Tomokazu Ohnishi

JNK Activity Is Essential for Atf4 Expression and Late-Stage Osteoblast Differentiation†

Osteoblasts differentiate from mesodermal progenitors and play a pivotal role in bone formation and mineralization. Several transcription factors including runt‐related transcription factor 2 (RUNX2), Osterix (OSX), and activating transcription factor4 (ATF4) are known to be crucial for the process, whereas the upstream signal transduction controlling the osteoblast differentiation sequence is largely unknown. Here, we explored the role of c‐jun N‐terminal kinase (JNK) in osteoblast differentiation using in vitro differentiation models of primary osteoblasts and MC3T3‐E1 cells with ascorbic acid/β‐glycerophosphate treatment. Terminal osteoblast differentiation, represented by matrix mineralization, was significantly inhibited by the inactivation of JNK with its specific inhibitor and exogenous overexpression of MKP‐M (MAP kinase phosphatase isolated from macrophages), which preferentially inactivates JNK. Conversely, enhanced mineral deposition was observed by inducible overexpression of p54JNK2, whereas it was not observed by the overexpression of p46JNK1 or p46JNK2, indicating a distinct enhancing role of p54JNK2 in osteoblast differentiation. Inactivation of JNK significantly inhibited late‐stage molecular events of osteoblast differentiation, including gene expression of osteocalcin (Ocn) and bone sialoprotein (Bsp). In contrast, earlier differentiation events including alkaline phosphatase (ALP) activation and osteopontin (Opn) expression were not inhibited by JNK inactivation. Although the expression levels of two transcription factor genes, Runx2 and Osx, were not significantly affected by JNK inactivation, induction of Atf4 mRNA during osteoblast differentiation was significantly inhibited. Taken together, these data indicate that JNK activity is specifically required for the late‐stage differentiation events of osteoblasts.

Involvement of Oxidative Stress in Tumor Cytotoxic Activity of Hepatocyte Growth Factor/Scatter Factor

In this study, we show thatN-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin trapsN-t-butyl-α-phenylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2′,7′-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.

Low-intensity pulsed ultrasound (LIPUS) induces RANKL, MCP-1, and MIP-1β expression in osteoblasts through the angiotensin II type 1 receptor

Constant mechanical stress is essential for the maintenance of bone mass and strength, which is achieved through the cooperative functions of osteoblasts and osteoclasts. However, it has not been fully elucidated how these cell types mediate mechanical signals. Low‐intensity pulsed ultrasound (LIPUS) therapy is a recently developed method for application of mechanical stress, and is used clinically to promote bone fracture healing. In the present study, we applied LIPUS to osteoblasts at different stages of maturation and analyzed their chemokine and cytokine expression. In comparison with their immature counterparts, mature osteoblasts expressed significantly higher levels of mRNAs for the receptor activator of nuclear factor kappa B ligand (RANKL), monocyte chemoattractant protein (MCP)‐1, and macrophage‐inflammatory protein (MIP)‐1β after a few hours of LIPUS treatment. Intriguingly, protein and mRNA expression of angiotensin II type 1 receptor (AT1), a known mechanoreceptor in cardiomyocytes, was detected in osteoblasts, and the level of expression increased significantly during cell maturation. Furthermore, LIPUS‐induced extracellular signal‐regulated kinase (ERK) phosphorylation and RANKL/chemokine expression was abrogated by a specific AT1 inhibitor. Thus, AT1 may play one of the essential roles in bone metabolism as a mechanoreceptor of osteoblasts. J. Cell. Physiol. 211: 392–398, 2007.

Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation.

Osteoblasts express Toll like receptor (TLR) 4 and produce osteoclast-activating cytokines in response to the stimulation by lipopolysaccharide (LPS). It has recently been reported that LPS exerts an inhibitory effect on osteoblast differentiation into osteocytes. However, the molecular mechanisms of this inhibitory effect remain ambiguous. The downstream signals of TLR4 are mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), leading to the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERKs), whose activation by LPS requires the upstream serine/threonine kinase, Cot/Tpl2. To determine the signal molecules responsible for the inhibitory effects of LPS on osteoblast differentiation, we examined the in vitro differentiation of the primary osteoblasts from myd88(-/-) and cot/tpl2(-/-) mice. The matrix mineralization by the wild-type and cot/tpl2(-/-) osteoblasts was significantly inhibited by LPS, whereas that of myd88(-/-) was not affected. During differentiation, LPS suppressed the mRNA expression of runt related transcription factor 2 (Runx2), osterix (Sp7), and activating transcription factor 4 (ATF4) in the wild-type, but not in the myd88(-/-) osteoblasts. The inhibitory effect of LPS on the mRNA expression of these transcription factors was absent in the early phase but partially impaired in the late phase of differentiation in the cot/tpl2(-/-) osteoblasts. Thus, the inhibitory effect of LPS on osteoblast differentiation is Myd88-dependent, whereas the degree of its requirement for Cot/Tpl2 varies depending on the differentiation phase.

