Figure 5.

PGE₂ regulates fibroblast responses to increasing stiffness. (A) Relative PGE₂ levels in NHLF cell culture supernatants with and without selective COX-2 inhibitor (NS-398). (B–D) NHLF accumulation in discrete stiffness wells (1% FBS) and modification by exogenous PGE₂, EP2 agonist (butaprost; Buta), and COX-2 inhibitor (NS-398), respectively. (E) Immunostaining for procollagen I in NHLFs (1% FBS). EP2 agonist (5 µM butaprost) abrogates the stiffness effect on procollagen I expression, whereas COX-2 selective inhibitor (3 µM NS-398) is unable to augment procollagen I expression at low stiffness. Bar, 50 µm. (F) Relative mRNA levels for COL1A1, assessed by qPCR, on 0.1 and 6.4 kPa substrates (0.1% FBS) with and without 5 µM butaprost or 3 µM NS-398. (A–D and F) Error bars indicate SD from three independent experiments for each panel. (G) COX-2 (PTGS2) mRNA levels decrease across stiffness, as measured by qPCR, in A549 human lung epithelial cells, primary human dermal, synovial and lung (NHLF) fibroblasts, and airway smooth muscle (ASM) cells, as well as CCL-151 and IMR90 lung fibroblast cell lines (0.1% FBS). (A–D and G) Colored triangles along the x axis are used to indicate the interquartile and median stiffness values of lung parenchyma from saline (cyan)- or bleomycin (red)-treated mice.