The protective effect of IL-12/23 neutralizing antibody in sarcopenia associated with dextran sulfate sodium-induced experimental colitis.

BACKGROUND: The improvement of colitis symptoms by treatment with IL-12/23 p40 neutralizing antibody should increase the muscle mass and the function of the sarcopenia phenotype. METHODS: An experimental colitis model was induced by oral administration of 2% dextran sulfate sodium (DSS) for 7 days. During induction of colitis, IL-12/23 p40 neutralizing antibody was injected twice on Days 3 and 5. The total body mass index was measured by dual-energy X-ray absorptiometry. The muscle function was measured by forelimb grip strength and fatigue running distance. The muscle fibre cross-sectional area (CSA) was calculated after the transverse section and haematoxylin and eosin staining, and gene expression was confirmed by RT-qPCR. Differentiated C2C12 cells were used as in vitro models and treated with recombinant IL12/23 proteins to mimic the enhanced cytokines in colitis. RESULTS: The symptoms of colitis were alleviated by injection of IL-12/23 p40 neutralizing antibody compared with phosphate-buffered saline (PBS), and the disease activity index score was significantly lower on Day 8 (0.0 ± 0.00 of cont. vs. 11.3 ± 0.9 of DSS + PBS, P < 0.0001; DSS + PBS vs. 7.7 ± 1.25 of DSS + p40Ab, P < 0.0001). The CSA of the gastrocnemius and tibialis anterior muscle fibres decreased in mice with DSS-induced colitis (gastrocnemius, 1258.2 µm2 ± 176.45 of cont. vs. 640.1 µm2 ± 59.83 of DSS + PBS, P < 0.0001; tibialis anterior, 1251.8 µm2 ± 331.48 of cont. vs. 678.9 µm2 ± 67.59 of DSS + PBS, P < 0.0001), and the treatment of IL-12/23 p40 neutralizing antibody partially restored CSA of the gastrocnemius (640.1 µm2 ± 59.83 of DSS + PBS vs. 1062.0 µm2 ± 83.41 of DSS + p40Ab, P < 0.0001) and tibialis anterior (678.9 µm2 ± 67.59 of DSS + PBS vs. 1105.3 µm2 ± 143.15 of DSS + p40Ab, P = 0.0003).vs. 640.1 µm2  ± 59.83 of DSS + PBS, P < 0.0001) and tibialis anterior (1251.8 µm2  ± 331.48 of cont. vs. 678.9 µm2  ± 67.59 of DSS + PBS, P < 0.0001), and the treatment of IL-12/23 p40 neutralizing antibody partially restored CSA of the gastrocnemius (640.1 µm2  ± 59.83 of DSS + PBS vs. 1062.0 µm2  ± 83.41 of DSS + p40Ab, P < 0.0001) and tibialis anterior (678.9 µm2  ± 67.59 of DSS + PBS vs. 1105.3 µm2  ± 143.15 of DSS + p40Ab, P = 0.0003). In the evaluation of muscle function, grip strength and fatigue distance decreased by colitis were partially restored (grip strength: 139.9 g ± 5.38 of cont. vs. 83.9 g ± 5.48 of DSS + PBS, P < 0.0001; DSS + PBS vs. 118.6 g ± 4.05 of DSS + p40Ab, P < 0.0001; fatigue distance: 872.5 m ± 104.01 of cont. vs. 58.2 m ± 107.72 of DSS + PBS, P < 0.0001; DSS + PBS vs. 328.0 m ± 109.71 of DSS + p40Ab, P = 0.0015) by injection of IL-12/23 p40 neutralizing antibody. CONCLUSIONS: Our study demonstrates that Il-12/23 acts directly on muscle to induce atrophy, and the IL-12/23 p40 neutralizing antibody is effective not only in suppressing colitis but also in maintaining muscle mass and improving muscle function in an experimental colitis model.

Changes in aquaporins expression due to acute water restriction in naturally aging mice

Aquaporins (AQPs) are water channels in the cell membrane that regulate osmosis in response to rapid changes in intracellular and extracellular fluid concentration caused by extrinsic factors. While there are so many studies on the association of AQPs with muscular atrophy, sarcopenia, and Duchenne muscular dystrophy (DMD), the expression of AQP has not been verified in naturally aging mice or humans. Notably, due to the characteristics of AQPs, the difference in function cannot be evaluated without extrinsic factors such as acute water restriction. The purpose of this study was to investigate the changes in AQPs expression and function due to natural aging under acute water restriction conditions in aging mice. The expression of AQP4 was shown to decrease with aging similar to previous studies. However, for the first time, this study results confirmed that AQP1 expression increased in aging mice. In addition, the expression of Aqp1 decreased in the acute water restricted group compared to the control group after acute water restriction in aging mice. These results suggest that although the expression of AQP1 increases with aging, its function is reduced. We also confirmed that overexpression of Aqp1 can inhibit myotube differentiation and that knockdown can promote myotube differentiation through in vitro experiments. In conclusion, based on our results, we suggest that the AQP1 is an important factor in sarcopenia caused by natural aging accompanied by chronic dehydration. (AU)

Changes in aquaporins expression due to acute water restriction in naturally aging mice.

Aquaporins (AQPs) are water channels in the cell membrane that regulate osmosis in response to rapid changes in intracellular and extracellular fluid concentration caused by extrinsic factors. While there are so many studies on the association of AQPs with muscular atrophy, sarcopenia, and Duchenne muscular dystrophy (DMD), the expression of AQP has not been verified in naturally aging mice or humans. Notably, due to the characteristics of AQPs, the difference in function cannot be evaluated without extrinsic factors such as acute water restriction. The purpose of this study was to investigate the changes in AQPs expression and function due to natural aging under acute water restriction conditions in aging mice. The expression of AQP4 was shown to decrease with aging similar to previous studies. However, for the first time, this study results confirmed that AQP1 expression increased in aging mice. In addition, the expression of Aqp1 decreased in the acute water restricted group compared to the control group after acute water restriction in aging mice. These results suggest that although the expression of AQP1 increases with aging, its function is reduced. We also confirmed that overexpression of Aqp1 can inhibit myotube differentiation and that knockdown can promote myotube differentiation through in vitro experiments. In conclusion, based on our results, we suggest that the AQP1 is an important factor in sarcopenia caused by natural aging accompanied by chronic dehydration.

Effects of lifelong spontaneous exercise on skeletal muscle and angiogenesis in super-aged mice.

There has been an increasing awareness of sarcopenia, which is characterized by a concomitant decrease in skeletal muscle mass and quality due to aging. Resistance exercise is considered more effective than aerobic exercise in terms of therapeutic exercise. To confirm the effect of long-term aerobic exercise in preventing sarcopenia, we evaluated the skeletal muscle mass, quality, and angiogenic capacity of super-aged mice that had undergone lifelong spontaneous exercise (LSE) through various experiments. Our findings show that LSE could maintain skeletal muscle mass, quality, and fitness levels in super-aged mice. In addition, ex vivo experiments showed that the angiogenic capacity was maintained at a high level. However, these results were not consistent with the related changes in the expression of genes and/or proteins involved in protein synthesis or angiogenesis. Based on the results of previous studies, it seems certain that the expression at the molecular level does not represent the phenotypes of skeletal muscle and angiogenesis. This is because aging and long-term exercise are variables that can affect both protein synthesis and the expression patterns of angiogenesis-related genes and proteins. Therefore, in aging and exercise-related research, various physical fitness and angiogenesis variables and phenotypes should be analyzed. In conclusion, LSE appears to maintain the potential of angiogenesis and slow the aging process to maintain skeletal muscle mass and quality. Aerobic exercise may thus be effective for the prevention of sarcopenia.

Two Types of Mouse Models for Sarcopenia Research: Senescence Acceleration and Genetic Modification Models.

Sarcopenia leads to loss of skeletal muscle mass, quality, and strength due to aging; it was recently given a disease code (International Classification of Diseases, Tenth Revision, Clinical Modification, M62.84). As a result, in recent years, sarcopenia-related research has increased. In addition, various studies seeking to prevent and treat sarcopenia by identifying the various mechanisms related to the reduction of skeletal muscle properties have been conducted. Previous studies have identified muscle synthesis and breakdown; investigating them has generated evidence for preventing and treating sarcopenia. Mouse models are still the most useful ones for determining mechanisms underlying sarcopenia through correlations and interventions involving specific genes and their phenotypes. Mouse models used to study sarcopenia often induce muscle atrophy by hindlimb unloading, denervation, or immobilization. Though it is less frequently used, the senescence-accelerated mouse can also be useful for sarcopenia research. Herein, we discuss cases where senescence-accelerated and genetically engineered mouse models were used in sarcopenia research and different perspectives to use them.

Comparative analysis of the association between various serum vitamin D biomarkers and sarcopenia.

BACKGROUND: Vitamin D status is associated with muscle strength and maintenance of muscle fibers. However, which serum vitamin D biomarker better reflects sarcopenia remains unclear. The aim of this study was to investigate associations between various serum vitamin D biomarkers (total 25-hydroxy vitamin D [25(OH)D], bioavailable 25(OH)D, 24,25-dihydroxyvitamin D [24,25(OH)2 D], and vitamin D metabolite ratio [VMR]) and sarcopenia. METHODS: The data for 83 hip fracture patients were finally included in the analysis. Sarcopenia was defined according to the Asia Working Group for Sarcopenia (AWGS) criteria. Measurements of 24,25(OH)2 D and 25(OH)D were made using solid-phase extraction (SPE) and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Vitamin D binding protein (VDBP) concentration was measured using an enzyme-linked immunosorbent assay. The VMR was calculated by dividing serum 24,25(OH)2 D by serum 25(OH)D and then multiplying by 100. Based on total 25(OH)D, VDBP, and albumin concentrations, bioavailable 25(OH)D concentrations were calculated using the equations from the other previous studies. RESULTS: Bioavailable 25(OH)D levels were significantly (p = 0.030) decreased in the sarcopenia group compared with the non-sarcopenia group. Results of ROC analysis for the diagnosis of sarcopenia using serum level of bioavailable of 25(OH)D revealed that the cutoff point for bioavailable 25(OH)D was 1.70 ng/ml (AUC = 0.649, p < 0.001). In the group with a bioavailable 25(OH)D less than 1.70 ng/ml, the incidence of sarcopenia increased by 3.3 times (odds ratio: 3.33, p = 0.013). CONCLUSION: We demonstrated that bioavailable 25(OH)D was associated with sarcopenia among the various serum vitamin D biomarkers. Bioavailable vitamin D might be helpful for assessing the risk of sarcopenia.

Protective Role of Limitrin in Experimental Autoimmune Optic Neuritis.

Purpose: This study investigated the role of limitrin in the pathogenesis of demyelinating optic neuritis using an experimental autoimmune optic neuritis (EAON) model. Methods: EAON was induced in mice via subcutaneous injection with myelin oligodendrocyte glycoprotein peptide. Limitrin protein and mRNA expression were examined in the optic nerve before and after EAON induction. Proinflammatory cytokine expression profiles and degree of glial activation were compared between wild-type (WT) and limitrin knockout mice by real-time PCR and histologic analysis, respectively, after EAON induction. Plasma limitrin levels in patients with optic neuritis and healthy controls were measured by ELISA. Results: Limitrin expression, observed in astrocytes in the optic nerve of WT mice, was lower in EAON-induced than in naïve WT mice. A comparative analysis of WT and limitrin knockout mice revealed that limitrin deficiency induced more severe neuroinflammation and glial hyperactivation in the optic nerve after EAON induction. Limitrin-deficient astrocytes were more chemotactically responsive to neuroinflammatory stimulation than WT astrocytes. Patients with optic neuritis demonstrated higher plasma limitrin levels than healthy controls (P = 0.0001), which was negatively correlated with visual acuity at the nadir of the optic neuritis attack (r = 0.46, P = 0.036). Conclusions: Limitrin deficiency induced severe neuroinflammation and reactive gliosis in the optic nerve after EAON induction. Our results imply that astrocyte-derived limitrin may protect against neuroinflammation by decreasing immune cell infiltration into the optic nerve. The plasma limitrin level may reflect the extent of blood-brain barrier disruption and provide a valuable biomarker reflecting the severity of optic neuritis.

Clinical utility of cerebrospinal fluid vitamin D-binding protein as a novel biomarker for the diagnosis of viral and bacterial CNS infections.

BACKGROUND: Rapid and accurate diagnosis of central nervous system (CNS) infections is important, and laboratory tests help diagnose CNS infections. Even when the patient has symptoms, laboratory tests often do not reveal any specific findings. The potential of vitamin D-binding protein (VDBP) to be used as a biomarker for viral and bacterial CNS infections was studied. METHODS: A total of 302 subjects with suspected CNS infection who underwent lumbar puncture were included. Clinical and laboratory data were collected retrospectively. VDBP levels were measured in the cerebrospinal fluid (CSF) samples. Genotyping for the GC gene encoding VDBP was also performed. VDBP levels were analyzed and compared by CNS infection, pathogen, CSF opening pressure, and GC genotype. RESULTS: A CNS infection group (n = 90) and a non-CNS infection group (n = 212) were studied. In terms of its receiver operating characteristic, CSF VDBP showed an area under the curve of 0.726 for the diagnosis of CNS infection. CSF VDBP levels were significantly different between the CNS infection and non-infection groups. The CNS infection group with enterovirus showed a statistically lower distribution of CSF VDBP levels than the other virus groups. The group with CSF opening pressure > 25 cmH2O showed higher CSF VDBP levels than the other groups. There was no significant difference in GC gene allele distribution between the CNS infection and non-infection groups. CONCLUSIONS: CSF VDBP levels were increased in patients with CNS infection. The CSF VDBP showed potential as a new biomarker for viral and bacterial CNS infections.

Hypoxia-inducible factor 2α is a novel inhibitor of chondrocyte maturation.

Hypoxic environment is essential for chondrocyte maturation and longitudinal bone growth. Although hypoxia-inducible factor 1 alpha (Hif-1α) has been known as a key player for chondrocyte survival and function, the function of Hif-2α in cartilage is mechanistically and clinically relevant but remains unknown. Here we demonstrated that Hif-2α was a novel inhibitor of chondrocyte maturation through downregulation of Runx2 stability. Mechanistically, Hif-2α binding to Runx2 inhibited chondrocyte maturation by Runx2 degradation through disrupting Runx2/Cbfß complex formation. The Hif-2α-mediated-Runx2 degradation could be rescued by Cbfß transfection due to the increase of Runx2/Cbfß complex formation. Consistently, mesenchymal cells derived from Hif-2α heterozygous mice were more rapidly differentiated into hypertrophic chondrocytes than those of wild-type mice in a micromass culture system. Collectively, these findings demonstrate that Hif-2α is a novel inhibitor for chondrocyte maturation by disrupting Runx2/Cbfß complex formation and consequential regulatory activity.

Transglutaminase-2 regulates Wnt and FoxO3a signaling to determine the severity of osteoarthritis.

Transglutaminase 2 (TG2), also known as tissue transglutaminase, is a calcium-dependent enzyme that has a variety of intracellular and extracellular substrates. TG2 not only increases in osteoarthritis (OA) tissue but also affects the progression of OA. However, it is still unclear how TG2 affects cartilage degradation in OA at the molecular level. Surgically induced OA lead to an increase of TG2 in the articular cartilage and growth plate, and it was dependent on TGFß1 in primary chondrocytes. The inhibition of TG2 enzymatic activity with intra-articular injection of ZDON, the peptide-based specific TG2 inhibitor, ameliorated the severity of surgically induced OA as well as the expression of MMP-3 and MMP-13. ZDON attenuated MMP-3 and MMP-13 expression in TGFß- and calcium ionophore-treated chondrocytes in a Runx2-independent manner. TG2 inhibition with ZDON suppressed canonical Wnt signaling through a reduction of ß-catenin, which was mediated by ubiquitination-dependent proteasomal degradation. In addition, TG2 activation by a calcium ionophore enhanced the phosphorylation of AMPK and FoxO3a and the nuclear translocation of FoxO3a, which was responsible for the increase in MMP-13. In conclusion, TG2 plays an important role in the pathogenesis of OA as a major catabolic mediator that affects the stability of ß-catenin and FoxO3a-mediated MMP-13 production.

Rodent Model of Muscular Atrophy for Sarcopenia Study.

The hallmark symptom of sarcopenia is the loss of muscle mass and strength without the loss of overall body weight. Sarcopenia patients are likely to have worse clinical outcomes and higher mortality than do healthy individuals. The sarcopenia population shows an annual increase of ~0.8% in the population after age 50, and the prevalence rate is rapidly increasing with the recent worldwide aging trend. Based on International Classification of Diseases, Tenth Revision, a global classification of disease published by the World Health Organization, issued the disease code (M62.84) given to sarcopenia in 2016. Therefore, it is expected that the study of sarcopenia will be further activated based on the classification of disease codes in the aging society. Several epidemiological studies and meta-analyses have looked at the correlation between the prevalence of sarcopenia and several environmental factors. In addition, studies using cell lines and rodents have been done to understand the biological mechanism of sarcopenia. Laboratory rodent models are widely applicable in sarcopenia studies because of the advantages of time savings, cost saving, and various analytical applications that could not be used for human subjects. The rodent models that can be applied to the sarcopenia research are diverse, but a simple and fast method that can cause atrophy or aging is preferred. Therefore, we will introduce various methods of inducing muscular atrophy in rodent models to be applied to the study of sarcopenia.

Serum vitamin D binding protein level, but not serum total, bioavailable, free vitamin D, is higher in 30-days survivors than in nonsurvivors with sepsis.

The prognostic value of 3 types (total, bioavailable, and free) of 25-hydroxy vitamin D [25(OH)D] and vitamin D binding protein (VDBP) in patients with sepsis is unknown. The aim of this study was to evaluate the association of levels of those 3 types of 25(OH) D and VDBP with 30-day mortality in patients with sepsis. From March to December 2018, patients diagnosed with sepsis and admitted to the medical intensive care unit were enrolled, prospectively. We measured total 25(OH)D and VDBP levels, performed GC genotyping for the polymorphisms rs4588 and rs7041, and calculated bioavailable and free 25(OH)D levels. Total, bioavailable, and free 25(OH)D levels did not differ in 30-days nonsurvivors and survivors. Serum VDBP level was significantly higher in survivors than nonsurvivors (138.6 ug/mL vs 108.2 ug/mL, P = .023) and was associated with 30-day mortality in univariate but not multivariate analysis. VDBP polymorphisms and allele frequencies were not statistically different between the groups. Serum VDBP level was significantly higher in survivors than nonsurvivors over 30-days mortality in septic patients. However, 3 types (total, bioavailable, and free) of 25(OH)D levels did not differ between the survivors and nonsurvivors group.

Degrading products of chondroitin sulfate can induce hypertrophy-like changes and MMP-13/ADAMTS5 production in chondrocytes.

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in articular cartilage and the loss of CS-GAG occurs early in OA. As a major component of perichondral matrix interacting directly with chondrocytes, the active turnover of CS can affect to break the homeostasis of chondrocytes. Here we employ CS-based 3-dimensional (3D) hydrogel scaffold system to investigate how the degradation products of CS affect the catabolic phenotype of chondrocytes. The breakdown of CS-based ECM by the chondroitinase ABC (ChABC) resulted in a hypertrophy-like morphologic change in chondrocytes, which was accompanied by catabolic phenotypes, including increased MMP-13 and ADAMTS5 expression, nitric oxide (NO) production and oxidative stress. The inhibition of Toll-like receptor 2 (TLR2) or TLR4 with OxPAPC (TLR2 and TLR4 dual inhibitor) and LPS-RS (TLR4-MD2 inhibitor) ameliorated these catabolic phenotypes of chondrocytes by CS-ECM degradation, suggesting a role of CS breakdown products as damage-associated molecular patterns (DAMPs). As downstream signals of TLRs, MAP kinases, NF-kB, NO and STAT3-related signals were responsible for the catabolic phenotypes of chondrocytes associated with ECM degradation. NO in turn reinforced the activation of MAP kinases as well as NFkB signaling pathway. Thus, these results propose that the breakdown product of CS-GAG can recapitulate the catabolic phenotypes of OA.

Idiopathic pulmonary arterial hypertension associated with a novel frameshift mutation in the bone morphogenetic protein receptor II gene and enhanced bone morphogenetic protein signaling: A case report.

RATIONALE: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by intense remodeling of small pulmonary arteries. Loss-of-function mutation of bone morphogenetic protein receptor II (BMPR2) gene and exaggerated activation of transforming growth factor (TGF)-ß signaling play a critical role in this process. PATIENT CONCERNS AND DIAGNOSIS: We report a novel frameshift mutation (c.117InsT, p.Y40fsX48) of the BMPR2 gene identified in a 19-year-old IPAH patient with syncope. Despite BMPR2 mutation, the phosphorylation of Smad2/3 and Samd1/5/8 was increased in the patient's peripheral blood mononuclear cells, and this event was accompanied by the upregulation of bone morphogenetic protein (BMP) signaling target genes, but not TGF-ß signaling target genes. Moreover, we observed an increased expression of other BMPRs, that is, anti-Mullerian hormone type-2 receptor and the activin receptor-like kinases (ALK) 1, ALK3, and ALK6. INTERVENTIONS AND OUTCOMES: The patient was prescribed a combination of macitentan, sildenafil, and nifedipine, which successfully controlled her symptom of syncope and normalized N-terminal pro-brain natriuretic peptide level after 3 months of medication. LESSONS: In light of these results, we propose a new pathogenetic mechanism for IPAH, based on enhanced BMP signaling via the functional replacement of mutated BMPR2 by other BMP receptors.

Enhanced Activation of Rac1/Cdc42 and MITF Leads to Augmented Osteoclastogenesis in Autosomal Dominant Osteopetrosis Type II.

The autosomal dominant osteopetrosis type II (ADOII) caused by the mutation of chloride channel 7 (ClC-7) gene is the most common form of adult-onset osteopetrosis. Despite dysfunctional bone resorption, an augmented osteoclast differentiation was reported recently in ADOII patients. DNA sequencing analysis of the ADOII patient's ClC-7 gene identified a known heterozygous mutation, c.643G>A in exon 7, encoding p.Gly215Arg. In vitro osteoclast differentiation from the ADOII patient's peripheral blood mononuclear cells (PBMCs) increased compared with control despite their dysfunctional bone resorbing capacity. Osteoclasts from the ADOII patient's PBMCs and ClC-7 knockdown bone marrow monocytes (BMMs) showed an enhanced Ser-71 phosphorylation of Rac1/Cdc42 and increase of the microphthalmia-associated transcription factor (MITF) and receptor activator of NF-κB (RANK) that can be responsible for the enhanced osteoclast differentiation. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Osteoclasts in the Inflammatory Arthritis: Implications for Pathologic Osteolysis.

The enhanced differentiation and activation of osteoclasts (OCs) in the inflammatory arthritis such as rheumatoid arthritis (RA) and gout causes not only local bone erosion, but also systemic osteoporosis, leading to functional disabilities and morbidity. The induction and amplification of NFATc1, a master regulator of OC differentiation, is mainly regulated by receptor activator of NF-κB (RANK) ligand-RANK and calcium signaling which are amplified in the inflammatory milieu, as well as by inflammatory cytokines such as TNFα, IL-1ß and IL-6. Moreover, the predominance of CD4+ T cell subsets, which varies depending on the condition of inflammatory diseases, can determine the fate of OC differentiation. Anti-citrullinated peptide antibodies which are critical in the pathogenesis of RA can bind to the citrullinated vimentin on the surface of OC precursors, and in turn promote OC differentiation and function via IL-8. In addition to adaptive immunity, the activation of innate immune system including the nucleotide oligomerization domain leucine rich repeat with a pyrin domain 3 inflammasome and TLRs can regulate OC maturation. The emerging perspectives about the diverse and close interactions between the immune cells and OCs in inflammatory milieu can have a significant impact on the future direction of drug development.

DICAM Attenuates Experimental Colitis via Stabilizing Junctional Complex in Mucosal Barrier.

BACKGROUND: Adhesion molecules maintain the intestinal barrier function that is crucial to prevent intestinal inflammation. Dual immunoglobulin domain-containing adhesion molecule (DICAM) has been recently identified and known for the involvement in cell-cell adhesion through homophilic interaction and heterophilic interaction with integrin αVß3. We tested whether the change of DICAM expression affects the severity of colonic inflammation. METHODS: Colitis was induced with oral administration of 2.5% dextran sulfate sodium (DSS) in 8-week-old male mice for 5 days. The function of DICAM under inflammatory condition was investigated using loss-of-function and gain-of-function models such as DICAM-deficient mice and adenoviral transduction of DICAM into Caco-2 colonic epithelial cells. RESULTS: DICAM increased in parallel with the degree of inflammation after 5-day administration of DSS and decreased with the resolution of inflammation. DICAM was expressed in the epithelial junctional complex and colocalized with ZO-1. Treatment with TNF-α or IFN-γ in Caco-2 cells significantly increased DICAM in protein and RNA level. The DICAM knockout mice showed more severe DSS-induced colitis compared with WT littermates. Adenoviral transduction of DICAM into Caco-2 cells significantly attenuated the inflammation-mediated decrease of adhesion molecules, including ZO-1 and occludin. Furthermore, Caco-2 cells with DICAM overexpression maintained intestinal barrier function under IFN-γ treatment as estimated by transepithelial electrical resistance. CONCLUSION: Our study demonstrates that DICAM which is increased in an inflammatory condition has a protective role in experimental colitis by stabilizing the integrity of junctional complex in the intestinal mucosal barrier.

Calcium-phosphate complex increased during subchondral bone remodeling affects earlystage osteoarthritis.

An activation of osteoclasts and subchondral bone remodeling is a major histologic feature of early-stage osteoarthritis (OA), which can be accompanied by an increase of calcium (Ca) and phosphate (Pi) level in the subchondral milieu. Considering articular cartilage gets most of nutrition from subchondral bone by diffusion, these micro-environmental changes in subchondral bone can affect the physiology of articular chondrocytes. Here, we have shown that Ca is increased and co-localized with Pi in articular cartilage of early-stage OA. The Ca-Pi complex increased the production of MMP-3 and MMP-13 in the hypertrophic chondrocytes, which was dependent on nuclear factor-kappa B (NF-kB), p38 and extracellular signal-regulated kinase (Erk) 1/2 mitogen-activated protein (MAP) kinase and Signal transducer and activator of transcription 3 (STAT3) signaling. The Ca-Pi complexes increased the expression of endocytosis markers, and the inhibition of the formation of the Ca-Pi complex ameliorated the Ca-Pi complex-mediated increases of MMPs expression in hypertrophic chondrocytes. Our data provide insight regarding the Ca-Pi complex as a potential catabolic mediator in the subchondral milieu and support the pathogenic role of subchondral bone in the early stages of cartilage degeneration.

Functional cooperation between vitamin D receptor and Runx2 in vitamin D-induced vascular calcification.

The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr(-/-)) and Runx2 carboxy-terminus truncated heterozygous (Runx2(+/ΔC)) mice. Treatment of VSMCs with active vitamin D3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D3 was completely inhibited in Vdr(-/-) or Runx2(+/ΔC) mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D3.

Role of interleukin-10 in endochondral bone formation in mice: anabolic effect via the bone morphogenetic protein/Smad pathway.

OBJECTIVE: Interleukin-10 (IL-10) is a pleiotropic immunoregulatory cytokine with a chondroprotective effect that is elevated in cartilage and synovium in patients with osteoarthritis. However, the role of IL-10 during endochondral bone formation and its mechanism of action have not been elucidated. METHODS: IL-10(-/-) mice and IL-10-treated tibial organ cultures were used to study loss and gain of IL-10 functions, respectively, during endochondral bone formation. Primary chondrocytes from the long bones of mouse embryos were cultured with and without IL-10. To assess the role of IL-10 in chondrogenic differentiation, we conducted mesenchymal cell micromass cultures. RESULTS: The lengths of whole skeletons from IL-10(-/-) mice were similar to those of their wild-type littermates, although their skull diameters were smaller. The tibial growth plates of IL-10(-/-) mice showed shortening of the proliferating zone. Treatment with IL-10 significantly increased tibial lengths in organ culture. IL-10 also induced chondrocyte proliferation and hypertrophic differentiation in primary chondrocytes in vitro. Mechanistically, IL-10 activated STAT-3 and the Smad1/5/8 and ERK-1/2 MAP kinase pathways and induced the expression of bone morphogenetic protein 2 (BMP-2) and BMP-6 in primary chondrocytes. Furthermore, the blocking of BMP signaling attenuated the IL-10-mediated induction of cyclin D1 and RUNX-2 in primary chondrocytes and suppressed Alcian blue and alkaline phosphatase staining in mesenchymal cell micromass cultures. CONCLUSION: These results indicate that IL-10 acts as a stimulator of chondrocyte proliferation and chondrogenic or hypertrophic differentiation via activation of the BMP signaling pathway.