Roles of epithelial cell-derived periostin in TGF-β activation, collagen production, and collagen gel elasticity in asthma

Abstract

Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial–mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-β signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-β–dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-β activation and collagen gel elasticity in asthma.

In high-density microarray studies of gene expression in the airway epithelium in asthmatic subjects and healthy controls, we previously found that periostin is among the most highly differentially expressed genes in asthma (1). Full-length periostin is a secreted 90-kDa disulfide-linked protein composed of a typical signal sequence followed by a cysteine-rich EMI domain (believed to be important in multimerization), a tandem repeat of four fascilin-like (FAS1) domains—an evolutionarily conserved domain considered important in adhesion—and a variable C-terminal region (2–5). Periostin is considered a matricellular protein with expression confined to cells in connective tissues, including the periodontal ligament, tendons, heart valves, myocardium, skin, and bone (6, 7). Despite lacking an RGD domain, periostin is thought to interact with multiple integrins, including αVβ3, αVβ5, and α6β4, to initiate a variety of biologic effects including cell proliferation, cell migration, epithelial to mesenchymal transformation, modulation of the biomechanical properties of connective tissues, and regeneration of cardiac myocytes after injury (4, 8, 9). Periostin also binds type 1 collagen to promote fibrillogenesis (10), and periostin-null mice show aberrant type 1 collagen fibrillogenesis in skin and poor integrity of the periodontal ligament in response to mechanical stress (11, 12).

Subepithelial fibrosis is an important pathologic feature of asthma, involving increased deposition of collagens and other ECM proteins in the lamina reticularis of the basement membrane zone (13–15). It is most prominent in patients with eosinophilia and severe disease (16, 17). Subepithelial fibrosis is thought to change the mechanical properties of the airway wall, alter the folding behavior of the airway mucosa, heighten bronchial reactivity, or narrow the airway and reduce airflow (18, 19). Multiple cytokines, growth factors, and adhesion molecules, including IL-13 and TGF-β, have been implicated in the pathophysiology of subepithelial fibrosis in asthma (20, 21). Although periostin has been considered a mediator of inflammation and fibrosis in allergic diseases, these studies have focused on periostin derived from fibroblasts (22, 23); to our knowledge, the expression of periostin by epithelial cells has not been considered in mechanisms of airway fibrosis in asthma.

The “epithelial mesenchymal trophic unit” (EMTU) is comprised of opposing layers of epithelial and mesenchymal cells separated by the basement membrane zone (24). Reciprocal signaling from epithelial to mesenchymal cells in the EMTU is considered important for normal airway homeostasis, and alterations in the EMTU may result in aberrant autocrine and paracrine responses that could promote subepithelial fibrosis in asthma (25, 26). Relatively few mediators of epithelial mesenchymal communication have been studied in airway disease models, but IL-1β, secreted by epithelial cells undergoing squamous differentiation, can induce a fibrotic response in adjacent airway fibroblasts (27). In considering our data for fourfold up-regulation of periostin in epithelial cells from asthmatics and the distinctive pattern of fibrosis and of periostin immunolocalization in asthma (1, 22), we considered the possibility that periostin is secreted basally by epithelial cells to influence fibrotic events in the underlying matrix. Furthermore, because periostin can bind to matrix proteins, we also investigated the effects of periostin on the biomechanical properties of these proteins.

Results

Periostin Expression in Airway Epithelial Cells in Asthmatic Subjects Is Correlated with Extent of Subepithelial Fibrosis.

We hypothesized that epithelial cell-derived periostin has a role in the mechanism of subepithelial fibrosis in asthma. To begin to examine this hypothesis, we determined the relationship between periostin gene expression in epithelial brushings (measured by real-time RT-PCR) and subepithelial fibrosis in biopsy specimens (measured by stereology) in asthmatic subjects. For this analysis we were able to use previously published periostin gene expression data and subepithelial fibrosis data from paired samples of epithelial brushings and bronchial biopsies from 38 steroid-naive asthmatic subjects with mild to moderate disease (1, 28). In directly analyzing the relationship between these outcomes, we found a strong and positive correlation between periostin gene expression in epithelial brushings and measures of subepithelial fibrosis in biopsy specimens from the same subjects (r = 0.52; P = 0.0008; Fig. 1A). This result supports our hypothesis that periostin secreted by activated epithelial cells plays a role in the pathophysiology of subepithelial fibrosis in asthma.

