Utilizing DREADD chemogenetic tools to identify beneficial GPCR signaling for fibrosis.

Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many FDA approved drugs. To identify new targets for fibrosis, we utilized a synthetic GPCR system named Designed Receptors Exclusively Activated by Designer Drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq and Gs-DREADDs abrogated the induction of TGFβ-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFβ. To investigate endogenous GPCR modulating TGFβ signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFβ signaling. Among which, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof-of-concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. Significance Statement Fibrosis is the hallmark of many end-stage cardiometabolic diseases and there is an unmet medical need to discover new anti-fibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes DREADD chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.


IRES
Internal ribosome entry site

Introduction
Fibrosis is an evolutionarily conserved mechanism developed by an organism to survive chronic injury (Wynn and Ramalingam, 2012). Excessive fibrosis, however, leads to disruption of organ function and is a common feature of many chronic diseases. Many progressive forms of cardiometabolic diseases, such as end stage renal disease (ESRD), nonalcoholic steatohepatitis (NASH) and heart failure, are characterized by the presence of extensive fibrosis (Friedman et al., 2013).  Armstrong et al., 2016;Ratziu et al., 2016;Friedman et al., 2018;Harrison et al., 2018). However, no new drug has yet been approved to stop fibrosis progression, relieve symptoms, or substantially improve patient survival. There is a huge unmet medical need to develop new anti-fibrosis therapies with clinically meaningful efficacy in patients.
GPCRs are important cell signaling molecules that respond to a variety of extracellular stimuli, including hormones, neurotransmitters, growth factors, chemokines, light, odor, taste, extracellular matrix, shear stress, etc. (Gilman, 1987;Hauser et al., 2017;Weis and Kobilka, 2018). GPCRs regulate nearly all physiological activities, such as blood pressure control, respiration, hormonal-neural regulation, and immune response (Premont and Gainetdinov, 2007). It has been estimated that GPCRs comprise approximately 34% of the molecular targets for FDA approved drugs and ~60 agents against novel GPCR targets are in clinical trials (Hauser et al., 2017). Several GPCR targeted therapies have been evaluated for fibrotic indications. The lysophosphatidic acid receptor LPAR1 has been found linking pulmonary fibrosis to lung injury (Tager et al., 2008). A LPAR1 antagonist BMS-986020 was recently examined in IPF patients (NCT01766817) and found effective at slowing down the decline of lung function (Palmer et al., 2018). A drug-related liver toxicity precluded BMS-986020 from further clinical development. Cannabinoid receptor 1 (CB1) antagonism has also attracted a considerable amount of interests for the treatment of NASH due to its role in central orexigenic effect, energy expenditure and fibrosis (Mallat et al., 2013;Patsenker and Stickel, 2016). Neuronal adverse effects of the known CB1 inverse agonist Rimonabant have prompted the development of a newer peripherally restricted-agent (Sam et al., 2011). Nonetheless, the interest to develop new GPCR therapies with better safety profiles for fibrosis is clearly warranted.
The activation of GPCRs under different conditions can have different biological outcomes depending on the cellular context and conditions (Gilman, 1987;Weis and Kobilka, 2018 Clozapine N-oxide (CNO) (Urban and Roth, 2015). Different DREADDs have been engineered to selectively activate Gq, Gs or Gi pathways. By using the DREADD chemogenetic tools, we found that Gq and Gs signaling decreases cellular response to TGFβ. Whole-genome transcriptome analysis of human primary fibroblasts and fibrosis models revealed 13 highly-dysregulated GPCRs that signal through either Gq or Gs pathways with known agonists. We further showed that among those GPCRs, activation of the prostaglandin receptors reduced fibrotic marker expression in fibroblasts. The combination of DREADD chemogenetic tools with transcriptome analysis represents a powerful tool for identifying potential GPCR targets for fibrosis.  for 1 hour at 37C. The plate was then set up in the FLIPR Tetra high-throughput cellular screen system, treated with CNO, and read immediately.

Cultured Cells and Reagents
NJ. Mouse UUO kidney fibrosis and CCl4-induced liver fibrosis models, as well as rat bile-duct ligation models were carried out as previously described (Zhang et al., 2020).

Gene Expression Taqman Analysis
Total RNA was extracted from cultured cells or tissues following the instruction of RNeasy Mini QIAcube Kit (Qiagen, #74116). 2ug RNA was then reverse-transcribed using SuperScript VILO cDNA Synthesis kit (Invitrogen, #11754-050). 2ul diluted cDNA was added to quantitative real-time PCR mix with TaqMan Universal PCR Master mix (Applied Biosystems, #4364338) and Taqman probes (Life Technologies). PCR amplification (one cycle at 50°C for 2 minutes, one cycle at 95°C for 10 minutes and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute) was done in an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystem). Ct value of each sample was normalized to GAPDH level and quantified based on 2e-delta (deltaCT) method (comparing with no treatment or sham group).

