Kevin K. Kim

Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix

Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. Although there is in vitro evidence of lung alveolar epithelial-to-mesenchymal transition (EMT), whether EMT occurs within the lung is currently unknown. Biopsies from fibrotic human lungs demonstrate epithelial cells with mesenchymal features, suggesting EMT. To more definitively test the capacity of alveolar epithelial cells for EMT, mice expressing β-galactosidase (β-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-β1. β-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-β1 expression. The increase in vimentin-positive cells within injured lungs was nearly all β-gal-positive, indicating epithelial cells as the main source of mesenchymal expansion in this model. Ex vivo, primary alveolar epithelial cells cultured on provisional matrix components, fibronectin or fibrin, undergo robust EMT via integrin-dependent activation of endogenous latent TGF-β1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-β1 pathway and, if exposed to active TGF-β1, undergo apoptosis rather than EMT. These data reveal alveolar epithelial cells as progenitors for fibroblasts in vivo and implicate the provisional extracellular matrix as a key regulator of epithelial transdifferentiation during fibrogenesis.

Epithelial cell α3β1 integrin links β-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis

Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-beta1. As the ECM critically regulates AEC responses to TGF-beta1, we explored the role of the prominent epithelial integrin alpha3beta1 in experimental fibrosis by generating mice with lung epithelial cell-specific loss of alpha3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through beta-catenin has been implicated in EMT; we found that in primary AECs, alpha3 integrin was required for beta-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654-beta-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654-beta-catenin/pSmad2 complexes and showed accumulation of pY654-beta-catenin in myofibroblasts. These findings demonstrate epithelial integrin-dependent profibrotic crosstalk between beta-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.

Integrin α3β1–dependent β-catenin phosphorylation links epithelial Smad signaling to cell contacts

Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, α3β1, were found to have a markedly blunted EMT response to TGF-β1. A mechanism for this defect was explored in α3-null cells reconstituted with wild-type (wt) α3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-β1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-β1 receptors, generate phospho-Smad2 (p-Smad2)–pY654–β-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2–pY654–β-catenin complexes do not form in the absence of α3 or when α3β1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that α3β1 coordinates cross talk between β-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-β1 activation.

Regulation of α5β1 integrin conformation and function by urokinase receptor binding

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with β1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the β-propeller of α3β1 empowers vitronectin adhesion by this integrin. How uPAR modifies other β1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds α5β1 and rather than blocking, renders fibronectin (Fn) binding by α5β1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of α5β1–uPAR to Fn type III repeats 12–15 in addition to type III repeats 9–11 bound by α5β1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall α5β1 conformation. A β1 peptide (res 224NLDSPEGGF232) that models near the known α-chain uPAR-binding region, or a β1-chain Ser227Ala point mutation, abrogated effects of uPAR on α5β1. Direct binding and regulation of α5β1 by uPAR implies a modified “bent” integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

The C2 Domains of Rabphilin3A Specifically Bind Phosphatidylinositol 4,5-Bisphosphate Containing Vesicles in a Ca2+-dependent Manner IN VITRO CHARACTERISTICS AND POSSIBLE SIGNIFICANCE

In the present study we investigated the lipid binding characteristics of the C2 domains of Rabphilin3a. We found that the tandem C2 domain of Rabphilin3a specifically bound lipid vesicles containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in a Ca2+-dependent manner. There was little binding to vesicles containing PtdIns(3,4)P2 in the presence or absence of Ca2+. Binding to phosphatidylinositol 3,4,5-triphosphate-containing vesicles was similar to binding to PtdIns(4,5)P2-containing vesicles. The presence of physiological amounts of phosphatidylserine (PS) greatly potentiated the ability of PtdIns(4,5)P2 to cause vesicle binding. As with the C2 domains together, the binding of individual C2 domain of Rabphilin3a was much greater to PtdIns(4,5)P2-containing vesicles than PtdIns(3,4)P2-containing vesicles. Both C2 domains also bound 29 mol % PS-containing vesicles in a Ca2+-dependent manner. Because of the importance of the C2B domain in the enhancement of secretion from chromaffin cells by Rabphilin3a, its biochemistry was further investigated. The mutation of aspartates 657 and 659 to asparagines in C2B decreased Ca2+-dependent and increased Ca2+-independent vesicle binding, indicating the Ca2+ dependence of the domain is provided by aspartic acid residues in the putative Ca2+-binding pocket. A peptide from the COOH-terminal region of the C2B domain specifically inhibited ATP-dependent secretion from permeabilized chromaffin cells and the binding of Rabphilin3a to phosphatidylcholine/PS/PtdIns(4,5)P2-containing lipid vesicles, suggesting a role of this sequence in secretion through its ability to interact with acidic lipid vesicles.

Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis

Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process.

Axin pathway activity regulates in vivo pY654-β-catenin accumulation and pulmonary fibrosis

Background: TGFβ1-induced pY654-β-catenin correlates with epithelial mesenchymal transition (EMT) and pulmonary fibrosis, but whether pY654-β-catenin is functionally important is unknown. Results: β-Catenin point mutants reveal that pY654 is critical to EMT, and pY654-β-catenin accumulation is blocked by axin-dependent β-catenin turnover. Conclusion: Raised axin levels in vivo attenuate EMT and fibrosis after bleomycin injury. Significance: Targeting axin levels could attenuate fibrosis without blocking TGFβ1 homeostatic functions. Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis, we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.

Inhibition of Myocardin-Related Transcription Factor/Serum Response Factor Signaling Decreases Lung Fibrosis and Promotes Mesenchymal Cell Apoptosis

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-β1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-β1-induced myofibroblast differentiation; and inhibited TGF-β1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-β1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Activated Alveolar Epithelial Cells Initiate Fibrosis through Secretion of Mesenchymal Proteins

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-β-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor

Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.