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Rheumatoid arthritis (RA) is a disease characterized by chronic joint inflammation, pain and joint destruction, leading to alteration in activities of daily living, yet pathological mechanisms underlying the condition are not fully clarified. To date, various therapeutic agents have been developed as RA therapy including DMARDs and/or biological agents that target inflammatory cytokines or inhibit JAK. Here we asked whether inhibiting signal transducer and activator of transcription 3 (Stat3) activity would antagonize RA. Stat3 forms dimers when activated and undergoes nuclear translocalization; thus we screened approximately 4.9 million small compounds as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening. We identified 15 as strong candidates as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening from those compounds. Four of the 15 significantly inhibited expression of IL-6 and RANKL , both of which are direct targets of Stat3, induced by IL-6. Among four, one compound, F0648-0027, significantly inhibited arthritis development without apparent adverse effects in vivo in collagen-induced arthritis model mice. F0648-0027 also significantly blocked Stat3 phosphorylation and nuclear localization following IL-6 stimulation of fibroblasts. These data suggest that Stat3 is a target for collagen-induced arthritis in mice, and that F0648-0027 could serve as a therapeutic reagent against comparable conditions in humans. • Stat3 inhibiting compounds were screened from approximately 4.9 million compounds. • F0648-0027 was isolated as the most effective Stat3 inhibitor in vitro and in vivo. • Phosphorylation of Stat3 by IL-6 stimulation was inhibited by F0648-0027. • Nuclear translocation of Stat3 by IL-6 stimulation was inhibited by F0648-0027. • Arthritis score in RA mouse models was significantly inhibited by F0648-0027. [ABSTRACT FROM AUTHOR]

Skeletal muscle is known to regulate bone homeostasis through muscle-bone interaction, although factors that control this activity remain unclear. Here, we newly established Smad3-flox mice, and then generated skeletal muscle-specific Smad2/Smad3 double conditional knockout mice (DcKO) by crossing Smad3-flox with skeletal muscle-specific Ckmm Cre and Smad2-flox mice. We show that immobilization-induced gastrocnemius muscle atrophy occurring due to sciatic nerve denervation was partially but significantly inhibited in DcKO mice, suggesting that skeletal muscle cell-intrinsic Smad2/3 is required for immobilization-induced muscle atrophy. Also, tibial bone atrophy seen after sciatic nerve denervation was partially but significantly inhibited in DcKO mice. Bone formation rate in wild-type mouse tibia was significantly inhibited by immobilization, but inhibition was abrogated in DcKO mice. We propose that skeletal muscle regulates immobilization-induced bone atrophy via Smad2/3, and Smad2/3 represent potential therapeutic targets to prevent both immobilization-induced bone and muscle atrophy. • Smad3 flox mice were newly established. • Skeletal muscle specific Smad2/3 conditional knockout (Smad2/3 cKO) were generated. • Smad2/3 cKO are resistant to denervation induced muscle and bone atrophy. • Denervation induced bone atrophy is promoted by Smad2/3 in muscles. [ABSTRACT FROM AUTHOR]

Auto-inflammatory syndromes are rare diseases characterized by arthritis and joint destruction, symptoms similar to but distinct from rheumatoid arthritis (RA). Therapeutic targets have not been well characterized for auto-inflammatory syndromes, although the E3 ligase Synoviolin was previously shown to be a novel therapeutic target for RA. Here, we show that Synoviolin loss has little impact on a model of auto-inflammatory diseases. We previously established such a model, the hIL-1 cTg mouse, in which IL-1 signaling was constitutively activated, and animals exhibit symptoms recapitulating auto-inflammatory syndromes such as major joint dominant arthritis. Here, we crossed hIL-1 cTg with Synoviolin flox'd mice to yield hIL-1 cTg/Synoviolin cKO mice. Synoviolin gene expression was ablated in adult hIL-1 cTg/Synoviolin cKO mice by injection of pIpC to activate Mx1 promoter-driven Cre recombinase. However, symptoms seen in hIL-1 cTg mice such as arthritis and joint destruction were not alleviated by targeting Synoviolin, ruling out Synoviolin as a therapeutic target for auto-inflammatory disease. Our results indicate that although similar, RA and auto-inflammatory diseases are different diseases, and treatment strategies should differ accordingly. • Synoviolin is a pathogenic factor for rheumatoid arthritis, an auto-immune diseases. •Synoviolin accumulates in synovial cells of joints in hIL-1 cTg mouse, an auto-inflammatory disease model. •Arthritis and joint destruction seen in hIL-1 cTg mice are unchanged by Synoviolin deficiency. •Synoviolin dose not promote auto-inflammatory disease development. [ABSTRACT FROM AUTHOR]

