Promotes Myoblast Proliferation Research Articles

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.

Construction of a eukaryotic expression vector for pEGFP-FST and its biological activity in duck myoblasts

article i nfo Background: Follistatin (FST), a secreted glycoprotein, is intrinsically linked to muscle hypertrophy. To explore the function of duck FST in myoblast proliferation and differentiation, the pEGFP-FST eukaryotic expression vector was constructed and identified. The biological activities of this vector were analyzed by transfecting pEGFP-FST into cultured duck myoblasts using Lipofectamine™ 2000 and subsequently determining the mRNA expression profiles of FST and myostatin (MSTN). Results: The duck pEGFP-FST vector was successfully constructed and was confirmed to have high liposome-mediated transfection efficiency in duck myoblasts. Additionally, myoblasts transfected with pEGFP-FST had a higher biological activity. Significantly, the overexpression of FST in these cells significantly inhibited the mRNA expression of MSTN (a target gene that is negatively regulated by FST). Conclusions: The duck pEGFP-FST vector has been constructed successfully and exhibits biological activity by promoting myoblast proliferation and differentiation in vitro.

The transient expression of miR-203 and its inhibiting effects on skeletal muscle cell proliferation and differentiation.

Previous studies have shown that miR-203 is a skin-specific microRNA (miRNA) with a profound role in skin cell differentiation. However, emerging microarray and deep sequencing data revealed that miR-203 is also expressed in embryonic skeletal muscle and myoblasts. In this study, we found that miR-203 was transiently upregulated in chicken embryos on days 10 to 16 (E10–E16) and was sharply downregulated and even not expressed after E16 in chicken embryonic skeletal muscle. Histological profiles and weight variations of embryo skeletal muscle revealed that miR-203 expression is correlated with muscle development. In vitro experiments showed that miR-203 exhibited downregulated expression during myoblast differentiation into myotubes. miR-203 overexpression inhibited myoblast proliferation and differentiation, whereas its loss-of-function increased myoblast proliferation and differentiation. During myogenesis, miR-203 can target and inhibit the expression of c-JUN and MEF2C, which were important for cell proliferation and muscle development, respectively. The overexpression of c-JUN significantly promoted myoblast proliferation. Conversely, knockdown of c-JUN by siRNA suppressed myoblast proliferation. In addition, the knockdown of MEF2C by siRNA significantly inhibited myoblast differentiation. Altogether, these data not only suggested that the expression of miR-203 is transitory during chicken skeletal muscle development but also showed a novel role of miR-203 in inhibiting skeletal muscle cell proliferation and differentiation by repressing c-JUN and MEF2C, respectively.

Substrate and stretch regulate muscle‐resident mesenchymal stem cells to promote myoblast proliferation (673.1)

Mesenchymal stem cells (MSCs) reside in a variety of tissues and provide a stromal role in regulating progenitor cell function. Current studies focus on identifying the specific factors in the niche that can alter the MSC secretome, ultimately determining effectiveness and timing of tissue repair. The purpose of the present study was to evaluate the extent to which substrate and mechanical strain regulate MSC quantity, gene expression and secretome. MSCs (Sca‐1+CD45‐) isolated from murine skeletal muscle via FACS were seeded onto laminin‐(LAM) or collagen‐1‐(COL) coated membranes and exposed to a single bout of mechanical strain (10%, 1 Hz, 20 min). MSC proliferation was not directly affected by substrate or strain; however, the genetic expression of growth and inflammatory factors and ECM proteins were down‐regulated in MSCs grown on COL in a manner independent of strain. In conditioned media (CM) experiments, the combination of strain and LAM induced secretion of factors from MSCs that increased myoblast quantity 24 hours post‐treatment (5.6 fold; p<0.05), while this response was delayed in myoblasts treated with CM from MSCs grown on COL. Here, we demonstrate that exposure to COL, the primary ECM component associated with tissue fibrosis, down‐regulates MSC gene expression, and delayed their ability to stimulate myoblast proliferation.Grant Funding Source: Supported by the Ellison Medical Foundation

