Nongenomic estrogen action in human lung myofibroblasts

Recent studies have suggested that the antiproliferative effects of E2 may be mediated through a nongenomic action. Herein, we asked whether nongenomic estrogen action regulates phosphorylation of Raf1 and ERK1/2 mitogen-activated protein (MAP) kinase in lung myofibroblasts. We demonstrated that lung myofibroblasts, incubated in the presence of E2, showed a rapid phosphorylation on serine-259 of Raf1 and tyrosine-204 of ERK1/2 MAP kinase at 15 min, by approximately 3- and 5-fold, respectively. This phosphorylation was followed by dephosphorylation between 30 and 60 min. Western blot analysis showed that E2 regulates tyrosine phosphorylation of four main cytoplasmic proteins in lung myofibroblasts, of 42, 44, 70 and 100 kDa. Furthermore, our results indicated that E2 inhibits cell proliferation (BrdU index) in lung myofibroblasts by approximately 30% (P < 0.01). These data provide evidence that nongenomic action of E2, regulates both serine and tyrosine phosphorylation of cytoplasmic proteins in lung myofibroblasts, including Raf1 and ERK1/2 MAP kinase, which may regulate proliferation in lung myofibroblasts.     

IL-6 induces neuroendocrine dedifferentiation and cell proliferation in non-small cell lung cancer cells

Interleukin-6 (IL-6) has been identified as an important growth regulator of lung cancer cells. Elevation of serum levels of IL-6 has been found in a subpopulation of lung cancer patients, but rarely in patients with benign lung diseases. Approximately 15% of non-small cell lung cancer (NSCLC) tumors exhibit neuroendocrine (NE) properties (NSCLC-NE) and have been suggested to have the biological characteristics similar to small cell lung cancer (SCLC) with early metastasis and initial responsiveness to chemotherapy. We recently showed that IL-6 promotes cell proliferation and downregulates the expression of neuron-specific enolase (NSE, one of the major NE markers) in NSCLC-NE cells. In this study, we show that IL-6 stimulates a transient increase of tyrosine phosphorylation of STAT3 in a dose-dependent fashion. Inhibition of STAT3 signaling pathway by either AG-490 (JAK2-specific inhibitor) or overexpression of STAT3Y705F (a dominant-negative STAT3) reverses NSE expression in IL-6- treated NSCLC-NE cells. In addition, IL-6 induces phosphorylation and activation of p38 MAPK. SB-203580, a p38 MAPK-specific inhibitor, inhibits IL-6-induced p38 MAPK phosphorylating activity and suppresses IL-6-stimulated cell proliferation. Together, our results indicate that STAT3 signaling pathway is involved in IL-6-induced NE differentiation and that p38 MAPK is associated with IL-6-stimulated growth regulation in NSCLC-NE cells. These data suggest that both kinase pathways play critical roles in the pathogenesis of NSCLC-NE malignancies, providing new molecular targets for future therapeutic approaches.     

BCR-ABL induces opposite phenotypes in murine ES cells according to STAT3 activation levels

The mechanisms by which p210-BCR-ABL determines hematopoietic stem cells fate remain poorly understood. To better understand the behavior of BCR-ABL in pluripotent stem cells, we previously developed a murine embryonic stem (ES) cell model transformed by p210-BCR-ABL and reported that BCR-ABL activates STAT3, a major protein involved in ES cells self-renewal, which leads specifically to inhibition of ES cells differentiation. We show here that BCR-ABL either inhibits differentiation or, unexpectedly, induces a rapid commitment to differentiation of murine ES cells, according to the intracellular levels of activated STAT3. We show that inhibition of endogenous STAT3 activation with an inducible STAT3 protein with dominant-negative activity (STAT3F) results in an early, rapid and complete differentiation of BCR-ABL-expressing ES cells, whereas control ES cells retain a more undifferentiated phenotype. This phenomenon could be totally abrogated by PD98059, a specific MEK1 inhibitor, suggesting the involvement of mitogen-activated protein kinase (MAP-Kinase)/ERK1/2 pathway, which was found constitutively phosphorylated in BCR-ABL-expressing cells. In addition, BCR-ABL-expressing ES cells harboring low levels of activated STAT3 committed more rapidly through hematopoietic differentiation, since embryoid bodies (EBs) derived from these cells were able to generate numerous hematopoietic progenitors 2 days early. Moreover, BCR-ABL-expressing ES cells cultured first with low levels of activated STAT3 before EBs derivation displayed a more rapid loss of pluripotency than controls and failed to generate hematopoietic progenitors. This phenomenon was partially abrogated when ES cells were first exposed to PD98059 or to the tyrosine kinase inhibitor imatinib mesylate. From this predictive model, we suggest that variations of the activation levels in BCR-ABL substrates such as STAT3 may represent "instructive" secondary cooperating events involved in the transformation of the leukemic cell phenotype during the course of CML.     

Attenuation of immunological symptoms of allergic asthma in mice lacking the tyrosine kinase ITK

Allergic asthma patients manifest airway inflammation and some show increases in eosinophils, T(H)2 cells, and cytokines, increased mucous production in the lung, and elevated serum IgE. This T(H)2-type response suggests a prominent role for T(H)2 cells and their cytokines in the pathology of this disease. The Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) has been shown to play a role in the differentiation and/or function of T(H)2-type cells, suggesting that ITK may represent a good target for the control of asthma. Using a murine model of allergic asthma, we show here that ITK is involved in the development of immunological symptoms seen in this model. We show that mice lacking ITK have drastically reduced lung inflammation, eosinophil infiltration, and mucous production following induction of allergic asthma. Notably, T cell influx into the lung was reduced in mice lacking ITK. T cells from ITK(-/-) mice also exhibited reduced proliferation and cytokine secretion, in particular IL-5 and IL-13, in response to challenge with the allergen OVA, despite elevated levels of total IgE and increased OVA-specific IgE responses. Our results suggest that the tyrosine kinase ITK preferentially regulates the secretion of the T(H)2 cytokines IL-5 and IL-13 and may be an attractive target for antiasthmatic drugs.     

Modulation of STAT5 interaction with LMW-PTP during early megakaryocyte differentiation

STATs are involved in a variety of cellular processes, including cell proliferation and differentiation. They are activated through tyrosine phosphorylation, which promotes their dimerization via SH2 domains. We have demonstrated previously that in DAMI megakaryoblastic cells LMW-PTP dephosphorylates STAT5, interacting with an essential sequence of nine amino acids in its C-terminal region. Here we characterize STAT5 tyrosine phosphorylation and its interaction with LMW-PTP during early phorbol-12-myristate-13-acetate-induced megakaryocyte differentiation; these processes show clear dependence on STAT5 threonine phosphorylation. Since protein kinase C inhibition prevents phorbol-12-myristate-13-acetate-induced STAT5 threonine phosphorylation and association with LMW-PTP, it follows that these processes depend on protein kinase C activity. By using a Thr757/Val mutant of STAT5 we also demonstrate that the 757 serine/threonine conserved residue, which is in the STAT5A region involved in the interaction with LMW-PTP, is essential for such an association, though its phosphorylation is not necessary.     

Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts

Transforming growth factor-beta (TGF-beta) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-beta1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-beta1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-beta1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-beta1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-beta1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-beta1 in tissue fibrosis and tumour-promotion.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.     

bFGF and LIF signaling activates STAT3 in proliferating myoblasts

Different mitogens elicit similar effects on growth and differentiation of skeletal muscle, suggesting that potential overlap exists in the signaling cascades activated by such factors. To investigate this possibility, we examined the status of STAT and ERK proteins in C2C12 myoblasts and myotubes following stimulation with bFGF or LIF. Both STAT1 and STAT3 as well as ERK1 and ERK2 proteins were detectable in extracts of myoblasts. LIF stimulation of myoblasts lead to rapid phosphorylation on tyrosine of STAT3 and of ERKs 1 and 2. Similarly, bFGF stimulation of myoblasts resulted in the tyrosine phosphorylation of STAT3. However, unlike LIF, the bFGF induced tyrosine phosphorylation of STAT3 appeared cyclical, with recurrent peaks of phosphorylation even after prolonged exposure. By contrast, STAT1 remained unphosphorylated in myoblasts treated with bFGF or LIF. In differentiated myotubes, LIF treatment resulted in the tyrosine phosphorylation of both STAT3 and STAT1, but ERK phosphorylation was not detectable, and bFGF treatment did not lead to STAT1 or STAT3 tyrosine phosphorylation. Therefore these observations suggest that disparate mitogens can activate similar downstream effectors in proliferating myoblasts. © 1996 Wiley-Liss, Inc.     

EGF and PDGF receptor tyrosine kinases as therapeutic targets for chronic lung diseases

Cell-surface receptor tyrosine kinases play pivotal roles in development, tissue repair, and normal cellular homeostasis. Aberrant expression or signaling patterns of these kinases has also been linked to the progression of a diversity of diseases, including cancer, atherosclerosis, asthma, and fibrosis. Two major families of receptor tyrosine kinases, the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) families, have received a great deal of attention as potential therapeutic targets for pulmonary diseases, as these receptors have been shown to play key roles in chronic tissue remodeling in asthma, bronchitis, and pulmonary fibrosis. The EGFR system on epithelial cells and underlying mesenchymal cells (fibroblasts, myofibroblasts, and smooth muscle cells) drives numerous phenotypic changes during the progression of these pulmonary diseases, including epithelial cell mucous cell metaplasia and mesenchymal cell hyperplasia, differentiation, and extracellular matrix production. The PDGFR system, located primarily on mesenchymal cells, transduces signals for cell survival, growth and chemotaxis. The variety of EGFR and PDGFR ligands produced by the airway epithelium or adjacent mesenchymal cells allows for intimate epithelial-mesenchymal cell communication. A full understanding of the complex mechanisms involving these receptors and ligands should lead to therapeutic strategies for the treatment of a wide range of fibroproliferative lung diseases.