G Protein-Coupled Receptor Kinase 2 Regulating ?2-Adrenergic Receptor Signaling In M2-Polarized Macrophages Contributes To Hepatocellular Carcinoma Progression

Hepatocellular carcinoma?HCC?accounts for about 90%of primary liver cancer and the second leading cause of cancer-associated mortality.Because the cause of HCC is not completely clear and lack of effective approaches to treatments,patients with HCC still have a poor prognosis with frequent recurrence and metastasis.Therefore,novel therapeutic targets are required to improve the survival of patients with HCC.Tumor microenvironment refers to local biological environment in which tumors develop,mainly including immune cells,neovascular cells,tumor-associated fibroblasts and extracellular matrix.Macrophages are mononuclear phagocytic leukocytes and are divided into two subtypes,M1-polarized macrophages and M2-polarized macrophages,according to their functions.Macrophages which infiltrated in the tumor microenvironment are called tumor associated macrophages?TAMs?.They resemble M2 subtype,producing large amounts of tumor-associated cytokines and chemokines,and differing from M1 cells.It has become increasingly evident that the presence of high numbers of intratumor macrophages is associated with the progression of various tumors.A large number of autonomic nerves are distributed in the liver,includingsympathetic and parasympathetic nerves.Norepinephrine and adrenaline which were released by the activated by sympathetic nervous system can active the?-adrenergic receptor??-AR?of tumor cells and host stromal cells to promote the growth and metastasis of various solid tumors.?2-AR is a subtype of ARs,belonging to the G protein-coupled receptor?GPCR?family with a common structural signature of seven membrane-spanning helices.?2-AR is overexpressed in HCC and activation of?2-AR leads to the HCC growth progression as demonstrated in various HCC cell lines,such as SMMC-7721,MHCC97L,HepG2 and MHCC97H.Furthermore,ARs are distributed on the surface of macrophages.Activation of the?2-AR modified the entire macrophage phenotype,resulting in M2-polarized macrophages in mice.However,the effect of?2-AR activation in M2-polarized macrophages on HCC cells remains unclear.G protein-coupled receptor kinase 2?GRK2?belongs to GRK family.GRK2 is expressed throughout the body and is primarily identified as the kinase that phosphorylates and desensitizes agonist-bound GPCR.Our previous study revealed that the inhibition of GRK2 significantly promotes HCC cell growth.Nevertheless,whether GRK2 regulates?2-AR in M2-polarized macrophages and contributes to the progression of HCC requires further investigation.In the present study,immunofluorescence was performed to study the expression of M2-polarized macrophages in HCC,co-expression of?2-AR and CD163,GRK2 and CD163 in HCC and the relationship between the co-expression and clinicopathological characteristics.Western blot was also performed to study the expression of M2-polarized macrophages in HCC.Next,the M2-polarized macrophages with?2-AR agitated were co-cultured with HepG2 and SMMC-7721 in vitro.Relevant assays were performed to detect the function of liver cancer cells.Furthermore,GRK2 siRNA was used to knock down the expression of GRK2 in M2-polarized macropahges and the function of liver cancer cells were detected once again.Last,the involved underlying mechanisms were investigated.OBJECTIVEThe paraffin sections of patients and tissues of patients with HCC were collected to detect the expression of M2-polarized macrophages in HCC,the co-expression of?2-AR and M2-polarized macrophages,GRK2 and M2-polarized macrophages.The relationship between the co-expression and clinicopathological characteristics was also detected.Furthermore,the present study was designed to investigate the role of activated?2-AR in M2-polarized macrophages in the proliferation,migration and invasion of HCC and the regulatory effect of GRK2,as well as the underlying mechanisms involved.METHODSParaffin-embeded sections of 80 patients with HCC and 10 patients with hepatolithiasis?normal control group?,fresh tissues of 20 patients with HCC and 6cases of adjacent normal tissues were included.The clinicopathological data from aforementioned patients were collected.The clinical stages of HCC were determined according to the Tumor Node Metastasis?TNM?classification system of the American Joint Committee on Cancer in 2010.There were 34 patients of?+?TNM stage and 46 patients of?+?TNM stage.Immunofluorescence double staining was performed to mark CD163 and CD206,?2-AR and CD163,GRK2 and CD163.Image-Pro Plus was used to calculate the co-expression of?2-AR and CD163,GRK2 and CD163.The co-expression among normal group,?+?stage group and?+?stage group and the relationship between the co-expression and clinicopathological characteristics were analyzed by SPSS 16.0.In vitro,human THP-1 cell line was used to induce M2-polarized macrophage and immunofluorescence double staining was performed to identify whether the induction was successful.The supernatants of?2-AR activated M2-polarized macrophages were collected to incubate with HepG2 and SMMC-7721 liver cancer cell lines.MTT,wound-healing and transwell assays were performed to examine the proliferative,migratory and invasive abilities of liver cancer cells.After transfected with GRK2siRNA,the supernatants of M2-polarized macrophages were incubated with liver cancer cells and the proliferative,migratory and invasive abilities of liver cancer cells were examined by MTT,wound-healing and transwell assays once again.Next,the supernatants of?2-AR activated M2-polarized macrophages with or without transfection with GRK2 siRNA were collected.Cytokine assays were performed to detect the cytokines and chemokines in the supernatant of M2-poarized macrophages.Western blot assay and flow cytometry were performed to detect the?2-AR on the membrane of M2-polarized macrophages with or without transfection with GRK2siRNA,followed by stimulation with Terb for several time points.Next,an ELISA kit was used to determine the cAMP level in M2-polarized macrophages with or without transfection with GRK2 siRNA,followed by stimulation with Terb for 10 min.And last,western blot analysis was performed to determine the PKA,CREB,p-CREB,STAT3 and p-STAT3 levels of M2-polarized macrophages with or without transfection with GRK2 siRNA,followed by stimulation with Terb for 10 min.RESULTS1.Expression of M2-polarized macrophages in HCCImmunofluorescence double staining showed that the co-expression of CD163 and CD206 was increased with the progression of HCC.Similarly,Western blot showed that the expression of CD163 and CD206 were increased with the progression of HCC.2.Co-expression of?2-AR and CD163,GRK2 and CD163 in HCC of early and late stage and the relationship between the co-expression and clinicopathological characteristicsImmunofluorescence double staining showed that co-expression of?2-AR and CD163 was elevated in patients with late stage of HCC while the co-expression of GRK2 and CD163 was decreased in patients with late stage of HCC.No significant differences were observed in the co-expression of GRK2 and CD163 or?2-AR and CD163,between male and female,young and old,complete and incomplete tumor capsule groups,with or without HBV or HCV infection groups.Furthermore,the co-expression of?2-AR and CD163 was increased in patients with late T stage and late TNM stage compared with early T stage and early TNM stage,respectively,while the co-expression of GRK2 and CD163 was decreased in patients with the aforementioned characteristics and patient with lymph node metastasis.3.Induction and identification of M2-polarized macrophagesAfter 48 h of induction,the round THP-1 cells became adherent and differentiated into a heterogeneous cell population comprising round and spindle-shaped cells.Immunofluorescence double staining confirmed that the induction was successful in more than 90%cells following treatments with PMA,IL-4 and IL-13.4.M2-polarized macrophages exhibit a higher phagocytic capacity compared with THP-1 cells and treatment with Terb reduces phagocytosisThe capacity of taking up FITC-dextran was significantly higher in M2-polarized macrophages compared with that in THP-1 cells.There was a significant reduction in phagocytosis following treatment with 10^-55 mol/L Terb compared with 10^-6 mol/L Terb.Nevertheless,when the Terb concentration increased to 10^-4 mol/L,no significant reduction was observed compared with the 10^-55 mol/L Terb group.5.Activation of?2-AR in M2-polarized macrophages promotes the proliferation, migration and invasion of HCC cells,and the regulatory effect of GRK2The proliferative,migratory and invasive abilities of HepG2 and SMMC-7721 liver cancer cell lines were elevated in the Terb stimulation group compared with non-treated group.After thansfection with GRK2 siRNA in M2-polarized macrophages,the