Inflammation following muscle injury promotes myoblast proliferation (LB808)

Objective: To determine the expression of IL‐1β and IL‐6 at several time points following injury, and their effects on myoblast proliferation. Experimental Design: Skeletal muscle was injured using BaCl2 in mice and tissue was collected 1, 5, 10, and 28 days following injury. Cytokine expression was determined using real time RT‐PCR. Mechanisms of cytokine‐induced proliferation were determined in cell culture using both primary and C2C12 myoblasts. Results: ~20‐fold increase in IL‐1β (p<0.05) and IL‐6 (p=0.06) expression was observed 5 days following injury. IL‐1β increased proliferation of both primary and C2C12 myoblasts ~25%. It was determined that IL‐1β not only stimulated NF‐B activation, but this signalling was required for the mitogenic effect of IL‐1β. These data demonstrate for the first time that IL‐1β alone can increase the mitogenic activity of primary skeletal muscle satellite cells and offers insight into the mechanisms dictating satellite cell function following injury.Grant Funding Source: Supported by NIH‐NIA (R03 AG‐034352, P.I., SJL), NIH‐NIAAA (K01 017190, P.I., JSO), NOHFC (P.I., SJL

Role of the TWEAK-Fn14-cIAP1-NF-κB Signaling Axis in the Regulation of Myogenesis and Muscle Homeostasis

Mammalian skeletal muscle maintains a robust regenerative capacity throughout life, largely due to the presence of a stem cell population known as “satellite cells” in the muscle milieu. In normal conditions, these cells remain quiescent; they are activated upon injury to become myoblasts, which proliferate extensively and eventually differentiate and fuse to form new multinucleated muscle fibers. Recent findings have identified some of the factors, including the cytokine TNFα-like weak inducer of apoptosis (TWEAK), which govern these cells’ decisions to proliferate, differentiate, or fuse. In this review, we will address the functions of TWEAK, its receptor Fn14, and the associated signal transduction molecule, the cellular inhibitor of apoptosis 1 (cIAP1), in the regulation of myogenesis. TWEAK signaling can activate the canonical NF-κB signaling pathway, which promotes myoblast proliferation and inhibits myogenesis. In addition, TWEAK activates the non-canonical NF-κB pathway, which, in contrast, promotes myogenesis by increasing myoblast fusion. Both pathways are regulated by cIAP1, which is an essential component of downstream signaling mediated by TWEAK and similar cytokines. This review will focus on the seemingly contradictory roles played by TWEAK during muscle regeneration, by highlighting the interplay between the two NF-κB pathways under physiological and pathological conditions. We will also discuss how myogenesis is negatively affected by chronic conditions, which affect homeostasis of the skeletal muscle environment.

Role of microRNA‐27a in myoblast differentiation

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that are critically involved in roles in various aspects of skeletal myogenesis. microRNA miR-27a promotes myoblast proliferation by targeting myostatin, a critical inhibitor of skeletal muscle development, but its mode of action in myoblast differentiation remains unclear. We have found that expression of miR-27a and myostatin were upregulated and downregulated, respectively, during myoblast differentiation. Overexpression of miR-27a increased the number of myosin heavy chain (MHC)-positive cells and upregulated mRNA and protein of MyoD and myogenin. These findings indicate that miR-27a plays a role in enhancing myoblast differentiation.

