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.

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.

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.

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.

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.