Estrogen deficiency can be caused by ovarian dysfunction in females. Mechanisms underlying osteoporosis in this condition have been characterized in animal models, such as ovariectomized mice and rats, although it remains unclear how hypothalamic dysfunction promotes osteoporosis. Here, we show that administration of a gonadotropin-releasing hormone antagonist (GnRHa) significantly decreases uterine weight, a manifestation of hypothalamic dysfunction, and promotes both cortical and trabecular bone loss in female mice in vivo. We also report that osteoclast number significantly increased in mice administered GnRHa, and that the transcription factor hypoxia inducible factor 1 alpha (HIF1α) accumulated in those osteoclasts. We previously reported that treatment of mice with the active vitamin D analogue ED71, also known as eldecalcitol, inhibited HIF1α accumulation in osteoclasts. We show here that in mice, co-administration of ED71 with GnRHa significantly rescued the reduced cortical and trabecular bone mass promoted by GnRHa administration alone. GnRHa-dependent HIF1α accumulation in osteoclasts was also blocked by co-administration of ED71. We conclude that hypothalamic dysfunction promotes HIF1α accumulation in osteoclasts and likely results in reduced bone mass. We conclude that treatment with ED71 could serve as a therapeutic option to counter osteoporotic conditions in humans. • Administration of a gonadotropin-releasing hormone antagonist (GnRHa) reduces cortical and trabecular bone mass in female mice. • Hypoxia inducible factor 1 alpha (HIF1α) accumulates in osteoclasts from mice administered GnRHa. • ED71 treatment significantly rescues reduced bone mass seen in GnRHa-treated mice. • HIF1α accumulation in osteoclasts of mice administered GnRHa is blocked by simultaneous ED71 treatment. [ABSTRACT FROM AUTHOR]

Low energy availability in female athletes often causes hypothalamic amenorrhea and osteoporosis, in turn promoting stress fractures. Mechanisms underlying these conditions remain unclear. Here we show that model mice subjected to food restriction (FR) or FR-plus-voluntary running exercise exhibit significantly reduced bone mineral density, cortical bone parameters and uterine weight than do control mice, and that these parameters worsen in the FR-plus-exercise group. Relative to controls, FR and FR-plus-exercise groups showed significantly lower mineral apposition rate and osteoclast number and significantly reduced serum insulin-like growth factor-1 (IGF1) levels. Outcomes were rescued by ED71 or 1.25(OH) 2 D 3 treatment. Thus, we conclude that administration of active vitamin D analogues represents a possible treatment to prevent these conditions. • Food restriction (FR) reduces cortical bone mass. • Exercise worsens reduced cortical bone mass by FR. • FR or FR-plus-exercise promotes a significantly lower mineral apposition rate (MAR) and decreases serum insulin-like growth factor-1 (IGF1) levels relative to controls. • Reduced cortical bone mass, MAR and serum IGF1 levels by FR are significantly restored by treatment with active vitamin D analogues. [ABSTRACT FROM AUTHOR]

Abstract Granulocyte-colony stimulating factor (G-CSF) is a cytokine crucially involved in the regulation of granulopoiesis and the mobilization of hematopoietic stem cells from bone marrow. However, emerging data suggest that G-CSF exhibits more diverse functions than initially expected, such as conferring protection against apoptosis to neural cells and stimulating mitogenesis in cardiomyocytes and skeletal muscle stem cells after injury. In the present study, we sought to investigate the potential contribution of G-CSF to the regulation of muscle volume. We found that the administration of G-CSF significantly enhances muscle hypertrophy in two different muscle overload models. Interestingly, there was a significant increase in the transcripts of both G-CSF and G-CSF receptors in the muscles that were under overload stress. Using mutant mice lacking the G-CSF receptor, we confirmed that the anabolic effect is dependent on the G-CSF receptor signaling. Furthermore, we found that G-CSF increases the diameter of myotubes in vitro and induces the phosphorylation of AKT, mTOR, and ERK1/2 in the myoblast-like cell line C2C12 after differentiation induction. These findings indicate that G-CSF is involved in load-induced muscle hypertrophy and suggest that G-CSF is a potential agent for treating patients with muscle loss and sarcopenia. Highlights • G-CSF enhances load-induced muscle hypertrophy in mice. • Transcripts of G-CSF and its receptor are upregulated in skeletal muscle under overload. • G-CSF increases the diameter of myotubes formed in vitro. • G-CSF induces the phosphorylation of AKT and mTOR. [ABSTRACT FROM AUTHOR]

It is shown that chalcogenide double-clad fibers (Ch-DCFs) have large potential to realize the highly coherent mid-infrared (MIR) supercontinuum generation. We design the Ch-DCFs with flatter chromatic dispersion and smaller effective mode diameter than chalcogenide step index fibers in the MIR region. We numerically investigate MIR supercontinuum generation in designed Ch-DCFs. It is numerically demonstrated that highly coherent MIR supercontinuum extending from 3.3 to 10.4 μm in the Ch-DCFs can be generated by pumping with hyperbolic secant pulse at 6 μm, the peak power of 2 kW, and the full width at half maximum of 300 fs. [ABSTRACT FROM AUTHOR]

Rapid increases in the number of elderly people have dramatically increased the number of female and male osteoporosis patients. Osteoporosis often causes bone fragility fractures, and males exhibit particularly poor prognosis after these fractures, indicating that control of osteoporosis is crucial to maintain quality of men's lives. However, osteoporosis therapies available for men have lagged behind advances available for women. Here, we show that three selective estrogen receptor modulators (SERMs), namely, raloxifene, bazedoxifene, and tamoxifen, plus the vitamin D analogue ED71, also called eldecalcitol, completely block orchiectomy-induced, testosterone-depleted bone loss in male mice in vivo . Patients treated with hormone deprivation therapy for prostate cancer also exhibit male osteoporosis, and bone management is critical for these patients. Given that androgen replacement therapy is not an option for these patients, our results represent a novel approach potentially useful to control male osteoporosis. [ABSTRACT FROM AUTHOR]

Both bone and muscle volume is concomitantly reduced under immobilization conditions; however, no single drug is currently available to block these outcomes simultaneously. Bisphosphonates are utilized clinically to inhibit osteoclast-dependent bone resorption, but their effects on muscle are largely unknown. Here we show that skeletal muscle is a direct target of the bisphosphonate ibandronate (IBN) and that reduced muscle volume and induction of Atrogin-1 and MuRF1 , both atrogenes, are significantly inhibited by IBN administration in vivo using a mouse model of muscle atrophy. IBN treatment also significantly blocked immobilization-induced bone loss in vivo . We also report that expression of Atrogin-1 and MuRF1 and accumulation of Smad2/3 proteins, which are upstream of atrogines, occurred following serum starvation of myogenic C2C12 cells in vitro , effects significantly inhibited by IBN treatment. Interestingly, IBN effects on C2C12 cells were abrogated by MG132, an ubiquitin/proteasome inhibitor, suggesting that IBN functions via the ubiquitin-proteasome system. Our findings lend new insight into the role of IBN in preventing muscle atrophy. [ABSTRACT FROM AUTHOR]

The number of osteoporosis patients is increasing not only in women but in men. Male osteoporosis occurs due to aging or androgen depletion therapies, leading to fractures. However, molecular mechanisms underlying male osteoporosis remain unidentified. Here, we show that hypoxia inducible factor 1 alpha (Hif1α) is required for development of testosterone deficiency-induced male osteoporosis. We found that in mice Hif1α protein accumulates in osteoclasts following orchidectomy (ORX) in vivo . In vitro , Hif1α protein accumulated in osteoclasts cultured in hypoxic conditions, but Hif1α protein rather than mRNA levels were suppressed by testosterone treatment, even in hypoxia. Administration of a Hif1α inhibitor to ORX mice abrogated testosterone deficiency-induced osteoclast activation and bone loss but did not alter osteoclast activities or bone phenotypes in sham-operated, testosterone-sufficient animals. We conclude that Hif1α protein accumulation due to testosterone-deficiency promotes development of male osteoporosis. Thus Hif1α protein could be targeted to inhibit pathologically-activated osteoclasts under testosterone-deficient conditions to treat male osteoporosis patients. [ABSTRACT FROM AUTHOR]

Bone mass is tightly controlled by a balance between osteoclast and osteoblast activities. Although these cell types mature via different pathways, some factors reportedly regulate differentiation of both. Here, in a search for factors governing osteoblastogenesis but also expressed in osteoclasts to control both cell types by one molecule, we identified B cell lymphoma 6 (Bcl6) as one of those factors and show that it promotes osteoblast differentiation. Bcl6 was previously shown to negatively regulate osteoclastogenesis. We report that lack of Bcl6 results in significant inhibition of osteoblastogensis in vivo and in vitro and in defects in secondary ossification center formation in vivo . Signal transducer and activator of transcription 1 (Stat1) reportedly attenuates osteoblast differentiation by inhibiting nuclear translocation of runt-related transcription factor 2 (Runx2), which is essential for osteoblast differentiation. We found that lack of Bcl6 resulted in significant elevation of Stat1 mRNA and protein expression in osteoblasts and showed that Stat1 is a direct target of Bcl6 using a chromatin immune-precipitation assay. Mice lacking both Bcl6 and Stat1 (DKO) exhibited significant rescue of bone mass and osteoblastic parameters as well as partial rescue of secondary ossification center formation compared with Bcl6-deficient mice in vivo . Altered osteoblastogenesis in Bcl6-deficient cells was also restored in DKO in vitro . Thus, Bcl6 plays crucial roles in regulating both osteoblast activation and osteoclast inhibition. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-1 is one of the most evolutionarily conserved cytokines and plays an essential role in the regulation of innate immunity. IL-1 binds to two different receptors, IL-1R1 and IL-1R2, which share approximately 28% amino acid homology. IL-1R1 contains a cytoplasmic domain and is capable of transducing cellular signals; by contrast, IL-1R2 lacks a functional cytoplasmic domain and serves as a decoy receptor for IL-1. Interestingly, IL-1R2 is proteolytically cleaved and also functions as a soluble receptor that blocks IL-1 activity. In the present study, we examined the shedding properties of IL-1R2 and demonstrate that ADAM17 is de facto the major sheddase for IL-1R2 and that introducing a mutation into the juxta-membrane domain of IL-1R2 significantly desensitizes IL-1R2 to proteolytic cleavage. IL-1R1 was almost insensitive to ADAM17-dependent cleavage; however, the replacement of the juxta-membrane domain of IL-R1 with that of IL-1R2 significantly increased the sensitivity of IL-1R1 to shedding. Furthermore, we demonstrate that ADAM17 indirectly enhances IL-1 signaling in a cell-autonomous manner by selectively cleaving IL-1R2. Taken together, the data collected in the present study indicate that ADAM17 affects sensitivity to IL-1 by changing the balance between IL-1R1 and the decoy receptor IL-1R2. [ABSTRACT FROM AUTHOR]

