Tenascin-C deficiency attenuates TGF-ß-mediated fibrosis following murine lung injury

Abstract

Tenascin-C (TNC) is an extracellular matrix glycoprotein of unknown function that is highly expressed in adult lung parenchyma following acute lung injury (ALI). Here we report that mice lacking TNC are protected from interstitial fibrosis in the bleomycin model of ALI. Three weeks after exposure to bleomycin, TNC-null mice had accumulated 85% less lung collagen than wild-type mice. The lung interstitium of TNC-null mice also appeared to contain fewer myofibroblasts and fewer cells with intranuclear Smad-2/3 staining, suggesting impaired TGF-β activation or signaling. In vitro, TNC-null lung fibroblasts exposed to constitutively active TGF-β expressed less α-smooth muscle actin and deposited less collagen I into the matrix than wild-type cells. Impaired TGF-β responsiveness was correlated with dramatically reduced Smad-3 protein levels and diminished nuclear translocation of Smad-2 and Smad-3 in TGF-β-exposed TNC-null cells. Reduced Smad-3 in TNC-null cells reflects both decreased transcript abundance and enhanced ubiquitin-proteasome-mediated protein degradation. Together, these studies suggest that TNC is essential for maximal TGF-β action after ALI. The clearance of TNC that normally follows ALI may restrain TGF-β action during lung healing, whereas prolonged or exaggerated TNC expression may facilitate TGF-β action and fibrosis after ALI.

Fig. 1.

Tenascin-C (TNC) induction and localization after lung injury. A: Western blot of whole lung extracts prepared before and 6, 14, or 21 days after bleomycin administration to wild-type and TNC-null mice (−/−). The 190-kDa “large” isoform of TNC predominates at all time points after acute lung injury (ALI). At ×100 magnification, TNC staining in the matrix is robust in areas of lung injury (D and E), but not in adjacent areas of normal lung (H and I). At ×250 magnification (F and G), TNC is primarily localized to the interstitium of thickened alveolar septae in areas of injury. There is minimal TNC staining in saline-injected wild-type lungs (B and C) or in bleomycin-treated TNC-null lungs (J and K). Bars in fluorescence images represent 50 μm.

Fig. 2.

Comparable mortality in TNC-deficient mice. Wild-type (♦) and TNC-deficient (◊) mice received bleomycin either by direct tracheal or transoral injection and were followed for 21 days. Survival was measured after direct tracheal instillation (A, n = 27 each genotype) or transoral instillation (B, n = 9 each genotype). All saline-treated control animals survived for 21 days (direct tracheal, n = 6 and transoral, n = 4 for each genotype).

Fig. 3.

Reduced collagen deposition in the lungs of TNC-deficient mice. Wild-type and TNC-deficient mice received either bleomycin or sterile saline via direct tracheal or transoral injection. Mice were killed after 21 days for measurement of right lung collagen content and wet weight (transoral, n = 8, direct tracheal, n = 6). A: following exposure to bleomycin, substantially more collagen was deposited in the lungs of wild-type mice (black bars), independent of the mode of administration. *P < 0.01. B–E: Masson's trichome staining of lung sections from mice who received direct tracheal bleomycin (collagen stains blue). Sections were obtained from wild-type and TNC-null animals at 10 days (B and C, respectively) and 21 days (D and C, respectively). Images are representative of sections from 3 mice of each genotype examined.

Fig. 4.

Reduced TGF-β activity in the lungs and fibroblasts of TNC-deficient mice. Wild-type and TNC-deficient mice received bleomycin by direct tracheal injection and were killed 10 days later. Fibroproliferative areas within the lungs of wild-type mice (A) appeared to contain a greater number of cells staining positive for P-Smad-2 and -3 (brown staining nuclei, arrows) than did lungs of TNC-deficient mice (B). The interstitium of wild-type lungs (C) also contained more α-smooth muscle actin staining (arrowheads) compared with TNC-null lungs (D).

Fig. 5.

Reduced TGF-β signaling in TNC-null fibroblasts. A: lung fibroblasts were exposed to varying concentrations of TGF-β, and the fraction of α-SMA-positive cells was determined for wild-type (■) and TNC-deficient (○) cultures. Each data point represents the mean of 4 wells from each of 2 independent cell lines for each genotype. B: collagen deposited by wild-type (black bars) and TNC-null (white bars) cells (n = 4). C: Western blots of Smad-2/3 (whole cell extract) and phospho-Smad-2/3 (nuclear fraction) after TGF-β stimulation. D: Western blots of whole cell extracts showing reduced Smad-3 expression in TNC-null fibroblasts compared with wild type. E and F: quantification of Smad-2 and Smad-3 in nuclear extracts after TGF-β stimulation (mean of 4 replicates). *P < 0.05; **P < 0.01; ***P < 0.001 in all panels.

Fig. 6.

Reduced Smad-3 mRNA and protein stability in TNC-null fibroblasts. A: quantitative RT-PCR analysis of TGF-β signaling components in wild-type and TNC-null fibroblasts. Mean and 95% confidence intervals for log2 expression ratios of TNC-null over wild-type mRNA are shown for each target. A positive ratio reflects increased expression in TNC-null vs. wild type, and negative ratio reflects decreased expression. Significance threshold (*) was reduced to P < 0.005 to compensate for multiple comparisons. N = 4 for each target. B: Smad-3 protein content in unstimulated wild-type and TNC-null fibroblasts in the presence and absence of the ubiquitin-protease system inhibitor, MG132. Smad-3 protein was normalized for β-actin content. N = 5 for each condition; *P < 0.05.