MyoD Over-Expression Rescues GST-bFGF Repressed Myogenesis.

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.

Development of a multifunctional bioreactor to evaluate the promotion effects of cyclic stretching and electrical stimulation on muscle differentiation.

A multifunctional bioreactor was fabricated in this study to investigate the facilitation efficiency of electrical and mechanical stimulations on myogenic differentiation. This bioreactor consisted of a highly stretchable conductive membrane prepared by depositing polypyrrole (PPy) on a flexible polydimethylsiloxane (PDMS) film. The tensile deformation of the PPy/PDMS membrane can be tuned by adjusting the channel depth. In addition, PPy/PDMS maintained its electrical conductivity under continuous cyclic stretching in the strain range of 6.5%-13% for 24 h. This device can be used to individually or simultaneously perform cyclic stretching and electrical stimulation. The results of single stimulation showed that either cyclic stretching or electrical stimulation upregulated myogenic gene expression and promoted myotube formation, where electrical stimulation improved better than cyclic stretching. However, only cyclic stretching can align C2C12 cells perpendicular to the stretching direction, and electrical stimulation did not affect cell morphology. Myosin heavy chain (MHC) immunostaining demonstrated that oriented cells under cyclic stretching resulted in parallel myotubes. The combination of these two stimuli exhibited synergetic effects on both myogenic gene regulation and myotube formation, and the incorporated electrical field did not affect the orientation effect of the cyclic stretching. These results suggested that these two treatments likely influenced cells through different pathways. Overall, the simultaneous application of cyclic stretching and electrical stimulation preserved both stimuli's advantages, so myo-differentiation can be highly improved to obtain abundant parallel myotubes, suggesting that our developed multifunctional bioreactor should benefit muscle tissue engineering applications.

Knee arthroscopy has limited effects on relieving local symptoms of knee osteoarthritis: an analysis of data from the Osteoarthritis Initiative.

BACKGROUND: Knee arthroscopy's efficacy in symptom improvement for knee osteoarthritis remains debated. In this study, we analyzed a multicenter database to investigate local symptom improvement. METHODS: We extracted and analyzed the data of 163 patients from the Osteoarthritis Initiative cohort who underwent unilateral knee arthroscopy (UKA) and were followed up for at least 24 months. UKA patients were matched to non-UKA patients (n = 163) according to sex, age, abdominal circumference, and Kellgren-Lawrence grade. The verified KOOS questionnaires (knee catching, locking, grinding, or clicking) and common local symptoms (frequent knee pain, aching, or stiffness) were set as outcomes. Furthermore, we built a binary logistic regression model to examine the relationship between UKA and local symptom improvement and new-onset symptoms, adjusting for conservative therapeutic covariables (injection of steroids or transparent acid into the knee joint, oral chondroitin sulfate, amino glucose, or analgesics). RESULT: Analysis showed that the UKA and non-UKA groups showed no obvious difference in the three knee symptoms, but the probability of new-onset grinding or clicking, and frequent knee pain, aching, or stiffness symptoms in the UKA group were respectively 5.82 and 5.65-fold higher than that in the non-UKA group. After analyzing conservative treatment data using a multiple imputation method, the results were consistent with previous regression analyses. CONCLUSION: Compared to the non-UKA group, the UKA group showed no noticeable differences in the improvement of the three knee symptoms and showed an increased the probability of new-onset grinding or clicking and frequent knee pain, aching, or stiffness symptoms. Key Points • Knee arthroscopy may increase the probability of new-onset grinding or clicking and frequent knee pain, aching, or stiffness symptoms. • We found no difference in the improvement of local knee symptoms (knee catching, locking, grinding, clicking or frequent pain, aching, or stiffness) improvement between the two groups with or without knee arthroscopy.

The cooperation of cis-elements during M-cadherin promoter activation.

M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47. Furthermore, MyoD/E47 heterodimer and MyoD ∼ E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM. We also found that GC boxes and CDE positively affected MyoD mediated activation. The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer. Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin. Using ChIP assay, we further found significant reduction in MyoD recruitment to M-cadherin promoter when CDE was deleted. Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.

Di-(2-ethylhexyl) phthalate limits the pleiotropic effects of statins in chronic kidney disease patients undergoing dialysis and endothelial cells.

The level of di-(2-ethylhexyl) phthalate (DEHP) is elevated in chronic kidney disease patients undergoing dialysis. However, statins are unable to reduce the cardiovascular events in chronic dialysis patients. In this study, we investigated the effects of DEHP on statin-conferred pleiotropic effects and the underlying molecular mechanism in peritoneal dialysis (PD) patients and endothelial cells (ECs). In PD patients with serum DEHP level ≥0.0687 µg/mL, statin treatment was not associated with lower risk of cardiovascular disease. In ECs, exposure to DEHP abrogated the simvastatin-induced NO bioavailability and EC-related functions. Additionally, DEHP abolished the anti-inflammatory effect of simvastatin on the tumor necrosis factor α-induced upregulation of adhesion molecules and monocyte adhesion to ECs. Mechanistically, DEHP blunted the activation of transient receptor potential vanilloid type 1 (TRPV1), which is required for NO production by simvastatin in ECs. Notably, DEHP increased the activity and expression of protein phosphatase 2B (PP2B), a negative regulator of TRPV1 activity. The effect of DEHP on PP2B activation was mediated by the activation of the NADPH oxidase/reactive oxygen species (NOX-ROS) pathway. Inhibition of PP2B activity by pharmacological antagonists prevented the inhibitory effects of DEHP on simvastatin-induced Ca2+ influx, NO bioavailability, and EC migration, proliferation, tube formation, and anti-inflammatory action. Collectively, DEHP activates the NOX-ROS-PP2B pathway, which in turns inhibits TRPV1/Ca2+-dependent signaling and abrogates the statin-conferred pleiotropic protection in ECs.

Thyroid hormone suppresses hepatocarcinogenesis via DAPK2 and SQSTM1-dependent selective autophagy.

Recent studies have demonstrated a critical association between disruption of cellular thyroid hormone (TH) signaling and the incidence of hepatocellular carcinoma (HCC), but the underlying mechanisms remain largely elusive. Here, we showed that disruption of TH production results in a marked increase in progression of diethylnitrosamine (DEN)-induced HCC in a murine model, and conversely, TH administration suppresses the carcinogenic process via activation of autophagy. Inhibition of autophagy via treatment with chloroquine (CQ) or knockdown of ATG7 (autophagy-related 7) via adeno-associated virus (AAV) vectors, suppressed the protective effects of TH against DEN-induced hepatic damage and development of HCC. The involvement of autophagy in TH-mediated protection was further supported by data showing transcriptional activation of DAPK2 (death-associated protein kinase 2; a serine/threonine protein kinase), which enhanced the phosphorylation of SQSTM1/p62 (sequestosome 1) to promote selective autophagic clearance of protein aggregates. Ectopic expression of DAPK2 further attenuated DEN-induced hepatoxicity and DNA damage though enhanced autophagy, whereas, knockdown of DAPK2 displayed the opposite effect. The pathological significance of the TH-mediated hepatoprotective effect by DAPK2 was confirmed by the concomitant decrease in the expression of THRs and DAPK2 in matched HCC tumor tissues. Taken together, these findings indicate that TH promotes selective autophagy via induction of DAPK2-SQSTM1 cascade, which in turn protects hepatocytes from DEN-induced hepatotoxicity or carcinogenesis.

Chemotherapy resistance and metastasis-promoting effects of thyroid hormone in hepatocarcinoma cells are mediated by suppression of FoxO1 and Bim pathway.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and systemic chemotherapy is the major treatment strategy for late-stage HCC patients. Poor prognosis following chemotherapy is the general outcome owing to recurrent resistance. Recent studies have suggested that in addition to cytotoxic effects on tumor cells, chemotherapy can induce an alternative cascade that supports tumor growth and metastasis. In the present investigation, we showed that thyroid hormone (TH), a potent hormone-mediating cellular differentiation and metabolism, acts as an antiapoptosis factor upon challenge of thyroid hormone receptor (TR)-expressing HCC cells with cancer therapy drugs, including cisplatin, doxorubicin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TH/TR signaling promoted chemotherapy resistance through negatively regulating the pro-apoptotic protein, Bim, resulting in doxorubicin-induced metastasis of chemotherapy-resistant HCC cells. Ectopic expression of Bim in hepatoma cells challenged with chemotherapeutic drugs abolished TH/TR-triggered apoptosis resistance and metastasis. Furthermore, Bim expression was directly transactivated by Forkhead box protein O1 (FoxO1), which was negatively regulated by TH/TR. TH/TR suppressed FoxO1 activity through both transcriptional downregulation and nuclear exclusion of FoxO1 triggered by Akt-mediated phosphorylation. Ectopic expression of the constitutively active FoxO1 mutant, FoxO1-AAA, but not FoxO1-wt, diminished the suppressive effect of TH/TR on Bim. Our findings collectively suggest that expression of Bim is mediated by FoxO1 and indirectly downregulated by TH/TR, leading to chemotherapy resistance and doxorubicin-promoted metastasis of hepatoma cells.

Double homeobox gene, Duxbl, promotes myoblast proliferation and abolishes myoblast differentiation by blocking MyoD transactivation.

Homeobox genes encode transcription factors that regulate embryonic development programs including organogenesis, axis formation and limb development. Previously, we identified and cloned a mouse double homeobox gene, Duxbl, whose homeodomain exhibits the highest identity (67 %) to human DUX4, a candidate gene of facioscapulohumeral muscular dystrophy (FSHD). Duxbl proteins have been shown to be expressed in elongated myocytes and myotubes of trunk and limb muscles during embryogenesis. In this study, we found that Duxbl maintained low expression levels in various adult muscles. Duxbl proteins were induced to express in activated satellite cells and colocalized with MyoG, a myogenic differentiating marker. Furthermore, Duxbl proteins were not detected in quiescent satellite cells but detected in regenerated myocytes and colocalized with MyoD and MyoG following cardiotoxin-induced muscle injury. Ectopic Duxbl overexpressions in C2C12 myoblast cells promoted cell proliferation through mainly enhancing cyclin D1 and hyper-phosphorylated retinoblastoma protein but reducing p21 expression. However, Duxbl overexpression in C2C12 cells inhibited myogenic differentiation by decreasing MyoD downstream gene expressions, including M-cadherin, MyoG, p21 and cyclin D3 but not MyoD itself. Duxbl overexpressions also promoted cell proliferation but blocked MyoD-induced myogenic conversion in multipotent mesenchymal C3H10T1/2 cells. In addition, results of a luciferase reporter assay suggest that Duxbl negatively regulated MyoG promoter activity through the proximal two E boxes. In conclusion, these results indicate that Duxbl may play a crucial role in myogenesis and postnatal muscle regeneration by activating and proliferating satellite and myoblast cells.

Using isothermal titration calorimetry to real-time monitor the heat of metabolism: a case study using PC12 cells and Aß(1-40).

Isothermal titration calorimetry (ITC) is one of the most powerful means for direct determination of thermodynamic information associated with most physiochemical and biological processes. The deposition and aggregation of ß-amyloid (Aß) on cell membranes was considered as one of the primary factors in having Alzheimer's disease (AD). Recently, a growing body of evidence has suggested that plasma membrane could accelerate the accumulation of Aß on the plasma membranes. However, the mechanism of AD is still a controversial issue. This study provided a biothermodynamic approach to real-time monitor the heat of metabolism involved in the co-incubation of PC12 cells and Aß(1-40) by ITC. The effects of Aß conformation and concentration of oligomeric Aß on cytotoxicity were successfully distinguished by ITC. This approach with rapid and direct measurement may provide not only real-time information for the effects of Aß species on living cells but also a platform for the screening of drug candidates for AD.

Thyroid hormone promotes cell invasion through activation of furin expression in human hepatoma cell lines.

The objective of this study was to identify genes regulated by thyroid hormone (T(3)) and associated with tumor invasion. The gene encoding furin, as previously identified by cDNA microarray, is known to be up-regulated by T(3) treatment, and stimulated furin production occurs in thyroidectomized rats after administration of T(3). Presently, by using serial deletion of the promoter and EMSAs, the T(3) response element on the furin promoter was localized to the -6317/-6302 region. T(3)-mediated furin up-regulation was cooperative with TGF-beta because T(3) induction increased after Smad3/4 addition. Furthermore, the invasiveness of HepG2-thyroid hormone receptor (TR) cells was significantly increased by T(3) treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. In addition, furin up-regulation either by stable overexpression or T(3) and/or TGF-beta induction was evident in severe-combined immune-deficient mice inoculated with HepG2-TRalpha1 cells. The HepG2-furin mice displayed a higher metastasis index and tumor size than HepG2-neo mice. Notably, the increased liver and lung tumor number or size in the hyperthyroid severe-combined immune-deficient mice as well as TGF-beta mice was attributed specifically to furin overexpression in the HepG2-TRalpha1 cells. Furthermore, this study demonstrated that furin overexpression in some types of hepatocellular carcinomas is TR dependent and might play a crucial role in the development of hepatocellular carcinoma. Thus, T(3) regulates furin gene expression via a novel mechanism or in cooperation with TGF-beta to enhance tumor metastasis in vitro and in vivo.

Regulation of fibronectin by thyroid hormone receptors.