Highlights: [•] A chalcogenide–tellurite microstructured optical fiber was fabricated. [•] The flattened chromatic dispersion can be achieved at λ =1.35–1.7μm. [•] Supercontinuum generation in this fiber was investigated. [ABSTRACT FROM AUTHOR]

Rehabilitative treatment, including treadmill training, is considered an important strategy for restoring motor function after spinal cord injury (SCI). However, many unexplained problems persist regarding the appropriate rehabilitative method and the mechanism underlying the beneficial effects of rehabilitation. Moreover, only a few preclinical studies have been performed on rehabilitative interventions for chronic SCI, although most patients have chronic injuries. In fact, several preclinical studies reported that rehabilitative training was less effective when applied during the chronic phase than when applied sooner. While numerous studies have examined the effects of treadmill training during the subacute phase, the training conditions vary considerably among preclinical reports. Therefore, establishing a standard training protocol is essential for achieving beneficial rehabilitation effects at the chronic stage. Since the difficulty of applying an appropriate training load hinders training at constant speeds, it is important to adjust the training intensity in accordance with the exercise tolerance of an individual animal to provide further functional recovery benefits. Here, we created a novel quadrupedal treadmill training protocol based on the overload principle for mice with incomplete thoracic SCI. We subjected SCI model mice to rehabilitative training according to the protocol for two consecutive weeks starting at 42 days after injury. We examined the treadmill speeds at which the mice were able to run based on the severity of paresis and investigated the impact of the protocol on functional recovery. Assessment of running speed changes during the treadmill training period revealed faster treadmill speeds for mice with mild paresis than for those with severe paresis. The training parameters, including the speed and distance traveled, were positively correlated with the changes in motor function. These results suggest that the most suitable running speed during treadmill training differs according to the level of motor dysfunction and that running longer distances has a positive impact on motor functional recovery. Based on this established protocol, we compared functional and histological results between the chronic SCI groups with and without rehabilitation. The gait analyses showed significantly better functional improvement in the rehabilitation group than in the nonrehabilitation group. Histological analyses revealed that the BDNF- and VGLUT1-positive areas of lumbar enlargement were significantly increased in the rehabilitation group. These findings implied that rehabilitation promoted not only motor performance but also motor control, including forelimb-hindlimb coordination, even in chronic SCI, resulting in functional improvement by treadmill training alone. Therefore, rehabilitative training based on the overload principle appears to be one of the appropriate treatment options for incomplete thoracic SCI, and evidence of its efficacy exists in actual clinical settings. • We developed a novel treadmill training protocol based on the overload principle for mice with incomplete thoracic SCI. • Spinal cord-injured mice with milder paresis could adapt to a faster treadmill speed than those with more severe paresis. • Motor functional recovery correlated with the distance traveled. • Treadmill training based on the overload principle had a positive impact on motor function and histological results. [ABSTRACT FROM AUTHOR]

Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis. [Copyright &y& Elsevier]

Abstract: The intervertebral disc (IVD) is composed of two avascular tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). IVDs is the largest avascular tissue in the human body, however, how these tissues are maintained without a blood supply is poorly understood. Here we show that vascular endothelial growth factor-A (VEGF-A) is highly expressed in NP and that VEGF-A plays a role in NP survival. High VEGF-A expression in NP was detected by microarray analysis, and NP was positive for the hypoxic probe pimonidazole and hypoxia-responsive genes. VEGF-A expression in NP was promoted by hypoxic conditions in vitro. NP cells also expressed the membrane-bound VEGF receptor-1 (VEGFR-1), and the number of apoptotic cells in cultured cell model of NP increased following treatment with VEGFR-1-Fc, which traps VEGF-A in NP. These results indicate that NP is a hypoxic tissue, and that VEGF-A functions in NP survival in an autocrine/paracrine manner. [Copyright &y& Elsevier]

Abstract: Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD. [Copyright &y& Elsevier]