RNA Isolation and Gene Expression Profiling
Gene expression analysis was performed as previously described (Schlessinger et al.

Data analysis
All data are presented as mean  standard error of the mean (SE). One or Two-way ANOVA post hoc Tukey analysis was used for data comparisons as indicated in the figure legends, together with group size. All analyses were done using Prism 7 software. A p-value of <0.05 was statistically significant.

Establishment of functional DREADDs expression system in primary human lung fibroblasts
DREADDs are synthetic GPCRs whose expression does not affect the biological system when not activated by their highly specific synthetic ligands. Gq-DREADD (hM3Dq) was created by directed molecular evolution of the human M3 muscarinic receptor (hM3) in yeast (Armbruster et al., 2007).
When activated by the ligand CNO, it stimulated calcium release into the cytoplasm. Gi-DREADD (hM4Di) was designed based on the human M4 muscarinic receptor; it decreased cAMP signaling and opens K + channels (GIRKs) in response to CNO (Nawaratne et al., 2008). A chimeric hM3Dq β-adrenergic receptor DREADD was created as Gs-DREADD, which activated cAMP production and PKA signaling (Guettier et al., 2009). Unlike hM3Dq and hM4Di, Gs-DREADD has a modest degree of constitutive activity, which can lead to basal phenotypes in certain cellular contexts.
BacMam is a highly effective viral expression system that enables protein expression in a large variety of cells, including human primary cells (Ames et al., 2007). Gq/Gs/Gi-DREADDs were cloned into a bi- cistronic BacMam vector that simultaneously expresses mCitrine fluorescent protein under the control of internal ribosome entry site (IRES) (Figure 1A). At 24hr after BacMam infection, the expression of DREADDs was confirmed by the co-expressed fluorescent signals in normal human lung fibroblasts (NHLFs) (Figure 1B). At the optimal viral titers, about 50% of cells were positive for fluorescence. To verify the function of exogenously-expressed DREADDs, we next examined calcium signaling in all cells.
As shown in the top panel of Figure 1C, CNO treatment induced a dose-dependent spike of calcium release into the cytoplasm of Gq-DREADD expressing fibroblasts. cAMP production was induced by CNO in Gs-DREADD expressing fibroblasts ( Figure 1C, middle panel). Conversely, activation of Gi-DREADD expressing fibroblasts by CNO led to an expected, modest reduction of the basal cAMP level ( Figure 1C, bottom panel). These results indicated that BacMam-delivered DREADD expression system was functional in those cells.

Activation of Gq and Gs signaling inhibits the expression TGFβ-induced fibrosis marker genes
Among many factors that regulate fibrosis, TGFβ plays an essential role in both myofibroblast activation and extracellular matrix remodeling (Akhurst and Hata, 2012); it has often been used to induce fibrotic response in cultured cells. To gain novel insight into the GPCR pathways modulating TGFβ, we next established a cell-based assay to examine TGFβ response (Figure 2A). At 24hr after TGFβ treatment, RT-qPCR analysis of fibrotic marker genes, such as α-smooth muscle actin (αSMA), PAI-1, and Collagen 1A1, in NHLFs revealed a robust and statistically significant response to TGFβ, which was almost completely abrogated in the presence of SB-525334, a TGFβ type I receptor (ALK5) inhibitor ( Figure 2A).
To test the effect of DREADD activation on fibrosis markers in those cells, we infected the NHLFs with DREADDs-expressing BacMAM viruses and treated NHLFs with various concentration of CNO in the presence of TGFβ. After treatment of 24hr, the cells were collected for mRNA extraction and RT-qPCR analysis. TGFβ induced transcription of αSMA, PAI-1, and Collagen 1A1 (Col1A1) was suppressed by activation of Gq and Gs DREADDs . CNO had no effect on these markers in NHLFs infected with Gi

DREADD. A clear dose dependent CNO effect was observed for all markers in Gs-DREADD expressing
NHLFs; while for Gq-DREADD expressing cells, a clear dose dependent effect was only observed for αSMA, only a minimal effect on Col1A1, and a saturated significant effect on PAI-1 ( Figure 2B).

RNA-seq analysis identifies dysregulated GPCRs in NHLFs upon TGFβ treatment.
Notably, many GPCRs function through Gq and Gs signaling, such as 5HT2 receptors and prostaglandin receptors (Fribourg et al., 2011). To identify endogenous GPCRs that modulate TGFβ signaling, we resorted to our previous genome-wide transcriptome analysis of human primary cells treated with TGFβ to uncover differentially-expressed GPCRs under TGFβ stimulation (manuscript accepted at Cell Reports Medicine, NCBI GEO accession number GSE152250) (Zhang et al., 2020). Upon TGFβ treatment, human primary cardiac fibroblasts, NHLFs, hepatic stellate cells (HSC), and renal proximal tubular epithelial cells (RPTEC) were collected for RNA-seq analysis. Among the four cell types, the expression of many nonsensory GPCR genes was significantly changed upon TGFβ treatment when compared to the vehicle group ( Figure 3A). To delineate this further, a volcano plot visualization of GPCR expression fold change in NHLFs is shown in Figure 3B. We highlighted 13 GPCRs that signal through Gq or Gs pathways and have known biological functions relevant to fibrosis and commercially-available agonists ( Table 1).
Among the selected GPCRs, S1PR3, HTR2B, BDKRB1, BDKRB2, TBXA2R, PTGER2, and GPER1 were significantly decreased upon TGFβ treatment in lung fibroblasts, whereas DRD1, PAR2 (F2RL1), and PTGER4 were significantly up-regulated ( Figure 3B). Furthermore, we examined the expression of this set of GPCRs in UUO (unilateral ureter obstruction) kidney, CCl4 (carbon tetrachloride) liver, and BDL (bile duct ligation) liver fibrosis models. As shown in the heatmap of Figure 3C, the expression of many GPCRs were reduced in primary cells treated with TGFβ, but were augmented in fibrotic tissues, suggesting additional layers of complexity for the regulation of GPCR.

Reversal of fibrosis by prostaglandin receptors in NHLFs.
We have found a subset of Gq or Gs-coupled GPCRs whose expression was differentially regulated in lung fibroblasts treated with TGFβ. We next sought to examine whether activation of those endogenous GPCRs would alter TGFβ signaling. To this end, we obtained 14 potent and selective agonists for those GPCRs with reported EC50 at low nM range ( Table 1). NHLFs were first treated with CNO at various concentration. Calcium signal and cAMP production were determined to ensure selective G protein pathway activation. As shown in Figure 4A, dose-dependent calcium release was detected in cells treated with Bradykinin and Oxytocin at a statistically significant level. These results suggested that BDKRB1/2 and OXTR receptor are the major Gq-coupled endogenous receptors in NHLFs. Moreover, cAMP production was substantially elevated in cells treated with A68930 (DRD1 agonist), Prostaglandin E2 (EP agonist), and TCS2510 (EP4 agonist), consistent with their role in Gs-mediated signaling ( Figure   4B). U46619 treatment induced a dose-dependent increase of cAMP level, albeit with lower overall signals, indicating an alternative signaling pathway than the previously reported Gq signaling.

