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Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.

Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein–coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1–4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy.Significance: This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy–resistant lung cancer to anti-PD-1 therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg. Cancer Res; 78(17); 4929–42. ©2018 AACR.

This file contains the characterization of M2 (Supplementary Fig.S1) and M1 (Supplementary Fig.S2) type polarization in both Lgr4 knockdown and overexpression cells; the expression and immune regulation of Lgr4 in tumor tissues (Supplementary Fig.S3); the toxicity of LGR4 ECD, Rspo1 antibody and BLZ945 on tumor cells and macrophages (Supplementary Fig.S4); the immune regulation of Rspo/Lgr4 blockade in B16F10 melanoma mouse model (Supplementary Fig.S5).

PDF file - 367K, Fig S1. Screening adhesion GPCR expression in breast cancer cells. Fig S2. The GPR116 expression in knockdown or rescued breast cancer cells. Fig S3. Knockdown of GPR116 had little effect on breast cancer cell growth and apoptosis. Fig S4. The expression of GPR116 in breast cancer cell lines was quantified by Real-time PCR. Fig S5. Overexpression of GPR116 increased the activitiy of RhoA and Rac1 but not Cdc42 in 293T cells as measured by GTPase pull-down assays. Fig S6. Regulation of actin stress fibers and lamellipodia by GPR116 is RhoA and Rac1 dependent. Fig S7. The effect of RGS domain of p115RGS and Gq CT-His on GPR116 induced activity of RhoA and Rac1. Fig S8. Two additional independent experiments of Fig. 4F and Fig. 4G. Table S1. Oligonucleotide sequences of PCR primers.

The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis.

Inflammasomes are essential for inflammation and pathogen elimination in response to microbial infection and endogenous danger signals. However, the mechanism of inflammasome activation by endogenous danger signals mediated posttranslational modification and the connection between inflammasomes and inflammatory diseases remains elusive. In this study, we found that ADP was highly released from injured colonic tissue as a danger signal during inflammatory bowel disease. Consequently, extracellular ADP activated the NLRP3 inflammasome through P2Y1 receptor-mediated calcium signaling, which led to the maturation and secretion of IL-1β and further aggravation of experimental colitis. Genetic ablation or pharmacological blockade of the P2Y1 receptor significantly ameliorated DSS-induced colitis and endotoxic shock through reducing NLRP3 inflammasome activation. Moreover, ERK5-mediated tyrosine phosphorylation of ASC was essential for activation of the NLRP3 inflammasome. Thus, our study provides a novel theoretical basis for posttranslational modification of ASC in NLRP3 inflammasome activation and revealed that ADP/P2Y1 is a potential drug target for inflammatory bowel disease.

Intestinal tumors mainly originate from transformed crypt stem cells supported by Wnt signaling, which functions through downstream critical factors enriched in the intestinal stem/progenitor compartment. Here, we show Uhrf2 is predominantly expressed in intestinal crypts and adenomas in mice and is transcriptionally regulated by Wnt signaling. Upregulated UHRF2 correlates with poor prognosis in colorectal cancer patients. Although loss of Uhrf2 did not affect intestinal homeostasis and regeneration, tumor initiation and progression were inhibited, leading to a markedly prolonged life span in Uhrf2 null mice on an ApcMin background. Uhrf2 deficiency also strongly reduced primary tumor organoid formation suggesting impairment of tumor stem cells. Moreover, ablation of Uhrf2 suppressed tumor cell proliferation through downregulation of the Wnt/β-catenin pathway. Mechanistically, Uhrf2 directly interacts with and sumoylates Tcf4, a critical intranuclear effector of the Wnt pathway. Uhrf2 mediated SUMOylation stabilized Tcf4 and further sustained hyperactive Wnt signaling. Together, we demonstrate that Wnt-induced Uhrf2 expression promotes tumorigenesis through modulation of the stability of Tcf4 for maintaining oncogenic Wnt/β-catenin signaling. This is a new reciprocal feedforward regulation between Uhrf2 and Wnt signaling in tumor initiation and progression.

