Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle α-actin (smα) in response to profibrotic agents such as TGFβ is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2 −/− ) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2 −/− MEF to express less smα than their corresponding wild-type (WT) MEF at baseline and in response to TGFβ. Furthermore, TGFβ causes downregulation of smα in MK2 −/− MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2 −/− MEF. Our results further suggest that downregulation of smα in MK2 −/− MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smα message stability in these cells. These results indicate that MK2 plays a key role in regulation of smα expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis. Keywords fibrosis; smooth muscle actin; cytoskeleton; MAPKAPK2; fibroblast; stress fibers Differentiation of fibroblasts into myofibroblasts is an important event in many conditions such as wound repair and fibrosis. For example, in pulmonary fibrosis (PF) myofibroblasts occur in areas of active fibrosis and are responsible for production and deposition of extracellular matrix proteins [Vyalov et al., 1993]. Myofibroblasts derive from fibroblasts through the action of growth factors, such as, TGFβ [Desmouliere et al., 1993;Ronnov-Jessen and Petersen, 1993;Yokozeki et al., 1997;Roy et al., 2001]. Several signaling pathways have been proposed to mediate the actions of TGFβ on fibroblasts including the MAP kinase p38. Furthermore inhibition of p38 reduced pulmonary [Underwood et al., 2000;Matsuoka et al., 2002] and renal [Stambe et al., 2004] fibrosis in animal models. Recently the role of differentiation of fibroblasts into myofibroblasts in the pathogenesis of pulmonary hypertension has also been highlighted [Stenmark et al., 2002;Short et al., 2004]. We launched this project to investigate the role of MAP kinase activated protein kinase 2 (MAPKAPK2 or MK2), which is a *Correspondence to: Usamah S. Kayyali, PhD, MPH, Pulmonary and Critical Care Division, Tufts-New England Medical Center, 750 Washington Street #257, Boston, MA 02111. E-mail: ukayyali@tufts-nemc.org. [Piguet et al., 1993;Pan et al., 1996] have been reported to be elevated in the lungs of IPF patients. The range of cytokines altered during fibrosis led researchers to propose that fibrosis is the result of...

Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pm-HSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia-and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27. permeability; edema; pulmonary; vascular; cytoskeleton HYPOXIA has been shown to alter the structure and function of endothelial cells in a manner that contributes to a variety of diseases. Signaling pathways that are activated by hypoxia such as p38 mitogen-activate protein (MAP) kinase and Rho kinase pathways alter the actin cytoskeleton and contractile proteins leading to changes in biomechanical forces in these cells (3). These forces likely play an important role in endothelial cell migration and interaction with leukocytes as well as in regulation of endothelial barrier permeability which is altered in hypoxia (10,14,19). Intermediate filaments constitute one of the three major components of the cytoskeleton which also include actin microfilaments and tubulin microtubules.These structures act in concert to control cell morphology and biomechanics. The role of intermediate filaments in regulation of endothelial function has not been sufficiently studied.We have focused on the regulation of vimentin filaments in rat pulmon...

This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia-induced contractility which was ROCK-dependent rather than HSP27-dependent. Results described here show that increased HSP27 phosphorylation not only does not underlie hypoxia-induced permeability, but may actually augment the endothelial barrier. Hypoxia causes gap formation between RPMEC and increases MLC2 phosphorylation. The phosphorylation of MYPT1, which inhibits MLC2 phosphatase, is also increased in hypoxia. In addition, FAK phosphorylation, which alters focal adhesion signaling, is increased in hypoxia. Overexpressing phospho-mimicking HSP27 (pmHSP27), which induces significant actin stress fiber formation, surprisingly renders RPMEC resistant to hypoxia-or TGFβ-induced permeability. siRNA against pmHSP27 reverses the increased actin stress fiber formation in pmHSP27-overexpressing cells, and disrupting actin stress fibers in pmHSP27-overexpressing RPMEC renders them more susceptible to hypoxia. Finally, hypoxia-induced gap formation, as well as phosphorylation of MLC2, MYPT1 and FAK are almost abolished by overexpressing pmHSP27 in RPMEC. These effects of pmHSP27 overexpression might represent decreased cytoskeletal plasticity and increased tethering which counteracts permeability-inducing contractility. Thus hypoxia activates two pathways one leading to contractility and increased permeability, the other leading to actin stress fibers, stronger adhesion, and reduced permeability. Altering HSP27 phosphorylation, which tips the balance towards decreased permeability, might be targeted in managing endothelial barrier dysfunction.