Fig. 1.

IL-13 induces gene expression and basal secretion of periostin in primary HBE cells. (A) Periostin gene expression in epithelial brushings from asthmatic subjects correlates with the thickness of the basement membrane zone (BMZ) in the same subjects (n = 38; r = 0.56; P = 0.0002). (B–E) In situ hybridization analysis confirms periostin mRNA expression in epithelial cells in tissue sections taken from asthmatic and healthy subjects. Periostin staining is more intense in asthma epithelium (C; arrows) versus healthy controls (B) and the submucosa does not differ in staining. Higher-power images for healthy controls (D) and asthmatic subjects (E). (Scale bars: 20 μm in B and C; 7 μm in D and E.) (F) Diagram of ALI model used for the culture of primary bronchial epithelial cells. (G) Bar graphs show increased periostin gene expression in HBE cells after 4 d of treatment with IL-13 (10 ng/mL) compared with CTL (no treatment) or TNF-α (10 ng/mL); data are averaged from experiments in HBE cells from five different donors (*P < 0.01 vs. control). Error bars indicate SD. (H) Western blot analysis showing changes in periostin levels in basal medium [25 μL conditioned medium (CM)] and cell lysates (10 μg total protein) from HBE cells after 1, 2, and 4 d of treatment with IL-13 (10 ng/mL) compared with CTL (untreated cells). GAPDH was used a loading control.

Airway Epithelial Cells Express Periostin and Secrete Periostin in a Basal Direction.

In previous immunostaining studies of airway biopsies from asthmatic subjects, we and others have reported that periostin staining is subepithelial with a decreasing gradient of expression into the adjacent stromal tissue (1, 22). Surprisingly, despite strong gene expression for periostin in airway epithelial brushings, we have not seen periostin immunostaining in epithelial cells themselves. This discordance between epithelial expression for periostin at the transcript and protein levels led us to hypothesize that periostin protein is rapidly secreted by airway epithelial cells. To test this hypothesis, we decided to confirm periostin gene expression in airway epithelial cells using in situ hybridization and to determine protein secretion by epithelial cells using human bronchial epithelial (HBE) cells grown in an air/liquid interface (ALI) on transwell permeable inserts (model depicted in Fig. 1F). In situ hybridization studies for periostin mRNA in tissue sections of airway biopsy specimens from asthmatic subjects and healthy controls clearly show that periostin is expressed in bronchial epithelial cells and that there is a significant increase in periostin transcripts in the epithelium of asthmatic subjects (Fig. 1 B–E and Fig. S1). Fewer periostin transcripts were detected in the subepithelial region of the airways and no obvious changes in signal were observed between asthmatic subjects and healthy controls in this region. These data show that, although airway epithelial cells are not the exclusive source of periostin in the airway mucosa, they are a major source. We have found previously that periostin gene expression is markedly up-regulated in HBE cells stimulated with recombinant IL-13 (1). Here we confirm and extend these earlier findings and show that IL-13 stimulation of primary HBE cells for 1, 2, or 4 d of treatment causes increases in periostin gene and protein expression at all three time points (Fig. S1). We were unable to detect periostin protein in apical washes from these cells, but we could easily detect abundant periostin protein levels in the basal medium (Fig. 1H). For later experiments we continued to use 4-d treatments in primary HBE cultures to model chronic asthma and to allow us to study mechanisms of remodeling.

Stable Transfection of BEAS2B Cells with a Human Recombinant Periostin Expression Vector.

To explore the function of periostin in airway epithelial cells, we established an epithelial cell model of periostin overexpression. Specifically, we stably transfected BEAS2B cells (a SV40 large-T antigen transformed, but nontumorigenic, bronchial epithelial cell line) with a human recombinant periostin expression vector (B2BPn) or control vector alone (B2BCTL). Periostin gene expression was typically eightfold higher in B2BPn cells than in B2BCTL and parental cells (Fig. S2A); Western blot analysis of basal medium from cells grown in ALI confirmed increased periostin protein (Fig. S2B).

Periostin Up-Regulates Type I Collagen Secretion in B2BPn Cells.

Previous studies have shown that periostin can regulate collagen expression in osteoblasts and in fibroblasts from various tissues (5, 10, 29). We therefore examined if periostin could induce changes in collagen expression in airway epithelial cells. We initially found increased acid-soluble collagen levels in B2BPn cell lysates as measured by the Sircol assay (Fig. S2C) and then more specifically detected increased gene expression for type I collagen in B2BPn cells (Fig. S2D), and increases in type I collagen protein by Western blotting (Fig. S2E). The induction of type 1 collagen in airway epithelial cells by periostin led us to examine whether this effect of periostin occurs in the context of epithelial to mesenchymal transition (EMT). In the B2B cells overexpressing periostin, we examined several markers of EMT, including cell shape, E-cadherin, β-catenin, vimentin, and α-smooth muscle actin (α-SMA) expression. As can be seen in Fig. S3, the B2BPn cells show multiple markers of EMT. Light microscopy studies revealed that B2BPn cells displayed a striking change in cell shape, becoming more elongated and having reduced cell–cell contacts. Immunostaining, Western blotting, and TaqMan studies showed that B2BPn cells exhibited a loss in the epithelial markers E-cadherin and β-catenin and a gain in the mesenchymal markers vimentin and α-SMA (Fig. S3).

Type 1 Collagen Expression in B2BPn Cells is TGF-β–Dependent.

TGF-β is implicated in mechanisms of collagen deposition in subepithelial fibrosis in asthma (30), and we therefore investigated the effects of periostin on the expression of TGF-β isoforms. We found that periostin overexpression in BEAS2B cells caused increased expression of all three TGF-β isoforms (Fig. 2 A–C). To establish that periostin promotes activation of TGF-β, we used the transformed mink lung cell (TMLC) reporter system (31) in coculture with B2B, B2BCTL, or B2BPn cells separated by a permeable membrane. This system uses cells stably transfected with a TGF-β–responsive plasminogen activator inhibitor 1 promoter/luciferase construct and the B2BPn cells displayed increased luciferase reporter activity (Fig. 2D). To determine whether periostin-induced expression of type I collagen is a functional consequence of TGF-β signaling, we used SB431542 to inhibit TGF-β tyrosine kinase activity. We found a significant inhibition of type I collagen gene expression in B2BPn cells (Fig. 2E); these gene expression results were confirmed at the protein level using Western blots (Fig. 2F).

Fig. 2.

Periostin up-regulates TGF-β signaling in BEAS2B cells. (A–C) B2BPn cells up-regulate gene expression of TGF-β isoforms 1, 2, and 3 compared with B2BCTL and untreated parental cells. Asterisk indicates a significant difference from controls for TGF-β isoforms 1 (P < 0.048), 2 (P < 0.02), and 3 (P < 0.02). (D) Increased luciferase reporter activity indicating higher levels of bioactive TGF-β in TMLC cocultured (24 h) with B2BPn cells compared with B2BCTL and parental cells (*P < 0.001 vs. control). (E) Collagen I gene expression in B2BPn cells is mediated by TGF-β. SB431542 (10 μM) down-regulates periostin-mediated collagen I gene expression (*P < 0.001 vs. control; **P < 0.001 vs. periostin without treatment). y axis reflects percent change relative to untreated and nontransfected BEAS2B cells. Error bars indicate SD. (F) Western blots confirm that collagen I levels are reduced in lysates (10 μg) of B2BPn cells treated with SB431542 (10 μM) for 2 d. Data in A–E are averaged from three separate experiments performed under the same conditions. y axis reflects percent change relative to untreated and nontransfected BEAS2B cells. Error bars indicate SD.

Primary HBE Cells Treated with Recombinant Periostin Display Increased TGF-β Bioactivity.

To confirm the data from the B2BPn cells indicating that periostin can up-regulate TGF-β expression and increase collagen in epithelial cells, we repeated key experiments using primary HBE cells grown at an ALI and stimulated with recombinant human periostin. Five different donors of primary HBE cells grown in an ALI were treated with recombinant periostin (2 μg/mL) for 4 d with or without SB431542. Treatment of HBE cells with recombinant human periostin led to statistically significant increases in gene expression of TGF-β isoform 1 (Fig. S4A), but no significant changes in gene expression were observed in TGF-β isoforms 2 and 3 (Fig. S4 B and C). Confirming results in the BEAS2B cells, we found that periostin treatment of HBE cells led to an increase in gene expression for type 1 collagen that was inhibited by SB431542 (Fig. 4D); these gene expression results were confirmed at the protein level using Western blots (Fig. S4E). We next examined if periostin induces EMT in primary HBE cells. For these experiments, we treated HBE cells from three donors with recombinant periostin (2 μg/mL) for 4 d. Of the three donors examined, one exhibited a clear loss in E-cadherin expression with periostin stimulation whereas gains in vimentin were observed from two of three donors (Fig. S5). These results are consistent with other studies in primary airway epithelial cells that show that EMT responses to TGF-β activation in primary airway epithelial cells are more variable than the responses seen in BEAS2B cells (32). It is not surprising that highly differentiated HBE cells are more resistant than BEAS2B cells to EMT induction.

Fig. 4.

TGF-β signaling mediates periostin-induced type I collagen production in airway fibroblasts cultured with conditioned medium (CM) from B2BPn cells. (A) Recombinant periostin treatment alone (2 μg/mL for 48 h) does not stimulate collagen I gene expression in airway fibroblasts (data pooled from five different primary fibroblast donors). (B) Collagen I gene expression is up-regulated in airway fibroblasts (data pooled from five donors) after culture with CM from B2BPn cells compared with B2BCTL and parental cells (48 h). However, in the presence of anti–pan-TGF-β blocking Ab (1D11; 40 μg/mL) or SB431542 (10 μM) collagen gene expression is decreased. (*P < 0.003, **P < 0.004, ^#P < 0.007). y axis reflects percent change relative to fibroblast cells treated with CM from B2B cells. (C) Typical Western blot confirming changes in collagen I levels in lysates (5 μg) from airway fibroblasts after treatment with CM. (D) Bar graph depicting densitometric analysis of changes in collagen levels in fibroblast lysates after treatment with B2B, B2BCTL, or B2BPn with or without anti–pan-TGF-β Ab (40 μg/mL) or SB431542 (10 μM). Data are presented as fold change from B2BCTL alone. Data in A, B, and D are averaged from experiments using human airway fibroblasts from five different donors.

Role of Integrins and Metalloproteinases in Periostin-Induced TGF-β Activation.

TGF-β can be activated by integrin up-regulation (αvβ6 and αvβ8) and also by metalloproteinases (MMPs), most often MMP-2 and MMP-9 (33, 34), and we investigated whether these mechanisms were relevant to periostin-induced TGF-β activation in airway epithelial cells. We examined αvβ6 and αvβ8 integrin expression levels in B2BPn cells. FACs analysis showed down-regulation of both αvβ6 and αvβ8 in these cells compared with B2BCTL cells (Fig. S6A). We also assessed the effect of blocking Abs to both αvβ6 and αvβ8 (20 μg/mL for 48 h) on markers of EMT (vimentin and α-SMA) and collagen I as surrogate read-outs of TGF-β activation in B2BPn cells. The integrin blocking Abs had no significant effect on the markers examined (Fig. S6B). Taken together, these data suggest that αvβ6 and αvβ8 are not involved in periostin-driven TGF-β activation in these cells.

In examining MMP-2 and MMP-9 expression in B2B cells, we found a marked up-regulation in gene expression for MMP-2 and MMP-9 (Fig. 3A). To determine if MMP-2 and MMP-9 are involved in mechanisms of TGF-β up-regulation by periostin, we measured TGF-β1 protein in conditioned media from B2BPn cells and controls. We found no TGF-β1 in the conditioned media of the control cells but average concentrations of 200 pg/ml in the B2BPn cells (Fig. 3). These data confirm gene expression data and activity data for TGF-β in Fig. 2. Notably, we found that a potent cyclic peptide inhibitor of MMP-2 and MMP-9 (MMP-2/MMP-9 inhibitor III) reduced TGF-β1 release from the B2BPn cells by approximately 50% (Fig. 3). Taken together, these data point to important roles for MMP-2 and MMP-9 in the mechanism of periostin-induced activation of TGF-β in airway epithelial cells.

Fig. 3.

MMP-2 and MMP-9 mediate periostin-induced TGF-β up-regulation in airway epithelial cells. (A) Bar graphs display increased MMP-2 and MMP-9 gene expression in B2BPn cells compared with B2BCTL and parental cells. Data are averaged from three separate experiments performed under the same conditions (*P < 0.0001 and P < 0.006 vs. control, respectively). (B) TGF-β1 ELISA shows a marked increase in TGF-β1 in conditioned media from B2BPn cells after 8 h of culture and significant inhibition by the MMP-2/9 inhibitor III (20 μM); in contrast, the MMP-8 inhibitor I (8 nM) increased TGF-β1 release under these conditions. Data are averaged from six separate experiments performed under the same conditions (*P < 0.0001 vs. B2BPn). Error bars indicate SD.

B2BPn Cells Can Induce Collagen Gene Expression in Airway Fibroblasts via TGF-β

Our discovery that periostin could induce a substantial increase in type 1 collagen expression in bronchial epithelial cells led us to examine whether it could stimulate type 1 collagen expression in airway fibroblasts. Fibroblasts are thought to be the principal sources of collagen in peribronchial fibrosis in asthma (20). Surprisingly, recombinant periostin (2 μg/mL for 48 h, the same dose of recombinant periostin used to successfully to activate primary HBE cells) did not cause any significant change in gene expression for type 1 collagen in primary lung fibroblasts (Fig. 4A). However, conditioned medium from B2BPn cells was able to elicit a significant up-regulation of type 1 collagen gene expression in fibroblasts from these donors (n = 5) at a 48-h time point (Fig. 4B), an effect that was attenuated by inhibitors of TGF-β (Fig. 4B). Western blot and densitometric analysis confirmed these changes in type 1 collagen protein levels (Fig. 4 C and D).

Periostin Increases the Elastic and Viscous Moduli of Collagen Gels.

The high concentrations of periostin protein in the basal conditioned medium of IL-13 activated epithelial cells also led us to consider the possible biological effect of periostin in the subepithelial matrix. Periostin has been shown to bind to matrix proteins, but the effects of this binding on the biomechanical properties of matrix proteins have not been directly measured. Newly synthesized collagens form fibrils that are greatly strengthened by the formation of covalent cross-links between lysine residues of the constituent collagen molecules. We considered the possibility that periostin acts as a cross-linker of collagen, and used rheologic measures to test this possibility in vitro. Rheological measurements of viscosity and elasticity elucidate the microstructure of fluids (35, 36), and rheometers work by measuring the response of a fluid to an imposed force or an imposed deformation (Fig. 5A). A fluid's capacity to respond to deformation by flowing reflects its viscosity, whereas its capacity to resist deformation by storing energy and recoiling reflects its elasticity. Increased cross-linking of polymers will be reflected by an increase in the elastic response. A representative set of rheological data from analyses of collagen mixed with periostin is shown in Fig. 5B. For collagen solutions (0.35 mL of 2.5 mg/mL solution), we found that the elastic modulus (G′) predominated over the viscous modulus (G′′). This rheological signature is characteristic of cross-linked collagen polymers in a gel; however, the very low values for both G′ and G′′ indicate a very lightly cross-linked gel. In type 1 collagen solutions mixed with periostin, the G′ again predominated over G′′, but the values for G′ and G′′ were markedly higher, and the G′ and G′′ curves become parallel over a broader range of frequency, indicating transformation of the lightly cross-linked collagen gel into one that is more densely cross-linked (Fig. 5B). The data for G′ and G′′ at one frequency in the sweep is summarized for multiple experiments in Fig. 5C, which shows that type 1 collagen mixed with periostin markedly increases both the viscous and elastic moduli of the collagen gel, whereas control proteins (albumin and fibronectin) have no effect. We hypothesize that this effect of periostin is a result of periostin dimers forming physical cross-links between adjacent collagen fibrils. Indeed, when we performed immunoblotting of the recombinant periostin used in these experiments, we found that it forms dimers in solution (Fig. 5D). However, it is also possible that the effect of periostin is a result of enhanced collagen fibrillogenesis. Although we favor the cross-linking mechanism, our methods do not distinguish between cross-linking and fibrillogenesis as the mechanism of the marked effect of periostin on the elasticity of the collagen gel.

Fig. 5.

Periostin alters the biomechanical properties of gels formed by type 1 collagen. (A) Measurement of viscoelastic properties of a gel using an AR2000 shear stress rheometer. (B) Frequency sweep for type I collagen alone (open triangles and squares) and for type I collagen mixed with periostin (closed triangles and squares). The vertical line indicates data at 1.0 Hz, which is used as a summary data point for C. (C) The addition of periostin increases the elastic modulus (G′) of the collagen gel by as much as 100-fold. In contrast, the addition of albumin or fibronectin to type I collagen has no effects on the G’ of the collagen gel (*P < 0.001 vs. all collagen albumin and fibronectin controls). (D) Recombinant periostin (Biovendor) is present in monomeric and dimeric form in solution. Lane 1 shows detection by silver staining, lane 2 shows detection by an antiperiostin rabbit polyclonal antibody.

Discussion

Previous genome-wide profiling studies by our group identified periostin as the second most highly expressed gene in airway epithelial brushings from asthmatic subjects (1). In the work presented here, we describe experiments designed to reveal the consequences of periostin overexpression in airway epithelial cells. We demonstrate that activated airway epithelial cells secrete large quantities of periostin basally into the underlying matrix, where it has autocrine effects on epithelial cell function, including MMP-2- and MMP-9–mediated activation of TGF-β and up-regulation of collagen in the context of an overall EMT. We also show that periostin secreted by epithelial cells causes TGF-β−mediated activation of airway fibroblasts. Finally, we show that recombinant periostin mixed with type I collagen causes marked increases in the elasticity of the collagen gel. These data identify periostin as a previously unsuspected regulator of TGF-β and as a protein product of airway epithelial cells that can influence events in the underlying matrix, including fibroblast activation and the biomechanical properties of matrix proteins (Fig. S7).

Periostin is considered to be expressed primarily in mesenchymal tissues or in malignant epithelial cells. Although intense immunostaining for periostin has been observed in the subepithelial region in asthma (1, 22), airway epithelial cells themselves have not shown positive staining. In this study, we demonstrate that airway epithelial cells are a major source of periostin. We validate prior microarray and TaqMan quantitative PCR results with in situ hybridization studies that clearly show that periostin mRNA signal is up-regulated in the bronchial epithelial cells of asthmatic subjects. Furthermore, by using an in vitro model of the airway epithelium, we show that IL-13–activated epithelial cells secrete large quantities of periostin in a basal direction. Thus, the difficulty that we and others have had in detecting periostin in epithelial cells is most likely a result of rapid basal secretion of periostin from epithelial cells. Importantly, however, we unequivocally identify periostin as a protein that is synthesized and secreted by nonmalignant airway epithelial cells, and we show that its secretion is markedly up-regulated by IL-13, a key cytokine in allergic asthma.

Subepithelial fibrosis is a characteristic of airway remodeling in asthma and TGF-β is considered an important mediator of this pathology (30, 37–39). Despite its importance, the mechanisms of TGF-β up-regulation in the airway in asthma are incompletely understood. Here we provide evidence that periostin is an upstream regulator of TGF-β activation. In both periostin-overexpressing epithelial cells and primary airway epithelial cells stimulated with recombinant periostin, we found indicators of TGF-β up-regulation, including changes in TGF-β gene expression, TGF-β1 protein, and TGF-β bioactivity. The mechanism of periostin-induced activation of TGF-β did not depend on integrins αvβ6 and αvβ8, but we did find evidence for roles for MMP-2 and MMP-9, at least in the BEAS2B cells. Furthermore, although 48-h treatments with periostin in the same dose that activated epithelial cells did not up-regulate expression of type I collagen in primary human airway fibroblasts, we found that fibroblasts incubated for 48-h with conditioned medium from B2BPn cells did, and that this effect was decreased, at least in part, by inhibitors of TGF-β. Taken together, these data show that epithelial-cell-derived periostin up-regulates MMP-2 and MMP-9 in an autocrine manner to activate TGF-β leading to up-regulation of collagen in airway epithelial cells and fibroblasts. However, the inability of inhibitors to TGF-β to fully block periostin-mediated collagen I expression in these cells suggest that other molecules are also involved in this pathway.

By using rheological methods, we found that recombinant periostin markedly increases the elastic modulus of gels formed by type I collagen. The elastic modulus is a measure of polymer cross-linking and entanglement, and we show that the addition of periostin to type I collagen transforms it from a lightly cross-linked gel to a more densely cross-linked gel. The importance of collagen crosslinking in modulating tissue fibrosis in cancer has recently been highlighted (40), and although others have shown that periostin can bind to collagen and other matrix proteins (10, 22), we show here that periostin can cross-link collagen, as evidenced by an increase in elastic modulus. Indeed, these data support a role for periostin as an epithelial cell protein that can alter the biomechanical properties of the subepithelial matrix. Thus, in the airway, the secretion of periostin by epithelial cells in response to activation signals could allow epithelial cells to regulate the stiffness of the subepithelial matrix and the distensibility of the airway. This mechanism provides a way in which airway epithelial cells—the first-line sensors of inhaled toxins—can regulate airway physiologic function to defend the airway. In diseases such as asthma, the persistence of epithelial cell activation because of persistent IL-13 signaling could lead to inappropriate and prolonged periostin secretion, which could account for chronic subepithelial fibrosis and chronic reductions in airway distensibility.

In summary, we identify periostin as an epithelial cell–derived protein that plays a pivotal role in the regulation of the EMTU. Periostin secreted by airway epithelial cells has autocrine effects, which include activation of TGF-β and up-regulation of type I collagen, and paracrine effects, which include TGF-β–mediated increases in type I collagen production in fibroblasts. In addition, periostin can increase the elastic modulus of gels formed by type 1 collagen, which could change the biomechanical properties of the airway. Persistent up-regulation of periostin in the airway epithelium in asthma is likely to contribute to mechanisms of increased airway fibrosis and decreased airway distensibility.

Methods

Reagents and Assays.

Antibodies and immunoreagents are described in SI Methods, as are methods for immunoassays, collagen assays, and gene expression profiling. TGF-β protein was assayed using the TGF-β1 DuoSet ELISA (R&D Systems)

Cell Culture.

Primary HBE cells were cultured as described in SI Methods, as are the culture methods for BEAS2B cells and B2BPn cells, which was previously described (41). Normal primary human airway fibroblasts were obtained from S. Nishimura (University of California, San Francisco, San Francisco, CA) (42). The TGF-β reporter cell line TMLC containing TGF-β–responsive plasminogen activator inhibitor 1 promoter/luciferase construct (gift from Daniel B. Rifkin, New York University, New York) was cultured as previously described (43).

In Situ Hybridization.

A standard nonradioactive in situ hybridization protocol was used as described in SI Methods.

FACS.

Details of FACS procedures are provided in SI Methods. Anti-αVβ6 (3G9) and αVβ8 (14E5) were a gift from Dean Sheppard (University of California, San Francisco, CA).

Rheology.

Type 1 collagen in a 2.5 mg/mL solution was mixed with periostin or control proteins (albumin or fibronectin) in ratios of approximately 1:10,000 (20 μg/mL) and approximately 1:1,000 (200 μg/mL). Mixing was done for 4 h at 37 °C and rheological measurements were made with a sensitive cone-and-plate rheometer (AR2000; TA Instruments) as described in SI Methods.

Statistics.

Details of statistics in the present study are provided in SI Methods.