The effects of endoplasmic reticulum stress response on duck decorin stimulate myotube hypertrophy in myoblasts

Decorin inhibits the transforming growth factor superfamily to stimulate skeletal muscle hypertrophy. The endoplasmic reticulum (ER) plays a crucial role in the synthesis and folding of proteins. The disruption of ER homeostasis affects protein folding and causes ER stress, which is associated with protein synthesis in muscle cells. The purpose of this study was to establish the mechanism by which decorin promotes myoblast proliferation and myotube hypertrophy as mediated by an ER stress-related pathway. Duck myoblasts were incubated for 24 h with tunicamycin. Our results show that tunicamycin triggered ER stress in myoblasts and led to an increase in protein synthesis via a decorin-dependent pathway. We then transfected the decorin gene into duck myoblasts in vitro, and the results demonstrated that overexpression of duck decorin increased myogenic determination factor and follistatin expression and decreased myogenin and myostatin expression. Moreover, the results demonstrated that overexpression of duck decorin led to higher expression of ER stress marker genes. This increased expression trend can be alleviated in vitro by 4-PBA, which is a chemical chaperone that inhibits palmitate-induced ER stress. Collectively, our data show that duck decorin promotes myoblast proliferation mediated by an ER stress-related pathway.

Regulation of IRS1/Akt insulin signaling by microRNA-128a during myogenesis

Skeletal muscle possesses a strong ability to regenerate following injury, a fact that has been largely attributed to satellite cells. Satellite cells are skeletal muscle stem cells located beneath the basal lamina of the myofiber, and are the principal cellular source of growth and regeneration in skeletal muscle. MicroRNAs (miRNAs) play key roles in modulating several cellular processes by targeting multiple mRNAs that comprise a single or multiple signaling pathway. Several miRNAs have been shown to regulate satellite cell activity, such as miRNA-489, which functions to maintain satellite cells in a quiescent state. Although muscle-specific miRNAs have been identified, many of the molecular mechanisms that regulate myogenesis that are regulated by miRNAs still remain unknown. In this study, we have shown that miR-128a is highly expressed in brain and skeletal muscle, and increases during myoblast differentiation. MiR-128a was found to regulate the target genes involved in insulin signaling, which include Insr (insulin receptor), Irs1 (insulin receptor substrate 1) and Pik3r1 (phosphatidylinositol 3-kinases regulatory 1) at both the mRNA and protein level. Overexpression of miR-128a in myoblasts inhibited cell proliferation by targeting IRS1. By contrast, inhibition of miR-128a induced myotube maturation and myofiber hypertrophy in vitro and in vivo. Moreover, our results demonstrate that miR-128a expression levels are negatively controlled by tumor necrosis factor α (TNF-α). TNF-α promoted myoblast proliferation and myotube hypertrophy by facilitating IRS1/Akt signaling via a direct decrease of miR-128a expression in both myoblasts and myotubes. In summary, we demonstrate that miR-128a regulates myoblast proliferation and myotube hypertrophy, and provides a novel mechanism through which IRS1-dependent insulin signaling is regulated in skeletal muscle.

Interleukin-6/Signal Transducer and Activator of Transcription 3 (STAT3) Pathway Is Essential for Macrophage Infiltration and Myoblast Proliferation during Muscle Regeneration

Inflammation and microenvironment play a crucial role in muscle regeneration. IL (interleukin)-6, as a multifunctional cytokine is involved in the processes. However, the causative effect of IL-6 in muscle regeneration remains unclear. In a mouse model of cardiotoxin-induced muscle injury/regeneration, infiltrated monocytes/macrophages produce a high level of IL-6 started on 1 day (24 h) after injury. In IL-6 knock-out (-/-) mice, the muscle regeneration procedure was impaired along with decreased myogenic determination factor (MyoD) and myogenin mRNA level and increased interstitial fibrosis. The IL-6(-/-) mice exhibited less macrophage infiltration, lower inflammatory cytokine (IL-1β, inducible NO synthase, Transforming growth factor (TGF)-β1, and IL-10) and chemokine (CCL2, CCL3, and CCL5) expression, and inhibited myoblast proliferation. In vitro, IL-6 deficiency or Signal Transducer and Activator of Transcription 3 (STAT3) knockdown in activated macrophage attenuated the expression of CCL2, CCL3, but not CCL5, which resulted in less macrophage migration. Moreover, inflammatory macrophages promoted myoblast proliferation in an IL-6-dependent manner. Finally, adoptive transfer IL-6(+/+) BM cells into IL-6(-/-) mice rescued the impaired regeneration with improved MyoD and myogenin expression. Taken together, IL-6 expression and the activated STAT3 signaling pathway in monocytes/macrophages is a critical mediator of macrophage migration and myoblast proliferation during muscle regeneration.

