Article Figures & Data
Figures
- Fig. 1.
Expression of DREADDs in NHLFs. (A) Schematic representation of DREADD-expressing BacMam plasmids. Different DREADDs were cloned into the BacMam vector followed by internal ribosome entry site (IRES)-mCitrine to enable quick assessment of infectivity. (B) Expression of DREADD-containing BacMam viruses in NHLFs. mCitrine-positive cells are shown after 24 hours of infection. Representative images from three independent experiments were obtained. (C) The overexpressed DREADDs were functional in NHLFs upon CNO induction, as shown by calcium release and cAMP signaling assays. N = 3, mean ± S.D. RFU, relative fluorescence unit.
- Fig. 2.
Activation of Gq and Gs signaling inhibits TGFβ-induced fibrosis marker genes in lung fibroblasts. (A) αSMA, Col1A1, and PAI-1 genes were markedly upregulated after TGFβ treatment (5 ng/ml for 24 hours) in NHLFs, which was almost completely reversed by the coadministration of activin receptor-like kinase 5 inhibitor SB-525334 (10 μM). N = 3, mean ± S.D. *P < 0.05; **P < 0.005; ***P < 0.001, compared with no CNO treatment. One-way ANOVA post hoc Tukey test. Three independent replicates were obtained. (B) Activation of Gq and, to a lesser extent, Gs by CNO inhibits αSMA, Col1A1, and PAI-1 expression in NHLFs. N = 3, mean ± S.D. *P < 0.05; **P < 0.005; **P < 0.001, compared with no CNO treatment. One-way ANOVA post hoc Tukey test. Three independent replicates were obtained.
- Fig. 3.
RNA-seq analysis identifies dysregulated GPCRs in NHLFs upon TGFβ treatment. (A) Expression profile of nonsensory GPCRs in human primary cells treated with vehicle or with TGFβ (5 ng/ml). The color gradient represents the fold change compared with vehicle-treated cells (−3.0- to 3.0-fold). N = 3 for each time point or treatment group. (B) Volcano plot of GPCR expression in NHLFs. (C) Gene expression ratios of selected GPCRs in human primary cells and fibrotic tissues. From left: NHLFs, cardiac fibroblasts, hepatic stellate cells (HSCs), renal proximal tubular epithelial cell (RPTEC), UUO kidney, CCl4 liver, and BDL liver. Heatmap was generated by using Morpheus software (https://software.broadinstitute.org/morpheus/). The color gradient represents fold change comparing treatment (TGFβ 24 hours, surgery, or carbon tetrachloride treatment) with control groups (−10- to 10-fold). Gray indicates below detection limit. EDNRB, endothelin receptor B; Veh, vehicle.
- Fig. 4.
Reversal of fibrosis by prostaglandin receptors in NHLFs. (A) Dose titration of various agonists to identify functional GPCRs for Gq activation in NHLFs. N = 3, mean ± S.D. *P < 0.05; **P < 0.005; ***P < 0.001, compared with no compound treatment group. Two-way ANOVA post hoc Tukey test. Three independent replicates were obtained. (B) Dose titration of various agonists to identify functional GPCRs for Gs or Gi activation in NHLFs. N = 3, mean ± S.D. *P < 0.05; **P < 0.005; ***P < 0.001, compared with no compound treatment group. Two-way ANOVA post hoc Tukey test. Three independent replicates were obtained. (C) Inhibition of TGFβ-induced fibrosis marker genes by prostaglandin receptor agonists in NHLFs. N = 3, mean ± S.D. *P < 0.05; **P < 0.005; ***P < 0.001, compared with TGFβ but no compound treated group (orange bar). Two-way ANOVA post hoc Tukey test. Three independent replicates were obtained. RFU, relative fluorescence unit.
Tables
- TABLE 1
Selected GPCRs that function through Gq and/or Gs pathways
Their agonists were reported with EC50 value.
GPCR Signaling Functions Agonists EC50 or Kd GPER1 (Gpr30) Gq Activated by estradiol and functions via Gq signaling (Revankar et al., 2005). A selective agonist G1 is beneficial in myocardial ischemia reperfusion injury model (Bologa et al., 2006; Deschamps and Murphy, 2009). G1 2 nM (Bologa et al., 2006) BDKRB1 Gq BDKRB1 is a Gq-coupled GPCR, whereas BDKRB2 signals through Gq and Gi. In vivo activation of BDKRB2 attenuated kidney fibrosis (Schanstra et al., 2002). Bradykinin 3.26 nM (Wiernas et al., 1997) DRD1 Gs Pleuropulmonary fibrosis was linked to the long-term use of dopamine agonist pergolide in patients with Parkinson (Tintner et al., 2005). A68930 2.5 nM (DeNinno et al., 1991) PTGER2 Gq PGE2 is a potent bioactive eicosanoid that strongly inhibits fibroblast proliferation, migration, and collagen secretion (Bozyk and Moore, 2011). PGE2 PTGER1, 25 nM; PTGER2, 13 nM; PTGER3, 3 nM; PTGER4, 3 nM (Markovič et al., 2017) PTGER4 Gs TCS2510 is a highly selective EP4 agonist. TCS2510 2.5 nM (Billot et al., 2003) TBXA2R Gq/Gs/G12/13 U46619 is a potent and stable TBXA2R agonist. U46619 35 nM (Coleman et al., 1981) OXTR Gq Oxytocin is a peptide hormone and neurotransmitter signaling through OXTR and Gq pathways (Gimpl and Fahrenholz, 2001). WAY-267464 is a potent, selective, nonpeptide OXTR agonist. Oxytocin 9.0–10.8 nM (Gruber et al., 2012) 9.0 nM (Ring et al., 2010) WAY267464 EDNRB Gq Endothelin 3 is a vasoconstrictor that preferentially binds to EDNRB. Endothelin 3 1 nM (Sumner et al., 1992) F2RL1 (PAR2) Gq/Gi PAR2 is implicated in lung, kidney, liver, and cardiac fibrosis (Palygin et al., 2016; Shearer et al., 2016; Sun et al., 2017; Friebel et al., 2019). AC264613 ∼31 nM (Gardell et al., 2008) S1PR3 Gq S1PR3 KO mice has attenuated inflammation and fibrosis upon bleomycin-induced lung injury (Murakami et al., 2014). CYM5541 72–132 nM (Jo et al., 2012) HTR2B (5-HT2B receptor) Gq Serotonin fibrosis in human and treatment of fenfluramine, a serotonin releasing agent, causes cardiac valve fibrosis in patients with obesity (Roth, 2007). 5-HT2B activation mediates valvular fibroblasts activation. BW723C86 5-HT2B, 1.1 nM; 5-HT2C, 93.3 nM (Porter et al., 1999) HTR7 (5-HT7 receptor) Gs AS-19 is a potent 5-HT7 receptor agonist. AS19 0.83 nM (Perez-Garcia and Meneses, 2005) MC1R Gs MC1R is anti-inflammatory and has been implicated in several inflammatory diseases. BMS470539 16.8 nM (Kang et al., 2006) EDNRB, endothelin receptor B; KO, knockout.
In this issue
GPCR Signaling in Fibrosis
