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We screened pre-approved drugs for the survival of the Hu5/KD3 human myogenic progenitors. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, promoted the proliferation and survival of Hu5/KD3 cells. Meclozine increased expression of MyoD, but reduced expression of myosin heavy chain and suppressed myotube formation. Withdrawal of meclozine, however, resumed the ability of Hu5/KD3 cells to differentiate into myotubes. We examined the effects of meclozine on mdx mouse carrying a nonsense mutation in the dystrophin gene and modeling for Duchenne muscular dystrophy. Intragastric administration of meclozine in mdx mouse increased the body weight, the muscle mass in the lower limbs, the cross-sectional area of the paravertebral muscle, and improved exercise performances. Previous reports show that inhibition of phosphorylation of ERK1/2 improves muscle functions in mouse models for Emery-Dreifuss muscular dystrophy and cancer cachexia, as well as in mdx mice. We and others previously showed that meclozine blocks the phosphorylation of ERK1/2 in cultured cells. We currently showed that meclozine decreased phosphorylation of ERK1/2 in muscles in mdx mice but not in wild-type mice. This was likely to be one of the underlying mechanisms of the effects of meclozine on mdx mice.

Different conformational strains of tau have been implicated in the clinicopathological heterogeneity of tauopathies. In this study, we hypothesized that distinct strains are degraded in a different manner. Lithium, a drug for bipolar disorder, had previously been reported to reduce aggregation-prone protein content by promoting autophagy. Here, we assessed the effects of lithium on tau aggregates using different tauopathy brain seeds. SH-SY5Y cells were transfected with C-terminal tau fragment Tau-CTF24 (residues 243-441), and Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) brain seeds were introduced. After 48-h lithium treatment, sarkosyl-insoluble fractions were prepared. Lithium treatment was found to reduce the amount of insoluble tau and p62, and increase LC3-II levels along with the number of autophagic vacuoles in AD-seeded cells. The effects were lower in case of CBD seeds, and comparable between PSP and AD seeds. An inhibitor of myo-inositol monophosphatase (IMPase) also demonstrated similar effects. Overall, the study suggested that aggregated tau protein is degraded by lithium-induced autophagy, influencing IMPase in a strain-specific manner.

Although gemcitabine is an effective chemotherapeutic for pancreatic cancer, severe side effects often accompany its use. Since we have discovered that locally administered C1B domain peptides effectively control tumor growth without any side effects, the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer was examined. Two- and three-dimensional cell culture studies clarified that a co-treatment with C1B5 peptide and gemcitabine significantly attenuated growth of PAN02 mouse and PANC-1 human pancreatic cancer cells in 2D and 3D cultures. Although treatment with the low dose of gemcitabine alone (76%) or the C1B5 peptide alone (39%) inhibited tumor growth moderately, a co-treatment with C1B5 peptide and a low dose of gemcitabine markedly inhibited the growth of PAN02 autografts in the mouse peritoneal cavity (94% inhibition) without any noticeable adverse effect. The number of peritoneal cavity-infiltrating neutrophils and granzyme B+ lymphocytes was significantly higher in the co-treatment group than in the control group. A significant increase of granzyme B mRNA expression was also detected in human T cells by the co-treatment. Taken together, the current study suggests that C1B5 peptide offers a remarkably effective combination treatment strategy to reduce side effects associated with gemcitabine, without losing its tumoricidal effect.

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments.

The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering.

Although excessive mechanical stress loading is known to induce articular cartilage degradation, the mechanism underlying this process is unclear. The interaction between hyaluronan (HA) and its primary receptor CD44 maintains the homeostasis of articular chondrocytes. CD44 cleavage and the generation of CD44-intracellular domain (ICD) can lead to the loss of extracellular matrices in chondrocytes. Here we studied the effects of cyclic tensile strain (CTS) loading, a representative mechanical stress, on CD44 cleavage. CTS loading (1 Hz and 20% elongation for 48 h) increased ADAM10 expression and CD44 cleavage in HCS-2/8 cells, a human chondrocytic cell line. Co-treatment with a chemical ADAM10 inhibitor significantly suppressed CTS loading-induced CD44 cleavage. Chemical inhibition of transient receptor potential vanilloid 4 (TRPV4) significantly suppressed CTS loading-induced ADAM10 expression and CD44 cleavage. Conversely, chemical activation of TRPV4 increased ADAM10 expression and enhanced CD44 cleavage. Our findings suggest that CTS loading significantly increases the expression of ADAM10, which in turn enhances CD44 cleavage in HCS-2/8 cells. The primary mechanoreceptor mediating this process is TRPV4. This signature event could provide an avenue for intervention in the prevention of cartilage degradation leading to OA.

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. In contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca(2+) signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage.

