Dopamine Receptor D1 Is Exempt from Transforming Growth Factor β-Mediated Antifibrotic G Protein-Coupled Receptor Landscape Tampering in Lung Fibroblasts

Abstract

Pulmonary fibroblasts are the primary producers of extracellular matrix (ECM) in the lungs, and their pathogenic activation drives scarring and loss of lung function in idiopathic pulmonary fibrosis (IPF). This uncontrolled production of ECM is stimulated by mechanosignaling and transforming growth factor beta 1 (TGF-β1) signaling that together promote transcriptional programs including Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). G protein-coupled receptors (GPCRs) that couple to G α s have emerged as pharmacological targets to inactivate YAP/TAZ signaling and promote lung fibrosis resolution. Previous studies have shown a loss of expression of "antifibrotic GPCRs"-receptors that couple to G α s, in IPF patient-derived fibroblasts compared with non-IPF samples. Of the 14 G α s GPCRs we found to be expressed in lung fibroblasts, the dopamine receptor D1 (DRD1) was one of only two not repressed by TGF-β1 signaling, with the β2-adrenergic receptor being the most repressed. We compared the potency and efficacy of multiple D1 and β2 receptor agonists +/- TGF-β1 treatment in vitro for their ability to elevate cAMP, inhibit nuclear localization of YAP/TAZ, regulate expression of profibrotic and antifibrotic genes, and inhibit cellular proliferation and collagen deposition. Consistently, the activity of β2 receptor agonists was lost, whereas D1 receptor agonists was maintained, after stimulating cultured lung fibroblasts with TGF-β1. These data further support the therapeutic potential of the dopamine receptor D1 and highlight an orchestrated and pervasive loss of antifibrotic GPCRs mediated by TGF-β1 signaling. SIGNIFICANCE STATEMENT: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with limited therapies. GPCRs have emerged as a primary target for the development of novel antifibrotic drugs; however, a challenge to this approach is the dramatic changes in GPCR expression in response to profibrotic stimuli. Here, we investigate the impact of TGF-β1 on the expression of antifibrotic GPCRs and show the D1 dopamine receptor expression is uniquely maintained in response to TGF-β1, further implicating it as a compelling target to treat IPF.

Fig. 1.

TGF-β signaling regulates transcription of G α s coupled GPCRs in lung fibroblasts. (A) Analysis of a previously published RNA sequencing dataset. IPF patient-derived lung fibroblasts stimulated for 12 hours with 2 ng/ml TGF-β1 prior to RNA isolation and analysis. Shown are the mean % changes in expression of all GPCRs that couple exclusively to G α s and are expressed in these datasets. N = 3 unique patient samples. GEO database series GSE136534. (B and C) RNA expression of DRD1 and ADRB2 in cultured human lung fibroblasts +/− 2 ng/ml TGF-β1 over a 72-hour time course. N = 4 independent experiments performed with unique donor samples (*P < 0.05; **P < 0.01; ****P < 0.0001 vs. time matched control; #P < 0.05 and ##P < 0.01 vs. treatment matched 24-hour time point). (D) Human lung fibroblasts cultured for 72 hours with the indicated concentration of the TGF-β type I receptor/ALK5 inhibitor SB431542 prior to RNA isolation and assessment of known TGF-β/SMAD target—COL1A1, and ADRB2, and DRD1. N = 4 independent experiments performed with unique donor samples (***P < 0.001; ****P < 0.0001 vs. DMSO-treated cells).

Fig. 2.

TGF-β treatment induces loss of β2 adrenergic receptor-stimulated cAMP elevation. Human lung fibroblasts were treated for 72 hours with (B and D) or without (A and C) 2 ng/ml TGF-β1. Cells were then stimulated with the indicated concentration of β2 adrenergic receptor agonists (A and B) or D1 dopamine receptor agonists (C and D) for 30 minutes prior to measuring cAMP. Data are expressed as mean fold changes relative to untreated control. N = 3 independent experiments performed with unique donor samples.

Fig. 3.

TGF-β treatment induces loss of β2 adrenergic receptor-mediated inhibition of YAP/TAZ nuclear localization. Human lung fibroblasts were treated for 72 hours with (B and D) or without (A and C) 2 ng/ml TGF-β1. Cells were then stimulated with the indicated concentration of β2 adrenergic receptor agonists (A and B) or D1 dopamine receptor agonists (C and D) for 120 minutes prior to measuring fixing and staining using an antibody that detects YAP and TAZ transcription cofactors. Image analysis is performed using automated software. Data are expressed as mean % changes relative to untreated control. N = 3 independent experiments performed with unique donor samples.

Fig. 4.

TGF-β treatment induces loss of β2 adrenergic receptor-regulated antifibrotic transcriptional reprograming. Human lung fibroblasts were treated for 60 hours with or without 2 ng/ml TGF-β1. Cells were then stimulated with D1 dopamine receptor agonists dihydrexidine (DHX) or A77636 (A7) (both 10 µM), or β2 adrenergic receptor agonists formoterol (For) or fenoterol (Fen) (both 1 µM) for 12 hours prior to isolating RNA to measure transcription changes in collagen genes COL1A1 and COL1A2, and collagen regulating genes LOXL2 and CTSK. N = 5 independent experiments performed with three unique donor samples (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. the indicated group).

Fig. 5.

Collagen deposition assay development. (A) Human lung fibroblasts were cultured +/− TAA2 for 3 days or media changed and cultured for another 3 days. Cells were fixed and immunostained for type I collagen and DAPI. Representative images from control (serum-starved media) compared with TAA2 stimulated cells on days 3 and 6. Proliferation and collagen intensity quantified by image analysis software. (B) Cells were plated and treated identical to above, prior to fixation the matrices were decellularized. Images were immunostained for type I collagen and DAPI. N = 3 independent experiments performed with three unique donor samples (*P < 0.05; **P < 0.01 vs. the indicated group).

Fig. 6.

D1 agonists inhibit fibroblast proliferation and collagen expression. Human lung fibroblasts were cultured +/− TAA2 for 3 days and media changed and cultured for another 3 days +/− TAA2 and the indicated compound: 1 µM fenoterol (Fen), 1 µM formoterol (For), 10 µM A 77636 (A7), and 10 µM dihydrexidine (DHX). (A) Cells were fixed and immunostained for type I collagen and DAPI, or (B) decellularized and immunostained for type I collagen. Representative images from each group are shown. Proliferation and collagen intensity quantified by image analysis software. N = 3 independent experiments performed with three unique donor samples (*P < 0.05 vs. the indicated group).