Serum response factor (SRF) regulates transcription of many serum-inducible and muscle-specific genes. Using a functional screen, we identified LIM kinase-1 as a potent activator of SRF. We show that SRF activation by LIM kinase-1 is dependent on its ability to regulate actin treadmilling. LIM kinase activity is not essential for SRF activation by serum, but signals depend on alterations in actin dynamics. Studies with actin-binding drugs, the actin-specific C2 toxin, and actin overexpression demonstrate that G-actin level controls SRF. Regulation of actin dynamics is necessary for serum induction of a subset of SRF target genes, including vinculin, cytoskeletal actin, and srf itself, and also suffices for their activation. Actin treadmilling provides a convergence point for both serum- and LIM kinase-1-induced signaling to SRF.

The mammalian target of rapamycin (mTOR) and Akt proteins regulate various steps of muscle development and growth, but the physiological relevance and the downstream effectors are under investigation. Here we show that S6 kinase 1 (S6K1), a protein kinase activated by nutrients and insulin-like growth factors (IGFs), is essential for the control of muscle cytoplasmic volume by Akt and mTOR. Deletion of S6K1 does not affect myoblast cell proliferation but reduces myoblast size to the same extent as that observed with mTOR inhibition by rapamycin. In the differentiated state, S6K1(-/-) myotubes have a normal number of nuclei but are smaller, and their hypertrophic response to IGF1, nutrients and membrane-targeted Akt is blunted. These growth defects reveal that mTOR requires distinct effectors for the control of muscle cell cycle and size, potentially opening new avenues of therapeutic intervention against neoplasia or muscle atrophy.

Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics. SRF activity can be inhibited by ectopic expression of ␤-actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations. To distinguish between these possibilities, we studied actin mutants. Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies. These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio. Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor. SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B. Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals. These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding. Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF. INTRODUCTIONSerum response factor (SRF) is a transcription factor that regulates many immediate-early and muscle-specific genes. Deletion of SRF in ES cells leads to alterations in cellular morphology and adhesion, and is lethal in mice at gastrulation owing to defects in mesoderm formation (Arsenian et al., 1998;Weinhold et al., 2000;Schratt et al., 2002). SRF activity is controlled by the Rho family of small GTPases (Hill et al., 1995), and recent studies have revealed a close connection between SRF activation and actin polymerization. Downstream of RhoA, both the ROCK-LIMK-cofilin and the mDia effector pathways can promote both F-actin accumulation and SRF activity (Sotiropoulos et al., 1999;Tominaga et al., 2000;Copeland and Treisman, 2002;Geneste et al., 2002). The ability of LIMK and mDia mutants to activate SRF correlates with their ability to promote F-actin accumulation, and interfering derivatives of these proteins can inhibit the activation of SRF by extracellular signals (Sotiropoulos et al., 1999;Tominaga et al., 2000;Copeland and Treisman, 2002;Geneste et al., 2002). Alterations in actin dynamics are required for RhoA-mediated SRF activation, which is inhibited upon treatment of cells with the G-actin binding drug latrunculin or C2 toxin (Sotiropoulos et al., 1999). The RhoA-actin pathway controls a subset of SRF target genes, including the immediate-early genes ␤-actin, vinculin, and srf, and the muscle-specific SM22 and SM ␣-actin genes (Sotiropoulos et al., 1999;Gineitis and Treisman, 2001;Mack et al., 2001).Several lines of evidence suggest that actin itself is intimately involved in the control o...

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a⌬772 supported prolactin-induced transcription of a ␤-casein promoter-reporter construct in COS7 cells; Stat5a⌬750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a⌬750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a⌬750 and Stat5b⌬754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.The Jak-Stat pathway relays cytokine and growth factor signals from the cell surface to the nucleus (19, 20, 36). Jak (Janus kinase) factors are a family of receptor-associated tyrosine kinases. Stat (signal transducer and activator of transcription) factors are transcription factors regulated by Jak-catalyzed phosphorylation events. The Stat gene family contains seven members which share structural similarities.First insights have been gained into the domain structure of the Stat factors. A distinctive feature of the Stat factors is an SH2 domain, a phosphotyrosine-binding domain. It mediates specific interactions of the Stat factors with the cytoplasmic region of cytokine receptors (14,16,45) and is required for Stat dimerization (43). Stat dimers translocate to the nucleus and bind to specific DNA sequences in the promoters of responsive genes. The use of chimeric Stat1-Stat3 or Stat1-Stat6 fusion molecules led to the identification of a DNA-binding domain. It is localized in a region between amino acid positions 400 and 500, distinct from the SH3 and SH2 domains (17, 38).The slightly different sequences of the Stat DNA-binding motifs, their arrangement in the promoter regions of individual genes, and the properties of the Stat dimers are determinants of the specificity of transcriptional regulation (23,38,42). Selective recruitment of Stats to the receptor, specificity of Stat DNA binding, and specific transactivation properties are mechanisms by which a specific cytokine response is elicited.Alignments show that sequence diversity among members of the Stat family is most pronounced in the carboxyl-termi...