Discussion
In the present study, we utilized DREADD chemogenetic tools to probe GPCR signaling pathway(s) essential for fibrotic response in fibroblasts. We discovered that activation of either Gq or Gs pathway inhibits TGFβ-induced fibrosis marker gene expression. Stimulation of the Gi DREADD did not affect transcription of these marker genes. However, the current experiments cannot completely rule out the possibility that low level of hM4D expression is the reason we could not see a response. The reduction of basal levels of cAMP we measured, although statistically significant, was small and may suggest low level of expression.
To identify endogenous Gq/Gs-coupled GPCR in fibroblasts, we performed datamining of our fibrosis profiling dataset, in which whole-genome transcriptome analyses were carried out in human primary cardiac fibroblasts, NHLFs, hepatic stellate cells, and renal proximal tubular epithelial cells treated with TGFβ, as well as UUO kidney, CCl4 liver, and BDL liver fibrosis models (Zhang et al., 2020). Among the highly differentially-expressed GPCRs, we selected 13 receptors that were signaling through Gq or Gs and have known agonist available. We further verified the expression and activation of those GPCRs by agonist stimulation in the fibroblasts. Among the 13 GPCRs, activation of prostaglandin receptors using the pan-agonist prostaglandin, generated the strongest anti-fibrotic effect. Activation of only one of the prostaglandin receptors (EP4) using TCS2510 resulted in a reduced antifibrotic effect, suggesting the requirement of multiple prostaglandin receptors for optimal anti-fibrotic effect.
Although we refer to the signaling pathways of the DREADDs based on the Gα subunit they interact with, the signaling pathways activated by them are likely more complicated and include components of βγ signaling, arrestin signaling and potentially other down-stream molecules that may affect transcription factors in a cumulative manner. The definition of receptors as coupled to Gq or Gs does not necessarily mean that this is the only Gα protein they interact with, but it is the most common one.
In many cases these definitions relay on the cellular effects seen by activation of a specific receptor based on cAMP or Ca 2+ response. The endogenous receptors may have different effects on some of the signaling pathways due to different coupling preference or alternative signaling pathways that were not monitored in the current experiment.
Signal intensity in cellular system is another factor that needs to be considered when discussing downstream physiological effects. It is important to understand that in an artificial system overexpressing a GPCR, the signal intensity is often higher than what is generated by activation of GPCRs naturally-expressed in those cells, due to stochiometric considerations of the signal producing process.
Both the intensity and subcellular location of the signal may play a role in the type and amount of physiological response. Even when considering the limitation of the DREADD system, our results have demonstrated that it is a valuable tool to investigate beneficial GPCR signaling for fibrosis and other physiological phenomena.
Many studies have investigated the molecular mechanisms underlying fibrosis and a wide range of targets have been tested for drug discovery and clinical development (Wynn and Ramalingam, 2012;Friedman et al., 2013;Duffield, 2014;Nanthakumar et al., 2015). Regardless of disease etiology, fibrosis in the heart, lung, kidney, liver, and skin share many similar features, such as increases in myofibroblast activation, collagen production and inflammation, suggesting the existence of common core pathways/mechanisms among all fibrotic tissues. Our study focused on probing common GPCR signaling in the fibroblasts and identified Gs signaling as an important pathway for fibrosis, consistent with previously reported modulation of TGFβ signaling by cAMP (Schiller et al., 2010). DREADDs are synthetic GPCRs activated by synthetic small molecules that exhibit no biological activity at native receptors, so that it can be engineered to transduce a specific signaling in response to otherwise inert ligands in vitro and in vivo. Identification of GPCR signaling pathways that have desired outcomes in a given phenotypic assay along with transcriptional analysis of the disease state may greatly facilitate target identification and enable more focused drug discovery efforts.
About 34% of the FDA approved drugs are targeting against GPCRs (Hauser et al., 2017). Fibrosis is associated with end-stage organ damage and worsening disease progression in NASH, ESRD, heart failure, and IPF patients. There is a huge unmet medical need to develop new fibrosis therapies which bring clinically meaningful efficacy to patients. The interest in discovering new GPCR target for fibrosis is conceivable. Our work highlighted the importance of prostaglandin signaling in the fibroblasts. PGE2 is a locally-active eicosanoid that modulates vascular tone, coagulation homeostasis, and inflammation.
Additionally, PGE2 has been found working through serum-response factor (SRF)-MRTF-A (myocardinrelated transcription factor-A) to regulate αSMA expression (Penke et al., 2014). EP2 agonist butaprost attenuates fibrosis in UUO kidneys and human kidney slices (Jensen et al., 2019). Recent highthroughput myofibroblast phenotypic screen assay also finds EP2 and EP4 agonists beneficial for the This article has not been copyedited and formatted. The final version may differ from this version.
JPET Fast Forward. Published on August 26, 2020 as DOI: 10.1124/jpet.120.000103 at ASPET Journals on August 28, 2020 jpet.aspetjournals.org Downloaded from treatment of lung fibrosis (Sieber et al., 2018). The role of PGE2 in inflammation is complexed, proinflammatory in the early stages of inflammation but promoting the resolution of inflammation at the later time point (Calder, 2009). The pleiotropic effect of PGE2 limits its applications in broader patient population and chronic usage. Side effects of PGE2 systemic administration include headache, uterine contraction, gastrointestinal disturbance, and diarrhea. As such, a substantial amount of effort has been put into developing subtype-selective prostanoid receptor agonists to obtain better risk/benefit profile (Flesch et al., 2013). EP4 agonist ONO-4819CD has been tested in a phase 2 ulcerative colitis trial, but failed to achieve statistically significant therapeutic effect (Nakase et al., 2010). To date, no EP2, EP4, or EP2/4 dual agonist (antagonist) has been approved for clinical use. Developing locally-delivered PGE2 modulator, such as via inhalation and topical administration, may represent a viable approach for the treatment of fibrosis.
Our work has demonstrated that delineating beneficial GPCR signaling pathway for fibrosis is valuable for new target identification. In our proof-of-concept study, we focused on in vitro cell-based system.
DREADDs chemogenetic tools have been previously used to manipulate neuronal activity and behavior in animals (Vardy et al., 2015). As such, it is conceivable that a similar approach can be used to probe GPCR signaling in multiple fibrosis animal models. Combining with transcriptome analysis of GPCR family, it is possible to identify novel GPCR target(s) for fibrosis.      to control (-10 to 10-fold). Grey, below detection limit. This article has not been copyedited and formatted. The final version may differ from this version. 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 2nM (Bologa et al., 2006) BDKRB1 (Bradykinin receptor B1) Gq BDKRB1 is a Gq-coupled GPCR, while BDKRB2 signals through Gq and Gi. In vivo activation of BDKRB2 attenuated kidney fibrosis (Schanstra et al., 2002) (Billot et al., 2003) TBXA2R (Thromboxane A2 receptor) Gq/Gs/G 12/13 U46619 is a potent and stable TBXA2R agonist U46619 35nM (Coleman et al., 1981) OXTR (Oxytocin receptor) 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.