Endochondral ossification is an essential process for reparative phase of fracture healing, which starts with the differentiation of mesenchymal cells into chondrocytes followed by substitution of bone tissue. It is strictly controlled by the expression of crucial transcriptional factors: SOX9 in the early phase and RUNX2 in the late phase. Screening of FDA-approved compounds revealed that an anti-allergic drug, tranilast, that has been used for more than 30 years in clinical practice, enhanced the SOX9 promoter in chondrogenic cells and the RUNX2 promoter in osteoblastic cells. We observed that tranilast increased mRNA expression of both Sox9 and Runx2 in differentiating ATDC5 chondrogenic progenitor cells. Tranilast upregulated mRNA expression of chondrogenic marker genes (Col2a1, Acan, Col10a1, and Mmp13) in differentiating ATDC5 cells. Moreover, tranilast upregulated mRNA expression of essential signaling molecules involved in endochondral ossification (Pthrp, Ihh, and Axin2). In the later phase of differentiation of ATDC5 cells, tranilast increased synthesis of matrix proteoglycans, induced the alkaline phosphatase activity, and tended to accelerate mineralization. Tranilast is a potential agent that accelerates fracture repair by promoting the regulatory steps of endochondral ossification.

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.

During osteoarthritis there is a disruption and loss of the extracellular matrix of joint cartilage, composed primarily of type II collagen, aggrecan and hyaluronan. In young patients, autologous chondrocyte implantation can be used to repair cartilage defects. However, for more elderly patients with osteoarthritis, such a repair approach is contraindicated because the procedure requires a large expansion of autologous chondrocytes in vitro leading a rapid, perhaps irreversible, loss of the chondrocyte phenotype. This study investigates whether osteoarthritic chondrocytes obtained from older patients can be expanded in vitro and moreover, induced to re-activate their chondrocyte phenotype. A decrease in chondrocyte phenotype markers, collagen II, aggrecan and SOX9 mRNA was observed with successive expansion of cells in monolayer culture. However, chondrogenic induction in three-dimensional pellet culture successfully rescued the expression of all three marker genes to native levels, even with 4th passage cells–cells representing an approximate 625-fold expansion in cell number. This data supports the use of osteoarthritic cells for autologous implantation repair. In addition, another set of gene products were explored as useful markers of the chondrocyte phenotype. Differentiated primary chondrocytes exhibited a common pattern of hyaluronan synthase isoforms that changed upon cell expansion in vitro and, reverted back to the original pattern following pellet culture. Moreover, the change in isoform pattern correlated with changes in the molecular size of synthesized hyaluronan.

Rheumatoid synovial fibroblasts (RSF) are activated by toll-like receptor (TLR) signaling pathways during the pathogenesis of rheumatoid arthritis (RA). Cathepsin K is highly expressed by RSF, and is known to play a key role in the degradation of type I and type II collagen. Cathepsin K is considered to be implicated in the degradation of bone and cartilage in RA. Recent observations have shown that hyaluronan (HA) is an important inhibitor of inflammation. In the present study, we show that lipopolysaccharide (LPS) stimulation significantly increases cathepsin K expression by real-time PCR and western blotting analysis via a TLR-4 signaling pathway. Furthermore, we demonstrate that HA suppresses LPS-induced cathepsin K expression, which is dependent on CD44 but not intercellular adhesion molecule-1 (ICAM-1) interaction. We also show that HA suppresses LPS-induced matrix metalloproteinase-1 (MMP-1) expression, which is dependent on both CD44 and ICAM-1 interaction. We conclude that the anti-inflammatory effect of HA occurs through crosstalk between more than one HA receptor. Our study provides evidence for HA mediated suppression of LPS-induced cathepsin K and MMP-1 expression, supporting a protective effect of HA in RA.

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine mediating inflammatory as well as cell death activities, and is thought to induce chondrocytic chondrolysis in inflammatory and degenerative joint diseases. Selective estrogen receptor modulators (SERMs), such as raloxifene, which are commonly used in clinical settings act as estrogen agonists or antagonists. It is assumed that estrogens have a potential role in cartilage protection; however, the precise molecular mechanism for the protective effects of estrogens is unclear. This study was designed to examine whether raloxifene inhibits TNF-α-induced apoptosis in human chondrocytes and to clarify the mechanisms involved. We also investigated the signaling pathways responsible for the anti-apoptotic effect of raloxifene. Apoptosis in chondrocytes was determined by DNA fragmentation assay and caspase-3 activation. Raloxifene significantly inhibited TNF-α-induced caspase-3 activation and cell DNA fragmentation levels in chondrocytes. The inhibitory effect of raloxifene was abolished by the estrogen receptor antagonist ICI 182,780. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates apoptosis, acting as an apoptotic or anti-apoptotic signal. TNF-α-induced apoptosis was significantly enhanced by the ERK1/2 pathway inhibitor PD98059. Raloxifene stimulated a further increase in ERK1/2 phosphorylation in TNF-α-treated chondrocytes. Furthermore, the anti-apoptotic effects of raloxifene were inhibited by PD98059. In addition, the anti-apoptotic effects of raloxifene were completely abolished in ERK1/2 siRNA-treated chondrocytes. These results suggest that raloxifene prevents caspase-3-dependent apoptosis induced by TNF-α in human chondrocytes by activating estrogen receptors and the ERK1/2 signaling pathway.

Aims The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts. Materials and methods Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively. Results Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by ∼50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively. Conclusions The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts.