Oxidative stress causes alveolar bone loss in metabolic syndrome model mice with type 2 diabetes

BACKGROUND AND OBJECTIVE Alveolar bone loss is caused by a host response to periodontal pathogens, and its progression is often enhanced by systemic conditions such as insulin resistance. Alveolar bone dehiscence has been observed in KK-A(y) mice, which are metabolic syndrome model mice with type 2 diabetes. The aim of this study was to investigate inducements responsible for alveolar bone dehiscence in the KK-A(y) mice. MATERIAL AND METHODS The expression of endothelial nitric oxide synthase in the mandibles of mice was detected using immunohistochemical staining and the reverse transcription-polymerase chain reaction. After administration of N-acetylcysteine, an antioxidant, to KK-A(y) mice, alveolar bone loss and the expression of endothelial nitric oxide synthase protein in gingival keratinocytes and of hydrogen peroxide concentrations in plasma, were analyzed. The effect of hydrogen peroxide on endothelial nitric oxide synthase expression in keratinocytes was examined using cultured keratinocytes. RESULTS The expression of endothelial nitric oxide synthase was decreased in gingival keratinocytes from KK-A(y) mice compared with gingival keratinocytes from control mice. Administration of N-acetylcysteine to the mice restored endothelial nitric oxide synthase expression in the gingival keratinocytes, suppressed the alveolar bone loss and decreased the hydrogen peroxide concentrations in plasma without the improvement of obesity or diabetes. In vitro, stimulation with hydrogen peroxide decreased the expression level of endothelial nitric oxide synthase in cultured keratinocytes, which was restored by the addition of N-acetylcysteine. CONCLUSION Reactive oxygen species, such as hydrogen peroxide, are responsible for the alveolar bone loss accompanied by decreased endothelial nitric oxide synthase expression in KK-A(y) mice. Therefore, we propose a working hypothesis that the generation of oxidative stress is an underlying systemic condition that enhances alveolar bone loss in periodontitis occurring as a complication of diabetes.

Osteoblast differentiation is functionally associated with decreased AMP kinase activity.

Osteoblasts, originating from mesenchymal stem cells, play a pivotal role in bone formation and mineralization. Several transcription factors including runt‐related transcription factor 2 (Runx2) have been reported to be essential for osteoblast differentiation, whereas the cytoplasmic signal transduction pathways controlling the differentiation process have not been fully elucidated. AMP‐activated protein kinase (AMPK) is a serine–threonine kinase generally regarded as a key regulator of cellular energy homeostasis, polarity, and division. Recent lines of evidence have indicated that the activity of the catalytic α subunit of AMPK is regulated through its phosphorylation by upstream AMPK kinases (AMPKKs) including LKB1. Here, we explored the role of AMPK in osteoblast differentiation using in vitro culture models. Phosphorylation of AMPKα was significantly decreased during osteoblastic differentiation in both primary osteoblasts and MC3T3‐E1, a mouse osteoblastic cell line. Conversely, the terminal differentiation of primary osteoblasts and MC3T3‐E1 cells, represented by matrix mineralization, was significantly inhibited by glucose restriction and stimulation with metformin, both of which are known activators of AMPK. Matrix mineralization of MC3T3‐E1 cells was also inhibited by the forced expression of a constitutively active form of AMPKα. Metformin significantly inhibited gene expression of Runx2 along with osteoblast differentiation markers including osteocalcin (Ocn), bone sialo protein (Bsp), and osteopontin (Opn). Thus, our present data indicate that differentiation of osteoblasts is functionally associated with decreased AMPK activity. J. Cell. Physiol. 221: 740–749, 2009.

Hepatocyte growth factor/scatter factor in development, inflammation and carcinogenesis: its expression and role in oral tissues

Hepatocyte growth factor (HGF) was discovered as a potent mitogen for adult hepatocytes from the plasma of patients with fulminant hepatic failure. It is now known to be a broad-spectrum, multi-functional mitogen, motogen and morphogen. The activities of HGF are mediated through the signalling pathway of its receptor, c-Met. During tooth development, HGF is expressed in the dental papilla and c-Met is expressed in the inner enamel epithelium. The expression of HGF and c-Met indicates that HGF is involved in morphogenesis of the tooth by mediating epithelial-mesenchymal interactions. In the mature tooth, HGF expression by fibroblasts is enhanced in pulpitis and mediated through the induction of prostaglandin (PG) E(2); it is induced not only by inflammatory cytokines, but also by components of oral bacteria. Consequently, concentrations of HGF in gingival crevicular fluid (GCF) increase in periodontitis. The mitogenic and other biological activities, such as angiogenesis, of HGF contribute towards wound healing. Both HGF and c-Met are expressed in the developing tongue, and the signalling pathway of the latter is shown to be essential for myogenesis. Dysregulation of c-Met signalling is observed in carcinogenesis, but HGF also has cytotoxic activity to certain tumour cells. The reason for the discrepancy between these observations is not clear at present.

