Angiotensin II type-2 receptor signaling facilitates liver injury repair and regeneration via inactivation of Hippo pathway.

The angiotensin II type 2 receptor (AT2R) is a well-established component of the renin-angiotensin system and is known to counteract classical activation of this system and protect against organ damage. Pharmacological activation of the AT2R has significant therapeutic benefits, including vasodilation, natriuresis, anti-inflammatory activity, and improved insulin sensitivity. However, the precise biological functions of the AT2R in maintaining homeostasis in liver tissue remain largely unexplored. In this study, we found that the AT2R facilitates liver repair and regeneration following acute injury by deactivating Hippo signaling and that interleukin-6 transcriptionally upregulates expression of the AT2R in hepatocytes through STAT3 acting as a transcription activator binding to promoter regions of the AT2R. Subsequently, elevated AT2R levels activate downstream signaling via heterotrimeric G protein Gα12/13-coupled signals to induce Yap activity, thereby contributing to repair and regeneration processes in the liver. Conversely, a deficiency in the AT2R attenuates regeneration of the liver while increasing susceptibility to acetaminophen-induced liver injury. Administration of an AT2R agonist significantly enhances the repair and regeneration capacity of injured liver tissue. Our findings suggest that the AT2R acts as an upstream regulator in the Hippo pathway and is a potential target in the treatment of liver damage.

ß-arrestin2 deficiency ameliorates S-100-induced autoimmune hepatitis in mice by inhibiting infiltration of monocyte-derived macrophage and attenuating hepatocyte apoptosis.

Autoimmune hepatitis (AIH) is a progressive hepatitis syndrome characterized by high transaminase levels, interface hepatitis, hypergammaglobulinemia, and the presence of autoantibodies. Misdiagnosis or delayed treatment of AIH can lead to cirrhosis or liver failure, which poses a major risk to human health. ß-Arrestin2, a key scaffold protein for intracellular signaling pathways, has been found to be involved in many autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. However, whether ß-arrestin2 plays a role in AIH remains unknown. In the present study, S-100-induced AIH was established in both wild-type mice and ß-arrestin2 knockout (Arrb2 KO) mice, and the experiments identified that liver ß-arrestin2 expression was gradually increased, and positively correlated to serum ANA, ALT and AST levels during AIH progression. Furthermore, ß-arrestin2 deficiency ameliorated hepatic pathological damage, decreased serum autoantibody and inflammatory cytokine levels. ß-arrestin2 deficiency also inhibited hepatocyte apoptosis and prevented the infiltration of monocyte-derived macrophages into the damaged liver. In vitro experiments revealed that ß-arrestin2 knockdown suppressed the migration and differentiation of THP-1 cells, whereas ß-arrestin2 overexpression promoted the migration of THP-1 cells, which was regulated by the activation of the ERK and p38 MAPK pathways. In addition, ß-arrestin2 deficiency attenuated TNF-α-induced primary hepatocyte apoptosis by activating the Akt/GSK-3ß pathway. These results suggest that ß-arrestin2 deficiency ameliorates AIH by inhibiting the migration and differentiation of monocytes, decreasing the infiltration of monocyte-derived macrophages into the liver, thereby reducing inflammatory cytokines-induced hepatocytes apoptosis. Therefore, ß-arrestin2 may act as an effective therapeutic target for AIH.

Effect of PLC-ß1/CaM signaling pathway mediated by AT1R on the occurrence and development of hepatocellular carcinoma.

