Naoshi Ogata

Regulation of bone formation by adiponectin through autocrine/paracrine and endocrine pathways

Since interaction between bone and lipid metabolism has been suggested, this study investigated the regulation of bone metabolism by adiponectin, a representative adipokine, by analyzing deficient and overexpressing transgenic mice. We initially confirmed that adiponectin and its receptors were expressed in osteoblastic and osteoclastic cells, indicating that adiponectin can act on bone not only through an endocrine pathway as a hormone secreted from fat tissue, but also through an autocrine/paracrine pathway. There was no abnormality in bone mass or turnover of adiponectin‐deficient (Ad−/−) mice, possibly due to an equivalent balance of the two pathways. In the culture of bone marrow cells from the Ad−/− mice, osteogenesis was decreased compared to the wild‐type (WT) cell culture, indicating a positive effect of endogenous adiponectin through the autocrine/paracrine pathway. To examine the endocrine action of adiponectin, we analyzed transgenic mice overexpressing adiponectin in the liver, and found no abnormality in the bone. Addition of recombinant adiponectin in cultured osteoprogenitor cells suppressed osteogenesis, suggesting that the direct action of circulating adiponectin was negative for bone formation. In the presence of insulin, however, this suppression was blunted, and adiponectin enhanced the insulin‐induced phosphorylations of the main downstream molecule insulin receptor substrate‐1 and Akt. These lines of results suggest three distinct adiponectin actions on bone formation: a positive action through the autocrine/paracrine pathway by locally produced adiponectin, a negative action through the direct pathway by circulating adiponectin, and a positive action through the indirect pathway by circulating adiponectin via enhancement of the insulin signaling. J. Cell. Biochem.

Insulin receptor substrate-1 in osteoblast is indispensable for maintaining bone turnover

Insulin receptor substrates (IRS-1 and -2) are essential for intracellular signaling by insulin and IGF-I, anabolic regulators of bone metabolism. Mice lacking the IRS-1 gene IRS-1(-/-) showed severe osteopenia with low bone turnover. IRS-1 was expressed in osteoblasts, but not in osteoclasts, of wild-type (WT) mice. IRS-1(-/-) osteoblasts treated with insulin or IGF-I failed to induce tyrosine phosphorylation of cellular proteins, and they showed reduced proliferation and differentiation. Osteoclastogenesis in the coculture of hemopoietic cells and osteoblasts depended on IRS-1 expression in osteoblasts and could not be rescued by IRS-1 expression in hemopoietic cells in the presence of not only IGF-I but also 1,25(OH)(2)D(3). In addition, osteoclast differentiation factor (RANKL/ODF) was not induced by these factors in IRS-1(-/-) osteoblasts. We conclude that IRS-1 deficiency in osteoblasts impairs osteoblast proliferation, differentiation, and support of osteoclastogenesis, resulting in low-turnover osteopenia. Osteoblastic IRS-1 is essential for maintaining bone turnover, because it mediates signaling by IGF-I and insulin and, we propose, also by other factors, such as 1,25(OH)(2)D(3).

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.

Klotho gene polymorphisms associated with bone density of aged postmenopausal women

Because mice deficient in klotho gene expression exhibit multiple aging phenotypes including osteopenia, we explored the possibility that the klotho gene may contribute to age‐related bone loss in humans by examining the association between klotho gene polymorphisms and bone density in two genetically distinct racial populations: the white and the Japanese. Screening of single‐nucleotide polymorphisms (SNPs) in the human klotho gene identified 11 polymorphisms, and three of them were common in both populations. Associations of the common SNPs with bone density were investigated in populations of 1187 white women and of 215 Japanese postmenopausal women. In the white population, one in the promoter region (G‐395A, p = 0.001) and one in exon 4 (C1818T, p = 0.010) and their haplotypes (p < 0.0001) were significantly associated with bone density in aged postmenopausal women (≥65 years), but not in premenopausal or younger postmenopausal women. These associations were also seen in Japanese postmenopausal women. An electrophoretic mobility shift analysis revealed that the G‐A substitution in the promoter region affected DNA‐protein interaction in cultured human kidney 293 cells. These results indicate that the klotho gene may be involved in the pathophysiology of bone loss with aging in humans.

Insulin receptor substrate-2 maintains predominance of anabolic function over catabolic function of osteoblasts

Insulin receptor substrates (IRS-1 and IRS-2) are essential for intracellular signaling by insulin and insulin-like growth factor-I (IGF-I), anabolic regulators of bone metabolism. Although mice lacking the IRS-2 gene (IRS-2 −/− mice) developed normally, they exhibited osteopenia with decreased bone formation and increased bone resorption. Cultured IRS-2 − / − osteoblasts showed reduced differentiation and matrix synthesis compared with wild-type osteoblasts. However, they showed increased receptor activator of nuclear factor κB ligand (RANKL) expression and osteoclastogenesis in the coculture with bone marrow cells, which were restored by reintroduction of IRS-2 using an adenovirus vector. Although IRS-2 was expressed and phosphorylated by insulin and IGF-I in both osteoblasts and osteoclastic cells, cultures in the absence of osteoblasts revealed that intrinsic IRS-2 signaling in osteoclastic cells was not important for their differentiation, function, or survival. It is concluded that IRS-2 deficiency in osteoblasts causes osteopenia through impaired anabolic function and enhanced supporting ability of osteoclastogenesis. We propose that IRS-2 is needed to maintain the predominance of bone formation over bone resorption, whereas IRS-1 maintains bone turnover, as we previously reported; the integration of these two signalings causes a potent bone anabolic action by insulin and IGF-I.

Association of klotho gene polymorphism with bone density and spondylosis of the lumbar spine in postmenopausal women.

Based on the fact that the klotho-deficient mouse exhibits multiple aging phenotypes, including osteopenia and subchondral sclerosis of joints, we explored the possibility of whether human klotho gene polymorphism is associated with two major age-related skeletal disorders: osteoporosis and spondylosis. Analysis of the CA repeat sequence downstream of the final exon of the klotho gene identified ten types of alleles in Japanese postmenopausal women (n = 377). We investigated the association of this microsatellite polymorphism with bone density and spondylosis score of the lumbar spine. None of the genotypes was associated with bone density in the overall population (n = 377; 754 alleles) nor in the subpopulation at not more than 10 years after menopause (<or=10 years, n = 131; 262 alleles). However, the type 5 allele was significantly associated with low bone density in aged subpopulations at 10-20 years after menopause (n = 144; 288 alleles, p = 0.035) and >20 years after menopause (n = 102; 204 alleles, p = 0.024). The type 7 allele was associated with high bone density in women more than 20 years after menopause (p = 0.042). The association study with spondylosis of postmenopausal women (n = 221) revealed that another distinct allele, type 8, was significantly associated with low spondylosis score at L-4/5 (p = 0.019) and L-5/S-1 (p = 0.048) levels in the subpopulation equal to or younger than the average age (<or=63 years old, n = 119; 238 alleles), but not in the older subpopulation. These findings indicate that the klotho gene may be a candidate for the genetic regulation of common age-related diseases like osteoporosis and spondylosis, and we provide the first evidence suggesting that this gene may be involved in the etiology of human diseases.

Distinct roles of Sox5, Sox6, and Sox9 in different stages of chondrogenic differentiation

In mammals, most skeletal elements are formed through endochondral bone formation, which is characterized by the initial formation of cartilage molds from mesenchymal condensations and their subsequent replacement by bones [1]. A small number of skeletal elements, including some craniofacial bones, are formed through intramembranous bone formation, which is characterized by direct transformation of mesenchymal condensations into bones. During the initial step common to both processes, the mesenchyme receives patterning signals that determine the shape, size, and number of mesenchymal condensations [2]. Molecules providing such patterning information are members of the Wnt, hedgehog, and fibroblast growth factor (FGF) families, and the TGF-b superfamily, a series of transcription factors of the Hox, Pax, homeodomain-containing, Forkhead, and basic helix–loop–helix (bHLH) families, and of cell adhesion molecules, including N-cadherin and NCAM [3]. During the endochondral process, cells in the mesenchymal condensations differentiate into chondrocytes, while cells at the periphery become perichondrial cells [4]. Chondrocytes, the primary cell type of cartilage, have a characteristic shape, express a characteristic genetic program, and deposit a characteristic extracellular matrix rich in type II collagen and the proteoglycan aggrecan. The cartilage enlarges through chondrocyte proliferation and matrix production. Subsequently, chondrocytes in the center of the cartilage stop proliferating, dramatically increase in size (hypertrophy), and change their genetic program to synthesize a matrix rich in type X collagen. Hypertrophic chondrocytes play a pivotal role in coupling chondrogenesis and osteogenesis in the endochondral process; they mineralize a surrounding matrix to provide a scaffold for osteoblasts, direct adjacent cells in the perichondrium and the primary spongiosa to become osteoblasts, and attract blood vessels and blood cells, including osteoclasts [5,6]. Hypertrophic chondrocytes then undergo apoptotic cell death. The pathways of chondrocyte and osteoblast differentiation are thus coordinated in time and space during endochondral bone formation. This review summarizes the roles of Sox5, Sox6, and Sox9 in the different steps of chondrogenic differentiation.

Distinct effects of PPARγ insufficiency on bone marrow cells, osteoblasts, and osteoclastic cells

J Bone Miner Metab (2005) 23:275–279

REGULATION OF OSTEOCLAST DIFFERENTIATION BY FIBROBLAST GROWTH FACTOR 2: STIMULATION OF RECEPTOR ACTIVATOR OF NUCLEAR FACTOR KAPPAB LIGAND/OSTEOCLAST DIFFERENTIATION FACTOR EXPRESSION IN OSTEOBLASTS AND INHIBITION OF MACROPHAGE COLONY-STIMULATING FACTOR FUNCTION IN OSTEOCLAST PRECURSORS

This study investigated the mechanism of direct and indirect actions of fibroblast growth factor 2 (FGF‐2) on osteoclast differentiation using two mouse cell culture systems. In the coculture system of osteoblasts and bone marrow cells, FGF‐2 stimulated osteoclast formation. This effect was decreased markedly by osteoprotegerin (OPG) or NS‐398, a selective cyclo‐oxygenase 2 (COX‐2) inhibitor. FGF‐2 (≥10−9 M) stimulated receptor activator of nuclear factor κB ligand/osteoclast differentiation factor (RANKL/ODF) messenger RNA (mRNA) expression from 2 h to 7 days in cultured osteoblasts. NS‐398 did not affect the early induction but decreased the later one, indicating that the later effect is mediated by COX‐2 induction in osteoblasts. To study the direct action of FGF‐2 on osteoclast precursors, we used mouse macrophage‐like cell line C7 cells that can differentiate into osteoclasts in the presence of soluble RANKL/ODF (sRANKL/ODF) and macrophage colony‐stimulating factor (M‐CSF). Although osteoblasts expressed all FGF receptors (FGFR‐1 to −4), only FGFR‐1 was detected in C7 cells at various differentiation stages. FGF‐2 alone or in combination with sRANKL/ODF did not induce osteoclastogenesis from C7 cells; however, FGF‐2 from lower concentrations (≥10−11 M) significantly decreased osteoclast formation induced by M‐CSF in the presence of sRANKL/ODF. FGF‐2 did not alter mRNA levels of M‐CSF receptor (Fms) or RANK in C7 cells. Immunoprecipitation/immunoblotting analyses revealed that tyrosine phosphorylation of several cellular proteins including Fms in C7 cells induced by M‐CSF was inhibited by FGF‐2 in the presence of sRANKL/ODF. We conclude that FGF‐2 regulates osteoclast differentiation through two different mechanisms: (1) an indirect stimulatory action via osteoblasts to induce RANKL/ODF partly through COX‐2 induction and prostaglandin production and (2) a direct inhibitory action on osteoclast precursors by counteracting M‐CSF signaling.

Insulin Secretory Response Is Positively Associated with the Extent of Ossification of the Posterior Longitudinal Ligament of the Spine

Background: Glucose intolerance is frequently found in patients with ossification of the posterior longitudinal ligament of the spine. This study was undertaken to examine the relationship between glucose intolerance and the extent of ossification in patients with ossification of the posterior longitudinal ligament. Methods: A total of 100 patients with ossification of the posterior longitudinal ligament (the overall study group), including fifty-two inpatients who were scheduled to have an operation (the inpatient group) and forty-eight outpatients who had undergone an operation, were analyzed. Indices of glucose metabolism—fasting plasma glucose and serum insulin levels, hemoglobin A1c level, and insulinogenic index (a ratio of the increment of the serum level of insulin to that of glucose)—as well as age and body-mass index were correlated with the extent of ossification, as determined by the number of vertebral levels affected with ossification of the posterior longitudinal ligament (extent of ossification), in the inpatient group. In addition, a similar analysis was performed in twenty-eight inpatients (the selected inpatient group) whose ages and body-mass indices were within one standard deviation of the mean values of those of the inpatient group. Association of a polymorphism in the gene of insulin receptor substrate-1, an essential substrate in insulin signaling, with the extent of ossification was evaluated with genomic DNA extracted from the overall study group. Results: Multiple-regression analysis revealed direct correlations of age (p = 0.038), body-mass index (p = 0.006), and insulinogenic index (p = 0.0003) with the extent of ossification of the posterior longitudinal ligament in the inpatient group. The fasting plasma glucose level, the hemoglobin A1c level, and the stage of glucose tolerance were not associated with the extent of ossification. In the analysis of the selected inpatient group, only the insulinogenic index was correlated with the extent of ossification (p = 0.002). However, no significant association was seen between the insulin receptor substrate-1 polymorphism and the extent of ossification. Conclusions: The insulin secretory response was associated with the extent of ossification of the posterior longitudinal ligament. Since insulin receptor substrate-1 is expressed both in the spinal ligament and in the tissues regulating glucose metabolism, we speculate that some other molecules related to insulin signaling that are impaired only in the tissues regulating glucose metabolism may be responsible for the progression of ossification. We also speculate that the upregulation of insulin production due to the impairment of insulin action may stimulate osteoprogenitor cells in the ligament to induce ossification. Clinical Relevance: The insulinogenic index may be useful as a serum marker for the prediction of progression of ossification of the posterior longitudinal ligament. This study may serve as a stimulus for evaluation of the use of various drugs that may improve the response to insulin in the tissues regulating glucose metabolism to prevent the progression of ossification.