MicroRNA-27a promotes myoblast proliferation by targeting myostatin

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3′UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

Inhibition of mammalian muscle differentiation by excretory secretory products of muscle larvae of Trichinella spiralis in vitro

The excretory-secretory products (ESP) released by muscle stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ESP modulate nurse cell formation remain unclear. In the present study, the ability of ESP of muscle larvae of T. spiralis (ML-ESP) to influence the proliferation and differentiation of murine myoblasts and the mechanisms were evaluated in vitro using C2C12 myoblast cell line, which were incubated for various times under grow or differentiation culture medium containing various concentrations of ML-ESP. The results indicated that ML-ESP promoted myoblast proliferation in a dose-dependent manner and increased the expression of the cell-cycle regulator cyclin D1 as well as that of proliferating cell nuclear antigen (PCNA). Conversely, ML-ESP inhibited the differentiation of these cells, which was evidenced by a reduction in the levels of MHC and MRFs expression (MyoD and myogenin) as well as that of p21. In addition, ML-ESP also inhibited the phosphorylation of p38 MAPK in differentiating C2C12 myoblast. Taken together, these results imply that certain critical mediators contained in ML-ESP inhibit myogenesis through enhancing skeletal myoblasts proliferation and down-regulating the expression of MRFs as well as involving p38 MAPK signalling pathway, which provides insight into the mechanisms utilised by T. spiralis to interfere normal wound repair in infected muscle cells and affect nurse cell formation.

CREB Is Activated by Muscle Injury and Promotes Muscle Regeneration

The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration.

Effect of in ovo feeding and its interaction with timing of first feed on glycogen reserves, muscle growth, and body weight

Chicks are commonly fasted for the first 36 to 72 h posthatch because of the logistics of commercial production. Fasting for 48 to 72 h posthatch results in retarded BW, delayed intestinal development, and lower pectoral muscle weight. This study is focused on the first 36 h of fasting and its interaction with feeding before hatch. Four treatment groups, differing in time of first feed, 6 h [early feeding (EF)] or 36 h [standard feeding procedure (SP)] posthatch, with or without in ovo feeding (IOF) with dextrin and β-hydroxy-β-methylbutyrate-calcium salt in a saline solution, were examined for glycogen status in the liver and pectoral muscle, myogenic cell proliferation, and myofiber diameter in embryos and chickens on various days posthatch. In addition, chicken BW, ADG, pectoral muscle weight, and pectoral muscle percentage of BW until 35 d of age were recorded. Results showed that delaying the first feed for 36 h posthatch (SP group) led to an irreversibly reduced growth rate compared with the EF group. However, IOF affected the growth of chickens in the SP group, whereas the control embryos had depleted glycogen reserves in the liver; IOF-treated embryos had elevated hepatic glycogen contents on embryonic day (E) 19, E20, and the day of hatch. In addition, on d 2 posthatch, although hatchlings in the SP group showed the predicted low levels of glycogen in their livers, birds in the EF group exhibited more than 30-fold and 3-fold increases in liver and muscle glycogen, respectively. In ovo-fed birds in the SP group also exhibited higher glycogen reserves, BW, pectoral muscle weight, and BW gain than control birds in the SP group. In ovo feeding had an immediate effect on promoting myoblast proliferation on E19, whereas on d 3 posthatch, the effect was pronounced only in the EF groups. On d 5, although myoblast proliferation in all groups declined, it remained higher in both IOF groups. These effects were expressed on d 3 and 35 by myofiber diameter. Together, IOF had a long-term supportive effect on BW and posthatch muscle growth when first feed was delayed by 36 h.