liver cancer cells incubated with conditioned media?CM?from M2-polarized macrophages in the GRK2 siRNA+Terb-CM group had a higher absorbance at 490 nm,an increasing wound closure and an elevated number of migrated HCC cells compared with the Terb-CM group.6.Activation of?2-ARs increase the IL-6,vascular endothelial growth factor?VEGF?and matrix metalloproteinase 9?MMP-9?levels secreted by M2-polarized macrophages and the regulatory effect of GRK2The levels of IL-6,VEGF,MMP-9,granulocyte-macrophage colony stimulating factor?GM-CSF?,IL-10 and monocyte chemotactic protein-1?MCP-1?were significantly elevated in the supernatant of Terb-stimulated M2-polarized macrophages compared with the control group.While transfection with GRK2 siRNA,the levels of IL-6,VEGF and MMP-9 secreted by M2-polarized macrophages were significantly elevated in the GRK2 siRNA+Terb-CM group compared with the Terb-alone group.7.Down-regulation of GRK2 delays the internalization of?2-AR in M2-polarized macrophagesThe results of Western blot assay and flow cytometry analysis indicated that the expression of?2-AR on membrane of M2-polarized macrophages reached a peak following Terb stimulation for 10 min in both Terb and GRK2 siRNA+Terb groups.However,in the GRK2 siRNA+Terb group,there was an increase in the expression of?2-ARs on macrophage membranes compared with the Terb-alone group.Furthermore,in GRK2 siRNA+Terb group,the expression of?2-ARs gradually decreased following Terb stimulation for 10 min to 20 min at a slower rate compared with the Terb-alone group.The results demonstrated that downregulation of GRK2increased the expression of?2-ARs on the macrophages membrane surface,and delayed the desensitization and internalizationof?2-ARs,resulting in the prolongation of its effective duration.8.Down-regulation of GRK2 in M2-polarized macrophages promotes the activation of?2-AR downstream signalingTo further analyze the?2-AR downstream signaling,ELISA kit and Western blot assay were performed.Following the stimulation of Terb for 5,10 or 15 min,the concentration of cAMP peaked after 10 min of GRK2 siRNA+Terb group,which was more than 1-fold increase compared with the peak of the Terb group,then gradually declining from 5 min to 15 min at a slower speed than Terb group.Furthermore,the expressions of PKA,p-CREB,p-STAT3 were higher in the GRK2 siRNA+Terb group compared with the Terb-alone group.These results indicated that compared with?2-AR-activatedM2-polarizedmacrophages,theactivationofthe?2-AR/cAMP/PKA/CREB and the?2-AR/cAMP/IL-6/STAT3 signaling pathways were significantly elevated following the knockdown of GRK2.CONCLUSIONS1.The co-expression of CD163 and CD206 were elevated in HCC scections with?+?TNM stage group compared with control group and in?+?TNM stage group the co-expression of CD163 and CD206 were also elevated compared with?+?TNM stage group.The expression of CD163 and CD206 were elevated in HCC patients tissues with?+?TNM stage group compared with control group.In?+?TNM stage group the expression of CD163 and CD206 were also elevated compared with?+?TNM stage group,suggesting that the number of M2-polarized macrophages were increased in late HCC.2.The co-expression of?2-AR and CD163 was increased,while the co-expression of GRK2 and CD163 was reduced in late stage HCC samples compared with the early stage group and control group.The co-expression of GRK2 and CD163 was decreased in patients with late T stage,lymph node metastasis and late TNM stage compared with early T stage,patients without lymph node metastasis and early TNM stage,respectively,while the co-expression of?2-AR and CD163 was elevated between the aforementioned characteristics except for lymph node metastasis.The results suggested that?2-AR and GRK2 were related to the progression of HCC.3.After the activation of?2-AR in M2-polarized macrophages,the proliferation,migration and invasion of co-cultured liver cancer cells were elevated.Inhibition of GRK2 using siRNA in?2-AR-activated M2-polarized macrophages elevated the proliferation,migration and invasion of co-cultured liver cancer cells significantly compared with non-siRNA-treated macrophages,indicating that GRK2 could regulate the?2-AR of M2-polarized macrophages contributing to the growth and metastasis of HCC.4.Down-regulation of GRK2 could regulate the?2-AR in M2-polarized macrophages through its engagement in the activation of downstream signaling,resulting in the progression of HCC.