Large Maf transcription factor family is a major regulator of fast type IIb myofiber determination

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Differentiation potential of primary myogenic cells derived from skeletal muscle of dystonia musculorum mice

The dystonia musculorum (dt) mouse has a mutation in the gene encoding the cytoskeletal crosslinker protein bullous pemphigoid antigen 1 (Bpag1). These mice have perturbations in the cytoarchitecture of skeletal muscle. Bpag1 has been hypothesized to be involved in the maintenance rather than the establishment of the muscle cell architecture given that cytoskeletal disruptions are observed in the muscle tissue of post-natal dt mice. Not known is whether Bpag1-deficiency affects the proliferative and differentiation potential of myogenic cells. In the present investigation, we show that the growth rate of cultured primary myogenic cells derived from dt mice, as assessed by BrdU incorporation, is similar to that of myogenic cells derived from wild-type littermates. The myogenic differentiation potential of dt versus wild-type cells was monitored by examining the expression of myosin heavy chain by immunofluorescence, and by analyzing the expression profiles of myogenic regulatory factors and myogenic differentiation markers by RT-PCR. In all instances, both dt and wild-type myogenic cells displayed a similar differentiation profile. Furthermore, the absence of any observable differences in the proliferation and differentiation rates of dt and wild-type cells was not due to an overexpression of plectin, another crosslinker protein, in dt cells. Together, these findings demonstrate that the early phases of myogenic differentiation occur independently of Bpag1.     

Community effect triggers terminal differentiation of myogenic cells derived from muscle satellite cells by quenching Smad signaling

A high concentration of bone morphogenetic proteins (BMPs) stimulates myogenic progenitor cells to undergo heterotopic osteogenic differentiation. However, the physiological role of the Smad signaling pathway during terminal muscle differentiation has not been resolved. We report here that Smad1/5/8 was phosphorylated and activated in undifferentiated growing mouse myogenic progenitor Ric10 cells without exposure to any exogenous BMPs. The amount of phosphorylated Smad1/5/8 was severely reduced during precocious myogenic differentiation under the high cell density culture condition even in growth medium supplemented with a high concentration of serum. Inhibition of the Smad signaling pathway by dorsomorphin, an inhibitor of Smad activation, or noggin, a specific antagonist of BMP, induced precocious terminal differentiation of myogenic progenitor cells in a cell density-dependent fashion even in growth medium. In addition, Smad1/5/8 was transiently activated in proliferating myogenic progenitor cells during muscle regeneration in rats. The present results indicate that the Smad signaling pathway is involved in a critical switch between growth and differentiation of myogenic progenitor cells both in vitro and in vivo. Furthermore, precocious cell density-dependent myogenic differentiation suggests that a community effect triggers the terminal muscle differentiation of myogenic cells by quenching the Smad signaling.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.     

Expression of Masticatory-specific Isoforms of Myosin Heavy-chain,Myosin-binding Protein-C and Tropomyosin in Muscle Fibers and Satellite Cell Cultures of Cat Masticatory Muscle

We test the hypothesis that cat jaw satellite cells belong to a distinct lineage preprogrammed to express masticatory-specific isoforms of myosin heavy-chain (m-MyHC), myosin-binding protein-C (m-MBP-C), and tropomyosin (m-Tm) during myogenesis in vitro. A monoclonal antibody (MAb) against m-MyHC and MAbs raised here against cat m-MBP-C and m-Tm were used to stain cryostat sections of cat masseter muscle and cultured myotubes derived from satellite cells of cat temporalis and limb muscles, using peroxidase immunohistochemistry. MAbs against m-MBP-C bound purified m-MBP-C in Western blots. MAbs against m-Tm failed to react with m-Tm in Western blots, but reacted with native m-Tm in gel electrophoresis–derived ELISA. In cat masseter sections, MAbs against m-MyHC, m-MBP-C, and m-Tm stained all masticatory fibers, but not the jaw-slow fibers. Cat jaw and limb muscle cultures mature significantly more slowly relative to rodent cultures. However, at 3 weeks, all three MAbs extensively stained temporalis myotubes, whereas they apparently stained isolated myotubes weakly in cat limb and rat jaw cultures. We conclude that satellite cells of masticatory fibers are preprogrammed to express these isoforms during myogenesis in vitro. These results consolidate the notion that masticatory and limb muscle allotypes are distinct. (J Histochem Cytochem 58:623–634, 2010)     

Reorganization of myosin and focal adhesion proteins in Swiss 3T3 fibroblasts induced by transforming growth factor beta

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF-beta). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell-adhesion molecules were redistributed in TGF-beta-treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western-blot analysis. Changes in cell morphology became apparent within 12 h of the addition of TGF-beta and new RNA and protein synthesis was necessitated by the changes. While TGF-beta induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF-beta treatment, with an increased amount. In addition, the cell-shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell-shape change, and acto-myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF-beta in Swiss 3T3 fibroblasts.     

Conservation of Brachyury, Mef2, and Snail in the myogenic lineage of jellyfish: a connection to the mesoderm of bilateria

One major difference between simple metazoans such as cnidarians and all the bilaterian animals is thought to involve the invention of mesoderm. The terms diploblasts and triploblasts are therefore, often used to group prebilaterian and bilaterian animals, respectively. However, jellyfish contain well developed striated and smooth muscle tissues that derive from the entocodon, a mesoderm-like tissue formed during medusa development. We investigated the hypothesis, that the entocodon could be homologous to the third germ layer of bilaterians by analyzing the structures and expression patterns of the homologues of Brachyury, Mef2, and Snail in the jellyfish Podocoryne carnea. These are regulatory genes from the T-box, MADS-box and zinc finger families known to play important roles in bilaterian mesoderm patterning and muscle differentiation. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. Based on these data we conclude that the common ancestor of the cnidarians and bilaterians not only shared genes that play a role in regulating myogenesis but already used them to develop and differentiate muscle systems similar to those of triploblasts.     

