Citri Reticulatae Pericarpium Limits TLR-4-Triggered Inflammatory Response in Raw264.7 Macrophages by Activating RasGRP3.

Inflammation is an important immune response to pathogen invasion, but excessive inflammation leads to tissue injury and even cytokine storm. Therefore, proper response is needed depending on the intensity of the infection. Ras guanine nucleotide releasing protein 3 (RasGRP3) is a regulator of the TLR-mediated response. In low-intensity inflammation, it negatively regulates production of pro-inflammatory cytokines, especially IL-6. Citri Reticulatae Pericarpium, the peel of Citrus reticulata Blanco, is a major medicinal herb in Korean medicine. The present study aims to investigate whether the Citri Reticulatae Pericarpium extract (CRE) has immunomodulatory activity using the Raw264.7 macrophage. Also, we investigated the effect of CRE on RasGRP3 expression. In the present study, CRE reduced IL-6 production in the low-LPS environment (1 ng/mL) and did not in the high-LPS environment (100 ng/mL). The suppression of IL-6 production in the low-LPS environment (1 ng/mL) was abolished after the pretreatment of RasGRP3 siRNA. The reduced RasGRP3 protein content by 100 ng/mL LPS treatment was increased by CRE treatment. Additionally, nobiletin, a major component of CRE showed a suppressive effect on IL-6 production in the low-LPS environment (1 ng/mL). The present results suggest that CRE alleviates inflammatory response via activating RasGRP3 expression in low-intensity inflammation.

Downregulation of AC092894.1 promotes oxaliplatin resistance in colorectal cancer via the USP3/AR/RASGRP3 axis.

BACKGROUND: Oxaliplatin resistance is a complex process and has been one of the most disadvantageous factors and indeed a confrontation in the procedure of colorectal cancer. Recently, long non-coding RNAs (lncRNAs) have emerged as novel molecules for the treatment of chemoresistance, but the specific molecular mechanisms mediated by them are poorly understood. METHODS: The lncRNAs associated with oxaliplatin resistance were screened by microarray. lncRNA effects on oxaliplatin chemoresistance were then verified by gain- and loss-of-function experiments. Finally, the potential mechanism of AC092894.1 was explored by RNA pull-down, RIP, and Co-IP experiments. RESULTS: AC092894.1 representation has been demonstrated to be drastically downregulated throughout oxaliplatin-induced drug-resistant CRC cells. In vivo and in vitro experiments revealed that AC092894.1 functions to reverse chemoresistance. Studies on the mechanism suggested that AC092894.1 served as a scaffold molecule that mediated the de-ubiquitination of AR through USP3, thereby increasing the transcription of RASGRP3. Finally, sustained activation of the MAPK signaling pathway induced apoptosis in CRC cells. CONCLUSIONS: In conclusion, this study identified AC092894.1 as a suppressor of CRC chemoresistance and revealed the idea that targeting the AC092894.1/USP3/AR/RASGRP3 signaling axis is a novel option for the treatment of oxaliplatin resistance.

Mutant NPM1 maintains RASGRP3 protein stability via interaction with MID1 to promote acute myeloid leukemia cell proliferation and autophagy.

Acute myeloid leukemia is a heterogeneous hematologic malignancy with high mortality in the world. NPM1 gene mutations are a frequent occurrence in acute myeloid leukemia, leading to abnormal autophagy, while the mechanism of NPM1 mutation-driven acute myeloid leukemia pathogenesis remains to be fully elucidated. GEO microarrays were used to screen for dysregulated autophagy-related genes in NPM1-mutant acute myeloid leukemia and analysis of RASGRP3 expression and prognosis. Next, we explored the potential molecular mechanisms relationship between RASGRP3 and NPM1 through utilizing immunoprecipitation, Western blot, and cycloheximide assay. Further, CCK8, EdU staining, immunofluorescence, and Western blot were performed to explore the effect of RASGRP3 on cell proliferation and apoptosis in NPM1-mutated acute myeloid leukemia. Finally, Western blot was used to study the mechanism of action of RASGRP3. RASGRP3 expression was upregulated in NPM1-mutated acute myeloid leukemia. Mislocalized NPM1-mA in the cytoplasm could bind to E3 ubiquitin-protein ligase MID1 to block degradation of the RASGRP3 protein. RASGRP3 could also activate the EGFR-STAT3 axis to promote proliferation and autophagy in acute myeloid leukemia. In conclusion, our results identified RASGRP3 as a proto-oncogene in NPM1-mutated acute myeloid leukemia. The RASGRP3-EGFR/STAT3 axis may be a promising therapeutic target for this unique leukemic subtype.

Identification of Differentially Expressed Genes and Prediction of Expression Regulation Networks in Dysfunctional Endothelium.

The detection of early coronary atherosclerosis (ECA) is still a challenge and the mechanism of endothelial dysfunction remains unclear. In the present study, we aimed to identify differentially expressed genes (DEGs) and the regulatory network of miRNAs as well as TFs in dysfunctional endothelium to elucidate the possible pathogenesis of ECA and find new potential markers. The GSE132651 data set of the GEO database was used for the bioinformatic analysis. Principal component analysis (PCA), the identification of DEGs, correlation analysis between significant DEGs, the prediction of regulatory networks of miRNA and transcription factors (TFs), the validation of the selected significant DEGs, and the receiver operating characteristic (ROC) curve analysis as well as area under the curve (AUC) values were performed. We identified ten genes with significantly upregulated signatures and thirteen genes with significantly downregulated signals. Following this, we found twenty-two miRNAs regulating two or more DEGs based on the miRNA-target gene regulatory network. TFs with targets ≥ 10 were E2F1, RBPJ, SSX3, MMS19, POU3F3, HOXB5, and KLF4. Finally, three significant DEGs (TOX, RasGRP3, TSPAN13) were selected to perform validation experiments. Our study identified TOX, RasGRP3, and TSPAN13 in dysfunctional endothelium and provided potential biomarkers as well as new insights into the possible molecular mechanisms of ECA.

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells.

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.

RasGRP3 in peripheral blood mononuclear cells is associated with disease activity and implicated in the development of systemic lupus erythematosus.

This study examined the relationship between the expression of Ras guanyl nucleotide-releasing protein 3 (RasGRP3) and disease activity in systemic lupus erythematosus (SLE) and explored the possible mechanisms in MRL/lpr mice. We detected the expression of RasGRP3 in peripheral blood mononuclear cells (PBMCs) of SLE patients (n=26) and healthy controls (n=20) by employing RT-PCR and studied the association between the mRNA expression of RasGRP3 in PBMCs and the clinical findings. We also measured the protein level of RasGRP3 in PBMCs by Western blotting (n=10). In addition, we isolated the B cells from PBMCs with magnetic bead separation and determined the RasGRP3 expression by RT-PCR (n=10). Furthermore, we extracted spleen B cells from MRL/lpr mice and knocked down RasGRP3 by siRNA transfection to study the role of RasGRP3 in the pathway of B cell receptor (BCR) activation and the production of pro-inflammatory cytokines. Compared with healthy volunteers, the expression of RasGRP3 was significantly elevated in PBMCs and purified B cells from SLE patients. The mRNA expression of RasGRP3 in PBMCs was positively correlated with SLE disease activity index (SLEDAI). Moreover, silencing RasGRP3 could inhibit Akt and Erk1/2 activation in marginal zone (MZ) and follicular (FO) B cells of MRL/lpr mice. Additionally, the production of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), was decreased whereas activation of caspase-3 cleavage was induced in vitro. In conclusion, over-expression of RasGRP3 is associated with disease activity and might be involved in the pathogenesis of SLE.

A somatic mutation of RasGRP3 decreases Na+/I- symporter expression in metastases of radioactive iodine-refractory thyroid cancer by stimulating the Akt signaling pathway.

Mutation profiles of advanced radioactive iodine (RAI)-refractory differentiated thyroid cancer have revealed the pathogenic roles of the established oncogenic mutations of BRAF and PI3KCA, but the involvement of other genes is presently unknown. In the present study, we performed whole-exome sequencing on 10 tissue samples of metastases of RAI-refractory differentiated thyroid cancers and identified a recurrent hot-spot mutation (c.1924G>T) in the RasGRP3 gene, which codes for Ras guanine nucleotide-releasing protein 3. This mutation was found to occur at a high frequency (20%) in samples of metastases of RAI-refractory differentiated thyroid cancers compared with other types of thyroid cancer. Overexpression of mutant RasGRP3 significantly promoted cell proliferation, migration, and invasiveness of 8505C and BHT101 cells compared with cells transfected with wild-type RasGRP3 or an empty vector. In addition, mutant RasGRP3 decreased the expression of sodium iodide symporter (NIS) and thyroid-stimulating hormone receptor (TSHR), reduced the iodine uptake ability, and increased Akt phosphorylation in thyroid cancer cells. Finally, we showed that LY294002, an inhibitor of PI3K/Akt signaling, attenuated the effects of mutant RasGRP3 on thyroid cancer cells. Thus, our study revealed that the c.1924G>T hot-spot mutation in RasGRP3 is a more frequent genetic alteration in metastases of RAI-refractory differentiated thyroid cancer. This mutant RasGRP3 activated the Akt pathway, promoted thyroid cancer cell proliferation and invasion, and reduced NIS expression and the iodine uptake ability.

GNA11 Q209L Mouse Model Reveals RasGRP3 as an Essential Signaling Node in Uveal Melanoma.

Uveal melanoma (UM) is characterized by mutually exclusive activating mutations in GNAQ, GNA11, CYSLTR2, and PLCB4, four genes in a linear pathway to activation of PLCß in almost all tumors and loss of BAP1 in the aggressive subset. We generated mice with melanocyte-specific expression of GNA11Q209L with and without homozygous Bap1 loss. The GNA11Q209L mice recapitulated human Gq-associated melanomas, and they developed pigmented neoplastic lesions from melanocytes of the skin and non-cutaneous organs, including the eye and leptomeninges, as well as at atypical sites, including the lymph nodes and lungs. The addition of Bap1 loss increased tumor proliferation and cutaneous melanoma size. Integrative transcriptome analysis of human and murine melanomas identified RasGRP3 to be specifically expressed in GNAQ/GNA11-driven melanomas. In human UM cell lines and murine models, RasGRP3 is specifically required for GNAQ/GNA11-driven Ras activation and tumorigenesis. This implicates RasGRP3 as a critical node and a potential target in UM.

RasGRP3, a Ras guanyl releasing protein 3 that contributes to malignant proliferation and aggressiveness in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide; however, clinical and pathological parameters have limited ability in discriminating between clinically significant and indolent ESCC. Since RasGRP3 transcript levels have prognostic value in discriminating ESCC with different clinical aggressiveness, we decided to investigate its putative oncogenic role in ESCC. We found that RasGRP3 was highly expressed in ESCC cells. Suppression of endogenous RasGRP3 expression in esophageal cell lines reduced Ras-GTP formation as well as AKT phosphorylation. RasGRP3 suppression also inhibited cell invasion and migration and reduced proliferation, demonstrating the importance of RasGRP3 for the transformed phenotype of melanoma cells. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth and migration, confirming the functional role of RasGRP3 in the altered behaviour of these cells. This suggests that RasGRP3 may function as a Ras activator in the phosphoinositide signalling pathway and may potentially serve as a new therapeutic target.

RasGRP3 controls cell proliferation and migration in papillary thyroid cancer by regulating the Akt-MDM2 pathway.

Accumulating evidence has shown that Ras guanylnucleotide releasing peptide 3 (RasGRP3) is up-regulated in several distinct cancer types; however, its role in papillary thyroid cancer (PTC) remains unclear. In this study, we demonstrate that RasGRP3 was overexpressed in PTC tissues and cell lines. Downregulation of RasGRP3 using small interfering (si) RNA significantly inhibited PTC cell proliferation and migration in vitro, and tumor growth in vivo, reflecting an oncogenic role of RasGRP3 in PTC. We subsequently identified that the expression of mouse double minute 2 homolog (MDM2) and phosphorylated Akt (p-Akt) was significantly decreased in RasGRP3-downregulated PTC cells. Overexpression of MDM2 attenuated the function of si-RasGRP3. Taken together, our data show that RasGRP3 exerts its oncogenic effect in PTC through Akt-mediated MDM2 activation. RasGRP3 may serve as a potential new therapeutic target for PTC.

Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3.

RasGRP comprises a family of guanine nucleotide exchange factors, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP1 possesses REM (Ras exchange), GEF (catalytic), EF-hand, C1, SuPT (suppressor of PT), and PT (plasma membrane-targeting) domains, among which the C1 domain drives membrane localization in response to diacylglycerol or phorbol ester and the PT domain recognizes phosphoinositides. The homologous family member RasGRP3 shows less plasma membrane localization. The objective of this study was to explore the role of the different domains of RasGRP3 in membrane translocation in response to phorbol esters. The full-length RasGRP3 shows limited translocation to the plasma membrane in response to PMA, even when the basic hydrophobic cluster in the PT domain, reported to be critical for RasGRP1 translocation to endogenous activators, is mutated to resemble that of RasGRP1. Moreover, exchange of the C-termini (SuPT-PT domain) of the two proteins had little effect on their plasma membrane translocation. On the other hand, while the C1 domain of RasGRP3 alone showed partial plasma membrane translocation, truncated RasGRP3 constructs, which contain the PT domain and are missing the REM, showed stronger translocation, indicating that the REM of RasGRP3 was a suppressor of its membrane interaction. The REM of RasGRP1 failed to show comparable suppression of RasGRP3 translocation. The marked differences between RasGRP3 and RasGRP1 in membrane interaction necessarily will contribute to their different behavior in cells and are relevant to the design of selective ligands as potential therapeutic agents.

RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.

Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11.

Gene polymorphisms and oral cancer risk in tobacco habitués.

Oral cancer incidence of 77,003 poses a major health concern in India, with 5-10 % tobacco habitués developing oral cancer. The current study examined the role of specific genomic variants in oral cancer. We examined five genomic variants represented as single nucleotide polymorphisms (SNPs) in genes associated with cell proliferation and cellular invasion. The SNPs rs2124437 (RASGRP3), rs1335022 (GRIK2), rs4512367 (PREX2), rs4748011 (CCDC3), and rs1435218 (LNX1) were analyzed in 500 histopathologically confirmed oral cancers and 500 healthy controls with a minimum of 10 years of tobacco usage. Allelic discrimination real-time PCR SYBR Green assay was used. The genotypic and allelic frequencies between cases and controls were analyzed using SPSS software (version 19) and odds ratio (OR) using Hutchon.net, indicating increased risk to oral cancers. A significant association of the SNPs in oral cancer was observed in RASGRP3 AA (rs2124437) (p < 0.000, OR 1.34, 95 % confidence interval (CI) 1.01-1.76), GRIK2 TT (rs1335022) (p = 0.008, OR 1.58, 95 % CI 1.23-2.03), PREX2 CC (p = 0.008, OR 1.56, 95 % CI 1.15-2.1), and TT (p < 0.000, OR 2.77, 1.68-4.57) genotypes, whereas the heterozygous genotypes showed higher frequencies in controls, i.e., GRIK2 CT (rs1335022) (p = 0.029, OR 0.68, 95 % CI 0.53-0.87) and PREX2 CT (p = 0.004, OR 0.49, 95 % CI 0.37-0.64), indicating protection. Coinheritance of the SNPs was associated with further increase in the risk. Thus, the SNP genotypes in the three genes, present singly or as a coinherited panel constituted "Predictive Biomarkers" indicating increased risk of oral cancer in tobacco habitués.

Pleiotropic effects of methionine adenosyltransferases deregulation as determinants of liver cancer progression and prognosis.

Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients' survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC.