Figure 5.

Alveolar epithelial cell-derived factors promote fibroblast activation. A: DDR2 phosphorylation of collagen I-deleted fibroblasts by BAL from control and SCcol mice collected 1 week after saline or bleomycin injury. Cells treated with 10 μg/mL exogenous type I collagen and cells treated with BAL from bleomycin-injured control mice have induction of DDR2 phosphorylation. B: Reverse transcription-PCR of primary lung fibroblasts from Col1a1^f/f mice treated with AdGFP, AdCre, or AdCre plus exogenously added type I collagen (collagen I-coated plus 10 μg/mL added to media). Loss of collagen I expression led to reduction in expression of several fibroblast activation markers; the expression of these genes was restored by exogenous collagen. C: Immunoblot of lung fibroblasts stimulated with plain media, CM from AECs cultured on Matrigel (AEC CM), CM from AECs cultured on FN (EMT CM), and media supplemented with 4 ng/mL TGF-β. TGF-β and EMT CM activate fibroblast expression of α-SMA and collagen I. TGF-β promotes smad2 phosphorylation but EMT CM does not. D: Immunoblot of lung fibroblasts stimulated with plain media, CM from WT AECs cultured on FN (WT CM), smad3-null AECs (Sm3-CM) cultured on FN, and WT AECs cultured on FN with addition of TGFβ receptor inhibitor SB431542 (SB), 10 μmol/L (WT + SB CM). E: Mouse fibrosis reverse transcription-PCR array of AECs cultured on Matrigel, FN, or FN with addition of SB. Expression of genes on FN versus Matrigel (black) and FN with SB versus Matrigel (red) are shown. Blue lines indicate fivefold differential expression. Several genes of interest are indicated. AdCre, adenovirus expressing Cre; AdGFP adenovirus expressing green fluorescent protein; AEC, alveolar epithelial cell; BAL, bronchoalveolar lavage; Bleo, bleomycin; CM, conditioned media; Col I, type I collagen; Col1a1^f/f, homozygous floxed Col1a1; Col1a2, collagen, type I, alpha 2; Col3a1, collagen, type III, alpha 1; ctgf, connective tissue growth factor; Ctrl, control; DDR2, discoidin domain receptor-2; EMT, epithelial-mesenchymal transition; FN, fibronectin; GAPDH, glyceraldehyde phosphate dehydrogenase; lox, lysyl oxidase; Sal, saline; SB, transforming growth factor β receptor inhibitor SB431542; SCcol, triple transgenic surfactant proteins-C promoter-reverse tetracycline transactivator/tetO-cytomegalovirus promoter-Cre recombinase/homozygous floxed collagen, type I, alpha 1; SMA, smooth muscle actin; sma, smooth muscle actin; TGF-β, transforming growth factor β; tsp, thrombospondin; WT, wild-type.