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Phosphorylation of GSK-3β by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes
Yosuke Kawasaki, … , Ung-il Chung, Hiroshi Kawaguchi
Yosuke Kawasaki, … , Ung-il Chung, Hiroshi Kawaguchi
Published June 12, 2008
Citation Information: J Clin Invest. 2008;118(7):2506-2515. https://doi.org/10.1172/JCI35243.
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Research Article Bone biology

Phosphorylation of GSK-3β by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes

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Abstract

cGMP-dependent protein kinase II (cGKII; encoded by PRKG2) is a serine/threonine kinase that is critical for skeletal growth in mammals; in mice, cGKII deficiency results in dwarfism. Using radiographic analysis, we determined that this growth defect was a consequence of an elongated growth plate and impaired chondrocyte hypertrophy. To investigate the mechanism of cGKII-mediated chondrocyte hypertrophy, we performed a kinase substrate array and identified glycogen synthase kinase–3β (GSK-3β; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3β was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3β. Analyses of mice with compound deficiencies in both protein kinases (Prkg2–/–Gsk3b+/–) demonstrated that the growth retardation and elongated growth plate associated with cGKII deficiency were partially rescued by haploinsufficiency of Gsk3b. We found that β-catenin levels decreased in Prkg2–/– mice, while overexpression of cGKII increased the accumulation and transactivation function of β-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3β. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3β by cGKII.

Authors

Yosuke Kawasaki, Fumitaka Kugimiya, Hirotaka Chikuda, Satoru Kamekura, Toshiyuki Ikeda, Naohiro Kawamura, Taku Saito, Yusuke Shinoda, Akiro Higashikawa, Fumiko Yano, Toru Ogasawara, Naoshi Ogata, Kazuto Hoshi, Franz Hofmann, James R. Woodgett, Kozo Nakamura, Ung-il Chung, Hiroshi Kawaguchi

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Figure 2

Identification of GSK-3β as a principal phosphorylation target of cGKII during chondrocyte hypertrophy.

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Identification of GSK-3β as a principal phosphorylation target of cGKII ...
(A) RT-PCR of 8 candidate genes that were identified by the serine/threonine kinase substrate array (Supplemental Table 1) in cultured ATDC5 cells in the prehypertrophic or hypertrophic differentiation stage. (B) COL10 promoter activity, as assessed by transfection of the 8 candidate genes or the empty vector (EV) in HuH-7 cells with the luciferase reporter gene construct containing a cloned 4.5-kb promoter fragment of COL10. Data are mean ± SD fold change relative to empty vector. *P < 0.01 versus control. (C) In vitro kinase assay of the phosphorylation of recombinant GSK-3β at Ser9 by recombinant cGKII with or without cGMP. Proteins were incubated in the presence of ATP, and the reaction products were analyzed by IB using the same antibody to Ser9-phosphorylated GSK-3β (p-GSK-3βSer9) as that used in Supplemental Table 1. (D) Phosphorylation of endogenous GSK-3β at Ser9 and GSK-3α at Ser21 by cGKII with or without cGMP in ATDC5 cells. Whole-cell lysates were incubated with recombinant cGKII or cGMP in the presence of ATP, and the reaction products were analyzed as in C. (E) Localization of cGKII, total GSK-3β, and Ser9-phosphorylated GSK-3β, as assessed by immunohistochemistry in the growth plate of proximal tibia in a 2-week-old mouse. Specific stainings were confirmed by immunohistochemistry by respective nonimmune serums (nonimmune control). Blue, green, and yellow bars indicate proliferative zone, hypertrophic zone, and primary spongiosa, respectively. Scale bars: 50 μm.

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