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. 2008 Jan 1;177(1):66-74.
doi: 10.1164/rccm.200706-963OC. Epub 2007 Oct 4.

Variable prostaglandin E2 resistance in fibroblasts from patients with usual interstitial pneumonia

Steven K Huang  1 , Scott H Wettlaufer, Cory M Hogaboam, Kevin R Flaherty, Fernando J Martinez, Jeffrey L Myers, Thomas V Colby, William D Travis, Galen B Toews, Marc Peters-Golden
Affiliations

Variable prostaglandin E2 resistance in fibroblasts from patients with usual interstitial pneumonia

Steven K Huang et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Prostaglandin (PG) E2, a cyclooxygenase-derived lipid mediator, is a potent down-regulator of fibroblast activation in normal lung fibroblasts. Although fibroblasts from patients with idiopathic pulmonary fibrosis are known to exhibit a defect in PGE2 synthesis, there is little information about their responsiveness to this lipid mediator.

Objectives: To compare responses to PGE2 in normal, usual interstitial pneumonia (UIP), and other diffuse parenchymal lung disease (DPLD) fibroblasts.

Methods: Fibroblasts were grown in vitro from well characterized control (n = 7), UIP (n = 17), or other DPLD (n = 13) lung tissue. The effects of PGE2 on fibroblast proliferation and collagen expression were determined.

Measurements and main results: Only 3 of 12 UIP fibroblast lines exhibited PGE2-mediated inhibition of both collagen synthesis and cell proliferation, as opposed to 6 of 6 nonfibrotic control cell lines. The degree of PGE2 resistance in DPLD fibroblasts was quite variable, with UIP cells exhibiting the greatest degree of resistance to PGE2, whereas other DPLD fibroblasts manifested a degree of resistance intermediate to control and UIP. The resistance to suppression of collagen expression correlated with worse lung function. Molecular mechanisms for resistance included altered E prostanoid receptor profiles and diminished expression of the downstream kinase, protein kinase A.

Conclusions: The recognition that UIP fibroblasts manifest variable refractoriness to PGE2 suppression sheds new light on the activation phenotype of these cells and on the pathogenesis of fibrotic lung disease.

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Figures

<b>Figure 1.</b>
Figure 1.
Usual interstitial pneumonia (UIP) fibroblasts exhibit variable resistance to prostaglandin (PG) E2 suppression of collagen expression and proliferation as compared with control fibroblasts. (A) Primary lung fibroblasts were treated with varying concentrations of PGE2 for 18 hours and assayed for proliferation ([3H]-thymidine incorporation) as described in Methods. Data are expressed as percent of untreated, no-PGE2 control. (B) The proliferative response of cells treated for 18 hours with 500 nM PGE2 (expressed as % of no-PGE2 control) for individual lines is shown. Numbers correspond to specific cell lines listed in Table 1; bars represent mean and SE. *P < 0.05 relative to control fibroblasts (analysis of variance [ANOVA]). (C) Primary lung fibroblasts were treated with PGE2 for 18 hours and collagen I expression was measured as described in Methods. Results are expressed as percent of no-PGE2 control. Numbers correspond to specific cell lines listed in Table 1; bars represent mean and SE (P = 0.26; ANOVA). (D) PGE2-mediated inhibition of collagen I expression and cell proliferation (both expressed as % of no-PGE2 control) is displayed for all cell lines that had data on both parameters. The degree of inhibition of collagen expression and cell proliferation for each individual line is expressed as scatter points. The dotted line marks the boundaries of the upper limits of inhibition seen in control fibroblasts.
<b>Figure 2.</b>
Figure 2.
PGE2 inhibition of fibroblast collagen I expression is inversely correlated with impairment in patients' lung function. Pulmonary function test results were obtained from patients just before lung biopsy. Patients' percent predicted TLC and diffusing capacity for carbon monoxide (DlCO) were correlated with the degree of PGE2 (500 nM) inhibition of collagen expression and proliferation (both expressed as % of no-PGE2 control). An inverse relationship was seen between TLC and PGE2 inhibition of collagen (A) (r2 = 0.37; P < 0.05), but not between TLC and PGE2 inhibition of proliferation (C) (r2 = 0.03; P = 0.57). A similar relationship was seen between DlCO and PGE2 inhibition of collagen (B) (r2 = 0.27; P = 0.08), but not between DlCO and PGE2 inhibition of proliferation (D) (r2 = 0.004; P = 0.81). Each number corresponds to the patients listed in Table 1.
<b>Figure 3.</b>
Figure 3.
Low E prostanoid (EP) 2 receptor expression accounts for defective PGE2 signaling in select usual interstitial pneumonia (UIP) fibroblast lines. (A) EP2 receptor protein expression for control and UIP fibroblast lines was analyzed by immunoblot. Densitometric analysis was performed relative to α-tubulin and is displayed numerically as arbitrary units below each lane. (B) Collagen expression in cells treated with the EP2-, EP3-, or EP4-specific agonists, butaprost free acid (500 nM), Ono-AE3–248 (100 nM), and Ono-AE1–329 (100 nM), respectively, are compared between UIP line 15 and a representative control fibroblast line (line 5). Representative immunoblot with densitometric analysis relative to α-tubulin is shown. (C) cAMP levels among control and UIP cells treated for 15 minutes with forskolin (100 μM) or PGE2 (500 nM) are shown. (D) In line 19, a UIP fibroblast line, iloprost (500 nM) suppressed collagen expression, whereas PGE2 (500 nM) resulted in increased collagen. Representative immunoblot and densitometric analysis relative to α-tubulin is shown.
<b>Figure 4.</b>
Figure 4.
Diminished protein kinase (PK) A expression and catalytic activity confers resistance to PGE2 in other usual interstitial pneumonia (UIP) patient cell lines. (A) Protein expression of the catalytic subunit of PKA for various control and UIP fibroblast lines was analyzed by immunoblot. Representative immunoblot is shown. Densitometric analysis was performed relative to α-tubulin. (B) Treatment with 500 nM PGE2 for 30 minutes resulted in cAMP-responsive element–binding protein (CREB) phosphorylation in lines 2 (control) and 18 (UIP), but not in line 14. Phosphorylated CREB and α-tubulin immunoblots are shown. (C) Collagen I expression in cells treated with PGE2 (500 nM), forskolin (100 μM), or the PKA agonist, 6-bnz-cAMP (125 μM) was assayed. Representative blots from UIP line 14 and control cell line 6 is shown. Densitometric analysis relative to α-tubulin expressed as a percent of untreated control is shown beneath each condition. (D) Treatment with the relatively specific serine-threonine phosphatase (PP2A) inhibitor, okadaic acid (40 nM), in line 14 resulted in diminished collagen expression; addition of PGE2 (500 nM) further potentiated collagen suppression. Representative immunoblot with densitometric analysis is shown.
<b>Figure 5.</b>
Figure 5.
Schematic representation of PGE2 signaling within fibroblasts and potential sites of defects conferring resistance to PGE2 in usual interstitial pneumonia (UIP) fibroblasts. In control lung fibroblasts, PGE2 suppression of collagen I expression and proliferation occurs through ligation of the EP2 receptor, resulting in activation of adenyl cyclase, increased cAMP production, and activation of cAMP-dependent signaling pathways, including PKA. Defects in EP2 receptor expression or PKA expression/activity may account for the lack of PGE2-mediated suppression of collagen synthesis and proliferation seen in select UIP fibroblasts. AC = adenyl cyclase.
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