PTHrP overexpression partially inhibits a mechanical strain-induced arthritic phenotype in chondrocytes D. Wang, J.M. Taboas, R.S. Tuan Osteoarthritis.

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PTHrP overexpression partially inhibits a mechanical strain-induced arthritic phenotype in chondrocytes D. Wang, J.M. Taboas, R.S. Tuan Osteoarthritis and Cartilage Volume 19, Issue 2, Pages 213-221 (February 2011) DOI: 10.1016/j.joca.2010.11.003 Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Cell viability at the end of experimentation was observed using LIVE/DEAD staining to detect live (green fluorescence) and dead (red fluorescence) cells. The majority of chondrocytes (>90%) remained viable after 48h of (A) No CTS and (B) CTS. Osteoarthritis and Cartilage 2011 19, 213-221DOI: (10.1016/j.joca.2010.11.003) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effects of 16% CTS on normal bovine articular chondrocytes. CTS increased gene expression of (A) MMPs, (B) ADAMTSs, (D) hypertrophic genes, and (F) iNOS and COX-2 and decreased gene expression of (C) Col2a1. CTS increased secretion of (D) NO and PGE2, which correlates with (E). Error bars represent ±95% CIs (N=3). Solid lines=CTS; Dotted lines=No CTS. *=P<0.05 and **=P<0.01 vs control. Specifically, MMP-13 48h P=0.0493, 24h P=0.0465; Col2a1 48h P=0.0498; Col10al P=0.0072, 24h P=0.0124, 12h P=0.0122; Runx2 48h P=0.0471; iNOS 48h P=0.0097, 24h P=0.0139; COX-2 48h P=0.0059, 24h P=0.0130, 12h P=0.0075; NO 48h P=0.0397, 24h P=0.0301; PGE2 48h P=0.0147, 24h P=0.0066. Osteoarthritis and Cartilage 2011 19, 213-221DOI: (10.1016/j.joca.2010.11.003) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Total PTHrP secretion by articular chondrocytes transfected with hPTHrP expression constructs. (A) Chondrocytes expressing hPTHrP constructs secrete more total PTHrP (bovine and human) under CTS and No CTS conditions (only CTS data shown). Error bars represent ±95% CIs (N=3). *=P<0.05 and **=P<0.01 vs control. # Denotes P=0.0102 vs No CTS control. (B) Endogenous PTHrP (bPTHrP) gene expression, as measured by RT-PCR using a primer specific for the bovine gene, was up-regulated by CTS. Chondrocytes expressing hPTHrP 1-141 and 1-173 demonstrated feedback inhibition of endogenous bPTHrP mRNA levels. Error bars represent ±95% CIs (N=3). **=P<0.01 control vs hPTHrP 1-141 and 1-173. Osteoarthritis and Cartilage 2011 19, 213-221DOI: (10.1016/j.joca.2010.11.003) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Inhibition of Col10a1 gene expression in articular chondrocytes by PTHrP overexpression. Expression of exogenous hPTHrP in chondrocytes suppressed (A) CTS-induced Col10a1 gene expression and (B) basal Col10a1 gene expression. hPTHrP overexpression had no significant effect on matrix remodeling genes (data not shown). Error bars represent ±95% CIs (N=3). *=P<0.05 and **=P<0.01 vs hPTHrP 1-141 and 1-173. Osteoarthritis and Cartilage 2011 19, 213-221DOI: (10.1016/j.joca.2010.11.003) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effects of hPTHrP overexpression on CTS-induced iNOS and COX-2 gene expression and NO and PGE2 production in articular chondrocytes. Overexpression of hPTHrP 1-141 augmented gene expression of inducible enzyme isoforms, (A) iNOS and (C) COX-2, and production of their respective inflammatory mediators, (B) NO and (D) PGE2. Chondrocytes overexpressing hPTHrP 1-173 did not show significant changes in iNOS and COX-2 gene expression or production of NO and PGE2 when compared to control. Error bars represent ±95% CIs (N=3). *=P<0.05 and **=P<0.01 vs control. Osteoarthritis and Cartilage 2011 19, 213-221DOI: (10.1016/j.joca.2010.11.003) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effects of hPTHrP overexpression on CTS-induced apoptotic gene expression in articular chondrocytes. Chondrocytes overexpressing either exogenous hPTHrP isoform did not show any significant change in CTS-induced (A) Bcl-2 and (B) Bax gene expression levels. (C) hPTHrP overexpression also did not significantly affect Bcl-2/Bax ratio. Error bars represent ±95% CIs (N=3). Osteoarthritis and Cartilage 2011 19, 213-221DOI: (10.1016/j.joca.2010.11.003) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions