Yusuke Shinoda

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.

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.

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.

PEDF regulates osteoclasts via osteoprotegerin and RANKL

Bone homeostasis is maintained through a balance between bone formation and resorption. Bone resorption is mainly carried out by a specific type of cell called the osteoclast (OCL). Previously, expression of pigment epithelium-derived factor (PEDF), the most potent endogenous inhibitor of angiogenesis, has been demonstrated in bone tissue and it known to induce differentiation in osteoblastic cells. Furthermore, therapeutic effects of PEDF on osteosarcoma, a prevalent primary bone tumor, with inhibition of bone destruction has been shown. Thus, PEDF is possibly involved in bone homeostasis as an inhibitor of bone resorption. To address this involvement, we studied the effect of PEDF on OCL function. OCL differentiation, RANKL-mediated survival and bone resorption activity were inhibited by PEDF in a dose-dependent manner. PEDF upregulated osteoprotegerin (OPG), which naturally blocks OCL maturation, in primary osteoblasts and OCL precursor cells. These results suggest that PEDF inhibits OCL function via regulating OPG expression, and thereby contributes to the maintenance of bone homeostasis.

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.

Systemic RANK‐Fc protein therapy is efficacious against primary osteosarcoma growth in a murine model via activity against osteoclasts

Objectives Osteosarcoma (OS) is the most common primary malignant bone tumour, and mainly affects adolescents and young adults. Although there has been substantial improvement in management of OS with surgery and chemotherapy, further survival increase has not been achieved over the past two decades.

Impact of hospital volume on postoperative complications and in-hospital mortality after musculoskeletal tumor surgery: analysis of a national administrative database.

BACKGROUND We are aware of only one report describing the relationship between operative volume and outcomes in musculoskeletal tumor surgery, although numerous studies have described such relationships in other surgical procedures. The aim of the present study was to use a nationally representative inpatient database to evaluate the impact of hospital volume on the rates of postoperative complications and in-hospital mortality after musculoskeletal tumor surgery. METHODS We used the Japanese Diagnostic Procedure Combination administrative database to retrospectively identify 4803 patients who had undergone musculoskeletal tumor surgery during 2007 to 2010. Patients were then divided into tertiles of approximately equal size on the basis of the annual hospital volume (number of patients undergoing musculoskeletal tumor surgery): low, twelve or fewer cases/year; medium, thirteen to thirty-one cases/year; and high, thirty-two or more cases/year. Logistic regression analyses were performed to examine the relationships between various factors and the rates of postoperative complications and in-hospital mortality adjusted for all patient demographic characteristics. RESULTS The overall postoperative complication rate was 7.2% (348 of 4803), and the in-hospital mortality rate was 2.4% (116 of 4803). Postoperative complications included surgical site infections in 132 patients (2.7%), cardiac events in sixty-four (1.3%), respiratory complications in fifty-one (1.1%), sepsis in thirty-one (0.6%), pulmonary emboli in sixteen (0.3%), acute renal failure in eleven (0.2%), and cerebrovascular events in seven (0.1%). The postoperative complication rate was related to the duration of anesthesia (odds ratio [OR] for a duration of more than 240 compared with less than 120 minutes, 2.44; 95% confidence interval [CI], 1.68 to 3.53; p < 0.001) and to hospital volume (OR for high compared with low volume, 0.73; 95% CI, 0.55 to 0.96; p = 0.027). The mortality rate was related to the diagnosis (OR for a metastatic compared with a primary bone tumor, 3.67; 95% CI, 1.66 to 8.09; p = 0.001), type of surgery (OR for amputation compared with soft-tissue tumor resection without prosthetic reconstruction, 3.81; 95% CI, 1.42 to 10.20; p = 0.008), and hospital volume (OR for high compared with low volume, 0.26; 95% CI, 0.14 to 0.50; p < 0.001). CONCLUSIONS We identified an independent effect of hospital volume on outcomes after adjusting for patient demographic characteristics. We recommend regionalization of musculoskeletal tumor surgery to high-volume hospitals in an attempt to improve patient outcomes.

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1−/−) or when osteoblasts were from PTH1R−/− mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010.

Gαq Signal in Osteoblasts Is Inhibitory to the Osteoanabolic Action of Parathyroid Hormone

This study examined the role of the Gαq signal constituted by Gαq and Gα11 (encoded by Gnαq and Gnα11, respectively), a major intracellular pathway of parathyroid hormone (PTH), in the PTH osteoanabolic action by the gain- and loss-of-function analyses. Transgenic mice with osteoblast-specific overexpression of the constitutively active Gnαq gene under the control of 2.3-kb type I collagen α1 chain (Col1a1) promoter exhibited osteopenia with decreased bone formation parameters and did not respond to the daily PTH treatment. We then established osteoblast-specific Gnαq and Gnα11 double-knock-out (cDKO) mice by crossing the 2.3-kb Col1a1 promoter-Cre recombinase transgenic mice and those with Gnαq gene flanked with loxP and global ablation of Gnα11 (Col1a1-Cre+/−;Gnaqfl/fl;Gna11−/−) and found that the cDKO and single knock-out littermates of Gnαq or Gnα11 exhibited normal bone volume and turnover under physiological conditions. With a daily injection of PTH, however, the cDKO mice, but not the single knock-out mice, showed higher bone volume and turnover than the wild-type littermates. Cultures of primary osteoblasts derived from cDKO and wild-type littermates confirmed enhancement of the PTH osteoanabolic action by the Gαq signal deficiency in a cell-autonomous mechanism, in association with the membrane translocation of protein kinase Cδ. This enhancement was reproduced by overexpression of regulator of G protein signaling-2, a Gαq signal inhibitor, in osteoblastic MC3T3-E1 cells. Hence, the Gαq signal plays an inhibitory role in the PTH osteoanabolic action, suggesting that its suppression may lead to a novel treatment in combination with PTH against osteoporosis.

Serum acid phosphatase can be a useful tumour marker for giant cell tumour of bone

IntroductionThe purpose of this study was to elucidate the clinical significance of acid phosphatase in giant cell tumour of bone.Patients and methodsSerum acid phosphatase levels were measured in 32 patients with this tumour both preoperatively and postoperatively.ResultsSerum acid phosphatase value before surgery was high in 15 patients, whereas it was within normal limits in 17 patients. The serum acid phosphatase values of all the 15 patients with high preoperative serum level fell within normal limits postoperatively. In the remaining 17 patients in whom preoperative serum acid phosphatase values were within normal limits, postoperative serum acid phosphatase levels were lower than that of preoperative ones in all the patients. In addition, there was a statistically significant positive correlation between the tumour volume and the preoperative serum acid phosphatase level.ConclusionIt is concluded that serum acid phosphatase is a useful tumour marker for giant cell tumour of bone.