Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes  Z. He, D.J. Leong, Z. Zhuo, R.J. Majeska, L. Cardoso, D.C. Spray, M.B. Goldring, N.J. Cobelli, H.B. Sun  Osteoarthritis and Cartilage  Volume 24, Issue 5, Pages 892-901 (May 2016) DOI: 10.1016/j.joca.2015.11.015 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Uniaxial CTS induces CITED2 expression in a loading magnitude-, frequency-, and duration-dependent manner. CITED2 mRNA levels were analyzed in C28/I2 chondrocytes subjected to uniaxial strain strains of various magnitudes (0–10%, 1 h, 1 Hz) (A), frequencies (0–2 Hz, 5%, 1 h) (B), and loading durations (0–3 h, 5%, 1 Hz) (C) and CITED2 protein was analyzed by western blotting (D). Data represent the mean fold-change relative to unloaded group (CTRL) and 95% confidence intervals. Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 CITED2 expression is associated with and required for uniaxial strain-induced suppression of MMP-1 and -13 in human primary and C28/I2 chondrocytes. Induction of CITED2 is associated with suppressed expression of MMP-1 and MMP-13 in human primary and C28/I2 chondrocytes at the mRNA level (A) and protein level (B). CITED2 knockdown abolished uniaxial strain-induced suppression of MMP-1 and -13 gene expression in human primary chondrocytes (C). The relative mRNA levels of CITED2 (A), MMP-1, and MMP-13 (B) were determined in C28/I2 and primary human chondrocytes subjected to 5% strain at 1 Hz for 1 h (A) or subjected to loading after the cells were transfected with the shRNA-CITED2 plasmid (C). Data represent the fold-change relative to the unloaded, nontransfected group (0% strain) and 95% confidence intervals. (B) CITED2, MMP-1, MMP-13 and β-actin as a gel loading control were analyzed by Western blotting in C28/I2 cells subjected to 5% uniaxial strain at 1 Hz for 1 h and extracted 1, 3, and 6 h(s) later. A representative blot from 3 experiments is shown. Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Strain-induced CITED2 expression is attenuated by impaired primary cilia, inhibition of P2 purinergic receptors, and antagonists of Ca2+ signaling. Representative photographs of primary cilia and percentage of cilia positive cells in primary and C28/I2 chondrocytes transfected with scramble siRNA or with IFT88 siRNA and subjected to uniaxial strain (5%, 1 Hz, 1 h) (A). Relative mRNA levels of IFT88, CITED2, MMP-1, and MMP-13 after transfection with scrambled or IFT88 siRNA and application of uniaxial strain (B) in the parallel experiment with (A). Relative mRNA expression of CITED2 in chondrocytes treated with chemical modulators apyrase, gadolinium, suramin, thapsigargin, and EGTA prior to and during loading (C). Expression of CITED2 and MMP-1 and MMP-13 are assayed by RT-qPCR. Cilia were counted by scoring acetylated α-tubulin-positive structures greater than 1 μm in length from at least five different views of each sample. The data represent the mean and 95% confidence intervals of the percentage or fold-change relative to control (CTRL) or to unloaded (0% strain) and scrambled siRNA-treated group. Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Impairing primary cilia by intra-articular knockdown diminished treadmill running-induced CITED2 expression and reduction of MMP-1 and MMP-13 in mice. Representative images of primary cilia (indicated by white arrowheads) and percentage of positive stained cilia, Cited2, Mmp1 and Mmp13 cells in articular cartilage in naïve mice and the mice intra-articularly transfected with scramble siRNA or IFT88 siRNA and subjected to treadmill running (10 m/min for 45 min) (A). Relative mRNA levels of Cited2, Mmp1, Mmp13, and Ift88 in the parallel experiment (B). The data represent the mean and 95% confidence intervals or percentage or fold-change relative to naïve mice. Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Activation of ERK1/2 is required for cilia-mediated uniaxial strain induction of CITED2 expression and transactivation. Inhibiting or knocking down ERK1/2, but not JNK or p38 abolished strain-induced (5%, 1 Hz, 1 h) CITED2 mRNA expression (A). Role of various MAPK on CITED2 mRNA expression was analyzed by RT-qPCR in C28/I2 chondrocytes transfected with siRNA-p38δ or siRNA-p38α, or treated with MAPK inhibitors (B). Phosphorylated and total ERK1/2 protein were assayed by western blotting (C). In A, B, the data represent the mean fold change and 95% confidence intervals relative to the unloaded, nontreated group (CTRL). C28/I2 chondrocytes were transfected with the wild-type CITED2 promoter reporter construct, without or with cotransfection with siIFT88, or treatment with ERK1/2 inhibitor (D). Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Presence of SSRE, Sp1 and HIF-1α are required for uniaxial strain-induced CITED2 transactivation. Schematic view of the CITED2 promoter showing the Sp1, HIF-1α, and SSRE sites (A). C28/I2 chondrocytes were transfected with the wild-type (control) CITED2 promoter reporter construct, without or with cotransfection with siSp1 or siHIF1α or both siRNA. The SSRE-deletion construct was cotransfected with siSp1, siHIF1α, or both siRNA (B). After application of strain (5%, 1 Hz, 1hr), luciferase reporter activity was measured. Significant P values vs control shown. Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Hypothesized CITED2 mediated-signaling pathway in regulation of MMP expression in chondrocytes. CITED2 is a critical mediator in the signaling generated by strain and transduced by primary cilia, extracellular ATP-induced purinergic receptors and calcium signaling, that leads to transactivation of CITED2. Strain-induced CITED2 transactivation requires HIF1α and Sp1 and intact SSRE and leads to the downregulation of MMP expression. Osteoarthritis and Cartilage 2016 24, 892-901DOI: (10.1016/j.joca.2015.11.015) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions