Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive lung disease in which fibroblasts accumulate in the alveolar wall within a type I collagen–rich matrix. Although lung fibroblasts derived from patients with IPF display durable pathological alterations in proliferative function, the molecular mechanisms differentiating IPF fibroblasts from their normal counterparts remain unknown. Polymerized type I collagen normally inhibits fibroblast proliferation, providing a physiological mechanism to limit fibroproliferation after tissue injury. We demonstrate that β1 integrin interaction with polymerized collagen inhibits normal fibroblast proliferation by suppression of the phosphoinositide 3-kinase (PI3K)–Akt–S6K1 signal pathway due to maintenance of high phosphatase activity of the tumor suppressor phosphatase and tensin homologue (PTEN). In contrast, IPF fibroblasts eluded this restraint, displaying a pathological pattern of β1 integrin signaling in response to polymerized collagen that leads to aberrant activation of the PI3K–Akt–S6K1 signal pathway caused by inappropriately low PTEN activity. Mice deficient in PTEN showed a prolonged fibroproliferative response after tissue injury, and immunohistochemical analysis of IPF lung tissue demonstrates activation of Akt in cells within fibrotic foci. These results provide direct evidence for defective negative regulation of the proliferative pathway in IPF fibroblasts and support the theory that the pathogenesis of IPF involves an intrinsic fibroblast defect

Low α2β1 Integrin Function Enhances the Proliferation of Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis by Activation of the β-Catenin Pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α2β1 integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3β and high levels of active β-catenin in the nucleus. Knockdown of β-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α2β1 integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3β and active β-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α2β1 integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the β-catenin pathway

(A) Western blot analysis of phospho-Akt and S6K1 in the absence or presence of the PI3K inhibitor wortmannin (WT) in response to control fibroblast attachment to monomeric collagen in the absence of serum

One representative example is shown. (B) Western blot analysis of phospho-Akt in control fibroblasts preincubated with the indicated integrin blocking antibody and plated on monomeric collagen. (C) Fibroblasts were plated on tissue culture plates coated with monomeric collagen and blocked by BSA. Cells were allowed to spread for 30 min. Cell areas of a random 150 cells were quantified by tracing the cell border using ImageJ software (available at ). (D) Western blot analysis of phospho-Akt and S6K1 in response to control fibroblast attachment to polymerized collagen in the absence of serum. (E) Western blot analysis of phospho-Akt and S6K1 in control fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media. One representative example is shown. (F) GD25, GD25 β1, and GD25 α2β1 fibroblasts were cultured on monomeric (left) or polymerized (right) collagen in the presence of serum, and cell numbers were quantified. (G) Proliferation assay of control fibroblasts expressing dominant-negative Akt (Ad-DN-Akt; left) or constitutively active p110 subunit of PI3K (Ad-PI3Kp110; right) and cultured on monomeric or polymerized collagen in growth factor–replete media, respectively. (top left) Western blot analyses of phospho- and total Akt. (bottom left) Shown is the percent change in cell growth. *, P < 0.01 and 0.001 for the percent change in fibroblast growth on monomeric collagen that was significantly less in control fibroblasts expressing dominant-negative Akt compared with cells expressing control vector at days 3 and 6, respectively. (top right) Western blot analyses of phospho- and total Akt. (bottom right) Shown is the percent change in cell growth. *, P < 0.01 and 0.0001 for the percent change in fibroblast growth on polymerized collagen that was significantly greater in control cells expressing constitutively active p110 subunit of PI3K compared with control vector at days 3 and 6, respectively. Error bars represent SEM. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

(A) Western blot analysis of total PTEN and phospho-Akt and S6K1 in IPF fibroblasts expressing wild-type PTEN (Ad-wtPTEN) or empty vector (Ad-GFP) and cultured on polymerized collagen in growth factor–replete media

(B) eIF4F activity (RLUC luminescence) in IPF fibroblasts expressing wild-type PTEN or control vector and cultured for 3 d on polymerized collagen. *, P < 0.0001 for significantly less eIF4F activity in IPF fibroblasts expressing wild-type PTEN compared with control. (C) Quantification of DNA synthesis by BrdU incorporation in IPF fibroblasts expressing wild-type PTEN or empty vector and cultured on polymerized collagen in growth factor–replete media for 3 d. Shown is the percentage of BrdU-positive IPF fibroblasts. *, P < 0.0001 for the percentage of BrdU-positive IPF fibroblasts expressing wild-type PTEN that was significantly less compared with control. Error bars represent SEM. Data are representative of three independent experiments from one IPF cell line.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

(A and B) Western blot analysis of total PTEN in control (A) or IPF (B) fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media

One representative sample out of five control and five IPF cell lines examined is shown. (C and D) PTEN phosphatase assay showing the percent change in PTEN activity in control ( = 3; C, left) or IFP ( = 3; D, left) fibroblasts cultured on polymerized or monomeric collagen in growth factor–replete media compared with cells cultured under proliferation-prohibitive conditions. *, P < 0.001 for PTEN activity in control fibroblasts on polymerized collagen that was significantly higher at day 3 compared with monomeric collagen; P < 0.01 for PTEN activity in IPF fibroblasts on polymerized collagen that was significantly less on day 3 compared with monomeric collagen. Shown is PI3K activity in control (C, right) and IPF (D, right) fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media for 3 d. *, P < 0.005 for PI3K activity in control fibroblasts cultured on polymerized collagen that was significantly less at day 3 compared with monomeric collagen; or P < 0.05 for PI3K activity in IPF fibroblasts cultured on polymerized collagen that was significantly higher at day 3 compared with monomeric collagen. Error bars represent SEM. (E) PTEN protein levels in membrane and cytosolic fractions of cell lysates were measured by Western blot analysis. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

Control fibroblasts were transfected with PTEN or control siRNA and cultured on polymerized collagen in serum

(A) Western blot analysis of total PTEN levels. (B) Western blot analysis showing PTEN protein expression in response to increasing concentrations of PTEN siRNA. (C) Proliferation assay of control fibroblasts on polymerized collagen in serum-replete media. After 6 d, there was a significant increase in proliferation on polymerized collagen in cells treated with PTEN siRNA compared with control siRNA (*, P < 0.04). Error bars represent SEM. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p

(A) Western blot analyses of phospho-Akt and S6K1 in response to IPF fibroblast attachment to monomeric collagen in the absence of serum

One representative example is shown. (B) Western blot analysis of phospho- and total Akt in response to treatment of control and IPF fibroblasts with TS2/16 β1 integrin activating antibody. (C) Control ( = 3) or IPF ( = 3) fibroblasts were stained with anti–β1 integrin antibodies and analyzed by FACS. Anti–β1 integrin antibodies used were against ligand-induced binding sites (9EG7), activating sites (TS2/16), or total β1 integrin regardless of activation state (P5D2). (D) Control and IPF fibroblasts were plated on monomeric collagen. Shown is a Western blot analysis of phosphorylated FAK (tyr397). Data are representative of three independent experiments from one control and one IPF cell line. (E) PI3K activity in control and IPF fibroblasts plated on tissue culture dishes and treated with TS2/16 β1 integrin activating antibody. Data are representative of three independent experiments from one IPF cell line. (F) Western blot analysis of phospho-Akt and S6K1 in response to IPF fibroblast attachment to polymerized collagen. One representative example is shown. (G) Western blot analysis of phospho-Akt in IPF fibroblasts preincubated with the indicated integrin blocking antibody and plated on polymerized collagen. Data are representative of three independent experiments from one representative IPF cell line. (H) Western blot analysis of phospho-Akt and S6K1 in IPF fibroblasts cultured on monomeric or polymerized collagen in growth factor–replete media. Data are representative of three independent experiments from five IFP cell lines. (I) Proliferation assay of IPF fibroblasts expressing dominant-negative Akt (left) or treated with 100 nM rapamycin (right) and cultured on polymerized collagen in growth factor–replete media. Shown is the percent change in cell growth. *, P < 0.01 and 0.0001 for the percent change in cell growth on polymerized collagen that was significantly less in IPF fibroblasts expressing dominant-negative Akt at days 3 and 6, respectively; or P < 0.002 or 0.001 for the percent change in cell growth on polymerized collagen that was significantly less in IPF fibroblasts treated with rapamycin at days 3 or 6, respectively. Error bars represent SEM. Data are representative of three independent experiments from one IPF cell line. (J) IPF fibroblasts were preincubated with the indicated integrin blocking antibody and plated on polymerized collagen in growth factor–replete media in the presence of antibody. BrdU staining as a measure of DNA synthesis was assessed at 24 h. Error bars represent SEM. Data are representative of three independent experiments from three IPF cell lines.<p><b>Copyright information:</b></p><p>Taken from "Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1659-1672.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442643.</p><p></p