Pathologic Caveolin-1 Regulation of PTEN in Idiopathic Pulmonary Fibrosis
2010; Elsevier BV; Volume: 176; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.091117
ISSN1525-2191
AutoresXia Hong, Wajahat Khalil, Judy Kahm, José Jessurun, Jill Kleidon, Craig A. Henke,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoIdiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder refractory to current pharmacological therapies. Fibroblasts isolated from IPF patients display pathological activation of PI3K/Akt caused by low PTEN phosphatase activity. This enables these cells to escape the negative proliferative properties of polymerized collagen. The mechanism underlying low PTEN activity in IPF fibroblasts is unclear, but our prior studies indicate that membrane-associated PTEN expression is decreased in these cells. Caveolin-1 is an integral membrane protein whose expression is decreased in IPF lung tissue, but how low caveolin-1 contributes to pathological fibrosis is incompletely understood. The objective of this study was to examine the hypothesis that caveolin-1 regulates PTEN function in IPF fibroblasts. Here we demonstrate that caveolin-1 expression is a determinant of membrane PTEN levels and show that PTEN interacts with caveolin-1 via its caveolin-1–binding sequence. We demonstrate that caveolin-1 expression is low in IPF fibroblasts and that this correlates with low membrane PTEN levels, whereas overexpression of caveolin-1 restores membrane PTEN levels, inhibits Akt phosphorylation, and suppresses proliferation. We demonstrate that caveolin-1 and PTEN expression are low in myofibroblasts within IPF fibroblastic foci. These data indicate that IPF fibroblasts display low caveolin-1 expression, which results in low membrane-associated PTEN expression. This creates a membrane microenvironment depleted of inhibitory phosphatase activity, facilitating the aberrant activation PI3K/Akt and pathological proliferation. Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder refractory to current pharmacological therapies. Fibroblasts isolated from IPF patients display pathological activation of PI3K/Akt caused by low PTEN phosphatase activity. This enables these cells to escape the negative proliferative properties of polymerized collagen. The mechanism underlying low PTEN activity in IPF fibroblasts is unclear, but our prior studies indicate that membrane-associated PTEN expression is decreased in these cells. Caveolin-1 is an integral membrane protein whose expression is decreased in IPF lung tissue, but how low caveolin-1 contributes to pathological fibrosis is incompletely understood. The objective of this study was to examine the hypothesis that caveolin-1 regulates PTEN function in IPF fibroblasts. Here we demonstrate that caveolin-1 expression is a determinant of membrane PTEN levels and show that PTEN interacts with caveolin-1 via its caveolin-1–binding sequence. We demonstrate that caveolin-1 expression is low in IPF fibroblasts and that this correlates with low membrane PTEN levels, whereas overexpression of caveolin-1 restores membrane PTEN levels, inhibits Akt phosphorylation, and suppresses proliferation. We demonstrate that caveolin-1 and PTEN expression are low in myofibroblasts within IPF fibroblastic foci. These data indicate that IPF fibroblasts display low caveolin-1 expression, which results in low membrane-associated PTEN expression. This creates a membrane microenvironment depleted of inhibitory phosphatase activity, facilitating the aberrant activation PI3K/Akt and pathological proliferation. Polymerized type I collagen suppresses normal fibroblast proliferation.1Xia H Diebold D Nho R Perlman D Kahm J Kleidon J Avdulov A Peterson M Bitterman PB Henke CA Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (176) Google Scholar, 2Schor SL Cell migration and proliferation on collagen substrata in vitro.J Cell Sci. 1980; 41: 159-175Crossref PubMed Google Scholar, 3Rhudy RW McPherson JM Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet-derived growth factor.J Cell Physiol. 1988; 137: 185-191Crossref PubMed Scopus (33) Google Scholar, 4Koyama H Raines EW Bornfedlt KE Roberts JM Ross R Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of cdk2 inhibitors.Cell. 1996; 87: 1069-1078Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar The mechanism involves inhibition of the PI3K/Akt signal by high PTEN phosphatase activity.1Xia H Diebold D Nho R Perlman D Kahm J Kleidon J Avdulov A Peterson M Bitterman PB Henke CA Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (176) Google Scholar This provides an effective physiological mechanism to limit fibroproliferation after tissue injury. In contrast, inappropriately low PTEN activity has been implicated in the pathogenesis of human fibroproliferative disorders, and PTEN-deficient mice display increased lung fibrosis in response to injury attributable to a durable fibroproliferative response.1Xia H Diebold D Nho R Perlman D Kahm J Kleidon J Avdulov A Peterson M Bitterman PB Henke CA Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (176) Google Scholar, 5White ES Atrasz RG Hu B Phan SH Stambolic V, Mak TW, Hogaboam CM, Flaherty KR, Martinez FJ, Kontos CD, Toews GB: negative regulation of myofibroblast differentiation by phosphatase and tensin homologue deleted on chromosome ten.Am J Respir Crit Care Med. 2006; 173: 112-121Crossref PubMed Scopus (177) Google Scholar Importantly, lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), a prototypical fibroproliferative disorder, display depleted membrane PTEN levels and consequently inappropriately low PTEN activity in response to their interaction with polymerized collagen via β1 integrin.1Xia H Diebold D Nho R Perlman D Kahm J Kleidon J Avdulov A Peterson M Bitterman PB Henke CA Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (176) Google Scholar This results in pathological activation of the integrin/PI3K/Akt signal pathway, which enables them to elude the proliferation-suppressive effects of polymerized collagen. The molecular mechanism underlying this inappropriately low PTEN function in IPF fibroblasts remains to be elucidated. PTEN is a dual lipid/protein phosphatase that negatively regulates proliferation by repressing the integrin–PI3K/Akt pathway.6Yamada KM Araki M Tumor suppressor PTEN: modulator of cell signaling, growth, migration and apoptosis.J Cell Science. 2002; 114: 2375-2382Google Scholar, 7Georgescu MM Kirsch KH Akagi T Shishido T Hanafusa H The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region.Proc Natl Acad Sci U S A. 1999; 96: 10182-10187Crossref PubMed Scopus (278) Google Scholar, 8Stambolic V Tsao MS Macpherson D Suzuki A Chapman WB Mak TW High incidence of breast and endometrial neoplasia resembling human Cowden Syndrome in pten+/- mice.Cancer Res. 2000; 60: 3605-3611PubMed Google Scholar, 9Lee JO Yang H Georgescu MM Cristofano AD Maehama T Shi Y Dixon JE Pandolfi P Pavletich NP Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association.Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar, 10Tamura M Gu J Danen EHJ Takino T Miyamoto S Yamada KM PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.J Biol Chem. 1999; 274: 20693-20703Crossref PubMed Scopus (325) Google Scholar, 11Stambolic V Suzuki A de la Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2103) Google Scholar, 12Lu Y Yu Q Liu JH Zhang J Wang H Koul D McMurray JS Fang X Yung WKA Siminovitch KA Mills GB Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidyinositol 3-kinase/Akt cascades.J Biol Chem. 2003; 278: 40057-40066Crossref PubMed Scopus (212) Google Scholar, 13Li DM Sun H TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor β.Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar It has relatively high constitutive phosphatase activity consistent with its function as a tumor suppressor. A current paradigm for PTEN regulation suggests that the phosphorylation of key serine/threonine residues in the PTEN C-terminal tail determine protein stability and activation. Evidence suggests that PTEN activation involves dephosphorylation of serine/threonine residues within the C terminus and translocation from the cytosol to the plasma membrane where it is in the right location to inhibit phosphoinositol 3,4,5-triphosphate.14Torres J Pulido R The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus.J Biol Chem. 2001; 276: 993-998Crossref PubMed Scopus (529) Google Scholar, 15Vazquez F Ramaswamy S Nakamura N Sellers WR Phosphorylation of the PTEN tail regulates protein stability and function.Mol Cell Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (653) Google Scholar, 16Wu X Hepner K Castelino-Prabhu Do D Kaye MB Yuan XJ Wood J Ross C Sawyers CL Whang YE Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2.Proc Natl Acad Sci U S A. 2000; 97: 4233-4238Crossref PubMed Scopus (338) Google Scholar However, the precise mechanism for PTEN localization to the plasma membrane and its activation remain incompletely understood. Caveolin-1 is an integral membrane protein that regulates a variety of cellular processes, including integrin turnover and signal transduction pathways controlling cell proliferation and apoptosis, such as the PI3K/Akt signal pathway.17Shi F Sottile J Caveolin-1-dependent beta1 integrin endocytosis is a critical regulator of fibronectin turnover.J Cell Sci. 2008; 121: 2360-2371Crossref PubMed Scopus (185) Google Scholar, 18Li L Ren CH Tahir SA Ren C Thompson TC Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A.Mol Cell Biol. 2003; 23: 9389-9404Crossref PubMed Scopus (257) Google Scholar Studies indicate that similar to PTEN haplo-insufficient mice, caveolin-1–deficient mice also display an increased propensity for the development of lung fibrosis.19Drab M Verkade P Elger M Kasper M Lohn M Lauterbach B Menne J Lindschau C Mende F Luft FC Schedl A Haller H Kurzchalia TV Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1312) Google Scholar, 20Galdo FD Sotgia F de Almeida CJ Jasmin JF Musick M Lisanti MP Jimenez SA decreased expression of caveolin-1 in patients with systemic sclerosis: crucial role in the pathogenesis of tissue fibrosis.Arthritis Rheum. 2008; 58: 2854-2865Crossref PubMed Scopus (140) Google Scholar Interestingly, caveolin-1 expression has been found to be low in fibrotic lung tissue from patients with IPF, but the mechanism by which caveolin-1 deficiency results in exaggerated fibrosis is still incompletely understood.21Wang XM Zhang Y Kim HP Zhou Z Feghali-Bostwick CA Lui F Ifedigbo E Xu X Oury TD Kaminiski N Choi AM Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis.J Exp Med. 2006; 203: 2895-2906Crossref PubMed Scopus (323) Google Scholar However, amino acid sequence analysis of PTEN indicates that PTEN contains the caveolin-1 consensus binding sequence ΦXΦXXXXΦ corresponding to amino acids 271-278 (FHFWVNTF), where Φ = aromatic amino acid phenylalanine (F).22Couet J Li S Okamoto T Ikezu T Lisanti MP Identification of peptide and protein ligands for the caveolin-scaffolding domain.J Biol Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar This suggests a relationship between caveolin-1 expression and PTEN function. Here we demonstrate that the level of caveolin-1 protein expression is a determinant of membrane-associated PTEN levels and activity. The mechanism involves direct interaction of caveolin-1 with PTEN via PTEN′s caveolin-1–binding sequence. We demonstrate that caveolin-1 protein expression is decreased in IPF fibroblasts cultured on polymerized collagen compared with control fibroblasts, and this corresponds to low membrane PTEN expression and augmented levels of phosphorylated Akt. However, overexpression of caveolin-1 in IPF fibroblasts augments PTEN levels, reduces the level of phospho-Akt, and suppresses their ability to proliferate. We show that there is a low level of caveolin-1 and PTEN expression in myofibroblasts comprising fibroblastic foci in human IPF lung tissue and that within the fibroblastic focus, the pattern of caveolin-1 expression correlates well with that of PTEN. Our data suggest that in IPF fibroblasts, reduced caveolin-1 expression at the plasma membrane creates a membrane microenvironment depleted of PTEN phosphatase activity and favorable for the pathological activation of the PI3K/Akt signal pathway. This confers IFP fibroblasts with the ability to elude the proliferation-suppressive effects of polymerized type I collagen. Eight primary fibroblast lines were established from IPF patients. Cells were obtained from lungs removed at the time of transplantation or death. The diagnosis of IPF was supported by history, physical examination, pulmonary function tests, and typical high-resolution chest computed tomography findings of IPF. In all cases, the diagnosis of IPF was confirmed by microscopic analysis of lung tissue and demonstrated the characteristic morphological findings of usual interstitial pneumonia. All patients fulfilled the criteria for the diagnosis of IPF as established by the American Thoracic Society and European Respiratory Society.23American Thoracic Society American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias.Am J Respir Crit Care Med. 2002; 165: 277-304Crossref PubMed Scopus (3385) Google Scholar Six nonfibrotic primary control adult human lung fibroblast lines were used. These lines were established from normal lung tissue (n = 3), or histologically normal lung tissue adjacent to carcinoid tumor (n = 2) or adjacent to radiation-induced fibrotic lung tissue (n = 1). Primary lung fibroblast lines were generated by explant culture and cultured in high-glucose DMEM containing 10% FCS. Fibroblasts were used between passages 5 and 8. Cells were characterized as fibroblasts as described.24Chen B Polunovsky V White J Blazar B Nakleh R Jessurun J Peterson M Bitterman P Mesenchymal cells isolated after acute lung injury manifest an enhanced proliferative phenotype.J Clin Invest. 1992; 90: 1778-1785Crossref PubMed Scopus (48) Google Scholar Use of human tissues was approved by the Institutional Review Board at the University of Minnesota. Primary lung fibroblasts (ATCC) were cultured in high glucose DMEM containing 10% fetal calf serum. Primary mouse lung fibroblasts, isolated from wild-type caveolin-1 (cav-1 wild-type) or caveolin-1 knock-out mice (cav-1 KO), were a gift from Augustine Choi (Harvard University, Cambridge, MA). PTEN wild-type and null fibroblasts were obtained from Eric White (University of Michigan, Ann Arbor, MI). The cells were cultured in high-glucose DMEM containing 10% fetal calf serum. The fibroblasts were used between passages 5 and 8 for all experiments. Anti-Cyclin D1, GAPDH, α-SMA antibodies were obtained from Santa Cruz Biotechnology Company (Santa Cruz, CA). Caveolin-1, keratin 8/18, PTEN, HA, and phospho-Akt (Ser473) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Three-dimensional polymerized collagen matrices (final concentration, 2 mg/ml) were prepared by neutralizing the collagen solution with 1/6 volume of 6× DMEM and diluting to a final volume with 1× DMEM to which fetal calf serum was added at a final concentration of 1% fetal calf serum. Gels formed after incubation of this solution at 37°C for 1 to 2 hours as described previously.9Lee JO Yang H Georgescu MM Cristofano AD Maehama T Shi Y Dixon JE Pandolfi P Pavletich NP Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association.Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar, 10Tamura M Gu J Danen EHJ Takino T Miyamoto S Yamada KM PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.J Biol Chem. 1999; 274: 20693-20703Crossref PubMed Scopus (325) Google Scholar, 11Stambolic V Suzuki A de la Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2103) Google Scholar Adenoviral vectors containing wild-type PTEN(Ad-wtPTEN), mutant PTEN(ad-mPTEN), and control (Ad-GFP) constructs were purified according to the manufacturer’s instructions (Takara Shuzo Co, Ltd., Kyoto, Japan). The adenoviral vector containing wild-type caveolin-1 was a gift from Augustine Choi (Harvard University). The cells were infected with adenoviral vectors at a multiplicity of infection of 1:20. Caveolin-1 and control siRNA were obtained from Invitrogen. Caveolin-1 siRNA is from Validated Stealth RNAi duoPak. Control siRNA is from Stealth RNA interference negative control duplexes. Transient transfection of normal lung fibroblasts was performed using FuGENE. HD siRNA transfection reagent was obtained from Roche Applied Science (Indianapolis, IN) and used according to the manufacturer’s instructions. Quantification of apoptosis was performed by DNA content assay and fluorescent-based assay for detection of active caspases in cells undergoing apoptosis (CHEMICON International, Inc., Billerica, MA). Briefly, the cells were fixed in ice-cold 70% alcohol overnight and stained with 2 μg/ml propidium iodide, 100 μg/ml DNase in 1× phosphate-buffered saline buffer for 60 minutes. The percentage of cells in each phase of the cell cycle was quantified by FACS analysis using FACSCalibur software. Serum-starved fibroblasts were plated on extracellular matrix-coated plates and lysed at the indicated times using cell lysis buffer containing 150 mmol/L NaCl, 1 mmol/L EGTA, 50 mmol/L Tris, pH 7.4, 1% Triton X-100, 1% Nonidet P-40, 1% sodium deoxycholate (SDS), with protease inhibitors (complete protease inhibitor mixture tablets; Roche Applied Science). To isolate the cell membrane fraction, cells were first lysed with 25 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Triton X-100, followed by centrifugation, and the pellets containing the membrane fraction lysed in lysis buffer containing Triton X-100, Nonidet P-40, and SDS (see above). Western analysis was performed on the resulting lysates. For immunoprecipitation assay, the cells were lysed in lysis buffer containing 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, protease, and phosphatase inhibitor cocktails. The samples were centrifuged at 20,000g for 15 minutes at 4°C, and the lysates were precleared for 1 hour at room temperature with protein A/G beads and immunoprecipitated for 16 hours at 4°C with the appropriate primary antibody. The samples were processed for Western analysis. Recombinant caveolin-GST protein was obtained from Abnova (Taipei, Taiwan). Recombinant PTEN-His protein was from R&D Systems (Minneapolis, MN). 0.5 μg of recombinant caveolin-1 and recombinant PTEN protein, either alone or mixed together, were added to the reaction buffer (120 mmol/L NaCl, 10 mmol/L KCl, 2 mmol/L KPO4, pH 7.4 in PBS) containing GST-beads and incubated (4°C, 16 hours). After centrifugation, the beads were washed 3× with wash buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.02% triton X-100). Western analysis was then performed. Fibroblasts were serum-starved for 2 days and then plated on monomeric (100 μg/ml) or on top of polymerized collagen (2 mg/ml) matrices in DMEM + 1% fetal calf serum. After the cells were replated onto polymerized collagen matrices, the media were replaced with DMEM + 10% FBS. The cells were incubated with 10 μmol/L BrdU for 5 hours before the cells were harvested at 24 hours. The cells were then stained with anti-BrdU antibody to quantify DNA synthesis and 7-amino actinomycin D as a measure of total DNA. DNA synthesis was quantified by assessing the percentage of BrdU-positive cells by FACS according to the manufacturer’s instructions (BD Biosciences, San Diego, CA). PTEN was immunoprecipitated from cell lysates with 4 μg of anti-PTEN antibody. 10 μl of PTEN enzyme assay buffer (100 mmol/L Tris-HCl, pH 8.0, and 2 mmol/L dithiothreitol) and 10 μl of phospholipid vesicles (PLV; 0.1 mmol/L diC8PIP3, 0.5 mmol/L dioleoyl phosphatidylserine in 20 mmol/L HEPES, pH 7.4, and 1 mmol/L EGTA in diC8PIP3 followed by sonication for 30 minutes) were added to 5 μl of immunoprecipitate and incubated. The enzyme reaction was terminated by adding 100 μl of Malachite Green solution. PTEN activity was measured using a microtiter plate reader at 630 nm. Immunostaining was performed on paraffin-embedded IPF and control lung tissue using a Zeiss Axiovert 200M Confocal Microscope. Immunofluorescence studies were performed on caveolin-1–null and wild-type fibroblasts cultured on glass coverslips. The cells were plated on glass coverslips and infected with the adenoviral vector containing wild-type caveolin-1 construct for 24 hours. The cells were then fixed, permeabilized, and incubated (60 minutes, room temperature) with the appropriate primary antibody (PTEN or Cav-1). The cells were then incubated (60 minutes, room temperature) with cy2- or cy3-conjugated secondary antibodies, followed by incubation (10 minutes, room temperature) with DAPI. Primary antibodies caveolin-1 and total PTEN (6H2.1) were obtained from Cell Signaling and Cascade Bioscience (Winchester, MA), respectively. Comparisons of data among each experiment were performed with the unipolar unpaired or paired Student t test. All experiments were replicated a minimum of three times. Data are expressed as mean ± SD. P < 0.05 were considered significant. The sentinel morphological lesion of IPF is the fibroblastic focus. Fibroblastic foci consist of a subepithelial accumulation of α smooth muscle actin expressing fibroblasts in a type I collagen–rich matrix. We have found that membrane PTEN levels are decreased in primary fibroblasts derived from patients with IPF, and a previous study has found that caveolin-1 expression is decreased in IPF lung tissue and fibroblasts.1Xia H Diebold D Nho R Perlman D Kahm J Kleidon J Avdulov A Peterson M Bitterman PB Henke CA Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (176) Google Scholar, 21Wang XM Zhang Y Kim HP Zhou Z Feghali-Bostwick CA Lui F Ifedigbo E Xu X Oury TD Kaminiski N Choi AM Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis.J Exp Med. 2006; 203: 2895-2906Crossref PubMed Scopus (323) Google Scholar Together, these data suggest a potential relationship between caveolin-1 and PTEN expression in IPF. To begin to address this issue, we analyzed the pattern of caveolin-1 and PTEN expression within IPF fibroblastic foci by immunofluorescence confocal microscopy and compared it to human control lung tissue (Figure 1A, H&E, L–N staining of a fibroblastic focus). α smooth muscle actin was used as a marker of myofibroblasts and keratin 8/18 as an epithelial marker. Several differences in caveolin-1 and PTEN staining features between the IPF fibroblastic focus and control lung tissue were noteworthy. First, when analyzing IPF lung tissue, we noted that prominent caveolin-1 and PTEN expression was displayed in cells overlying the fibroblastic foci (Figure 1B for caveolin-1 and F for PTEN). These cells stained positive for the epithelial cell marker keratin 8/18, suggesting that they are epithelial (Figure 1, J and K). α smooth muscle actin staining was not apparent in the epithelial cells overlying the fibroblastic foci. In contrast, cells constituting the fibroblastic foci, especially those in a zone just beneath the overlying epithelial cells, stained prominently for α smooth muscle actin indicating that these cells are myofibroblasts (Figure 1, C and G). Myofibroblasts within the fibrotic foci displayed relatively low intensity expression of both caveolin-1 and PTEN compared with the overlying epithelial cells (Figure 1, merged panels D and E showing caveolin-1 and α-smooth muscle actin and merged panels H and I showing PTEN and α-smooth muscle actin). Second, when we analyzed control lung tissue, we found that both caveolin-1 and PTEN expression were very prominent in cells lining the alveolar airspace (Figure 1, O–R), many of which stained positive for the epithelial cell marker keratin 8/18. The vast majority of caveolin-1 and PTEN staining cells lining the normal alveolar airspace did not stain for α smooth muscle actin (see supplemental Figure 1 at http://ajp.amjpathol.org). Rare cells within the alveolar wall stained for both vimentin and caveolin-1, suggesting that interstitial fibroblasts may stain for cavolin-1 (see supplemental Figure 2 at http://ajp.amjpathol.org). Taken together, these data demonstrate that myofibroblasts within the fibroblastic focus display relatively low intensity expression of caveolin-1 and PTEN compared with epithelial cells overlying the fibroblastic focus or lining the normal alveolar airspace. In addition, the level of caveolin-1 and PTEN expression in the myofibroblasts correlates well with one another. We have previously found that membrane PTEN levels are decreased in IPF fibroblasts.1Xia H Diebold D Nho R Perlman D Kahm J Kleidon J Avdulov A Peterson M Bitterman PB Henke CA Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.J Exp Med. 2008; 205: 1659-1672Crossref PubMed Scopus (176) Google Scholar To discern whether PTEN levels correlate with caveolin-1 levels in our primary IPF fibroblast lines, we analyzed caveolin-1 expression in primary lung fibroblasts derived from IPF patients (n = 6) and from control patients (n = 6) by Western analysis. We found that caveolin-1 expression was decreased in primary IPF fibroblast cell lines compared with controls (Figure 2), consistent with a prior report.21Wang XM Zhang Y Kim HP Zhou Z Feghali-Bostwick CA Lui F Ifedigbo E Xu X Oury TD Kaminiski N Choi AM Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis.J Exp Med. 2006; 203: 2895-2906Crossref PubMed Scopus (323) Google Scholar PTEN inhibits the integrin/PI3K/Akt signal pathway. Its activation is believed to involve translocation from the cytoplasm to the membrane where it is activated and in the right location to inhibit PI3K/Akt. Because caveolin-1 is an integral membrane protein, which also regulates signal transduction including the PI3K/Akt pathway, this suggested that low caveolin-1 expression may be a determinant of low membrane PTEN levels in IPF fibroblasts. To analyze whether caveolin-1 expression regulates membrane-associated PTEN levels, we used caveolin-1 wild-type and null lung fibroblasts. We found that caveolin-1–null fibroblasts displayed low levels of membrane-associated PTEN compared with wild-type controls, and this was associated with an augmented level of phospho-Akt (Figure 3A). Consistent with this immunofluorescence analysis showed that caveolin-1–null fibroblasts displayed a faint cytoplasmic distribution of PTEN, whereas PTEN expression was more intense in wild-type cells (data not shown). We next examined the effect of reconstituting caveolin-1 into caveolin-1–null fibroblasts using an adenoviral vector containing a wild-type caveolin-1 construct on membrane PTEN levels. Caveolin-1–null cells reconstituted with caveolin-1 now displayed caveolin-1 expression. Caveolin-1–null cells transfected with an empty vector did not express caveolin-1. Caveolin-1–null fibroblasts reconstituted with caveolin-1 displayed increased levels of membrane-associated PTEN and a lower level of phospho-Akt compared with cells transfected with empty vector (Figure 3B). These data indicate that the level of caveolin-1 expression is a determinant of the amount of PTEN associated with the membrane. Because caveolin-1 is a determinant of PTEN expression and the pattern of caveolin-1 expression correlates with PTEN expression, we next sought to assess whether PTEN associates with caveolin-1. We first analyzed whether we could detect a binding association between endogenous PTEN and caveolin-1 in control and IPF fibroblasts. An endogenous caveolin-1/PTEN complex could be detected in control fibroblasts (Figure 4A; see also Figure 5B), where endogenous caveolin-1 and membrane PTEN levels are relatively high. In contrast, both membrane PTEN and caveolin-1 levels are
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