LIF is a contraction-induced myokine stimulating human myocyte proliferation

The cytokine leukemia inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of myoblasts. We hypothesized that LIF is a contraction-induced myokine functioning in an autocrine fashion to activate gene regulation of human muscle satellite cell proliferation. Skeletal muscle LIF expression, regulation, and action were examined in two models: 1) young men performing a bout of heavy resistance exercise of the quadriceps muscle and 2) cultured primary human satellite cells. Resistance exercise induced a ninefold increase in LIF mRNA content in skeletal muscle, but LIF was not detectable in plasma of the subjects. However, electrically stimulated cultured human myotubes produced and secreted LIF, suggesting that LIF is a myokine with local effects. The well established exercise-induced signaling molecules PI3K, Akt, and mTor contributed to the regulation of LIF in cultured human myotubes as chemical inhibition of PI3K and mTor and siRNA knockdown of Akt1 were independently sufficient to downregulate LIF. Human myoblast proliferation was increased by recombinant exogenous LIF and decreased by siRNA knockdown of the endogenous LIF receptor. Finally, the transcription factors JunB and c-Myc, which promote myoblast proliferation, were induced by LIF in cultured human myotubes. Indeed, both JunB and c-Myc were also increased in skeletal muscle following resistance exercise. Our data suggest that LIF is a contraction-induced myokine, potentially acting in an autocrine or paracrine fashion to promote satellite cell proliferation.

Ornithine decarboxylase is upregulated by the androgen receptor in skeletal muscle and regulates myoblast proliferation

The aim of this study is to determine if the Odc1 gene, which encodes ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is directly regulated by the androgen receptor (AR) in skeletal muscle myoblasts and if Odc1 regulates myoblast proliferation and differentiation. We previously showed that expression of Odc1 is decreased in muscle from AR knockout male mice. In this study, we show in vivo that Odc1 expression is also decreased >60% in muscle from male muscle-specific AR knockout mice. In normal muscle homeostasis, Odc1 expression is regulated by age and sex, reflecting testosterone levels, as muscle of adult male mice expresses high levels of Odc1 compared with age-matched females and younger males. In vitro, expression of Odc1 is 10- and 1.5-fold higher in proliferating mouse C(2)C(12) and human skeletal muscle myoblasts, respectively, than in differentiated myotubes. Dihydrotestosterone increases Odc1 levels 2.7- and 1.6-fold in skeletal muscle cell myoblasts after 12 and 24 h of treatment, respectively. Inhibition of ODC activity in C(2)C(12) myoblasts by α-difluoromethylornithine decreases myoblast number by 40% and 66% following 48 and 72 h of treatment, respectively. In contrast, overexpression of Odc1 in C(2)C(12) myoblasts results in a 27% increase in cell number vs. control when cells are grown under differentiation conditions for 96 h. This prolonged proliferation is associated with delayed differentiation, with reduced expression of the differentiation markers myogenin and Myf6 in Odc1-overexpressing cells. In conclusion, androgens act via the AR to upregulate Odc1 in skeletal muscle myoblasts, and Odc1 promotes myoblast proliferation and delays differentiation.

Carbamylated erythropoietin does not alleviate signs of dystrophy in mdx mice

Erythropoietin promotes myoblast proliferation and inhibits fibrosis and thus it could impede the pathogenesis of muscle degenerative diseases. However, its stimulation of erythropoiesis limits its use as a therapeutic agent. An erythropoietin analog, carbamylated erythropoietin (C-EPO), retains these protective actions, yet it does not interact with the erythropoietin receptor. To determine whether treatment with C-EPO alleviates the signs of muscular dystrophy in an animal model of Duchenne muscular dystrophy, we treated mdx mice with intraperitoneal injections of 50 μg/kg and 100 μg/kg C-EPO for 4 and 12 weeks, and we monitored weight, serum creatine kinase levels, and changes in muscle histology. Moderate histological improvement was observed at 4 weeks, which did not translate into a significantly decreased level of serum creatine kinase. At the doses tested, C-EPO is not an effective therapeutic for the treatment of a mouse model of Duchenne muscular dystrophy.