Research highlights: {yields} Salmon proteoglycan suppresses IL-10{sup -/-} cell transfer-induced colitis progression. {yields} Salmon proteoglycan suppresses Th1- and Th17-related factors in colitis mice. {yields} Salmon proteoglycan enhances Foxp3 expression. -- Abstract: Proteoglycans (PGs) are complex glycohydrates which are widely distributed in extracellular matrix (ECM). PGs are involved in the construction of ECM, cell proliferation and differentiation. ECM components are involved in transduction of proinflammatory responses, but it is still unknown whether PGs are involved in inflammatory response. In this study, we investigated the effect of PG extracted from salmon cartilage on the progression of experimental colitis-induced in severe combined immunodeficiency mice by cell transfer from interleukin-10 (IL-10){sup -/-} mice. IL-10{sup -/-} cell-transferred mice showed weight loss, colon shortening and histological appearance of mild colitis. Daily oral administration of PG attenuated the clinical progression of colitis in a dose-dependent manner. Colitis-induced mice showed the elevated expression of IFN-{gamma}, IL-12, TNF-{alpha}, IL-21, IL-23p19, IL-6, IL-17A and retinoic acid-related orphan receptor {gamma}t (ROR{gamma}t) in lamina propria mononuclear cells (LPMCs) and oral administration of PG suppressed the expression of these factors. Conversely, expression of Foxp3 that induces CD4{sup +}CD25{sup +} regulatory T cells in LPMCs was enhanced by PG administration. These findings suggested thatmore » salmon PG attenuated the progression of colitis due to suppression of inflammatory response by enhancement of regulatory T cell induction.« less

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and β-D-glucopyranosides, such as salicin share a common binding site of hTAS2R16.

Proteoglycans bearing keratan sulfate (KS), such as aggrecan, are components of the human cartilage extracellular matrix (ECM). However, the role of KS in influencing cartilage degradation associated with arthritis remains to be completely understood. KS side chains of the length found in human cartilage are not found in murine skeletal tissues. Using a murine model of inflammatory polyarthritis and cartilage explants exposed to interleukin-1α (IL-1α), we examined whether administering KS could influence intraarticular inflammation and cartilage degradation. Acute arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail, followed by an intraperitoneal injection of lipopolysaccharide. This treatment was followed by an intraperitoneal KS administration in half of the total mice to evaluate the therapeutic potential of KS for ameliorating arthritis. To investigate the therapeutic potential ex vivo, we examined cartilage fragility by measuring IL-1α-induced aggrecan release from cartilage explants treated with or without KS. Intraperitoneal KS administration ameliorated arthritis in DBA/1J mice. The aggrecan release induced by IL-1α was less in cartilage explants containing media with KS than in those without KS. Our data indicate that exogenous KS ameliorated arthritis in vivo and suppressed cartilage degradation ex vivo. KS may have important therapeutic potential in the treatment of inflammatory arthritis. The mechanism responsible for this requires further investigation, but KS may become a novel therapeutic agent for treating inflammatory diseases such as rheumatoid arthritis.

Prion protein plays a key role in the pathogenesis of transmissible spongiform encephalopathies. Because changes in expression of the prion protein gene (PRNP) alter the incubation time and severity of prion diseases. Our previous work revealed a strong association between the promoter (spanning base pairs (bp) -88 to -30) and intron 1 (spanning bp +114 to +892) that leads to optimum expression of the bovine PRNP. Here, we employed two mutation analysis strategies (deletion and insertion) and two reporter assay systems (luciferase and GFP expression) to define the regulatory domains within intron 1 and further elucidate its role in regulating the promoter activity of the bovine prion protein gene. We identified DNA sequences with potential suppressor and enhancer activities within the 5' end of intron 1. Moreover stability analyses for PRNP mRNAs demonstrated that splicing sites and mechanism are critical for bovine PRNP expression.

To provide further insights into non-genomic action of thyroid hormone (T3), we investigated whether Src is under control of T3 in primary calvarial osteoblasts prepared from neonatal mice. Treatment of the cells with T3 rapidly decreased Src Y416 autophosphorylation, followed by the decrease of phosphorylated extracellular signal-regulated kinases, suggesting that T3 non-genomically suppresses Src activity. Furthermore, this T3 effect was rapid and persistent, and was associated with the increased expression of osteocalcin (OC). To confirm the contribution of Src to the effect of T3 on OC expression, a constitutively active Src (Y527F) was overexpressed in osteoblasts. In such cells, Y416 phosphorylation was markedly increased even in the presence of T3, and T3-dependent expression of OC was markedly attenuated. The present study demonstrates a novel, non-genomic action of T3 in primary mouse osteoblasts, by which T3 suppresses Src thereby stimulating OC expression.

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204. Crystal structure demonstrated that the mono-phosphorylated ERK1 kinase possessed a novel conformation distinguishable from the un-phosphorylated (inactive) and the dual-phosphorylated (full-active) forms. The characteristic structural features in both the C-helix and the activation loop likely contribute to the basal activity of the mono-phosphorylated ERK1. The structural dissection of ERK1 compared to ERK2 suggests that the structural differences in the D-motif binding site and in the backside binding site are putative targets for development of selective ERK1/ERK2 inhibitors.