical College of Nankai University, Tianjin 300071, PR China
² Department of Orthopaedics, Center for Musculoskeletal Research, University of Rochester School of Medicine, Rochester, NY 14642, USA
³ Spine Research Institute, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, PR China

Author for correspondence (e-mail: di_chen{at}urmc.rochester.edu)

Accepted 27 December 2009

In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myc-tagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GST-Runx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SA-Runx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2 cells, SA-Runx3 had a greater effect on reporter activity than SE-Runx3. The same was true for ALP activity in these cells. To investigate the role of cyclin D1 in chondrocyte proliferation and hypertrophy, we analyzed the growth plate morphology and expression of chondrocyte differentiation marker genes in Ccnd1-knockout mice. The proliferating and hypertrophic zones were significantly reduced and expression of chondrocyte differentiation marker genes and ALP activity were enhanced in 2-week-old Ccnd1-knockout mice. PTHrP significantly suppressed protein levels of both Runx2 and Runx3 in primary chondrocytes derived from wild-type mice. By contrast, the suppressive effect of PTHrP on Runx2 and Runx3 protein levels was completely abolished in primary chondrocytes derived from Ccnd1-knockout mice. Our findings demonstrate that the cell cycle proteins cyclin D1 and CDK4 induce Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. PTHrP suppresses Runx2 and Runx3 protein levels in chondrocytes through cyclin D1. These results suggest that PTHrP might prevent premature hypertrophy in chondrocytes, at least in part by inducing degradation of Runx2 and Runx3 in a cyclin-D1-dependent manner.

Key words: PTHrP, Cyclin D1, Runx2, Runx3, Chondrocyte

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

Related articles in JCS:

Boning up on cyclin D1

JCS 2009 122: 903. [Full Text]  

This article has been cited by other articles:

				Y. Yan, D. Tang, M. Chen, J. Huang, R. Xie, J. H. Jonason, X. Tan, W. Hou, D. Reynolds, W. Hsu, et al. Axin2 controls bone remodeling through the {beta}-catenin-BMP signaling pathway in adult mice J. Cell Sci., October 1, 2009; 122(19): 3566 - 3578. [Abstract] [Full Text] [PDF]

				J.H. Jonason, G. Xiao, M. Zhang, L. Xing, and D. Chen Post-translational Regulation of Runx2 in Bone and Cartilage Journal of Dental Research, August 1, 2009; 88(8): 693 - 703. [Abstract] [Full Text] [PDF]