Epigenetic abnormalities in DNA hydroxymethylation (5hmC) have been detected in patients with myeloid neoplasms, suggesting that 5hmC might act as a valuable epigenetic mark to reflect the disease status of myeloid neoplasms. Here, we report systematic genome-wide mapping of the DNA hydroxymethylomes in over 70 patients with myeloid neoplasms. Our integrative analysis leads to the identification of distinct 5hmC signatures that can sensitively discriminate patients from healthy individuals. At the molecular level, we unveiled dynamic 5hmC changes within key transcription factor (e.g., the CEBP family) binding motifs that are essential for hematopoiesis and myeloid lineage specification. 5hmC redistribution was found to alter the genome-wide binding of CEBP-α, thereby reprogramming transcriptional outputs to affect leukemia cell survival and stemness. Taken together, we provide a comprehensive 5hmC atlas representative of myeloid neoplasms, which sets the stage for future exploration on the epigenetic etiology of hematological malignancies. Mechanistically, our study further furnishes important insights into how abnormal 5hmC distribution in patients directly interrupts the binding of transcription factors to reshape transcriptional landscapes and aggravate leukemogenesis.

Osteoclasts were over-activated as we age, which leads to bone loss. Src-deficient mice lead to only one phenotype -severe osteopetrosis due to functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCR) have been targets for ∼35% of approved drugs. However, how Src kinase activity is negatively regulated by GPCRs remains largely elusive. Herein we report that Src is dephosphorylated at Tyr 416 by Dusp18 upon GPR54 activation by its natural ligand Kp-10. Mechanically, both active Src and the Dusp18 phosphatase are recruited by GPR54 through the proline/arginine-rich motif (PR motif) in the C terminus, which is dependent on the Gαq signal pathway. As such, Kiss1, Gpr54, Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss. Accordingly, Kp-10 abrogated bone loss by suppressing osteoclasts activity in vivo. Therefore, Kiss1/Gpr54 is a promising therapeutic strategy governing bone resorption through Src dephosphorylation by Dusp18.

In article number 2100424, Gang Han, Yubin Zhou, Ji Jing, and co‐workers introduce a set of optogenetic tools, designated LiPOPs, that enable photoswitchable control over key molecular steps involved in necroptosis or pyroptosis, including cell membrane perforation, extracellular ions influx, and cell suicide. LiPOPs are coupled with either upconversion nanoparticles (UCNPs) with NIR‐to‐blue emission (left corner) or NanoLuc‐catalyzed bioluminescence‐based optogenetic stimulation (NanoLOGS; right corner) to achieve wireless nano‐optogenetic or chemo‐optogenetic manipulation of cell fate with high spatiotemporal precision in living animals. LiPOPs open new avenues to study the role of non‐apoptotic cell death during microbial infection, inflammatory response and cancer immunotherapy. [Image: see text]

Hepatocellular carcinoma (HCC) is the sixth most common cancer and fourth leading cause of cancer-related death worldwide. The number of HCC cases continues to rise despite advances in screening and therapeutic inventions. More importantly, HCC poses two major health disparity issues. First, HCC occurs more commonly in men than women. Second, with the global increase in non-alcoholic fatty liver diseases (NAFLD), it has also become evident that HCC is more prevalent in some races and/or ethnic groups compared to others, depending on its predisposing etiology. Most studies on HCC in the past have been focused on genetic factors as the driving force for HCC development, and the results revealed that genetic mutations associated with HCC are often heterogeneous and involve multiple pathogenic pathways. An emerging new research field is epigenetics, in which gene expression is modified without altering DNA sequences. In this article, we focus on reviewing current knowledge on HCC-related DNA methylation changes that show disparities among different sexes or different racial/ethnic groups, in an effort to establish a point of departure for resolving the broader issue of health disparities in gastrointestinal malignancies using cutting-edge epigenetic approaches.

Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non‐apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo‐optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non‐apoptotic cell death pathways during microbial infection and anti‐tumor immunity., A new class of genetically‐encoded actuators, designated LiPOPs, are designed to enable photoswitchable necroptosis or pyroptosis. LiPOPs are coupled with upconversion nanoparticles (UCNPs) or NanoLuc‐mediated luminescence‐aided optogenetic stimulation (NanoLOGS) to achieve wireless nano‐optogenetic or chemo‐optogenetic control of cell death in vivo with superior spatiotemporal precision. LiPOPs can also be exploited as safety switches to facilitate the development of safer cell‐based therapies.