UPA and macrophages in muscle regeneration

The plasminogen system and macrophages play important roles in tissue repair following injury. Mice deficient in the urokinase‐type plasminogen activator (uPA) exhibit impaired accumulation of macrophages following muscle injury and severely impaired muscle fiber regeneration. We hypothesized that macrophage expression of uPA promotes macrophage migration into damaged muscle and subsequent muscle regeneration. To test this hypothesis, we cross‐bred macrophage‐specific uPA overexpressing mice with uPA null mice to generate mice that express uPA only in macrophages. Such macrophage‐only uPA expression was sufficient to restore uPA activity, macrophage accumulation and muscle fiber regeneration in otherwise uPA null mice. In addition, macrophage‐specific overexpression of uPA appears to enhance muscle fiber regeneration in wild‐type mice. In vitro studies demonstrated that uPA promotes macrophage migration through both proteolytic and non‐proteolytic mechanisms, which may include the proteolytic activation of HGF. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity and this process was inhibited by an HGF blocking antibody. In summary, macrophage expression of uPA plays a critical role in muscle regeneration.Supported by the Department of Defense Peer Reviewed Medical Research Program.

Urokinase-type plasminogen activator increases hepatocyte growth factor activity required for skeletal muscle regeneration

AbstractThe plasminogen system plays a crucial role in the repair of a variety of tissues, including skeletal muscle. We hypothesized that urokinase-type plasminogen activator (uPA) promotes muscle regeneration by activating hepatocyte growth factor (HGF), which, in turn, stimulates proliferation of myoblasts required for regeneration. In our studies, levels of active HGF and phosphorylation of the HGF receptor c-met were increased after muscle injury in wild-type mice. Compared with wild-type animals, mice deficient in uPA (uPA−/−) had markedly reduced HGF levels and c-met activation after muscle damage. This reduced HGF activity in uPA−/− animals was associated with decreased cell proliferation, myoblast accumulation, and new muscle fiber formation. On the other hand, HGF activity was enhanced at early time points in PAI-1−/− mice compared with wild-type mice and the PAI-1−/− animals exhibited accelerated muscle fiber regeneration. Furthermore, administration of exogenous uPA rescued HGF levels and muscle regeneration in uPA−/− mice, and an HGF-blocking antibody reduced HGF activity and muscle regeneration in wild-type mice. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity, and this process was inhibited by an HGF-blocking antibody. Together, our findings demonstrate that uPA promotes muscle regeneration through HGF activation and subsequent myoblast proliferation.

Leukotriene B4 regulates proliferation and differentiation of cultured rat myoblasts via the BLT1 pathway.

Skeletal muscle regeneration is a highly orchestrated process initiated by activation of adult muscle satellite cells. Upon muscle injury, the inflammatory process is always accompanied by muscle regeneration. Leukotriene B(4) is one of the essential inflammatory mediators. We isolated and cultured primary satellite cells. RT-PCR showed that myoblasts expressed mRNA for LTB(4) receptors BLT1 and BLT2, and LTB4 promoted myoblast proliferation and fusion. Quantitative real-time PCR and immunoblotting showed that LTB(4) treatment expedited the expression process of differentiation markers MyoD and M-cadherin. U-75302, a specific BLT1 inhibitor, but not LY2552833, a specific BLT2 inhibitor, blocked proliferation and differentiation of myoblasts induced by LTB(4), which implies the involvement of the BLT1 pathway. Overall, the data suggest that LTB(4) contributes to muscle regeneration by accelerating proliferation and differentiation of satellite cells.