Long-term insulin treatment leads to a change in myosin heavy chain fiber distribution in OLETF rat skeletal muscle

The objective of this study was to investigate molecular and physiological changes in response to long-term insulin glargine treatment in the skeletal muscle of OLETF rats. Male Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats aged 24 weeks were randomly allocated to either treatment with insulin for 24 weeks or no treatment, resulting in three groups. Insulin glargine treatment in OLETF rats (OLETF-G) for 24 weeks resulted in changes in blood glucose levels in intraperitoneal glucose tolerance tests compared with age-matched, untreated OLETF rats (OLETF-C), and the area under the curve was significantly decreased for OLETF-G rats compared with OLETF-C rats (P < 0.05). The protein levels of MHC isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of myosin heavy chain type I and II fibers were lower and higher, respectively, in OLETF-G compared with OLETF-C rats. Activation of myokines (IL-6, IL-15, FNDC5, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF-G compared with OLETF-C rats ( P < 0.05). MyoD and myogenin levels were decreased, while IGF-I and GLUT4 levels were increased, in the skeletal muscle of OLETF-G rats ( P < 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of AMPK, SIRT1, and PGC-1α. Together, our results suggested that changes in the distribution of fiber types by insulin glargine could result in downregulation of myokines and muscle regulatory proteins. The effects were likely associated with activation of the AMPK/SIRT1/PGC-1α signaling pathway. Changes in these proteins may at least partly explain the effect of insulin in skeletal muscle of diabetes mellitus.     

Apoptosis in v-myc-transfected MSU-1.1 fibroblasts is induced by cell-matrix contact and differs from that of normal dermal fibroblasts

In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3-4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF. MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors alpha1beta1 and alpha2beta1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.l fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.     

Regeneration of reinnervated rat soleus muscle is accompanied by fiber transition toward a faster phenotype.

The functional recovery of skeletal muscles after peripheral nerve transection and microsurgical repair is generally incomplete. Several reinnervation abnormalities have been described even after nerve reconstruction surgery. Less is known, however, about the regenerative capacity of reinnervated muscles. Previously, we detected remarkable morphological and motor endplate alterations after inducing muscle necrosis and subsequent regeneration in the reinnervated rat soleus muscle. In the present study, we comparatively analyzed the morphometric properties of different fiber populations, as well as the expression pattern of myosin heavy chain isoforms at both immunohistochemical and mRNA levels in reinnervated versus reinnervated-regenerated muscles. A dramatic slow-to-fast fiber type transition was found in reinnervated soleus, and a further change toward the fast phenotype was observed in reinnervated-regenerated muscles. These findings suggest that the (fast) pattern of reinnervation plays a dominant role in the specification of fiber phenotype during regeneration, which can contribute to the long-lasting functional impairment of the reinnervated muscle. Moreover, because the fast II fibers (and selectively, a certain population of the fast IIB fibers) showed better recovery than did the slow type I fibers, the faster phenotype of the reinnervated-regenerated muscle seems to be actively maintained by selective yet undefined cues.     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

川芎及两种主要药效成分对废用性肌萎缩的影响

目的：探讨川芎及川芎中起活血作用的两种主要药效成分（阿魏酸钠和川芎嗪）对后肢去负荷大鼠比目鱼肌萎缩的影响与作用。方法：尾部悬吊法建立大鼠废用性肌萎缩模型,用免疫组化技术及血液流变学方法观察药物对比目鱼肌各项指标的影响。结果：与后肢去负荷大鼠相比①高剂量的阿魏酸钠和川芎嗪使比目鱼肌I型肌纤维横截面积分别增加了37.3%和39.4%（P〈0.05）;②三种药物均能明显抑制梭外肌纤维MHCII表达水平的升高（P〈0.01）;③使肌梭内核袋2纤维MHCII的表达由阳性转变为阴性;④并能明显降低低切变率下的全血粘度。结论：川芎及两种主要药效成分阿魏酸钠与川芎嗪均能不同程度地对抗废用性肌萎缩的发生,以高剂量川芎嗪与阿魏酸钠的药效最为明显。     

Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4⁺ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28±0.5 pg/ml interleukin-6, (IL-6) in vitro but was negative for interferon , IL-1, IL-2, IL-4, tumor necrosis factor , granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca²⁺ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 °C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50–70 kDa, as detemined by gel filtration.This work was supported in part by the Pathology Education and Research Foundation and American Cancer Society grant IM-696 to TLW     

Purification and characterization of a 200[emsp4 ]kDa fructosyllysine-specific binding protein from cell membranes of U937 cells

Amadori-modified proteins are bound by macrophages and monocytes via fructosyllysine-specific receptors. Detergent extracts from U937 cell membranes were used to purify the binding proteins by affinity purification on glycated polylysine coated magnetic beads followed by SDS-PAGE. Two proteins of 200 and 100[emsp4 ]kDa were isolated. MS-analysis of the 200[emsp4 ]kDa protein showed high homologies with cellular myosin heavy chain, type A. Both fructosyllysine specific binding proteins, cellular myosin heavy chain and nucleolin, are glycosylated.