Background & aims Pregnant women may transmit their metabolic phenotypes to their offspring, enhancing the risk for nonalcoholic fatty liver disease (NAFLD); however, the molecular mechanisms remain unclear. Methods Prior to pregnancy female mice were fed either a maternal normal-fat diet (NF-group, "no effectors"), or a maternal high-fat diet (HF-group, "persistent effectors"), or were transitioned from a HF to a NF diet before pregnancy (H9N-group, "effectors removal"), followed by pregnancy and lactation, and then offspring were fed high-fat diets after weaning. Offspring livers were analysed by functional studies, as well as next-generation sequencing for gene expression profiles and DNA methylation changes. Results The HF, but not the H9N offspring, displayed glucose intolerance and hepatic steatosis. The HF offspring also displayed a disruption of lipid homeostasis associated with an altered methionine cycle and abnormal one-carbon metabolism that caused DNA hypermethylation and L-carnitine depletion associated with deactivated AMPK signalling and decreased expression of PPAR-α and genes for fatty acid oxidation. These changes were not present in H9N offspring. In addition, we identified maternal HF diet-induced genes involved in one-carbon metabolism that were associated with DNA methylation modifications in HF offspring. Importantly, the DNA methylation modifications and their associated gene expression changes were reversed in H9N offspring livers. Conclusions Our results demonstrate for the first time that maternal HF diet disrupted the methionine cycle and one-carbon metabolism in offspring livers which further altered lipid homeostasis. CpG islands of specific genes involved in one-carbon metabolism modified by different maternal diets were identified.

Immune checkpoint blockade (ICB) has a limited effect on colorectal cancer, underlining the requirement of co‐targeting the complementary mechanisms. Here, we identified prostaglandin E2 (PGE2) receptor 4 (EP4) as the master regulator of immunosuppressive myeloid cells (IMCs), which are the major driver of resistance to ICB therapy. PGE2‐bound EP4 promotes the differentiation of immunosuppressive M2 macrophages and myeloid‐derived suppressor cells (MDSCs) and reduces the expansion of immunostimulated M1 macrophages. To explore the immunotherapeutic role of EP4 signaling, we developed a novel and selective EP4 antagonist TP‐16. TP‐16 effectively blocked the function of IMCs and enhanced cytotoxic T‐cell‐mediated tumor elimination in vivo. Cell co‐culture experiments revealed that TP‐16 promoted T‐cell proliferation, which was impaired by tumor‐derived CD11b+ myeloid cells. Notably, TP‐16 and anti‐PD‐1 combination therapy significantly impeded tumor progression and prolonged mice survival. We further demonstrated that TP‐16 increased responsiveness to anti‐PD‐1 therapy in an IMC‐related spontaneous colorectal cancer mouse model. In summary, this study demonstrates that inhibition of EP4‐expressing IMCs may offer a potential strategy for enhancing the efficacy of immunotherapy for colorectal cancer., Immunosuppressive myeloid cells (IMCs) are a prominent driver of immunotherapy resistance in colorectal cancer. This study identifies EP4 as a master regulator of IMCs and highlights blockade of EP4 as a novel therapeutic strategy for enhancing immunotherapy in colorectal cancer.

It is well known that free fatty acids (FFAs) have beneficial effects on the skeletal system, however, which fatty acid sensing GPCR(s) and how the GPCR(s) regulating cartilage development and osteoarthritis (OA) pathogenesis is largely unknown. In this study, we found Gpr84, a receptor for medium-chain FFAs (MCFA), was the only FFA-sensing GPCR in human and mouse chondrocytes that exhibited elevated expression when stimulated by interleukin (IL)-1β. Gpr84-deficiency upregulated cartilage catabolic regulator expression and downregulated anabolic factor expression in the IL-1β-induced cell model and the destabilization of the medial meniscus (DMM)-induced OA mouse model. Gpr84-/- mice exhibited an aggravated OA phenotype characterized by severe cartilage degradation, osteophyte formation and subchondral bone sclerosis. Moreover, activating Gpr84 directly enhanced cartilage extracellular matrix (ECM) generation while knockout of Gpr84 suppressed ECM-related gene expression. Especially, the agonists of GPR84 protected human OA cartilage explants against degeneration by inducing cartilage anabolic factor expression. At the molecular level, GPR84 activation inhibited IL-1β-induced NF-κB signaling pathway. Furthermore, deletion of Gpr84 had little effect on articular and spine cartilaginous tissues during skeletal growth. Together, all of our results demonstrated that fatty acid sensing GPCR (Gpr84) signaling played a critical role in OA pathogenesis, and activation of GPR84 or MCFA supplementation has potential in preventing the pathogenesis and progression of OA without severe cartilaginous side effect.

Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gαq and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor–related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.

Synthetic biology, when combined with immunoengineering (designated synthetic immunology), has enabled the invention of an arsenal of genetically encoded synthetic devices and systems to reprogram cells for therapeutic purposes. The engineered intelligent cells can serve as ‘living’ drugs to treat a wide range of human diseases including cancer, disorders of the immune system, and infectious diseases. As the most successful example, cells of the immune system engineered with chimeric antigen receptors (CARs) have shown curative potentials for the treatment of hematological malignancies. We present herein emerging approaches of designing smart CARs to improve their safety, specificity and efficacy in cellular immunotherapy, and describe latest advances in applying CAR-engineered immune cells to target cancer and autoimmune diseases.

Adhesion GPCR Gpr126/Adgrg6 regulates mouse body length and bone mass by controlling osteoblast differentiation., Adhesion G protein–coupled receptor G6 (Adgrg6; also named GPR126) single-nucleotide polymorphisms are associated with human height in multiple populations. However, whether and how GPR126 regulates body height is unknown. In this study, we found that mouse body length was specifically decreased in Osx-Cre;Gpr126fl/fl mice. Deletion of Gpr126 in osteoblasts resulted in a remarkable delay in osteoblast differentiation and mineralization during embryonic bone formation. Postnatal bone formation, bone mass, and bone strength were also significantly affected in Gpr126 osteoblast deletion mice because of defects in osteoblast proliferation, differentiation, and ossification. Furthermore, type IV collagen functioned as an activating ligand of Gpr126 to regulate osteoblast differentiation and function by stimulating cAMP signaling. Moreover,the cAMP activator PTH(1–34), could partially restore the inhibition of osteoblast differentiation and the body length phenotype induced by Gpr126 deletion.Together, our results demonstrated that COLIV-Gpr126 regulated body length and bone mass through cAMP-CREB signaling pathway.

Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein–coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1–4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy. Significance: This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy–resistant lung cancer to anti-PD-1 therapy. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg. Cancer Res; 78(17); 4929–42. ©2018 AACR.

CD4+ T helper cells, especially T helper 17 (TH17) cells, combined with immune regulatory network dysfunction, play key roles in autoimmune diseases including multiple sclerosis (MS). Betulinic acid (BA), a natural pentacyclic triterpenoid, has been reported to be involved in anti-inflammation, in particular having an inhibitory effect on proinflammatory cytokine interleukin 17 (IL-17) and interferon-γ (IFN-γ) production. In this study, we screened BA derivatives and found a BA derivative, SH479, that had a greater inhibitory effect on TH17 differentiation. Our further analysis showed that SH479 had a greater inhibitory effect on TH17 and TH1, and a more stimulatory effect on regulatory T (Treg) cells. To evaluate the effects of SH479 on autoimmune diseases in vivo, we employed the extensively used MS mouse model experimental autoimmune encephalomyelitis (EAE). Our results showed that SH479 ameliorated clinical and histologic signs of EAE in both prevention and therapeutic protocols by regulating the TH17/Treg balance. SH479 dose-dependently reduced splenic lymphocyte proinflammatory factors and increased anti-inflammatory factors. Moreover, SH479 specifically inhibited splenic lymphocyte viability from EAE mice but not normal splenic lymphocyte viability. At the molecular level, SH479 inhibited TH17 differentiation by regulating signal transducer and activator of transcription-3 (STAT3) phosphorylation, DNA binding activity, and recruitment to the Il-17a promoter in CD4+ T cells. Furthermore, SH479 promoted the STAT5 signaling pathway and inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Together, our data demonstrated that SH479 ameliorated EAE by regulating the TH17/Treg balance through inhibiting the STAT3 and NF-κB pathways while activating the STAT5 pathway, suggesting that SH479 is a potential novel drug candidate for autoimmune diseases including MS.

The superfamily of G protein-coupled receptors (GPCRs) contains immense structural and functional diversity and mediates a myriad of biological processes upon activation by various extracellular signals. Critical roles of GPCRs have been established in bone development, remodeling, and disease. Multiple human GPCR mutations impair bone development or metabolism, resulting in osteopathologies. Here we summarize the disease phenotypes and dysfunctions caused by GPCR gene mutations in humans as well as by deletion in animals. To date, 92 receptors (5 glutamate family, 67 rhodopsin family, 5 adhesion, 4 frizzled/taste2 family, 5 secretin family, and 6 other 7TM receptors) have been associated with bone diseases and dysfunctions (36 in humans and 72 in animals). By analyzing data from these 92 GPCRs, we found that mutation or deletion of different individual GPCRs could induce similar bone diseases or dysfunctions, and the same individual GPCR mutation or deletion could induce different bone diseases or dysfunctions in different populations or animal models. Data from human diseases or dysfunctions identified 19 genes whose mutation was associated with human BMD: 9 genes each for human height and osteoporosis; 4 genes each for human osteoarthritis (OA) and fracture risk; and 2 genes each for adolescent idiopathic scoliosis (AIS), periodontitis, osteosarcoma growth, and tooth development. Reports from gene knockout animals found 40 GPCRs whose deficiency reduced bone mass, while deficiency of 22 GPCRs increased bone mass and BMD; deficiency of 8 GPCRs reduced body length, while 5 mice had reduced femur size upon GPCR deletion. Furthermore, deficiency in 6 GPCRs induced osteoporosis; 4 induced osteoarthritis; 3 delayed fracture healing; 3 reduced arthritis severity; and reduced bone strength, increased bone strength, and increased cortical thickness were each observed in 2 GPCR-deficiency models. The ever-expanding number of GPCR mutation-associated diseases warrants accelerated molecular analysis, population studies, and investigation of phenotype correlation with SNPs to elucidate GPCR function in human diseases.

Inflammasomes are essential for inflammation and pathogen elimination in response to microbial infection and endogenous danger signals. However, the mechanism of inflammasome activation by endogenous danger signals mediated posttranslational modification and the connection between inflammasomes and inflammatory diseases remains elusive. In this study, we found that ADP was highly released from injured colonic tissue as a danger signal during inflammatory bowel disease. Consequently, extracellular ADP activated the NLRP3 inflammasome through P2Y

Aberrant DNA hydroxymethylcytosine (5hmC) levels have been reported in patients with myelodysplastic syndromes (MDS). The dysregulation of 5hmC is not only observed in patients with TET2 mutations, but also those bearing other genetic defects, suggesting that 5hmC might act as a valuable epigenetic mark to reflect the status of MDS. We report herein a highly-sensitive 5hmC analysis method using low input DNA (10 ng), which was used to obtain a comprehensive atlas of the DNA hydroxymethylome in MDS patients. Our systematic bioinformatic analysis unveiled that 5hmC signatures could be used to separate patients with good and poor clinical outcomes. At the molecular level, we observed dynamic changes of 5hmC within several key transcription factor binding motifs that are essential for hematopoiesis and myeloid lineage specification, which might result in impaired transcriptional outputs during leukemogenesis. Interestingly, we also observed massive changes in 5hmC and transcriptomes in patient showing response to hypomethylating agent (HMA) treatment, suggesting the prognostic value of 5hmC in evaluating epigenetic therapy. Overall, our highly-sensitive 5hmC analysis method provides a useful means to potentially facilitate the clinical evaluation of MDS patients. Disclosures Garcia-Manero: AbbVie: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Onconova: Research Funding; Amphivena Therapeutics: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Jazz Pharmaceuticals: Consultancy; Acceleron Pharmaceuticals: Consultancy, Honoraria; H3 Biomedicine: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding.

The metabolite-sensing G protein–coupled receptors (GPCRs) bind to various metabolites and transmit signals that are important for proper immune and metabolic functions. However, the roles of metabolite-sensing GPCRs in viral infection are not well characterized. Here, we identified metabolite-sensing GPCR TGR5 as an interferon (IFN)-stimulated gene (ISG) which had increased expression following viral infection or IFN-β stimulation in a STAT1-dependent manner. Most importantly, overexpression of TGR5 or treatment with the modified bile acid INT-777 broadly protected host cells from vesicular stomatitis virus (VSV), newcastle disease virus (NDV) and herpes simplex virus type 1 (HSV-1) infection. Furthermore, VSV and HSV-1 replication was increased significantly in Tgr5-deficient macrophages and the VSV distribution in liver, spleen and lungs was increased in Tgr5-deficient mice during VSV infection. Accordingly, Tgr5-deficient mice were much more susceptible to VSV infection than wild-type mice. Mechanistically, TGR5 facilitates type I interferon (IFN-I) production through the AKT/IRF3-signaling pathway, which is crucial in promoting antiviral innate immunity. Taken together, our data reveal a positive feedback loop regulating IRF3 signaling and suggest a potential therapeutic role for metabolite-sensing GPCRs in controlling viral diseases.

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4+/- mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4-/- ) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4-/- ) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.

Osteoporosis is a global bone disease characterized by reduced bone mineral density (BMD) and increased risk of fractures. The risk of developing osteoporosis increases with aging, especially after menopause in women. Discovering the signaling pathways that play a significant role in aging- and menopause-induced osteoporosis should accelerate osteoporosis drug discovery. In this study, we found that bile acid membrane receptor Tgr5 knockout C57BL/6J mice had similar bone mass as wild-type mice during early and middle-age (before 4 months old) bone remodeling; however, Tgr5-/- markedly decreased bone mass in aged (more than 7 months old) and ovariectomized (OVX) mice compared with wild-type mice. Moreover, Tgr5 knockout strongly induced osteoclast differentiation but had no effect on osteoblast activity. Treatment with different TGR5 agonists consistently inhibited osteoclast differentiation. Importantly, our results showed that Tgr5 regulates osteoclastogenesis by the AMP-activated protein kinase (AMPK) signaling pathway, which is a central metabolic pathway involved in the pathophysiology of aging and age-related diseases. The bile acid nuclear receptor FXR is an established regulator of bone metabolism. We screened the derivatives of betulinic acid (BA), a known TGR5 agonist, to identify novel dual agonists of FXR and TGR5. The derivative SH-479, a pentacyclic triterpene acid, could activate both TGR5 and FXR, with a better inhibitory effect on osteoclastogenesis compared with agonists solely activating FXR or TGR5 and additionally enhanced osteoblastogenesis. Furthermore, SH-479 therapeutically abrogated bone loss in C57BL/6J mice through the bone remodeling pathways. Together, our results demonstrate that dual targeting the bile acid membrane receptor TGR5 and nuclear receptor FXR is a promising strategy for osteoporosis. © 2018 American Society for Bone and Mineral Research.

Blocking signaling by the neuropeptide hormone kisspeptin enhances antiviral immune responses., G protein–coupled receptor 54 (GPR54), the key receptor for the neuropeptide hormone kisspeptin, plays essential roles in regulating puberty development and cancer metastasis. However, its role in the antiviral innate immune response is unknown. We report that virus-induced type I interferon (IFN-I) production was significantly enhanced in Gpr54-deficient cells and mice and resulted in restricted viral replication. We found a marked increase of kisspeptin in mouse serum during viral infection, which, in turn, impaired IFN-I production and antiviral immunity through the GPR54/calcineurin axis. Mechanistically, kisspeptin/GPR54 signaling recruited calcineurin and increased its phosphatase activity to dephosphorylate and deactivate TANK [tumor necrosis factor receptor-associated factor (TRAF) family member-associated NF-κB activator]–binding kinase 1 (TBK1) in a Ca2+-dependent manner. Thus, our data reveal a kisspeptin/GPR54/calcineurin-mediated immune evasion pathway exploited by virus through the negative feedback loop of TBK1 signaling. These findings also provide insights into the function and cross-talk of kisspeptin, a known neuropeptide hormone, in antiviral innate immune response.

