Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway.

In response to transforming growth factor beta1 (TGFbeta) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFbeta has been shown to increase the expression of specific alphav integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFbeta1 increased both beta3 integrin subunit mRNA and protein levels as well as surface expression of alphavbeta3 in human lung fibroblasts. TGFbeta1-induced alphavbeta3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of beta3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or alphavbeta3 blocking antibody prevented the increase in beta3 but not beta5 integrin expression. In addition, echistatin inhibited TGFbeta1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFbeta1-induced expression of alphavbeta3 and p38 MAPK phosphorylation but not beta5 integrin expression and Smad3 activation. The TGFbeta1-induced alphavbeta3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-kappaB-dependent pathways. Our results demonstrate that TGFbeta1 induces alphavbeta3 integrin expression via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFbeta1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

alpha(v)beta(3) Integrin interacts with the transforming growth factor beta (TGFbeta) type II receptor to potentiate the proliferative effects of TGFbeta1 in living human lung fibroblasts.

The alpha(v)beta(3) integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether alpha(v)beta(3) regulates transforming growth factor beta (TGFbeta) 1-induced responses, we investigated the interaction between alpha(v)beta(3) and TGFbeta type II receptor (TGFbetaIIR) in primary human lung fibroblasts. We report that TGFbeta1 up-regulates cell surface and mRNA expression of alpha(v)beta(3) in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFbetaRII associates and clusters with alpha(v)beta(3), following TGFbeta1 exposure. This association was not observed with alpha(v)beta(5) or alpha(5)beta(1). We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFbeta1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds alpha(v)beta(3), did not induce a substantial BRET signal. Co-exposure to tenascin and TGFbeta1 produced no further increases in BRET than TGFbeta1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFbeta1 and tenascin, and as a consequence proliferation induced by TGFbeta1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFbetaRII and alpha(v)beta(3) and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks alpha(v)beta(3), also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the alpha(v)beta(3) integrin, the TGFbetaIIR, in which TGFbeta1-induced responses are potentiated in the presence alpha(v)beta(3) ligands. Our data provide a novel mechanism by which TGFbeta1 may contribute to abnormal wound healing and tissue fibrosis.

Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis.

It is increasingly recognized that the morphological changes of apoptosis vary between cell types. This heterogeneity reflects the wide range of cellular proteins and enzymes involved in apoptotic pathways. Fibroblast apoptosis is crucial to the regression of scars and the restitution of healthy tissue during wound repair and may be aberrant in diseases such as idiopathic pulmonary fibrosis (IPF). The biochemical and morphological changes characterizing fibroblast apoptosis are unknown and may provide insights into the specific enzymatic mediators activated in these cells. This study aimed to examine the morphological changes of fibroblast apoptosis in both primary normal lung fibroblasts (normal-Fb) and fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF-Fb) and to correlate these changes with conventional biochemical markers. Transmission electron microscopy (TEM) and video time-lapse microscopy demonstrated no difference in the duration of fibroblast apoptosis in response to FasL (6 +/- 0.3 h in normal-Fb and 6.4 +/- 0.2 h in IPF-Fb). However, IPF-Fb were more resistant to FasL-induced apoptosis compared with normal-Fb. Although the majority of morphological changes of normal-Fb and IPF-Fb were similar, the formation of filopodia and condensation of the cytoskeletal bundles in IPF-Fb, and more prominent vacuolation in normal-Fb, were the significant differences between these cell subtypes. Loss of the mitochondrial membrane potential occurred prior to caspase-3 activation, while phosphatidylserine expression, cytokeratin-18 cleavage, and DNA fragmentation commenced after caspase-3 activation. These observations not only suggest that specific enzymatic effectors may be preferentially activated during fibroblast apoptosis, but also provide potential insights into the pathogenesis of IPF.

Fibroblasts isolated from normal lungs and those with idiopathic pulmonary fibrosis differ in interleukin-6/gp130-mediated cell signaling and proliferation.

Interleukin (IL)-6 and IL-11 are elevated in a variety of lung conditions and may impact on repair mechanisms in chronic inflammatory disorders. However, the mechanisms by which these cytokines influence fibroblast proliferation in normal and disease states have not been previously addressed. We examined the effect of these cytokines on proliferation and cell-cycle kinetics of primary human lung fibroblasts obtained from normal patients and patients with idiopathic pulmonary fibrosis (IPF). IL-6 inhibited the proliferation of normal fibroblasts due to the sustained phosphorylation of STAT-3 and production of the cyclin-dependent kinase inhibitor p19(INK4D). In contrast IL-6 was mitogenic for IPF fibroblasts due to the sustained activation of MAPK, which in turn inhibited the production of p27(Kip1), allowing activation of cyclin D(1) and hyperphosphorylation of retinoblastoma protein. IL-11 was mitogenic for both normal and IPF fibroblasts. These results provide strong evidence for a fundamental abnormality in a cytokine-signaling pathway, as opposed to alterations in cytokine production, in the pathogenesis of IPF.

Inverse effects of interleukin-6 on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs.

Fibroblast apoptosis is crucial to the resolution of fibrosis. However, the mechanisms by which these cells undergo apoptosis are not well known. Because interleukin (IL)-6 and IL-11 may alter repair and remodeling processes, we hypothesized that they may play a role in idiopathic pulmonary fibrosis (IPF). We investigated the effects of these cytokines on Fas-induced apoptosis using primary lung fibroblasts from three patients with IPF (IPF-Fb) and three subjects without lung disease (normal-Fb). IPF-Fb were resistant to Fas-induced apoptosis compared with normal-Fb (P < 0.01). Using RNase protection assays, we showed that IL-6 enhanced Fas-induced apoptosis and expression of Bax in normal-Fb, but inhibited apoptosis and induced expression of Bcl-2 in IPF-Fb. Densitometry of Western blots revealed a Bcl-2/Bax ratio 0.15 +/- 0.01 in normal-Fb compared with 12.05 +/- 1.0 in IPF-Fb. Upregulation of Bcl-2 in normal-Fb and Bax in IPF-Fb were both STAT-3-dependent. Inhibition of extracellular signal-regulated kinase had no effect in normal-Fb, but reversed the antiapoptotic effect of IL-6 in IPF-Fb. IL-11 inhibited Fas-induced apoptosis and increased Bcl-2 expression in both normal-Fb and IPF-Fb. These results suggest that altered IL-6 signaling in IPF-Fb may enhance the resistance of these cells to apoptosis and contribute to a profibrotic effect of IL-6 in IPF.

Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts.

1. Oncostatin M (OSM), a member of the interleukin-6 (IL-6) cytokine family, acts on a variety of cells and elicits diversified biological responses, suggesting potential roles in the regulation of cell survival, differentiation and proliferation. 2. We have examined the effect of OSM on the regulation of human lung fibroblast proliferation, collagen production and spontaneous apoptosis. The proliferative effects of OSM (0.5 - 100 ng ml(-1)) were assessed using a MTS assay as well as [(3)H]-thymidine incorporation and cell counts at 24 and 48 h. Hydroxyproline was measured as an index of procollagen production by high pressure liquid chromotography (HPLC). Apoptosis was determined by annexin staining. 3. OSM enhanced the mitotic activity of lung fibroblasts in a time and dose dependent manner. Maximum proliferation of 57% above control was observed after incubation for 48 h with 2 ng ml(-1) OSM (P<0.05). 4. Incubation with the mitogen activated protein kinase (MAPK) kinase inhibitor, PD98059 or the tyrosine kinase inhibitor, genestein both significantly reduced the mitogenic effect of OSM (P<0.05). 5. In contrast, proliferation in response to OSM was not regulated by induction of cyclo-oxygenase and subsequent prostaglandin E(2) (PGE(2)) release or by IL-6. 6. OSM also stimulated fibroblasts to synthesize pro-collagen by a maximum of 35% above control levels after 48 h (P<0.05). 7. OSM significantly inhibited the spontaneous apoptosis of fibroblasts at 24 and 48 h. 8. These results provide evidence that OSM has pro-fibrotic properties and suggest that it may play a role in normal lung wound repair and fibrosis.