OBJECTIVE: To study the roles of AT1R, PLC-ß1, CaM and other related signal molecules in the formation and development of hepatocellular carcinoma (HCC) and their correlation. METHODS: ELISA and immunohistochemistry were used to analyze the expressions of target proteins in serum and liver tissue of HCC patients, and the correlation between AT1R, PLC-ß1 and CaM and postoperative survival status of patients was followed up and determined. CCK-8 method was used to screen the doses of Ang II and candesartan sensitive to HepG2 and HCCLM3 cells. Transwell experiment was used to observe the effects of different drugs on the migration and invasion activity of HCC cells. Meanwhile, flow cytometry and Western blot were used to detect the expression levels of AT1R, PLC-ß1 and CaM in the cells. Then PLC-ß1 siRNA was selected to transfect HCC cells, so as to further clarify the mechanism of the above signal proteins. HepG2 cells were inoculated under the hepatic capsule of mice to induce the formation of HCC in situ. Ang II and candesartan were used to stimulate HCC mice to observe the difference in liver appearance and measure the liver index. Finally, ELISA and immunofluorescence experiments were selected to analyze the levels of target proteins in mouse serum and liver tissue. RESULTS: The expression levels of target proteins in serum and liver tissue of HCC patients were significantly increased, and the postoperative survival time of patients with high expression of AT1R, PLC-ß1 or CaM was obviously shortened. Ang II and candesartan could significantly promote and inhibit the motility of HCC cells, and had different effects on the levels of AT1R, PLC-ß1 and CaM in cells. However, in hepatocellular carcinoma cells transfected with PLC-ß1 siRNA, the intervention ability of drugs was obviously weakened. Ang II could significantly promote the formation and progression of mouse HCC, while candesartan had the opposite effect. Meanwhile, medications could affect the expressions of target proteins in mouse serum and liver tissue. CONCLUSION: AT1R, PLC-ß1 and CaM may be risk factors affecting the formation and prognosis of HCC, and the PLC-ß1/CaM signaling pathway mediated by AT1R is an important way to regulate the migration and invasion activity of HCC cells.

ß-Arrestin2 deficiency attenuates oxidative stress in mouse hepatic fibrosis through modulation of NOX4.

Hepatic fibrosis is a disease characterized by excessive deposition of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is responsible for most of ECM production. Oxidative stress and reactive oxygen species (ROS) may be important factors leading to liver fibrosis. NADPH oxidase 4 (NOX4) is the main source of ROS in hepatic fibrosis, but the mechanism by which NOX4 regulates oxidative stress is not fully understood. ß-Arrestin2 is a multifunctional scaffold protein that regulates receptor endocytosis, signaling and trafficking. In this study, we investigated whether ß-arrestin2 regulated oxidative stress in hepatic fibrosis. Both ß-arrestin2 knockout (Arrb2 KO) mice and wild-type mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce hepatic fibrosis. Arrb2 KO mice showed significantly attenuated liver fibrosis, decreased ROS levels and NOX4 expression, and reduced collagen levels in their livers. In vitro, NOX4 knockdown significantly inhibited ROS production, and decreased expression of alpha-smooth muscle actin in angiotensin II-stimulated human HSC cell line LX-2. Through overexpression or depletion of ß-arrestin2 in LX-2 cells, we revealed that decreased ß-arrestin2 inhibited ROS levels and NOX4 expression, and reduced collagen production; it also inhibited activation of ERK and JNK signaling pathways. These results demonstrate that ß-arrestin2 deficiency protects against liver fibrosis by downregulating ROS production through NOX4. This effect appears to be mediated by ERK and JNK signaling pathways. Thus, targeted inhibition of ß-arrestin2 might reduce oxidative stress and inhibit the progression of liver fibrosis.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

Depletion of ß-arrestin 2 protects against CCl4-induced liver injury in mice.

Acute liver injury can be caused by oxidative stress within a short period and is a common pathway to many liver diseases. The liver is vulnerable to reactive oxygen species (ROS) and free radical-mediated disorders. ß-arrestin2 was initially discovered to be a negative regulator of G protein-coupled receptor signaling. Recently, ß-arrestin2 has been found to act as a multifunctional adaptor protein and play new roles in regulating intracellular signaling networks. However, the role of ß-arrestin2 in the pathogenesis of acute liver injury is unclear. In this study, we hypothesize that ß-arrestin2 regulates acute liver injury via modulation of oxidative stress. ß-arrestin2 knockout mice were used to investigate the impacts of ß-arrestin2 on carbon tetrachloride (CCl4)-induced acute liver injury and oxidative stress. Results here suggested that ß-arrestin2 deficiency decreased serum activities of aminotransferase and alleviated liver injury induced by CCl4 injection as compared with wildtype mice. ß-arrestin2 knockout mice exhibited stronger tolerance in oxidative stress compared with wild-type mice, which was demonstrated by decreased ROS level and increased superoxide dismutase (SOD) and glutathione (GSH) in the liver. Furthermore, ß-arrestin2 deficiency significantly inhibited NOX4 (a major source of ROS) expression and the activation of the extracellular regulated kinase (ERK) and, c-Jun NH2-terminal kinase (JNK) pathways. These results suggest that ß-arrestin2 deficiency protects against CCl4-induced acute liver injury through attenuating oxidative damage and decreased ERK and JNK phosphorylation.

The role of G protein-coupled receptor kinases in the pathology of malignant tumors.

G protein-coupled receptor kinases (GRKs) constitute seven subtypes of serine/threonine protein kinases that specifically recognize and phosphorylate agonist-activated G protein-coupled receptors (GPCRs), thereby terminating the GPCRs-mediated signal transduction pathway. Recent research shows that GRKs also interact with non-GPCRs and participate in signal transduction in non-phosphorylated manner. Besides, GRKs activity can be regulated by multiple factors. Changes in GRKs expression have featured prominently in various tumor pathologies, and they are associated with angiogenesis, proliferation, migration, and invasion of malignant tumors. As a result, GRKs have been intensively studied as potential therapeutic targets. Herein, we review evolving understanding of the function of GRKs, the regulation of GRKs activity and the role of GRKs in human malignant tumor pathophysiology.

Emerging Roles of G Protein-Coupled Receptors in Hepatocellular Carcinoma.

Among a great variety of cell surface receptors, the largest superfamily is G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors. GPCRs can modulate diverse signal-transduction pathways through G protein-dependent or independent pathways which involve β-arrestins, G protein receptor kinases (GRKs), ion channels, or Src kinases under physiological and pathological conditions. Recent studies have revealed the crucial role of GPCRs in the tumorigenesis and the development of cancer metastasis. We will sum up the functions of GPCRs—particularly those coupled to chemokines, prostaglandin, lysophosphatidic acid, endothelin, catecholamine, and angiotensin—in the proliferation, invasion, metastasis, and angiogenesis of hepatoma cells and the development of hepatocellular carcinoma (HCC) in this review. We also highlight the potential avenues of GPCR-based therapeutics for HCC.

The emerging roles of ß-arrestins in fibrotic diseases.

ß-Arrestins and ß-arrestin2 are important adaptor proteins and signal transduction proteins that are mainly involved in the desensitization and internalization of G-protein-coupled receptors. Fibrosis is characterized by accumulation of excess extracellular matrix (ECM) molecules caused by chronic tissue injury. If highly progressive, the fibrotic process leads to organ malfunction and, eventually, death. The incurable lung fibrosis, renal fibrosis and liver fibrosis are among the most common fibrotic diseases. Recent studies show that ß-arrestins can activate signaling cascades independent of G-protein activation and scaffold many intracellular signaling networks by diverse types of signaling pathways, including the Hedgehog, Wnt, Notch and transforming growth factor-ß pathways, as well as downstream kinases such as MAPK and PI3K. These signaling pathways are involved in the pathological process of fibrosis and fibrotic diseases. This ß-arrestin-mediated regulation not only affects cell growth and apoptosis, but also the deposition of ECM, activation of inflammatory response and development of fibrotic diseases. In this review, we survey the involvement of ß-arrestins in various signaling pathways and highlight different aspects of their regulation of fibrosis. We also discuss the important roles of ß-arrestins in the process of fibrotic diseases by regulating the inflammation and deposit of ECM. It is becoming more evident that targeting ß-arrestins may offer therapeutic potential for the treatment of fibrotic diseases.

TGF-ß signaling pathway as a pharmacological target in liver diseases.

Transforming growth factor ß (TGF-ß) belongs to a class of pleiotropic cytokines that are involved in the processes of embryonic development, wound healing, cell proliferation, and differentiation. Moreover, TGF-ß is also regarded as a central regulator in the pathogenesis and development of various liver diseases because it contributes to almost all of the stages of disease progression. A range of liver cells are considered to secrete TGF-ß ligands and express related receptors and, consequently, play a crucial role in the progression of liver disease via different signal pathways. In this manuscript, we review the role of the TGF-ß signaling pathway in liver disease and the potential of targeting the TGF-ß signaling in the pharmacological treatment of liver diseases.

Insight into the regulatory mechanism of ß-arrestin2 and its emerging role in diseases.

ß-arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein-coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that ß-arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post-transcriptional modifications can affect the expression of ß-arrestin2. Furthermore, post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation affect the cellular localization of ß-arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of ß-arrestin2. This review summarizes the structure and function of ß-arrestin2 and reveals the mechanisms involved in the regulation of ß-arrestin2 at multiple levels. Additionally, recent studies on the role of ß-arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting ß-arrestin2.

Depletion of ß-arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway.

ß-Arrestins are multifunctional adaptor proteins. Recently, some new roles of ß-arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of ß-arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of ß-arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of ß-arrestin1, ß-arrestin2, and α-smooth muscle actin (α-SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of ß-arrestin2 in the hepatic tissues, but not ß-arrestin1. Further, as hepatic fibrosis worsens, ß-arrestin2-expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of ß-arrestin2 had a significant positive correlation with the expression of α-SMA, an activated HSCs marker. In vitro studies, the dynamic expression of ß-arrestin1 and ß-arrestin2 in platelet derived growth factor-BB (PDGF-BB) stimulated HSCs was assessed by Western blotting. The expression of ß-arrestin2 was remarkably increased in PDGF-BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of ß-arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting ß-arrestin2 mRNA (siß-arrestin2) into HSCs led to a 68% and 70% reduction of ß-arrestin2 mRNA and protein expression, respectively. siß-arrestin2 abolished the effect of PDGF-BB on the proliferation of HSCs. In addition, siß-arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the ß-arrestin2 in the pathogenesis of hepatic fibrosis. The ß-arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF-BB. Selective targeting of ß-arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis.

Etanercept attenuates collagen-induced arthritis by modulating the association between BAFFR expression and the production of splenic memory B cells.

Anti-tumour necrosis factor-α (TNF-α) drugs are approved for the treatment of rheumatoid arthritis (RA). Many studies have investigated the effect of these drugs on the T cell response; however, some clues have indicated that it may also target B cells. This study was carried out to explore the potential effects and mechanisms of etanercept, a soluble TNF-α receptor, on the function of B cells and their development into memory B cells in type II collagen (CII)-induced arthritis (CIA). Beginning on day 24 after CII immunisation, the mice were evaluated every 2-3 days to determine two clinical parameters: their arthritis global assessment and swollen joint count (SJC). The serum concentrations of IgG1, IgG2a and anti-CII antibodies and the splenic pathology and proliferation of B cells were measured. The percentage of total memory B cells in the spleen was analysed with flow cytometry. BAFFR was detected by immunohistochemistry. In CIA mice, etanercept markedly suppressed the arthritis global assessment and the SJC, reduced the production of anti-CII, IgG1 and IgG2a antibodies, and prevented spleen histopathology to varying degrees; however, it had no obvious effect on splenic B cell proliferation. Etanercept also decreased the percentage of total CD27(+) memory B cells in the spleen. Treatment with etanercept was associated with a further increase in BAFFR expression, a significant reduction in CD27 expression, and a negative correlation between the levels of BAFFR and the percentage of memory B cells. Our findings showed that increased BAFFR expression has a regulatory effect on the activation of B cells and the generation of memory B cells, which may be one of the mechanisms of the therapeutic effects of etanercept.

Dextran sulfate sodium-induced gut microbiota dysbiosis aggravates liver injury in mice with S100-induced autoimmune hepatitis.

Recently, the incidence of autoimmune hepatitis (AIH) has gradually increased, and the disease can eventually develop into cirrhosis or even hepatoma if left untreated. AIH patients are often characterized by gut microbiota dysbiosis, but whether gut microbiota dysbiosis contributes to the progression of AIH remains unclear. In this study, we investigate the role of gut microbiota dysbiosis in the occurrence and development of AIH in mice with dextran sulfate sodium salt (DSS) induced colitis. C57BL/6J mice were randomly divided into normal group, S100-induced AIH group, and DSS+S100 group (1 % DSS in the drinking water), and the experimental cycle lasted for four weeks. We demonstrate that DSS administration aggravates hepatic inflammation and disruption of the intestinal barrier, and significantly changes the composition of gut microbiota in S100-induced AIH mice, which are mainly characterized by increased abundance of pathogenic bacteria and decreased abundance of beneficial bacteria. These results suggest that DSS administration aggravates liver injury of S100-induced AIH, which may be due to DSS induced gut microbiota dysbiosis, leading to disruption of the intestinal barrier, and then, the microbiota translocate to the liver, aggravating hepatic inflammation.

Paeoniflorin inhibits proliferation of fibroblast-like synoviocytes through suppressing G-protein-coupled receptor kinase 2.

