Naohiro Kawamura

Akt1 in osteoblasts and osteoclasts controls bone remodeling.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-κB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

GSK-3β Controls Osteogenesis through Regulating Runx2 Activity

Despite accumulated knowledge of various signalings regulating bone formation, the molecular network has not been clarified sufficiently to lead to clinical application. Here we show that heterozygous glycogen synthase kinase-3β (GSK-3β)-deficient mice displayed an increased bone formation due to an enhanced transcriptional activity of Runx2 by suppressing the inhibitory phosphorylation at a specific site. The cleidocranial dysplasia in heterozygous Runx2-deficient mice was significantly rescued by the genetic insufficiency of GSK-3β or the oral administration of lithium chloride, a selective inhibitor of GSK-3β. These results establish GSK-3β as a key attenuator of Runx2 activity in bone formation and as a potential molecular target for clinical treatment of bone catabolic disorders like cleidocranial dysplasia.

Patched1 haploinsufficiency increases adult bone mass and modulates Gli3 repressor activity.

Hedgehog (Hh)-Patched1 (Ptch1) signaling plays essential roles in various developmental processes, but little is known about its role in postnatal homeostasis. Here, we demonstrate regulation of postnatal bone homeostasis by Hh-Ptch1 signaling. Ptch1-deficient (Ptch1+/-) mice and patients with nevoid basal cell carcinoma syndrome showed high bone mass in adults. In culture, Ptch1+/- cells showed accelerated osteoblast differentiation, enhanced responsiveness to the runt-related transcription factor 2 (Runx2), and reduced generation of the repressor form of Gli3 (Gli3rep). Gli3rep inhibited DNA binding by Runx2 in vitro, suggesting a mechanism that could contribute to the bone phenotypes seen in the Ptch1 heterozygotes. Moreover, systemic administration of the Hh signaling inhibitor cyclopamine decreased bone mass in adult mice. These data provide evidence that Hh-Ptch1 signaling plays a crucial role in postnatal bone homeostasis and point to Hh-Ptch1 signaling as a potential molecular target for the treatment of osteoporosis.

Dnm3os, a non‐coding RNA, is required for normal growth and skeletal development in mice

Dnm3os, a gene that is transcribed into a non‐coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR‐199a, miR‐199a*, and miR‐214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os+/lacZ heterozygous embryos showed β‐galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3oslacZ/lacZ homozygous pups died within one month of birth. After birth, Dnm3oslacZ/lacZ mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR‐199a, miR‐199a*, and miR‐214 was significantly down‐regulated in Dnm3oslacZ/lacZ embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA‐encoding gene that is indispensable for normal skeletal development and body growth in mammals. Developmental Dynamics 237:3738–3748, 2008.

C/EBPβ Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Transactivation of p57Kip2

Background Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) in chondrocytes during endochondral ossification. Methodology/Principal Findings Mouse embryos with homozygous deficiency in C/EBPβ (C/EBPβ−/−) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPβ−/− chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPβ in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPβ. In fact, a DNA cell cycle histogram revealed that the C/EBPβ overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57Kip2 as the transcriptional target of C/EBPβ. p57Kip2 was co-localized with C/EBPβ in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPβ deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPβ in the p57Kip2 promoter between −150 and −130 bp region containing a putative C/EBP motif. The knockdown of p57Kip2 by the siRNA inhibited the C/EBPβ-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPβ+/− littermates, the C/EBPβ insufficiency caused resistance to joint cartilage destruction. Conclusions/Significance C/EBPβ transactivates p57Kip2 to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPβ-p57Kip2 signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis.

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

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-3beta (GSK-3beta; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3beta was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3beta. 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 beta-catenin levels decreased in Prkg2(-/-) mice, while overexpression of cGKII increased the accumulation and transactivation function of beta-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3beta. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3beta by cGKII.

Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type x collagen gene

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 co-localized 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 beta 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.

Can laminoplasty maintain the cervical alignment even when the C2 lamina is contained

Study Design. Retrospective case-controlled study of cervical laminoplasty. Objective. To evaluate the alignment and clinical result by laminoplasty when the C2 lamina is contained or retained. Summary of Background Data. Resection of the C2 lamina was reported to progress to kyphosis after laminectomy. Laminoplasty was reported to inhibit kyphosis. But no study has ever shown if the alignment is retained when laminoplasty also included the C2 lamina. Methods. Seventy-two patients with cervical spondylotic myelopathy undergoing laminoplasty were analyzed. Follow-up averaged 4.0 years. The outcome was assessed by the Cobb angle between C2 and C7, and the motor function scores of the upper and lower extremities for cervical myelopathy were made by the Japanese Orthopedic Association. Patients were stratified into three groups depending on the handling of the C2 lamina: fully split (S group; n = 17), C2 dome-like laminotomy (D group; n = 19), and intact (I group; n = 36). Change of the C2–C7 angle was compared by the analysis of variance and post hoc test. The association between the alignment and the motor scores was analyzed. Results. Upper/lower score increased from 2.4/2.0 to 3.4/2.9, respectively. The C2–C7 angle decreased in S group: −8.3°, D group: −5.2°, and I group: −1.5°. The cervical alignment deteriorated significantly in S group compared with the I group (P < 0.01). The C2–C7 angle change or postoperative C2–C7 angle had no significant correlation with the postoperative upper and lower m-JOA scores or score change. Conclusions. Subaxial laminoplasty maintained the alignment. But if laminoplasty included the C2 lamina, the alignment worsened.

Association of microsomal prostaglandin E synthase 1 deficiency with impaired fracture healing, but not with bone loss or osteoarthritis, in mouse models of skeletal disorders

OBJECTIVE Prostaglandin E synthase (PGES) functions as the terminal enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)) and is a potent regulator of bone and cartilage metabolism. Among the 3 isozymes of PGES, microsomal PGES-1 (mPGES-1) is known to play the most critical role in the production of PGE(2) in pathophysiologic events. This study investigated the roles of mPGES-1 under normal physiologic and pathophysiologic conditions in the skeletons of mPGES-1-deficient (mPGES-1(-/-)) mice. METHODS Skeletons of mPGES-1(-/-) mice and their wild-type littermates were compared by radiologic and histologic analyses. Four models of skeletal disorders were created: bone loss induced by ovariectomy, bone loss induced by hind limb unloading, osteoarthritis (OA) induced by instability in the knee joint, and bone fracture by osteotomy at the tibial midshaft. Expression of the PGES enzymes was examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction. The cellular mechanism of fracture healing was examined in ex vivo cultures of costal cartilage chondrocytes. RESULTS Microsomal PGES-1(-/-) mice had unaffected skeletal phenotypes under normal physiologic conditions. In the bone fracture model, fracture healing was impaired by the mPGES-1 deficiency, with half of the mice remaining in a non-bone union state even after 21 days; normal fracture healing was restored by adenoviral reintroduction of mPGES-1. The other skeletal disorders were not affected by the mPGES-1 deficiency. In vivo and ex vivo analyses revealed an impaired proliferation of chondrocytes in cartilage with the mPGES-1 deficiency, at an early stage of fracture healing. CONCLUSION In these mouse models of skeletal disorders, mPGES-1 was indispensable for bone repair through chondrocyte proliferation, but was not essential for the skeleton under normal physiologic conditions, nor did it play a role in the pathophysiologic conditions of bone loss due to ovariectomy, bone loss due to unloading, or stress-induced OA.

Kruppel-like factor 5 causes cartilage degradation through transactivation of matrix metalloproteinase 9.

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.