EGR1基因在牛骨骼肌卫星细胞分化不同时期的表达及定位

目的:研究EGR1基因在牛骨骼肌卫星细胞(MDSCs)分化过程的表达、定位及入核机制。方法:以牛的MDSCs为实验材料,在分化培养基中分别分化培养1 d、3 d和5 d,每组3个重复,检测不同分化时间的MDSCs中EGR1基因的表达和EGR1蛋白的定位情况;采用 CRISPRi方法干扰内源EGR1的表达,结合定点突变和激光共聚焦方法初步探索了EGR1蛋白入核的机制。结果:qRT-PCR和Western blot检测结果显示随着分化时间的进行,EGR1 基因在转录水平和蛋白水平的表达都显著高于未分化的细胞,并随时间的延长而表达逐渐升高,分化第3日时表达量最高,随后开始下降。免疫荧光检测到EGR1蛋白主要在分化的MDSCs中表达,并随肌管数量增多而表达量增加。共聚焦结果显示随着细胞分化的进行,部分EGR1蛋白转移进入细胞核。定点突变EGR1蛋白S533A后,分化的MDSCs细胞核内没有检测到EGR1蛋白。结论:在牛骨骼肌卫星细胞分化过程中,EGR1基因转录表达水平升高,部分EGR1蛋白转移入细胞核,且EGR1蛋白C端第533位丝氨酸磷酸化是入核所必需的。     

A novel lncRNA promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1

Myogenesis, the process of skeletal muscle formation, is a highly coordinated multistep biological process. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are emerging as a gatekeeper in myogenesis. Up to now, most studies on muscle development-related lncRNAs are mainly focussed on humans and mice. In this study, a novel muscle highly expressed lncRNA, named lnc23, localized in nucleus, was found differentially expressed in different stages of embryonic development and myogenic differentiation. The knockdown and over-expression experiments showed that lnc23 positively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Then, TMT 10-plex labelling quantitative proteomics was performed to screen the potentially regulatory proteins of lnc23. Results indicated that lnc23 was involved in the key processes of myogenic differentiation such as cell fusion, further demonstrated that down-regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells. Furthermore, RNA pulldown/LC-MS and RIP experiment illustrated that PFN1 was a binding protein of lnc23. Further, we also found that lnc23 positively regulated the protein expression of RhoA and Rac1, and PFN1 may negatively regulate myogenic differentiation and the expression of its interacting proteins RhoA and Rac1. Hence, we support that lnc23 may reduce the inhibiting effect of PFN1 on RhoA and Rac1 by binding to PFN1, thereby promoting myogenic differentiation. In short, the novel identified lnc23 promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.     

miR-101a靶向EZH2促进山羊骨骼肌卫星细胞的分化

本实验室前期研究发现,miR-101a对山羊骨骼肌卫星细胞(skeletal muscle satellite cells, SMSCs)分化有促进作用,但其具体作用机制并不清楚。本研究利用PicTar、TargetScan和miRanda软件在线预测miR-101a的靶基因,并通过双荧光素酶报告基因进行实验验证;检测了山羊SMSCs分化不同时期miR-101a和靶基因的表达关系,同时分析了超表达和抑制miR-101a对靶基因表达水平的影响。结果证实,zeste增强子同源物2(enhancer of zeste homologue 2, EZH2)基因mRNA的3°UTR具有miR-101a结合位点,是miR-101a的一个靶基因。在SMSCs分化过程中,随着miR-101a表达水平的升高,EZH2的表达在mRNA和蛋白水平均下调。抑制miR-101a后,EZH2的表达极显著升高(P<0.01),但是在超表达miR-101a条件下,EZH2表达变化在mRNA和蛋白水平均不显著(P>0.05)。以上研究结果表明,miR-101a能通过抑制EZH2的表达来促进山羊SMSCs分化,为进一步阐明miR-101a对SMSCs的调控机制提供了理论依据。     

Atx regulates skeletal muscle regeneration via LPAR1 and promotes hypertrophy

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Leptin expression in human primary skeletal muscle cells is reduced during differentiation

We found leptin to be strongly expressed in undifferentiated human myoblasts derived from biopsies of the thigh (Musculus vastus lateralis). Both mRNA expression and secretion of leptin were reduced during in vitro differentiation into primary myotubes. However, the expression of the leptin receptor (OB-Rb) mRNA, was unchanged during differentiation of the muscle cells. Administration of recombinant leptin had no effect on leptin, myogenin, myoD, or GLUT4 mRNA expressions during the period of cellular differentiation. A functional leptin receptor was demonstrated by an acute leptin-induced 1.5-fold increase in ERK activity (P = 0.029). Although mRNA expression of regulation of suppressor of cytokine signaling-3 (SOCS-3) mRNA expression was unaltered, leptin significantly stimulated fatty acid oxidation after 6 h measured as acid soluble metabolites (ASM). Palmitic acid (PA), oleic acid (OA), and eicosapentaenoic acid (EPA), known to modulate leptin expression in other tissues, had no effect on mRNA expression or secretion of leptin from human myotubes. In conclusion, we demonstrate that leptin is highly expressed in undifferentiated human myoblasts and the expression is reduced during differentiation to mature myotubes. The role of leptin in these cells needs to be further characterized.     

In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.     

