Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts
Hiroaki Seto, Satoshi Kamekura, Toshiki Miura, Aiichiro Yamamoto, Hirotaka Chikuda, Toru Ogata, Hisatada Hiraoka, Hiromi Oda, Kozo Nakamura, Hisashi Kurosawa, Ung-il Chug, Hiroshi Kawaguchi, Sakae Tanaka
Hiroaki Seto, Satoshi Kamekura, Toshiki Miura, Aiichiro Yamamoto, Hirotaka Chikuda, Toru Ogata, Hisatada Hiraoka, Hiromi Oda, Kozo Nakamura, Hisashi Kurosawa, Ung-il Chug, Hiroshi Kawaguchi, Sakae Tanaka
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Article Bone biology

Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts

Abstract

The role of TGF-β/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector–mediated gene transduction system. Expression of constitutively active activin receptor–like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.

Authors

Hiroaki Seto, Satoshi Kamekura, Toshiki Miura, Aiichiro Yamamoto, Hirotaka Chikuda, Toru Ogata, Hisatada Hiraoka, Hiromi Oda, Kozo Nakamura, Hisashi Kurosawa, Ung-il Chug, Hiroshi Kawaguchi, Sakae Tanaka

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Induction of Alizarin red staining and type X collagen in MKK6-transduce...
Induction of Alizarin red staining and type X collagen in MKK6-transduced SFs in pellet cultures. (A–F) SFs infected with MKK6CA virus alone (A, C, and E) or together with Smad1 virus (B, D, and F) were subjected to pellet culture. Cultures were fixed with 3.7% formaldehyde 3 weeks later, and then stained with Alcian blue (A and B), Alizarin red (C and D) or anti–type X collagen (E and F). Note the increased Alcian blue staining and the reduced Alizarin red activity and type X collagen immunoactivity with Smad1 coexpression. Scale bars:100 μm (A–D) and 50 μm (E and F).