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.

Objective. Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells.Methods. Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation.Results. RUNX-2 and type X collagen were colocalized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor ␤ in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between ؊89 and ؊60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells.Conclusion. Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA.Hypertrophic differentiation of chondrocytes during endochondral ossification is an essential step in skeletal growth and repair (1,2). We and others have reported that chondrocyte hypertrophy also contributes to cartilage degradation during the development of osteoarthritis (OA) (3-5). Type X collagen is a short, network-forming collagen specifically expressed by hypertrophic chondrocytes (6). The physiologic importance of type X collagen has been shown by the impairment of endochondral ossification and skeletal growth that results from loss of function of the type X collagen gene in mice (7-9). Similarly, mutations in the carboxyterminal domain of the human type X collagen gene (COL10A1) cause a severe skeletal disorder called Schmid-type metaphyseal chondrodysplasia, with growth retardation, waddling gait, and OA (10-12). Hence, elucidation of the mechanisms regulating the type X collagen gene will contribute to understanding the molecular backgrounds of skeletal growth and repair and OA not only in mice, but also in humans.

We have identified RelA as a transcriptional factor for SOX9 induction and chondrogenic differentiation via binding to an NF-kappaB binding motif in the SOX9 promoter.

Objective. To examine the role of the phosphoinositide-dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification.Methods. Skeletal phenotypes of homozygous Akt1-deficient (Akt1 ؊/؊ ) mice and their wild-type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1.Results. Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1 ؊/؊ chondrocytes, indicating a dominant role of Akt1. Akt1 ؊/؊ mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild-type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1 ؊/؊ joints. Calcification was significantly suppressed in cultures of Akt1 ؊/؊ chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/ phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1. Conclusion. Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA.Endochondral ossification is an essential process not only in physiologic skeletal growth, but also in pathologic disorders such as osteophyte formation during osteoarthritis (OA) progression (1,2). After chondrocytes proliferate and differentiate into mature hypertrophic cells, the cells calcify the surrounding matrix and recruit blood vessels, leading to progressive replacement of cartilage by bone. Inorganic pyrophosphate (PPi) plays a crucial role in the regulation of calcification by suppressing it through antagonizing the ability of inorganic phosphate (Pi) ions to crystallize with calcium (3).Extracellular PPi accumulation is regulated by a transmembrane protein progressive ankylosis (ANK) for extracellular channeling, nucleotide pyrophosphatase/ phosphodiesterase 1 (NPP1) for generation from nucle-

Although degradation of cartilage matrix has been suggested to be a rate-limiting step for endochondral ossification during skeletal development, little is known about the transcriptional regulation. This study investigated the involvement of KLF5 (Krüppel-like factor 5), an Sp/KLF family member, in the skeletal development. KLF5 was expressed in chondrocytes and osteoblasts but not in osteoclasts. The heterozygous deficient (KLF5 ؉/؊ ) mice exhibited skeletal growth retardation in the perinatal period. Although chondrocyte proliferation and differentiation were normal, cartilage matrix degradation was impaired in KLF5 ؉/؊ mice, causing delay in replacement of cartilage with bone at the primary ossification center in the embryonic limbs and elongation of hypertrophic chondrocyte layer in the neonatal growth plates. Microarray analyses identified MMP9 (matrix metalloproteinase 9) as a transcriptional target, since it was strongly up-regulated by adenoviral transfection of KLF5 in chondrogenic cell line OUMS27. The KLF5 overexpression caused gelatin degradation by stimulating promoter activity of MMP9 without affecting chondrocyte differentiation or vascular endothelial growth factor expression in the culture of chondrogenic cells; however, in osteoclast precursors, it affected neither MMP9 expression nor osteoclastic differentiation. KLF5 dysfunction by genetic heterodeficiency or RNA interference was confirmed to cause reduction of MMP9 expression in cultured chondrogenic cells. MMP9 expression was decreased in the limbs of KLF5 ؉/؊ embryos, which was correlated with suppression of matrix degradation, calcification, and vascularization. We conclude that KLF5 causes cartilage matrix degradation through transcriptional induction of MMP9, providing the first evidence that transcriptional regulation of a proteinase contributes to endochondral ossification and skeletal development.Endochondral ossification is an essential process for skeletal development and growth (1). During the process, chondrocytes undergo proliferation and hypertrophic differentiation. The hypertrophic chondrocytes then secrete a specialized extracellular matrix rich in type X collagen (COL10), 2 which is replaced by bone matrix. The ossification begins with chondrocyte apoptosis, cartilage matrix degradation, calcification, vascular invasion from perichondrium and bone marrow, and deposition of bone matrix by osteoblasts (2). Among these individual steps, previous studies have indicated that degradation of cartilage matrix is particularly crucial (3-6). This step requires proteolytic breakdown by a variety of proteinases, among which members of the matrix metalloproteinase (MMP) family are of special interest due to their ability to cleave collagens and aggrecan, the two principal matrix components of cartilage (7,8). However, little is known about transcriptional regulation of MMPs in the endochondral ossification process.Members of the Krüppel-like factor (KLF) family are important transcription factors that regulate development, cellular dif...