Low Intensity Pulsed Ultrasound (LIPUS) Influences the Multilineage Differentiation of Mesenchymal Stem and Progenitor Cell Lines through ROCK-Cot/Tpl2-MEK-ERK Signaling Pathway

Background: Low intensity pulsed ultrasound (LIPUS) is a mechanical stimulus clinically used to promote bone fracture healing. Results: LIPUS suppresses adipogenesis and promotes osteogenesis of mesenchyme stem/progenitor cell lines by inhibiting PPARγ2 through ROCK-Cot/Tpl2-MEK-ERK pathway. Conclusion: LIPUS influences multilineage differentiation of mesenchymal stem and progenitor cells. Significance: LIPUS may be a new clinical approach to chronic bone metabolic disorders, including osteoporosis. Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into multilineage cell types, including adipocytes and osteoblasts. Mechanical stimulus is one of the crucial factors in regulating MSC differentiation. However, it remains unknown how mechanical stimulus affects the balance between adipogenesis and osteogenesis. Low intensity pulsed ultrasound (LIPUS) therapy is a clinical application of mechanical stimulus and facilitates bone fracture healing. Here, we applied LIPUS to adipogenic progenitor cell and MSC lines to analyze how multilineage cell differentiation was affected. We found that LIPUS suppressed adipogenic differentiation of both cell types, represented by impaired lipid droplet appearance and decreased gene expression of peroxisome proliferator-activated receptor γ2 (Pparg2) and fatty acid-binding protein 4 (Fabp4). LIPUS also down-regulated the phosphorylation level of peroxisome proliferator-activated receptor γ2 protein, inhibiting its transcriptional activity. In contrast, LIPUS promoted osteogenic differentiation of the MSC line, characterized by increased cell calcification as well as inductions of runt-related transcription factor 2 (Runx2) and Osteocalcin mRNAs. LIPUS induced phosphorylation of cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, which was essential for the phosphorylation of mitogen-activated kinase kinase 1 (MEK1) and p44/p42 extracellular signal-regulated kinases (ERKs). Notably, effects of LIPUS on both adipogenesis and osteogenesis were prevented by a Cot/Tpl2-specific inhibitor. Furthermore, effects of LIPUS on MSC differentiation as well as Cot/Tpl2 phosphorylation were attenuated by the inhibition of Rho-associated kinase. Taken together, these results indicate that mechanical stimulus with LIPUS suppresses adipogenesis and promotes osteogenesis of MSCs through Rho-associated kinase-Cot/Tpl2-MEK-ERK signaling pathway.

LPS‐induced chemokine expression in both MyD88‐dependent and ‐independent manners is regulated by Cot/Tpl2‐ERK axis in macrophages

LPS signaling is mediated through MyD88‐dependent and ‐independent pathways, activating NF‐κB, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS‐mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS‐induced chemokine expression by studying myd88 −/− and cot/tpl2 −/− macrophages. Among the nine LPS‐responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88‐independent pathway. Notably, Cot/Tpl2‐ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS‐induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88‐dependent manner. The Cot/Tpl2‐ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages.

Increased hepatocyte growth factor level in cerebrospinal fluid in Alzheimer's disease.

Background & Objective– Hepatocyte growth factor (HGF), also known as the scatter factor, is a potent mitogen for mature hepatocytes, and also has multifunctional effects on some cells in various organs. Recently, we have found expression and localization of HGF in white matter astrocytes in human brain tissues. Furthermore, immunohistochemistry using anti‐HGF antibody revealed more intense immunolabeling in Alzheimers disease (AD) than control brains. The aim of the study is to investigate the level of HGF in cerebrospinal fluid (CSF) from patients with AD. Material and methods– We examined the level of HGF in CSF from 34 AD and 15 age‐matched disease control patients by highly sensitive enzyme‐linked immunoabsorbent assay (ELISA) system. Results– Consistent with the immunohistochemical data, a significantly higher concentration of HGF in AD CSF was found as compared with controls. A significant correlation was also seen between CSF HGF levels and white matter high‐signal foci determined on brain magnetic resonance imaging (MRI) in AD patients. Conclusion– These results indicate that CSF HGF levels correspond with the white matter damage in AD brain.