Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-beta1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-beta1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-beta1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-beta receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-beta1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-beta1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-beta1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-beta1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-beta1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-beta1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.     

Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

Senescence of alveolar type 2 (ATII) cells, progenitors of the alveolar epithelium, is implicated in the pathogeneses of idiopathic pulmonary fibrosis (IPF), an aging‐related progressive fatal lung disorder with unknown etiology. The mechanism underlying ATII cell senescence in fibrotic lung diseases, however, remains poorly understood. In this study, we report that ATII cells in IPF lungs express higher levels of serpine 1, also known as plasminogen activator inhibitor 1 (PAI‐1), and cell senescence markers p21 and p16, compared to ATII cells in control lungs. Silencing PAI‐1 or inhibition of PAI‐1 activity in cultured rat ATII (L2) cells leads to decreases in p53 serine 18 phosphorylation (p53^S18P), p53 and p21 protein expressions; an increase in retinoblastoma protein phosphorylation (ppRb); and a reduction in the sensitivity to bleomycin‐ and doxorubicin‐induced senescence. Silencing p53, on the other hand, abrogates PAI‐1 protein‐stimulated p21 expression and cell senescence. In vivo studies, using ATII cell‐specific PAI‐1 conditional knockout mouse model generated recently in this laboratory, further support the role of PAI‐1 in the activation of p53‐p21‐Rb cell cycle repression pathway, ATII cell senescence, and lung fibrosis induced by bleomycin. This study reveals a novel function of PAI‐1 in regulation of cell cycle and suggests that elevation of PAI‐1 contributes importantly to ATII cell senescence in fibrotic lung diseases.     

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.

Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.     

Aging,antagonistic pleiotropy and fibrotic disease

Tissue fibrosis is most often referred to in its pathological context and is a major cause of progressive organ failure and death. Here, we consider fibrosis as an evolutionarily conserved, adaptive tissue response to injury. The role of NADPH oxidase 4 (Nox4) as a novel pro-fibrogenic mediator is highlighted. The concept that Nox4 may function as an antagonistically pleiotropic gene in age-associated fibrotic disorders is discussed.     

Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-beta-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-beta1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype.     

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions

Members of the peroxidase-cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase-cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase-cyclooxygenase family, including the catalysis of dityrosine formation.     

Reversible differentiation of myofibroblasts by MyoD

Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the reparative response and restoration of tissue structure and function. The current paradigm on normal tissue repair is the apoptotic clearance of terminally differentiated myofibroblasts; while, the accumulation of activated myofibroblasts is associated with progressive human fibrotic disorders. The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for myofibroblast deactivation has not been examined. In this report, we have uncovered a role for MyoD in the induction of myofibroblast differentiation by transforming growth factor-β1 (TGF-β1). Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. We propose a model of reciprocal signaling between TGF-β1/ALK5/MyoD and mitogen(s)/ERK-MAPK/CDKs that regulate myofibroblast differentiation and de-differentiation, respectively. Our studies provide the first evidence for MyoD in controlling myofibroblast activation and deactivation. Restricted capacity for de-differentiation of myofibroblasts may underlie the progressive nature of recalcitrant human fibrotic disorders.