Oncostatin M (OSM) is a cytokine from the interleukin-6 family and

Oncostatin M (OSM) is a cytokine from the interleukin-6 family and plays important functions during inflammation. OSM-induced signaling. Moreover we found that STAT1 actually associated with MEF2 and repressed its transcriptional activity which could account for the OSM-mediated repression of MEF2. Although undetectable in normal muscle tissue are quickly induced on injury 29 30 31 However the role of OSM in myoblast differentiation and muscle mass regeneration remains unexplored. OSM is usually a 28-kDa glycoprotein secreted mainly by macrophages neutrophils and T cells 32. In human cells OSM can use both LIFR/gp130 and OSM receptor (OSMR)/gp130 for signaling. In mouse cells OSM has long been thought to transmission only through OSMR/gp130 33. However this view was recently challenged 34. We provide evidence here showing that OSM potently inhibits myoblast differentiation by selectively activating the JAK1/STAT1/STAT3 pathway. In addition we show that STAT1 actually associates with MEF2 and represses its transcriptional activity. Moreover we show that OSM was transiently induced in muscle tissue on injury and that prolonged expression of OSM in muscle tissue delayed muscle mass regeneration gene expression Torin 2 54 55 While OSM did not affect the expression of MyoD it obviously reduced the appearance of MEF2 in both principal myoblasts and C2C12 cells (Body 4A). Consistently the experience of gene encoding its DNA-binding area (i actually.e. aa 1-147). In this assay the activity of the reporter gene was mainly dependent on the transactivation function of MyoD or MEF2. In C2C12 cells cotransfected with and a construct encoding either Gal4-MyoD/or Gal4-MEFgene expression 54 55 We cotransfected C2C12 cells with a STAT1-expressing vector together with either Flag-MyoD or Flag-MEF2C with or Rabbit Polyclonal to hnRNP H. without OSM treatment. When we immunoprecipitated Flag-MyoD or Flag-MEF2C from your same amount of whole cell extracts (WCE) we found that STAT1 specifically coprecipitated with Flag-MEF2C but not with Flag-MyoD (Physique 5A). Moreover OSM treatment further enhanced the binding between STAT1 and Flag-MEF2C. Consistently we found that MEF2 but Torin 2 not MyoD preferentially associated with phospho-STAT1 as phospho-STAT1 was known to be enriched in the nucleus (Supplementary information Physique S3A). We then immunoprecipitated the endogenous MEF2 from WCE prepared from either proliferating or differentiating C2C12 cells and found that the endogenous STAT1 preferentially associated with MEF2 in proliferating myoblasts even though there was less MEF2 in proliferating myoblasts (Physique 5B). An anti-HA antibody failed to pull down STAT1. To further map the region in MEF2 that interacts with STAT1 we generated two truncated MEF2 constructs: one missing a 90-aa N-terminal MADS/MEF2 domain name (i.e. Δ90) the other missing a 148-aa C-terminus (i.e. 1 Different MEF2 constructs were transfected into C2C12 cells together with a Torin 2 construct expressing STAT1. Coimmunoprecipitation assays were performed to assess the conversation between MEF2 and STAT1. Flag-MyoD was used as a negative control. We found that the N-terminal 90 amino acids are required for MEF2 to specifically interact with STAT1 (Physique 5C). In addition we also mapped the region in STAT1 that Torin 2 interacts with MEF2. We first incubated the purified recombinant His-MEF2 with C2C12 extracts expressing either the full-length or different fragments of STAT1. Using Talon beads (BD Biosciences) to pull down His-tagged MEF2 we showed that two N-terminal STAT1 fragments including aa 1-317 and aa 1-576 failed to interact with His-MEF2 (Physique 5D). In contrast both the full-length and an N-terminus-truncated STAT1 (i.e. aa 317-750) interacted with His-MEF2 (Physique 5D) suggesting that this C-terminal portion of STAT1 is usually involved in interacting with MEF2. To uncover the functional significance of MEF2 conversation with STAT1 we first measured the effect of a constitutively active STAT1 (i.e. STAT1c) on two MEF2-dependent luciferase reporter genes: one is used in Physique 3 and the other is usually a luciferase gene driven by 133-bp proximal mouse myogenin promoter (i.e. G133-luc) 57 62 As shown in Physique 5E STAT1c inhibited the activity Torin 2 of both MEF2-dependent reporters. To find out whether STAT1c repressed the transcriptional activity of MEF2 we tested its effect on Gal4-MEF2 using as a reporter gene. As shown in Body 5E STAT1c could repress the transcriptional directly.