WISP3‐dependent regulation of type II collagen and aggrecan production in chondrocytes

Abstract

Objective. WISP3 (Wnt-1-inducible secreted protein 3) is a member of the CCN (connective tissue growth factor, cysteine-rich 61, nephroblastoma overexpressed) family of connective tissue growth factors. WISP3 mutations have been linked to progressive pseudorheumatoid dysplasia (PPRD). The present study was conducted to investigate whether WISP3 is responsible for the expression of cartilage-specific molecules.Methods. WISP3 expression in human cartilage was assessed by immunostaining with anti-WISP3 antibody. The effect of WISP3 on chondrocyte-specific gene regulation was determined by transfecting human chondrocyte lines C-28/I2 and T/C-28a2 with a WISP3 expression vector. Alterations in WISP3-mediated messenger RNA and protein expression of cartilage-specific molecules were assessed by reverse transcriptasepolymerase chain reaction and immunoblotting.Results. Immunohistochemistry experiments demonstrated that WISP3 protein is expressed in the midzone chondrocytes of normal adult articular cartilage, in chondrocyte clusters of osteoarthritic cartilage, and in the zone of proliferating chondrocytes of fetal growth cartilage. Human chondrocyte lines C-28/I2 and T/C-28a2 transfected with a WISP3 expression vector produced increased amounts of the cartilage-specific matrix molecules type II collagen and aggrecan, in part via activation of the sex-determining region Y-type high mobility group box (SOX) family of transcription factors. In contrast, a mutant WISP3, previously found to be associated with PPRD, had impaired effects on cartilage-specific gene expression.Conclusion. Our experimental results suggest that WISP3 supports cartilage integrity by regulating the expression of type II collagen and aggrecan, and mutations linked with PPRD can compromise this function and produce cartilage loss.

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

“…Reactions were run in triplicate with 40 cycles of amplification on an ABI Prism 7000 real-time PCR machine (Applied Biosystems, Foster City, CA). The sequences of primers used were as follows: aggrecan, sense (5Ј-AGAATCCACCACCA-CCAG-3Ј) and antisense (5Ј-ATGCTGGTGCTGATGACA-3Ј); TATA box-binding protein, sense (5Ј-TGCACAGGAG-CCAAGAGTGAA-3Ј) and antisense (5Ј-CACATCACAGC-TCCCCACCA-3Ј) (28); collagen II, sense (5Ј-TGCTGCCC-AGATGGCTGGAGGA-3Ј) and antisense (5Ј-TGCCTTGA-AATCCTTGAGGCCC-3Ј) (29); Sox9, sense (5Ј-CACAC-TACAGCCCCTCCTAC-3Ј) and antisense (5Ј-CCTCCTC-AAGGTCGAGTGAG-3Ј) (30). The expression levels of aggrecan, collagen II, and Sox9 mRNA were normalized relative to the expression of TATA box-binding protein measured in parallel samples.…”

WISP3 - IGF1 interaction regulates chondrocyte hypertrophy

“…Prior investigations revealed that WISP3 promotes expression of the cartilage specific proteins collagen-II and aggrecan, controls the accumulation of reactive oxygen species (ROS) and regulates metalloprotease expression, corroborating that WISP3 sustains cartilage differentiation in vivo (Sen et al, 2004;Davis et al, 2006;Miller and Sen, 2007;Baker et al, 2012). Other studies have reported that WISP3 interacts with BMP and components of the Wnt1 signaling pathway including LRP6 and Fz8 (Nakamura et al, 2007).…”

“…The disease manifestations linked to WISP3 mutations suggest that WISP3 is required for proper skeletal growth and maintenance of cartilage integrity (Hurvitz et al, 1999;Delague et al, 2005). Although the detailed molecular mechanism of the action of WISP3 is not resolved yet, existing functional studies justifying a role of WISP3 in skeletal development and cartilage maintenance are in accordance with the clinical and radiographic picture associated with WISP3 mutations (Hurvitz et al, 1999;Sen et al, 2004;Delague et al, 2005;Lamb et al, 2005;Davis et al, 2006;Miller and Sen, 2007;Nakamura et al, 2007;Baker et al, 2012;Garcia Segarra et al, 2012).…”

CCN6 as a profibrotic mediator that stimulates the proliferation of lung fibroblasts via the integrin .BETA.1/focal adhesion kinase pathway

“…The C57BL/6 and MLg lung fibroblasts (8!10 3 cells per well) were cultured in 96-well flat-bottomed tissue plates in RPMI1640 supplemented with 10% FBS, and then serum-starved in medium containing 0.1% FBS overnight (19,20,22) [19,27,28]. Next, the cells were treated with various doses (1 to 200 ng/ml) of CCN6, and PDGF-BB (10 ng/ml) as a positive control for 72 hours, and labeled with 3 H -thymidine deoxyribose (TdR) (6.7 Ci/mmol ; Amersham Corp., Arlington Heights, IL) at 1 μCi/ well for the final 18 hours.…”

“…The ECM plays a vital early role in the repair process, orchestrating the modulation and transition of cell phenotype and function (26). It has been shown that CCN6 stimulates collagen type II, aggrecan, and superoxide dismutase expression and activity in chondrocytes (27,28). To investigate whether CCN6 affects on ECM production, we treated C57BL/6 cells with CCN6 and examined the mRNA expression of fibronectin and type I collagen by real-time RT-PCR analysis.…”