Novel GPR43 Agonists Exert an Anti-Inflammatory Effect in a Colitis Model.

GPR43 (also known as FFAR2), a metabolite-sensing G-protein-coupled receptor stimulated by short-chain fatty acid (SCFA) ligands is involved in innate immunity and metabolism. GPR43 couples with Gαi/o and Gαq/11 heterotrimeric proteins and is capable of decreasing cyclic AMP and inducing Ca2+ flux. The GPR43 receptor has additionally been shown to bind ß-arrestin 2 and inhibit inflammatory pathways, such as NF-ΚB. However, GPR43 shares the same ligands as GPR41, including acetate, propionate, and butyrate, and determination of its precise functions in association with endogenous ligands, such as SCFAs alone, therefore remains a considerable challenge. In this study, we generated novel synthetic agonists that display allosteric modulatory effects on GPR43 and downregulate NF-ΚB activity. In particular, the potency of compound 187 was significantly superior to that of preexisting compounds in vitro. However, in the colitis model in vivo, compound 110 induced more potent attenuation of inflammation. These novel allosteric agonists of GPR43 clearly display anti-inflammatory potential, supporting their clinical utility as therapeutic drugs.

The Short-Chain Fatty Acid Receptor GPR43 Modulates YAP/TAZ via RhoA.

GPR43 (also known as FFAR2 or FFA2) is a G-protein-coupled receptor primarily expressed in immune cells, enteroendocrine cells and adipocytes that recognizes short-chain fatty acids, such as acetate, propionate, and butyrate, likely to be implicated in innate immunity and host energy homeostasis. Activated GPR43 suppresses the cAMP level and induces Ca2+ flux via coupling to Gαi and Gαq families, respectively. Additionally, GPR43 is reported to facilitate phosphorylation of ERK through G-protein-dependent pathways and interacts with ß-arrestin 2 to inhibit NF-κB signaling. However, other G-protein-dependent and independent signaling pathways involving GPR43 remain to be established. Here, we have demonstrated that GPR43 augments Rho GTPase signaling. Acetate and a synthetic agonist effectively activated RhoA and stabilized YAP/TAZ transcriptional coactivators through interactions of GPR43 with Gαq/11 and Gα12/13. Acetate-induced nuclear accumulation of YAP was blocked by a GPR43-specific inverse agonist. The target genes induced by YAP/TAZ were further regulated by GPR43. Moreover, in THP-1-derived M1-like macrophage cells, the Rho-YAP/TAZ pathway was activated by acetate and a synthetic agonist. Our collective findings suggest that GPR43 acts as a mediator of the Rho-YAP/TAZ pathway.

Sjögren Syndrome antigen B regulates LIN28-let-7 axis in Caenorhabditis elegans and human.

LIN28 protein and let-7 family micro RNAs (miRNAs) that are an evolutionarily conserved from nematodes to humans are the important regulators of developmental timing by dynamically interacting with each other. However, regulators of LIN28 remain largely elusive. Here, we show the evidences that Sjögren Syndrome antigen B (SSB) protein associates and cooperates with LIN28A and LIN28B, mammalian orthologues of Caenorhabditis elegans lin-28, proteins in the nucleus. Knockdown of SSB in HEK293 cell line resulted in the decrease of the amount of LIN28B mRNAs and proteins, and the increase of the level of mature let-7 miRNAs. Furthermore, RNA interference of ssb-1 gene, a worm SSB orthologue, was sufficient to cause a heterochronic defect in seam cells of C. elegans, recapitulating the phenotype of lin-28 downregulation. Collectively, we suggest that SSB is an important regulator for the LIN28-let-7 axis.

Phosphorylation of REPS1 at Ser709 by RSK attenuates the recycling of transferrin receptor.

RalBP1 associated EPS domain containing 1 (REPS1) is conserved from Drosophila to humans and implicated in the endocytic system. However, an exact role of REPS1 remains largely unknown. Here, we demonstrated that mitogen activated protein kinase kinase (MEK)-p90 ribosomal S6 Kinase (RSK) signaling pathway directly phosphorylated REPS1 at Ser709 upon stimulation by epidermal growth factor (EGF) and amino acid. While REPS2 is known to be involved in the endocytosis of EGF receptor (EGFR), REPS1 knockout (KO) cells did not show any defect in the endocytosis of EGFR. However, in the REPS1 KO cells and the KO cells reconstituted with a non-phosphorylatable REPS1 (REPS1 S709A), the recycling of transferrin receptor (TfR) was attenuated compared to the cells reconstituted with wild type REPS1. Collectively, we suggested that the phosphorylation of REPS1 at S709 by RSK may have a role of the trafficking of TfR. [BMB Reports 2021; 54(5): 272-277].

Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized ß-transducin repeat-containing protein 1 (ßTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

Selective novel inverse agonists for human GPR43 augment GLP-1 secretion.

GPR43/Free Fatty Acid Receptor 2 (FFAR2) is known to be activated by short-chain fatty acids and be coupled to Gi and Gq family of heterotrimeric G proteins. GPR43 is mainly expressed in neutrophils, adipocytes and enteroendocrine cells, implicated to be involved in inflammation, obesity and type 2 diabetes. However, several groups have reported the contradictory data about the physiological functions of GPR43, so that its roles in vivo remain unclear. Here, we demonstrate that a novel compound of pyrimidinecarboxamide class named as BTI-A-404 is a selective and potent competitive inverse agonist of human GPR43, but not the murine ortholog. Through structure-activity relationship (SAR), we also found active compound named as BTI-A-292. These regulators increased the cyclic AMP level and reduced acetate-induced cytoplasmic Ca(2+) level. Furthermore, we show that they modulated the downstream signaling pathways of GPR43, such as ERK, p38 MAPK, and NF-κB. It was surprising that two compounds augmented the secretion of glucagon-like peptide 1 (GLP-1) in NCI-H716 cell line. Collectively, these novel and specific competitive inhibitors regulate all aspects of GPR43 signaling and the results underscore the therapeutic potential of them.

Perspectives on the therapeutic potential of short-chain fatty acid receptors.

There is rapidly growing interest in the human microbiome because of its implication in metabolic disorders and inflammatory diseases. Consequently, understanding the biology of short chain fatty acids and their receptors has become very important for identifying novel therapeutic avenues. GPR41 and GPR43 have been recognized as the cognate receptors for SCFAs and their roles in metabolism and inflammation have drawn much attention in recent years. GPR43 is highly expressed on immune cells and has been suggested to play a role in inflammatory diseases such as inflammatory bowel disease. Both GPR41 and GPR43 have been implicated in diabetes and obesity via the regulation of adipose tissue and gastrointestinal hormones. So far, many studies have provided contradictory results, and therefore further research is required to validate these receptors as drug targets. We will also discuss the synthetic modulators of GPR41 and GPR43 that are critical to understanding the functions of these receptors.

Leucine-rich repeat-containing G-protein coupled receptor 5/GPR49 activates G12/13-Rho GTPase pathway.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5/GPR49) is highly expressed in adult stem cells of various tissues, such as intestine, hair follicles, and stomach. LGR5 is also overexpressed in some colon and ovarian tumors. Recent reports show that R-spondin (RSPO) family ligands bind to and activate LGR5, enhancing canonical Wnt signaling via the interaction with LRP5/6 and Frizzled. The identity of heterotrimeric G-proteins coupled to LGR5, however, remains unclear. Here, we show that Rho GTPase is a downstream target of LGR5. Overexpression of LGR5 induced SRF-RE luciferase activity, a reporter of Rho signaling. RSPOs, ligands for LGR4, LGR5, and LGR6, however, did not induce SRF-RE reporter activity in the presence of LGR5. Consistently, LGR5-induced activity of the SRF-RE reporter was inhibited by Rho inhibitor C3 transferase and RhoA N19 mutant, and knockdown of Gα12/13 genes blocked the reporter activity induced by LGR5. In addition, focal adhesion kinase, NF-κB and c-fos, targets of Rho GTPase, were shown to be regulated by LGR5. Here, we have demonstrated, for the first time, that LGR5 is coupled to the Rho pathway through G12/13 signaling.

ß-Arrestin 2 mediates G protein-coupled receptor 43 signals to nuclear factor-κB.

G-protein coupled receptor 43 (GPR43) serves as a receptor for short-chain fatty acids (SCFAs), implicated in neutrophil migration and inflammatory cytokine production. However, the intracellular signaling pathway mediating GPR43 signaling remains unclear. Here, we show that ß-arrestin 2 mediates the internalization of GPR43 by agonist. Agonism of GPR43 reduced the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB), which was relieved by short interfering RNA (siRNA) of ß-arrestin 2. Subsequently, mRNA expression of proinflammatory cytokines, interleukin (IL)-6 and IL-1ß, was downregulated by activation of GPR43 and knockdown of ß-arrestin 2 recovered the expression of the cytokines. Taken together, these results suggest that GPR43 may be a plausible target for a variety of inflammatory diseases.