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. Here we show by snRNA-seq and snATAC-seq that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we show that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a previously undefined mechanism of coordination of gene expression in a syncytium.

SUMMARY Mechanisms governing muscle satellite cell withdrawal from cell cycle to enter into quiescence remain poorly understood. We studied the role of angiopoietin 1 (Ang1) and its receptor Tie-2 in the regulation of myogenic precursor cell (mpc) fate. In human and mouse, Tie-2 was preferentially expressed by quiescent satellite cells in vivo and reserve cells (RCs) in vitro. Ang1/Tie-2 signaling, through ERK1/2 pathway, decreased mpc proliferation and differentiation, increased the number of cells in G0, increased expression of RC-associated markers (p130, Pax7, Myf-5, M-cadherin), and downregulated expression of differentiation-associated markers. Silencing Tie-2 had opposite effects. Cells located in the satellite cell neighborhood (smooth muscle cells, fibroblasts) up-regulated RC-associated markers by secreting Ang1 in vitro. In vivo, Tie-2 blockade and Ang1 overexpression increased the number of cycling and quiescent satellite cells, respectively. We propose that Ang1/ Tie-2 signaling regulates mpc self-renewal by controlling the return to quiescence of a subset of satellite cells.

S6 kinase (S6K) deletion in metazoans causes small cell size, insulin hypersensitivity, and metabolic adaptations; however, the underlying molecular mechanisms are unclear. Here we show that S6K-deficient skeletal muscle cells have increased AMP and inorganic phosphate levels relative to ATP and phosphocreatine, causing AMP-activated protein kinase (AMPK) upregulation. Energy stress and muscle cell atrophy are specifically triggered by the S6K1 deletion, independent of S6K2 activity. Two known AMPK-dependent functions, mitochondrial biogenesis and fatty acid beta-oxidation, are upregulated in S6K-deficient muscle cells, leading to a sharp depletion of lipid content, while glycogen stores are spared. Strikingly, AMPK inhibition in S6K-deficient cells restores cell growth and sensitivity to nutrient signals. These data indicate that S6K1 controls the energy state of the cell and the AMPK-dependent metabolic program, providing a mechanism for cell mass accumulation under high-calorie diet.

Activation of AMP-activated protein kinase (AMPK) inhibits protein synthesis through the suppression of the mammalian target of rapamycin complex 1 (mTORC1), a critical regulator of muscle growth. The purpose of this investigation was to determine the role of the AMPKalpha1 catalytic subunit on muscle cell size control and adaptation to muscle hypertrophy. We found that AMPKalpha1(-/-) primary cultured myotubes and myofibers exhibit larger cell size compared with control cells in response to chronic Akt activation. We next subjected the plantaris muscle of AMPKalpha1(-/-) and control mice to mechanical overloading to induce muscle hypertrophy. We observed significant elevations of AMPKalpha1 activity in the control muscle at days 7 and 21 after the overload. Overloading-induced muscle hypertrophy was significantly accelerated in AMPKalpha1(-/-) mice than in control mice [+32 vs. +53% at day 7 and +57 vs. +76% at day 21 in control vs. AMPKalpha1(-/-) mice, respectively]. This enhanced growth of AMPKalpha1-deficient muscle was accompanied by increased phosphorylation of mTOR signaling downstream targets and decreased phosphorylation of eukaryotic elongation factor 2. These results demonstrate that AMPKalpha1 plays an important role in limiting skeletal muscle overgrowth during hypertrophy through inhibition of the mTOR-signaling pathway.