Paeoniflorin (Pae) is a monoterpene glucoside and the main component of the total glucosides of paeony (TGP) extracted from the roots of Paeonia lactiflora. Its anti-inflammatory effect is associated with regulating G-protein-coupled receptors (GPCRs) signaling. The aim of this study was to explore the expression change of G-protein-coupled receptor kinase 2 (GRK2) in fibroblast-like synoviocytes (FLS) and the effect of Pae. Pae was obtained and purified from the roots of Paeonia lactiflora. We investigated the expression of GRK2 in synovium during the inflammatory process and assessed the effects of a specific GRK2 inhibitor and Pae on proliferation, cAMP level, and protein kinase A (PKA) activity of FLS in vitro. Additionally, the effect of Pae on GRK2 expression in FLS was detected in vitro. Expression of GRK2 in synovium from CIA rats increased during the inflammatory process. The specific GRK2 inhibitor suppressed proliferation and increased the cAMP level as well as PKA activity of FLS, and Pae had the same effects. Furthermore, Pae decreased GRK2 expression in FLS in vitro. Our results indicate that a chronic inflammatory process in CIA induces upregulation of GRK2 expression in FLS, and Pae can reverse this change, which might be one of the important mechanisms for Pae regulating GPCRs signaling and suppressing the proliferation of FLS in CIA.

CP-25 exerts a protective effect against ConA-induced hepatitis via regulating inflammation and immune response.

Hepatitis is a complex multifactorial pathological disorder, which can eventually lead to liver failure and even potentially be life threatening. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has proven to have critical anti-inflammatory effects in arthritis. However, the effects of CP-25 in the pathogenesis of hepatitis remains unclear. In this experiment, mice were intragastrically administered with CP-25 (25, 50 and 100 mg/kg), and then ConA (25 mg/kg) was intravenous injected to establish hepatitis model in vivo. CP-25 administration attenuated liver damage and decreased ALT and AST activities in mice with hepatitis. Besides, CP-25 modulated immune responses including down-regulated the proportions of activated CD4+, activated CD8+ T cells, and ratio of Th1/Th2 in ConA-injected mice. Furthermore, ConA-mediated production of reactive oxygen species (ROS), release of inflammatory cytokines including IFN-γ, TNF-α, activation of MAPK pathways and nuclear translocation of nuclear factor-kappaB (NF-κB) were significantly decreased in CP-25 administrated mice. In ConA-stimulated RAW264.7 cells, CP-25 suppressed inflammatory cytokines secretion and reduced ROS level, which were consistent with animal experiments. Otherwise, the data showed that CP-25 restrained phosphorylation of ERK, JNK and p38 MAPK pathways influenced by ROS, accompanied with inhibiting NF-κB nuclear translocation. In conclusion, our findings indicated that CP-25 protected against ConA-induced hepatitis may through modulating immune responses and attenuating ROS-mediated inflammation via the MAPK/NF-κB signaling pathway.

Inflammasome as an Effective Platform for Fibrosis Therapy.

Fibrosis is the final stage of the development of chronic inflammation. It is characterized by excessive deposition of the extracellular matrix, leading to tissue structure damage and organ dysfunction, which is a serious threat to human health and life. However, the molecular mechanism of fibrosis is still unclear. Inflammasome is a molecular complex of proteins that has been becoming a key innate sensor for host immunity and is involved in pyroptosis, pathogen infection, metabolic syndrome, cellular stress, and tumor metastasis. Inflammasome signaling and downstream cytokine responses mediated by the inflammasome have been found to play an important role in fibrosis. The inflammasome regulates the secretion of IL-1ß and IL-18, which are both critical for the process of fibrosis. Recently, researches on the function of inflammasome have attracted extensive attention, and data derived from these researches have increased our understanding of the effects and regulation of inflammasome during fibrosis. In this review, we emphasize the growing evidence for both indirect and direct effects of inflammasomes in triggering fibrosis as well as potential novel targets for antifibrotic therapies.

ß-arrestin2 regulating ß2-adrenergic receptor signaling in hepatic stellate cells contributes to hepatocellular carcinoma progression.

Background: ß-arrestin2 and ß2-adrenergic receptor (ß2-AR) have important roles in malignant tumors, the present study aims to investigate the role of activated ß2-AR in hepatic stellate cells (HSCs) during hepatocellular carcinoma (HCC) progression and the regulatory effect of ß-arrestin2. Methods: Immunofluorescence and Western blot were used to detect the expression of ß-arrestin2 and ß2-AR in HSCs of liver tissues from human HCC samples and diethylnitrosamine (DEN)-induced HCC model mice. We next used ß-arrestin2-/- mice to demonstrate the regulatory role of ß-arrestin2 in DEN mice. The subsets of T cells were quantified by flow cytometry. MTT and wound healing assay were applied to detect the proliferation and migration of cells. Co-immunoprecipitation assay was used to detect the link of ß-arrestin2 and ß2-AR in HSCs. Effect of ß-arrestin2 overexpression on ß2-AR downstream signaling pathway was verified by Western blot. The secretion of CCL2 was detected by ELISA. Results: The expression of ß2-AR was significantly increased, while ß-arrestin2 was decreased in HSCs of HCC tissues. And ß-arrestin2 deficiency exacerbates DEN-induced HCC accompanied with increased ß2-AR expression. The results of flow cytometry showed that the percentage of activated T cells decreased gradually after DEN injection. ß-arrestin2 knockout down-regulated the ratio of activated T cells. In vitro, selective activation of ß2-AR in HSCs promoted the proliferation and migration of HCC cells. ß-arrestin2 overexpression enhanced co-immunoprecipitation of ß-arrestin2 and ß2-AR in activated HSCs, and decreased its downstream Akt phosphorylation. Akt inhibitor decreased secretion of CCL2 in activated HSCs. Conclusion: Our study demonstrated that ß2-AR activation in HSCs induces the proliferation and migration of HCC cells may be through Akt signaling, and this effect appears to be regulated by ß-arrestin2.

G Protein-Coupled Receptor Kinase 2 as Novel Therapeutic Target in Fibrotic Diseases.

G protein-coupled receptor kinase 2 (GRK2), an important subtype of GRKs, specifically phosphorylates agonist-activated G protein-coupled receptors (GPCRs). Besides, current research confirms that it participates in multiple regulation of diverse cells via a non-phosphorylated pathway, including interacting with various non-receptor substrates and binding partners. Fibrosis is a common pathophysiological phenomenon in the repair process of many tissues due to various pathogenic factors such as inflammation, injury, drugs, etc. The characteristics of fibrosis are the activation of fibroblasts leading to myofibroblast proliferation and differentiation, subsequent aggerate excessive deposition of extracellular matrix (ECM). Then, a positive feedback loop is occurred between tissue stiffness caused by ECM and fibroblasts, ultimately resulting in distortion of organ architecture and function. At present, GRK2, which has been described as a multifunctional protein, regulates copious signaling pathways under pathophysiological conditions correlated with fibrotic diseases. Along with GRK2-mediated regulation, there are diverse effects on the growth and apoptosis of different cells, inflammatory response and deposition of ECM, which are essential in organ fibrosis progression. This review is to highlight the relationship between GRK2 and fibrotic diseases based on recent research. It is becoming more convincing that GRK2 could be considered as a potential therapeutic target in many fibrotic diseases.

GRK2 Suppresses Hepatocellular Carcinoma Metastasis and Invasion Through Down-Regulation of Prostaglandin E Receptor 2.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive form of human liver cancer and the fifth most common malignancy worldwide. Novel effective treatment strategies for HCC are urgently in clinical because of its poor response to conventional therapies. G protein-coupled receptor kinases (GRKs), including GRK2 and GRK3, are known that involves in various essential cellular processes and regulates numerous signaling pathways. However, the role of GRK2/3 in invasion and metastasis of HCC still remains unclear. MATERIALS AND METHODS: Immunohistochemistry, Western blot, laser confocal microscopy and qRT-PCR were used to detect the expression of GRK2/3 and EP2 in liver tissues of HCC patients and DEN-induced HCC mice. Wound healing and transwell assay were applied to measure the migration and invasion of HCC cells after transfected with GRK2 siRNA. The downstream pathway of Akt and ERK was verified by Western blot. RESULTS: The expression of GRK2 was significantly decreased, while GRK3 was not significantly changed in HCC tissues compared with noncancerous tissues of HCC patients. Moreover, GRK2 expression was reduced during liver tumorigenesis in diethylnitrosamine-induced liver tumor model. In addition, our in vitro study showed that GRK2 expression was gradually decreased with increasing HCC cell line metastatic potential, and GRK2 knockdown significantly promoted the migration and invasion of HCC cells. Furthermore, low GRK2 expression was associated with increased expression of EP2 receptor translocation to HCC cell membrane, and the activation of Akt pathway. CONCLUSION: These data suggest that GRK2 inhibits HCC metastasis and invasion may be through regulating EP2 receptor translocation, and this effect appears to be mediated by Akt pathway.