Figure 4.

OGR1 overexpression reduces the ability of TGF-β to induce myofibroblast differentiation. Human fibroblasts (healthy: A–D, IPF: E–H) were transfected with a plasmid expression GFP (con) or OGR1 and were subsequently treated with either TGF-β1 or DMSO as a control. Protein lysates were then harvested and western blotting was performed with antibodies against OGR1, α-SMA, collagen 1A1, and beta-tubulin (as a loading control). Representative western blots are presented in (A and E, respectively), and densitometry analysis is graphed below. OGR1 plasmids were successfully expressed in both healthy (B, ** p = 0.0020 and 0.0065) and IPF-derived fibroblasts (F, *** p = 0.0002 and 0.0004). As expected, TGF-β1 significantly increased expression of α-SMA in both healthy (C, **** p < 0.0001) and IPF-derived fibroblasts (G, **** p < 0.0001). However, overexpression of OGR1 attenuated TGF-β1-induced α-SMA expression in healthy fibroblasts (C, ** p = 0.0017) and IPF fibroblasts (G, difference between con plasmid + TGF-β1 and OGR plasmid p = 0.0025 by Student’s t-test). Again, as expected, treatment with TGF-β1 caused increased collagen1A1 expression in healthy (D, * p = 0.0364) and IPF fibroblasts (H, *** p = 0.0008). Again, OGR1 overexpression led to significant reductions in TGF-β induced collagen in healthy (D, p = 0.009 by Student’s t-test) and IPF fibroblasts (H, * p = 0.0027 by student t-test). Interestingly, in IPF fibroblasts, OGR1 overexpression decreased collagen expression in an unstimulated state (H, ** p = 0.0084). Data represent mean expression ± SEM.