MicroRNA调控哺乳动物骨骼肌发育

MicroRNA (miRNA)是一类长度大约为22 bp的小分子非编码RNA,广泛存在于哺乳动物中,部分miRNA表达具有时空和组织特异性。哺乳动物中miRNA主要通过与靶基因3° UTR区结合抑制其翻译,调控机体生物学功能。miRNA在哺乳动物骨骼肌发育中发挥重要调节作用。哺乳动物骨骼肌发育是一个复杂的生物学过程,包括骨骼肌干细胞增殖、迁移、分化,成肌细胞增殖、分化、肌管融合,肌纤维肥大,能量代谢,纤维类型转换等。miRNA参与骨骼肌发育的各个环节,通过靶向各个时期的关键因子调控骨骼肌发育。本文对miRNA在骨骼肌发育中的调控作用进行了综述,以期为深入理解骨骼肌发育规律提供参考。     

BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.     

Thiazolidinedione induces the adipose differentiation of fibroblast-like cells resident within bovine skeletal muscle.

To investigate the role of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte formation within the skeletal muscle of beef cattle, fibroblast-like cells were isolated from the longissimus muscle of cattle and cultured with activators of murine PPARgammaA thiazolidinedione T-174, which is a specific ligand for PPARgamma, stimulated adipose differentiation (evaluated by counting differentiated adipocytes under microscopic observation) in a dose-dependent fashion. A peroxisome proliferator Wy14,643 which strongly activates the alpha isoform of murine PPAR also stimulated differentiation but its potency was weaker than that of T-174. Unexpectedly, 15-deoxy-Delta12,14-prostaglandin J2, which is believed to be an endogenous ligand for PPARgamma, could not induce adipose differentiation in doses which have been found to be effective on rodent cells. Immunoblotting analysis confirmed the significant expression of PPARgamma protein in fibroblast-like cell cultures prepared from bovine skeletal muscle. In conclusion, bovine skeletal muscle contains adipose precursor cells expressing functionally active PPARgamma.     

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2′-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7^highMyoD^lowEdU^pos) and activated satellite cells (Pax7^highMyoD^highEdU^pos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.     

TCP11L2 promotes bovine skeletal muscle-derived satellite cell migration and differentiation via FMNL2

T-complex 11 like 2 (TCP11L2) is a protein containing a serine-rich region in its N-terminal region. However, the function of TCP11L2 is unclear. Here, we showed that TCP11L2 expression gradually increased during muscle-derived satellite cell (MDSC) differentiation in vitro, reaching a peak on Day 3, which is the migration and fusion stage of MDSCs. Using CRISPR/dCas9 gene-editing technology to elevate or repress the expression of TCP11L2, we also showed that TCP11L2 promoted MDSC differentiation. Moreover, wound-healing assays showed that TCP11L2 promoted the migration of MDSCs during differentiation. Additionally, immunofluorescence analyses showed that TCP11L2 was mainly distributed around the microfilament and microtubules. Furthermore, the expression of TCP11L2 affected the expression of actin-related protein 2/3 (ARP2/3) complex. Co-immunoprecipitation assays and immunofluorescence analysis showed that TCP11L2 interacted with formin-like 2 (FMNL2). This protein promoted migration of bovine MDSCs by affecting the expression of ARP2/3. Finally, the activities of TCP11L2 during MDSC differentiation and migration were blocked when FMNL2 was inhibited. Taken together, our data established that TCP11L2 interacted with FMNL2 to promote MDSC migration and differentiation.     

大鼠骨骼肌卫星细胞诱导分化为胰岛素生成细胞的研究

目的探讨大鼠骨骼肌卫星细胞（MDSCs）定向诱导分化为胰岛素生成细胞（IPCs）,为1型糖尿病的干细胞治疗提供一种新的研究思路。 方法通过二次酶消化法和差速贴壁培养法分离、培养大鼠MDSCs,利用不同的诱导培养液使MDSCs定向分化为IPCs,并对诱导后细胞进行形态观察,通过双硫腙染色和免疫组化染色对MDSCs-IPCs形态进行鉴定,采用Q-PCR和Western Blot方法检测MDSCs-IPCs中C-peptide和Insulin的表达,通过胰岛素释放实验检测MDSCs-IPCs的生物学功能,β细胞和MDSCs-IPCs两组间比较采用t检验。 结果MDSCs在接种4 h后开始贴壁部分细胞伸出小的突起,48 h后绝大多数细胞贴壁呈梭形、胞浆丰富、折光度高。随着培养时间的延长,细胞的梭形形状更为明显且生长迅速。免疫组化结果显示细胞表达Desmin、α-Sarcomeric Actinin、MyoD1、Myf5和PAX7。成胰诱导后MDSCs形成胰岛样的圆形细胞团,双硫腙染色呈猩红色,Insulin免疫组化染色阳性。Q-PCR结果显示MDSCs-IPCs中C-peptide和Insulin mRNA表达量分别是β细胞的0.73倍（P > 0.05）和0.79倍（P > 0.05）。胰岛素释放实验显示,5.6 mmol/L和16.7 nmlol/L葡萄糖刺激培养2 h后,β细胞和MDSCs-IPCs分泌胰岛素量分别为[（20.3±4.2）mU/L]、[（16.1±3.7）mU/L]、[（60.5±9.3）mU/L]和[（40.9±7.3）mU/L],葡萄糖可调节MDSCs-IPCs胰岛素的分泌。 结论MDSCs易于分离培养、增殖能力强,体外可诱导分化为有功能的IPCs,适合作为再生医学的种子细胞。     

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant‐derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast‐CD56⁺ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56‐purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non‐human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H₂O₂)‐induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non‐toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.     

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C₂C₁₂) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. © 1996 Wiley-Liss, Inc.     

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1β secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1β expression, it suppressed the secretion of cleaved IL-1β. In contrast, HGF decreased active IL-1β secretion without affecting pro-IL-1β expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1β expression level and active IL-1β secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.