Enhanced myofibroblastic differentiation and survival in Thy-1(-) lung fibroblasts

Abstract

Thy-1 is a glycosylphosphatidyl-inositol-linked cell surface glycoprotein whose exact biological role remains unclear. Differential expression of Thy-1 affects fibroblast proliferation and fibrogenic signaling. In idiopathic pulmonary fibrosis, the proliferating myofibroblasts within the fibroblastic foci are Thy-1(-), whereas normal lung fibroblasts are predominantly Thy-1(+). In this study, we used rat lung fibroblasts sorted for Thy-1 expression to examine myofibroblastic differentiation in response to fibrogenic stimuli. We examined the effects of transforming growth factor-beta, endothelin-1, and connective tissue growth factor on the expression of myofibroblast proteins and myogenic regulatory factors by real-time RT-PCR and immunoblotting. Thy-1(-) cells have significantly higher myofibroblast and myogenic regulatory factor gene and protein expression compared with Thy-1(+) cells, confirmed by immunofluorescence. We also used floating collagen matrix contraction assays to assess the functional differentiation of the fibroblasts. At baseline and after stimulation with transforming growth factor-beta and endothelin-1, Thy-1(-) cells caused significantly greater collagen contraction than did Thy-1(+) cells, supporting the hypothesis that Thy-1(-) cells are more fully differentiated myofibroblasts. Because apoptosis has been implicated in the regression of myofibroblasts, we examined the percentage of apoptotic cells in the contracted collagen matrices at baseline and after stimulation with fibrogenic agents. A significantly greater proportion of Thy-1(+) cells underwent apoptosis in all conditions compared with Thy-1(-) fibroblasts. Transfection of Thy-1 into Thy-1(-) cells inhibits collagen matrix contraction and reduces cell survival. Our data indicate that Thy-1 regulates myogenic gene expression, myofibroblastic differentiation, and survival in lung fibroblasts.

Figure 1.

Histogram of real-time RT-PCR. (A) α-SMA. (B) Sarcomeric myosin. (C) Desmin. (D) MyoD. (E) Myocardin. (F) Myogenin. Rat lung fibroblasts were sorted into Thy-1(+) (open bars) and Thy-1(−) (solid bars). mRNA levels were measured by real-time RT-PCR. The cells were quiescent in 0.4% FBS-MEM for 48 h after reaching 80% confluence. Cells were treated with TGF-β (50 ng/ml), ET-1 (100 nm), CTGF (100 ng/ml), or 0.4% FBS-MEM (control) for 4 h. Experiments were performed in triplicate, and data are expressed as the ratio to GAPDH. *P < 0.05 versus same cell subset control. ^†P < 0.05 versus Thy-1(+) under the same conditions.

Figure 2.

Immunoblots. (A) α-SMA. (B) Sarcomeric myosin. (C) MyoD. Thy-1(+) (white bars) and Thy-1(−) (black bars) rat lung fibroblasts were made quiescent in 0.4% FBS-MEM for 48 h, followed by the indicated treatments. Cell lysates were collected and subjected to electrophoresis in 10% SDS-PAGE gels under reducing conditions. Levels of α-SMA (A), sarcomeric myosin (B), and MyoD (C) expression were detected by immunoblotting as described in Materials and Methods. Blots were stripped and reprobed with β-actin for normalization. Relative protein levels were determined by scanning densitometry and normalized to β-actin. Histograms indicate the means (± SD) of three independent experiments. *P < 0.05 versus same cell subset control. ^†P < 0.05 versus Thy-1(+) under the same conditions.

Figure 3.

Immunofluorescence for α-SMA (red, upper eight panels) and MyoD (green, lower eight panels) in Thy-1(+) and Thy-1(−) primary lung fibroblasts (left panels) or in RFL-6 cells transfected with empty vector (ev) or Thy-1.2 (CD90) (right panels) as described in Materials and Methods. Fibroblasts were cultured in serum-free medium or stimulated with TGF-β (5 ng/ml × 48 h). Hoechst (blue) staining was used to visualize nuclei.

Figure 4.

Histograms of gel area (means ± SD) and representative gel photos of floating fibroblast-populated collagen matrices after 24 h (A) and 48 h (B) of stimulation with serum-free medium, CTGF, ET-1, or TGF-β as described in Materials and Methods. *P < 0.001 versus mean area of Thy-1(+) under same conditions. ^†P < 0.001 versus same subset serum-free medium control. The dashed line represents the starting gel area of 1.33 cm².

Figure 5.

Representative flow cytometry dot plots of Thy-1(+) and Thy-1(−) rat lung fibroblasts cultured in floating collagen matrices followed by digestion in 2 mg/ml collagenase at 37°C for 60 min. Digestions were stopped by adding 10% FBS-MEM. Cells were collected and washed followed by Annexin V-FITC apoptosis detection kit. (a) Live cells. (b) Early apoptotic cells. (c) Late apoptotic cells. (d) Necrotic cells. See Table 4 for percentages of live and apoptotic cells in all conditions and Figure 6 for graphic representation.

Figure 6.

Percentage (mean ± SD) of fibroblasts surviving after 48-h culture in floating collagen matrices exposed to serum-free medium, CTGF, ET-1, or TGF-β as described in Materials and Methods. (A) Sorted primary rat lung fibroblasts. (B) RFL-6 cells transfected with empty vector (ev) or Thy-1.2 (CD90). *P ⩽ 0.05 versus mean percent surviving Thy-1(+) fibroblasts under the same conditions. ^†P = 0.012 versus same subset serum-free medium control.