Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways.

BACKGROUND: G-protein coupled receptors (GPCRs) are recognized as attractive targets for drug therapy. However, it remains poorly understood how GPCRs, except for a few chemokine receptors, regulate the progression of liver fibrosis. Here, we aimed to reveal the role of GPR65, a proton-sensing receptor, in liver fibrosis and to elucidate the underlying mechanism. METHODS: The expression level of GPR65 was evaluated in both human and mouse fibrotic livers. Furthermore, Gpr65-deficient mice were treated with either bile duct ligation (BDL) for 21 d or carbon tetrachloride (CCl4) for 8 weeks to investigate the role of GPR65 in liver fibrosis. A combination of experimental approaches, including Western blotting, quantitative real-time reverse transcription­polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and rescue studies, were used to explore the underlying mechanisms of GPR65's action in liver fibrosis. Additionally, the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated. RESULTS: We found that hepatic macrophages (HMs)-enriched GPR65 was upregulated in both human and mouse fibrotic livers. Moreover, knockout of Gpr65 significantly alleviated BDL- and CCl4-induced liver inflammation, injury and fibrosis in vivo, and mouse bone marrow transplantation (BMT) experiments further demonstrated that the protective effect of Gpr65 knockout is primarily mediated by bone marrow-derived macrophages (BMMs). Additionally, in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited, while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-ß (TGF-ß), all of which subsequently promoted the activation of hepatic stellate cells (HSCs) and the damage of hepatocytes (HCs). Mechanistically, GPR65 overexpression, the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-α and IL-6 via the Gαq-Ca2+-JNK/NF-κB pathways, while promoted the expression of TGF-ß through the Gαq-Ca2+-MLK3-MKK7-JNK pathway. Notably, pharmacological GPR65 inhibition retarded the development of inflammation, HCs injury and fibrosis in vivo. CONCLUSIONS: GPR65 is a major regulator that modulates the progression of liver fibrosis. Thus, targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

A novel peptide stapling strategy enables the retention of ring-closing amino acid side chains for the Wnt/ß-catenin signalling pathway.

The all-hydrocarbon peptide stapling strategy has recently been extensively explored in drug discovery. There remains the potential for improvement regarding the retention of the amino acid side chains at the stapled positions. Herein, we describe a new series of amino acids that not only contain the native side chains, but also carry the alkenyl arms that are needed for the ring-closing stapling chemistry. We incorporate the new amino acids into a ß-catenin-binding domain of Axin (469-482) and develop a new category of stapled peptides with the retention of the native side chains. These stapled peptides exhibit high α-helicity, strong proteolytic stability and good cell permeability. Biochemical experiments demonstrate that these stapled peptides can activate ß-catenin more efficiently than canonical stapled peptides due to the presence of extra side chains. We expect that the new side-chain-retention stapling method would expand the scope of the all-hydrocarbon stapled peptide strategy by retaining some important peripheral residues for protein-protein interactions or preserving key hydrophilic side chains to improve solubility.

TGFß induced factor homeobox 1 promotes colorectal cancer development through activating Wnt/ß-catenin signaling.

Colorectal cancer (CRC) is one of the most common cancers, but the mechanisms underlying its initiation and progression are largely unknown. TGIF1 (TGFB induced factor homeobox 1) is a transcriptional corepressor that belongs to the three-amino acid loop extension (TALE) superclass of atypical homeodomains. It has been reported that TGIF1 is highly expressed in mammary cancer and non-small cell lung cancer and can enhance tumor progression. However, the role of TGIF1 in colorectal cancer remains unknown. Here, we report that TGIF1 is significantly upregulated in colorectal cancers, and its high expression predicts poor prognosis. Overexpression of TGIF1 markedly promotes the proliferation of colorectal cancer cells both in vivo and in vitro. In addition, TGIF1 activates Wnt/ß-catenin signaling, and the homeodomain is indispensable for Wnt activation and ß-catenin interaction. Taken together, our results suggest that TGIF1 is a novel colorectal tumor promoter and indicate that TGIF1 enhances colorectal cancer tumorigenesis through activating Wnt signaling.