The fourth member of the leucine-rich repeat-containing GPCR family (LGR4, frequently referred to as GPR48) and its cognate ligands, R-spondins (RSPOs) play crucial roles in the development of multiple organs as well as the survival of adult stem cells by activation of canonical Wnt signaling. Wnt/β-catenin signaling acts to regulate breast cancer; however, the molecular mechanisms determining its spatiotemporal regulation are largely unknown. In this study, we identified LGR4 as a master controller of Wnt/β-catenin signaling-mediated breast cancer tumorigenesis, metastasis, and cancer stem cell (CSC) maintenance. LGR4 expression in breast tumors correlated with poor prognosis. Either Lgr4 haploinsufficiency or mammary-specific deletion inhibited mouse mammary tumor virus (MMTV)- PyMT- and MMTV- Wnt1-driven mammary tumorigenesis and metastasis. Moreover, LGR4 down-regulation decreased in vitro migration and in vivo xenograft tumor growth and lung metastasis. Furthermore, Lgr4 deletion in MMTV- Wnt1 tumor cells or knockdown in human breast cancer cells decreased the number of functional CSCs by ∼90%. Canonical Wnt signaling was impaired in LGR4-deficient breast cancer cells, and LGR4 knockdown resulted in increased E-cadherin and decreased expression of N-cadherin and snail transcription factor -2 ( SNAI2) (also called SLUG), implicating LGR4 in regulation of epithelial-mesenchymal transition. Our findings support a crucial role of the Wnt signaling component LGR4 in breast cancer initiation, metastasis, and breast CSCs.-Yue, Z., Yuan, Z., Zeng, L., Wang, Y., Lai, L., Li, J., Sun, P., Xue, X., Qi, J., Yang, Z., Zheng, Y., Fang, Y., Li, D., Siwko, S., Li, Y., Luo, J., Liu, M. LGR4 modulates breast cancer initiation, metastasis, and cancer stem cells.

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues to regulate cell growth and survival through various mechanisms. However, how mTORC1 responds to acute inflammatory signals to regulate bowel regeneration is still obscure. In this study, we investigated the role of mTORC1 in acute inflammatory bowel disease. Inhibition of mTORC1 activity by rapamycin treatment or haploinsufficiency of Rheb through genetic modification in mice impaired intestinal cell proliferation and induced cell apoptosis, leading to high mortality in dextran sodium sulfate– and 2,4,6-trinitrobenzene sulfonic acid–induced colitis models. Through bone marrow transplantation, we found that mTORC1 in nonhematopoietic cells played a major role in protecting mice from colitis. Reactivation of mTORC1 activity by amino acids had a positive therapeutic effect in mTORC1-deficient Rheb+/− mice. Mechanistically, mTORC1 mediated IL-6–induced Stat3 activation in intestinal epithelial cells to stimulate the expression of downstream targets essential for cell proliferation and tissue regeneration. Therefore, mTORC1 signaling critically protects against inflammatory bowel disease through modulation of inflammation-induced Stat3 activity. As mTORC1 is an important therapeutic target for multiple diseases, our findings will have important implications for the clinical usage of mTORC1 inhibitors in patients with acute inflammatory bowel disease.

International audience; Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics.

G protein-coupled receptor 124 (GPR124; also called tumor endothelial marker 5, TEM5) is highly expressed in tumor vasculature. While recent studies have revealed a role in vasculogenesis, evidence for GPR124 function in tumor angiogenesis is lacking. Here, we demonstrate that GPR124 is required for VEGF-induced tumor angiogenesis. GPR124 silencing in human endothelial cells inhibited mouse xenograft tumor angiogenic vessel formation and tumor growth. GPR124 regulated VEGF-induced tumor angiogenic processes in vitro including cell-cell interaction, permeability, migration, invasion, and tube formation. Therefore, GPR124 plays a key role in VEGF-induced tumor angiogenesis.

AGR syndrome (the clinical triad of aniridia, genitourinary anomalies, and mental retardation, a subgroup of WAGR syndrome for Wilm's tumor, aniridia, genitourinary anomalies, and mental retardation) is a rare syndrome caused by a contiguous gene deletion in the 11p13-14 region. However, the mechanisms of WAGR syndrome pathogenesis are elusive. In this study we provide evidence that LGR4 (also named GPR48), the only G-protein-coupled receptor gene in the human chromosome 11p12-11p14.4 fragment, is the key gene responsible for the diseases of AGR syndrome. Deletion of Lgr4 in mouse led to aniridia, polycystic kidney disease, genitourinary anomalies, and mental retardation, similar to the pathological defects of AGR syndrome. Furthermore, Lgr4 inactivation significantly increased cell apoptosis and decreased the expression of multiple important genes involved in the development of WAGR syndrome related organs. Specifically, deletion of Lgr4 down-regulated the expression of histone demethylases Jmjd2a and Fbxl10 through cAMP-CREB signaling pathways both in mouse embryonic fibroblast cells and in urinary and reproductive system mouse tissues. Our data suggest that Lgr4, which regulates eye, kidney, testis, ovary, and uterine organ development as well as mental development through genetic and epigenetic surveillance, is a novel candidate gene for the pathogenesis of AGR syndrome.

Prostate cancer is a highly penetrant disease among men in industrialized societies, but the factors regulating the transition from indolent to aggressive and metastatic cancer remain poorly understood. We found that men with prostate cancers expressing high levels of the G protein–coupled receptor LGR4 had a significantly shorter recurrence-free survival compared with patients with cancers having low LGR4 expression. LGR4 expression was elevated in human prostate cancer cell lines with metastatic potential. We therefore generated a novel transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model to investigate the role of Lgr4 in prostate cancer development and metastasis in vivo. TRAMP Lgr4−/− mice exhibited an initial delay in prostate intraepithelial neoplasia formation, but the frequency of tumor formation was equivalent between TRAMP and TRAMP Lgr4−/− mice by 12 weeks. The loss of Lgr4 significantly improved TRAMP mouse survival and dramatically reduced the occurrence of lung metastases. LGR4 knockdown impaired the migration, invasion, and colony formation of DU145 cells and reversed epithelial–mesenchymal transition (EMT), as demonstrated by up-regulation of E-cadherin and decreased expression of the EMT transcription factors ZEB, Twist, and Snail. Overexpression of LGR4 in LNCaP cells had the opposite effects. Orthotopic injection of DU145 cells stably expressing shRNA targeting LGR4 resulted in decreased xenograft tumor size, reduced tumor EMT marker expression, and impaired metastasis, in accord with our findings in TRAMP Lgr4−/− mice. In conclusion, we propose that Lgr4 is a key protein necessary for prostate cancer EMT and metastasis.

In the original publication the grant number is incorrectly published. The correct grant number should be read as "17140901600". The corrected contents are provided in this correction article. This work was partially supported by grants from the National Natural Science Foundation of China (Nos. 81670470 and 81600149), a grant from the Shanghai Municipal Commission for Science and Technology (17140901600, 18411953500 and 15JC1400201) and a grant from National Key Research and Development Program (2016YFC0905100).

Adhesion G-protein–coupled receptors (GPCR), which contain adhesion domains in their extracellular region, have been found to play important roles in cell adhesion, motility, embryonic development, and immune response. Because most adhesion molecules with adhesion domains have vital roles in cancer metastasis, we speculated that adhesion GPCRs are potentially involved in cancer metastasis. In this study, we identified GPR116 as a novel regulator of breast cancer metastasis through expression and functional screening of the adhesion GPCR family. We found that knockdown of GPR116 in highly metastatic (MDA-MB-231) breast cancer cells suppressed cell migration and invasion. Conversely, ectopic GPR116 expression in poorly metastatic (MCF-7 and Hs578T) cells promoted cell invasion. We further showed that knockdown of GPR116 inhibited breast cancer cell metastasis in two mammary tumor metastasis mouse models. Moreover, GPR116 modulated the formation of lamellipodia and actin stress fibers in cells in a RhoA- and Rac1-dependent manner. At a molecular level, GPR116 regulated cell motility and morphology through the Gαq-p63RhoGEF-RhoA/Rac1 pathway. The biologic significance of GPR116 in breast cancer is substantiated in human patient samples, where GPR116 expression is significantly correlated with breast tumor progression, recurrence, and poor prognosis. These findings show that GPR116 is crucial for the metastasis of breast cancer and support GPR116 as a potential prognostic marker and drug target against metastatic human breast cancer. Cancer Res; 73(20); 6206–18. ©2013 AACR.

The key signaling networks regulating mammary stem cells are poorly defined. The leucine-rich repeat containing G protein-coupled receptor (Lgr) family has been implicated in intestinal, gastric, and epidermal stem cell functions. We investigated whether Lgr4 functions in mammary gland development and mammary stem cells. We found that Lgr4−/− mice had delayed ductal development, fewer terminal end buds, and decreased side-branching. Crucially, the mammary stem cell repopulation capacity was severely impaired. Mammospheres from Lgr4−/− mice showed decreased Wnt signaling. Wnt3a treatment prevented the adverse effects of Lgr4 loss on organoid formation. Chromatin immunoprecipitation analysis indicated that Sox2 expression was controlled by the Lgr4/Wnt/β-catenin/Lef1 pathway. Importantly, Sox2 overexpression restored the in vivo mammary regeneration potential of Lgr4−/− mammary stem cells. Therefore, Lgr4 activates Sox2 to regulate mammary development and stem cell functions via Wnt/β-catenin/Lef1.