The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-β signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1−/− mice at various ages (1–12 mo). Cav1−/− mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1−/− mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1−/− mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1α2 and col3α1 gene expression in lung tissues, which correlated tightly with increased TGF-β/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-β signaling pathway, in the regulation of the pulmonary ECM

SMAD-independent down-regulation of caveolin-1 by TGF-β: effects on proliferation and survival of myofibroblasts.

Transforming growth factor-β (TGF-β) mediates growth-inhibitory effects on most target cells via activation of the canonical SMAD signaling pathway. This growth-inhibitory activity may be coupled with cellular differentiation. Our studies demonstrate that TGF-β1 inhibits proliferation of primary, non-transformed human lung fibroblasts in association with the induction of myofibroblast differentiation. Differentiated myofibroblasts maintain the capacity to proliferate in response to exogenous mitogenic stimuli and are resistant to serum deprivation-induced apoptosis. These proliferative and anti-apoptotic properties of myofibroblasts are related, in part, to the down-regulation of caveolin-1 (Cav-1) by TGF-β1. Cav-1 down-regulation is mediated by early activation of p38 MAPK and does not require SMAD signaling. In contrast, myofibroblast differentiation is dependent on activation of the SMAD pathway, but not on p38 MAPK. Thus, combinatorial signaling by TGF-β1 of myofibroblast differentiation and down-regulation of Cav-1 by SMAD and p38 MAPK pathways, respectively, confer proliferative and apoptosis-resistant properties to myofibroblasts. Selective targeting of this SMAD-independent, p38-MAPK/Cav-1-dependent pathway is likely to be effective in the treatment of pathological conditions characterized by TGF-β signaling and myofibroblast activation

Blocking p38 MAPK inhibits myofibroblast proliferation.

<p><b><i>A</i></b>, IMR-90 cells stably expressing a p38 MAPK dominant negative (pcDNA-p38KM) and cells stably transfected with an empty vector (pcDNA) were serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h (n = 6 per group), data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pcDNA “fibroblasts”. **indicates <i>p</i> < 0.01 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. <b><i>B</i></b>, Cells described in (A) were grown in 96-well plates and serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% fetal bovine serum (n = 6 per group), data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pcDNA “fibroblasts”. **indicates <i>p</i> < 0.01 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. <b><i>C</i></b>, IMR-90 cells stably expressing SMAD2 <i>sh</i>RNA and cells stably transfected with an empty vector (pSU6H) were serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h (n = 6 per group) data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts” (no TGF-β1 pre-treatment). **indicates <i>p</i> < 0.05 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). <b><i>D</i></b>, Cells described in (C) were grown in 96-well plates and serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% fetal bovine serum (n = 6 per group) data shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts” (no TGF-β1 pre-treatment). **indicates <i>p</i> < 0.05 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated).</p

<i>sh</i>RNA knock-down of Cav-1 enhances fibroblast/myofibroblast proliferation and protects against serum deprivation-induced apoptosis.

<p><b><i>A</i></b>, IMR-90 cells stably transfected with a plasmid encoding <i>sh</i>RNA targeted against Cav-1 (pSU6H-<i>sh</i>Cav1) or with control plasmid (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and Western blots for Cav-1, α-smooth muscle actin (α-SMA) and β-tubulin performed. <b><i>B</i></b>, Stably-transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by stimulation with 10% FBS for 24 h. Cell counts were assessed both prior to and after serum stimulation with an automated Coulter counter (n = 6 per group) shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts”. **indicates <i>p</i> < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. <b><i>C</i></b>, Stably transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% FBS (n = 6 per group) shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts”. Similar results were obtained from 3 independent experiments. <b><i>D</i></b>, Quiescent stably-transfected cells described in (<b><i>A</i></b>) were treated with/without TGF-β1 (2 ng/ml) for 5 days and apoptotic assay for <i>ss</i>DNA performed as described in “Methods” (n = 6 per group) shown as mean±S.E.M. *indicates <i>p</i> < 0.05 vs. control pSU6H “fibroblasts”. **indicates <i>p</i> < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments.</p

Down-regulation of Cav-1 by TGF-β1 is mediated by p38 MAPK-dependent and SMAD-independent mechanisms.

<p><b><i>A</i></b>, IMR-90 cells were treated with inhibitors of p38 MAPK (SB203580; 6 μM) or ALK5 (SB431542; 0.5 μM) for 30 min prior to treatment with or without TGF-β1 (2 ng/ml) for a period of 48 h. Cell lysates were extracted and Western immunoblotting performed with an antibody against Cav-1; the blot was then stripped and probed for β-tubulin. <b><i>B</i></b>, Densitometric analyses of blots in (A) showed as % inhibition of baseline Cav-1 protein expression levels treated with TGF-β1. *indicates effect of SB203580 to completely block the inhibitory effect of TGF-β1 on Cav-1 expression. Results are averages of at least three independent experiments. Data are presented as mean±S.E.M. <b><i>C</i></b>, IMR-90 cells stably transfected with a kinase-deficient p38 MAPK (pcDNA-p38KM) or control vector (pcDNA) were treated with or without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to SDS-PAGE and immunoblotted for Cav-1 and α-smooth muscle actin (α-SMA); blots were stripped and probed for β-tubulin. <b><i>D</i></b>, IMR-90 cells stably expressing SMAD2 <i>sh</i>RNA (pSU6H-<i>sh</i>SMAD2) or control vector (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were immunoblotted for SMAD2, Cav-1 and α-SMA. The blots were stripped and probed for β-tubulin.</p

Schematic representation of TGF-β1-activated signaling pathways mediating mesenchymal cell growth -suppressive and -promoting effects.

<p>TGF-β1 activates the cell surface TGF-β receptor(s) complex that leads to rapid activation of the canonical SMAD pathway as well as the SMAD-<i>in</i>dependent p38 MAPK pathway. Activation of the SMAD pathway is required for the induction of a cellular program of growth-arrest and myofibroblast differentiation. In contrast, activation of the p38 MAPK pathway, independently of SMAD2/3, is required the down-regulation of Cav-1 by TGF-β1. Down-regulation of Cav-1 by TGF-β1 “primes” differentiated myofibroblasts for enhanced proliferative responses to mitogens and resistance to apoptosis. These divergent TGF-β signaling pathways may explain, in part, the contextual effects of TGF-β1 as both a growth-inhibitor and –promoter on the same target (mesenchymal) cells.</p