Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway.

The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the development and differentiation of several tissue types. Here, we examine the role of NFATC2 in skeletal muscle by analyzing adult NFATC2(-/)- mice. These mice exhibit reduced muscle size due to a decrease in myofiber cross-sectional area, suggesting that growth is blunted. Muscle growth was examined during regeneration after injury, wherein NFATC2-null myofibers form normally but display impaired growth. The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes. Retroviral-mediated expression of NFATC2 in the mutant cells rescues this cellular phenotype. Myonuclear number is similarly decreased in NFATC2(-/)- mice. Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth. We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.

A calcineurin- and NFAT-dependent pathway regulates Myf5 gene expression in skeletal muscle reserve cells.

Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. We propose that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. For these studies, we used myotube cultures that contain both multinucleated myotubes and quiescent, mononucleated cells termed 'reserve' cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an approximately 4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. We used immunohistochemical analyses and fractionation of the cell populations to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. We conclude that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation.

Calcineurin activity is required for the initiation of skeletal muscle differentiation.

Differentiation of skeletal muscle myoblasts follows an ordered sequence of events: commitment, cell cycle withdrawal, phenotypic differentiation, and finally cell fusion to form multinucleated myotubes. The molecular signaling pathways that regulate the progression are not well understood. Here we investigate the potential role of calcium and the calcium-dependent phosphatase calcineurin in myogenesis. Commitment, phenotypic differentiation, and cell fusion are identified as distinct calcium-regulated steps, based on the extracellular calcium concentration required for the expression of morphological and biochemical markers specific to each of these stages. Furthermore, differentiation is inhibited at the commitment stage by either treatment with the calcineurin inhibitor cyclosporine A (CSA) or expression of CAIN, a physiological inhibitor of calcineurin. Retroviral-mediated gene transfer of a constitutively active form of calcineurin is able to induce myogenesis only in the presence of extracellular calcium, suggesting that multiple calcium-dependent pathways are required for differentiation. The mechanism by which calcineurin initiates differentiation includes transcriptional activation of myogenin, but does not require the participation of NFAT. We conclude that commitment of skeletal muscle cells to differentiation is calcium and calcineurin-dependent, but NFAT-independent.

Activation and cellular localization of the cyclosporine A-sensitive transcription factor NF-AT in skeletal muscle cells.

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT-mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT-responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in acrosomal matrix-zona pellucida interactions during and immediately following the acrosome reaction in the mouse.

The sperm acrosomal matrix contains a novel member of the pentaxin family of calcium-dependent binding proteins.

The sperm acrosome is a regulated secretory granule that undergoes exocytosis during fertilization. To elucidate the structural organization of the contents within the acrosome, guinea pig sperm acrosomal apical segments were isolated and mapped by two-dimensional polyacrylamide gel electrophoresis (PAGE). Although complex, the two-dimensional PAGE map was dominated by two M(r) 50,000 polypeptides (p50 and proacrosin), a M(r) 67,000 polypeptide (p67), and a M(r) 32,000 polypeptide (sp32). Proacrosin (pI > 8.0), p67, and sp32 were extracted from apical segments by 1 M NaCl. Protein p50, a relatively acidic polypeptide, was not extracted in 1 M NaCl and/or 1% Triton X-100 at 4 degrees C, but was solubilized with 6 M urea. Protein p50 was purified from the urea extract by elution from DEAE-Sephacel with 100 mM guanidine HCl and appeared homogeneous by SDS-PAGE. Antibodies to p50 were monospecific as judged by Western blot analysis. Indirect immunofluorescence indicated that p50 was restricted to the acrosomal apical segment. Incubation of apical segments at pH 7.5 in the presence of 1 mM EDTA at 37 degrees C resulted in the release of p50 into the 200,000 x g supernatant fluid, a process that was reversed by a subsequent incubation with 1.5 mM CaCl2, but not with MgCl2. The Ca(2+)-dependent reassociation of p50 with the acrosomal apical segments was reversed by the addition of 2.0 mM EGTA, indicating that p50 binding is dependent on free Ca2+ concentrations. When acrosomal matrices were purified following Triton X-100 extraction, p50 was the major component, with p67, proacrosin, and sp32 as less prominent constituents. Molecular cloning demonstrated that p50 is a unique, testis-specific member of the pentaxin family of calcium-dependent binding proteins.