Thyroid hormones regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors and associated pathways. We investigated the triiodothyronine (T3) modulation of gene expression, in human hepatocellular carcinoma cell lines, via a PCR-based cDNA subtraction method. Here we present further data on one of the T3-upregulated genes, fibronectin (FN). We demonstrate that the induction of FN protein expression by T3 in TRalpha1 and TRbeta1 over-expressing cells was time and dose-dependent at the mRNA and protein levels. Blockade of protein synthesis by cycloheximide almost completely inhibited the concomitant induction of FN mRNA by T3, indicating that T3 indirectly regulates FN. Furthermore, nuclear-run on and FN promoter assay clearly can specifically increase the number of FN transcriptional demonstrated that the presence of T3 initiations. In addition, we further confirmed that the up-regulation of FN by T3 was mediated, at least in part, by transforming growth factor-beta (TGF-beta), because the induction of FN was blocked in a dose-dependent manner by the addition of TGF-beta neutralizing antibody. In an effort to elucidate the we demonstrated the involvement of the signaling pathways involved in the activation of FN by T3, mitogen activated protein kinase/c-Jun N-terminal kinase/p38 MAPK (MAPK/JNK/p38) pathway. Although T3 induces the expression of TGF-beta, neither wild-type nor dominant-negative Smad3 or Smad4 over-expression affected the activation of FN by T3. Thus, we demonstrate that T3 regulates FN gene expression indirectly at the transcriptional level, with the participation of the MAPK/JNK/p38 pathway and the TGF-beta signaling pathway but independent of Smad3/4.

Thyroid hormone receptor-dependent transcriptional regulation of fibrinogen and coagulation proteins.

Thyroid hormone (T(3)) regulates growth, development, and differentiation. These activities are mediated by the nuclear thyroid hormone receptors (TRs), which belong to the steroid/TR superfamily of ligand-dependent transcription factors. The effect of T(3) treatment on target gene regulation was investigated in a TRalpha-overexpressing hepatoma cell line (HepG2-TRalpha), by performing cDNA microarrays. We demonstrate that 148 of the 7597 genes represented were up-regulated by T(3), including fibrinogen and several other components of the coagulation factor system. To confirm the microarray results, fibrinogen and a small number of the blood clotting components were further investigated using quantitative RT-PCR. The T(3)-induction ratios observed with quantitative RT-PCR for factors such as thrombin (8-fold), coagulation factor X (4.9-fold), and hepatoglobin (30-fold) were similar to those observed by the cDNA microarray analysis. Further investigation, using HepG2-TRalpha (cell lines, revealed a 2- to 3-fold induction of fibrinogen transcription after 24 h of T(3) treatment. In addition, T(3) treatment increased the level of fibrinogen protein expression 2.5- to 6-fold at 48 h. The protein synthesis inhibitor, cycloheximide, did not inhibit the induction of fibrinogen by T(3), indicating that this regulation was direct. Furthermore, transcription run-on experiments indicate that the induction of fibrinogen by T(3) is regulated largely at the level of transcription. Similar observations were made on the regulation of fibrinogen by T(3) using rats that received surgical thyroidectomy (TX) as an in vivo model. These results suggest that T(3) plays an important role in the process of blood coagulation and inflammation and may contribute to the understanding of the association between thyroid diseases and the misregulation of the inflammatory and clotting profile evident in the circulatory system of these patients.

P53 is a regulator of the metastasis suppressor gene Nm23-H1.

p53, a tumor suppressor gene involved in the G1 cell cycle checkpoint, is also the most frequently mutated gene in human cancer. In addition, p53 modifies the ability of tumor cells to metastasize. The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase, was previously identified in cells with low metastatic potential. Although p53 and Nm23-H1 proteins play an important part in regulating the progression of cancer, any functional relationship between these two proteins is currently unknown. Here we report an association between p53 levels and expression of the Nm23-H1 gene. Our data indicate that wild-type (wt) p53 upregulated the expression of Nm23-H1 at protein and mRNA levels in MCF-7 and J7B cells. This capacity of wt p53 to regulate expression of Nm23-H1 was not only dependent on the endogenous but also the exogenous origin of p53, and could not be reproduced with mutant p53. Subsequently, the invasive ability of MCF-7 and J7B cells was suppressed upon induction of the Nm23-H1 protein by p53. In contrast, increased levels of p53 downregulated the expression of Nm23-H1 at the protein and mRNA levels in RKO and H1299 cells and, as a consequence, increased the invasive ability of both cell types. Thus, our results implicated the differential regulation of Nm23-H1 by p53 in different cell types as an important component in the molecular mechanisms of tumor metastasis.