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...

Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-beta1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-beta1-induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-beta receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide-inducible clone-5 (Hic-5) are rearranged in response to TGF-beta1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-beta1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-beta type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-beta receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-beta1-induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.

Chemo-irradiation induced oxidative damage to vascular endothelium may contribute to pulmonary complications of hematopoietic stem cell transplantation (HSCT). We measured antioxidants, markers of oxidative stress and plasma antioxidant capacity in plasma or serum from 24 subjects at day 7 before HSCT and 20 control subjects. The plasma concentration of extracellular glutathione peroxidase (GPX-3) was significantly reduced in the HSCT subjects compared with controls (HSCT: 98742 lg/ml, control: 169756 lg/ml, Po0.0001). The concentration of c-tocopherol was significantly higher in the HSCT subjects compared with controls (HSCT: 2077103 lg/dl; Control: 98752 lg/dl; P ¼ 0.0002). The plasma concentrations of protein carbonyl, nitrotyrosine, malondialdehyde, a-tocopherol, vitamin A, homocysteine, cysteine and cysteinylglycine did not differ between HSCT and control subjects. Plasma from HSCT subjects was as effective as control plasma in quenching menadione-induced intracellular reactive oxygen species production in human microvascular endothelial cells. In summary, subjects before HSCT have significantly reduced plasma concentrations of GPX-3, elevated plasma c-tocopherol yet retains the ability to quench an acute oxidative stress. These changes may play a role in chronic oxidative stress in the HSCT population.