Involvement of Embryo-Derived and Monocyte-Derived Intestinal Macrophages in the Pathogenesis of Inflammatory Bowel Disease and Their Prospects as Therapeutic Targets.

Inflammatory bowel disease (IBD) is a group of intestinal inflammatory diseases characterized by chronic, recurrent, remitting, or progressive inflammation, which causes the disturbance of the homeostasis between immune cells, such as macrophages, epithelial cells, and microorganisms. Intestinal macrophages (IMs) are the largest population of macrophages in the body, and the abnormal function of IMs is an important cause of IBD. Most IMs come from the replenishment of blood monocytes, while a small part come from embryos and can self-renew. Stimulated by the intestinal inflammatory microenvironment, monocyte-derived IMs can interact with intestinal epithelial cells, intestinal fibroblasts, and intestinal flora, resulting in the increased differentiation of proinflammatory phenotypes and the decreased differentiation of anti-inflammatory phenotypes, releasing a large number of proinflammatory factors and aggravating intestinal inflammation. Based on this mechanism, inhibiting the secretion of IMs' proinflammatory factors and enhancing the differentiation of anti-inflammatory phenotypes can help alleviate intestinal inflammation and promote tissue repair. At present, the clinical medication of IBD mainly includes 5-aminosalicylic acids (5-ASAs), glucocorticoid, immunosuppressants, and TNF-α inhibitors. The general principle of treatment is to control acute attacks, alleviate the condition, reduce recurrence, and prevent complications. Most classical IBD therapies affecting IMs function in a variety of ways, such as inhibiting the inflammatory signaling pathways and inducing IM2-type macrophage differentiation. This review explores the current understanding of the involvement of IMs in the pathogenesis of IBD and their prospects as therapeutic targets.

IGFBP2 drives epithelial-mesenchymal transition in hepatocellular carcinoma via activating the Wnt/ß-catenin pathway.

Metastasis has emerged as a major impediment to achieve successful therapeutic outcomes in hepatocellular carcinoma (HCC). Nonetheless, the intricate molecular mechanisms governing the progression of HCC remain elusive. Herein, we present evidence highlighting the influence exerted by insulin-like growth factor-binding protein 2 (IGFBP2) as a potent oncogene driving the malignant phenotype. Our investigation reveals a marked elevation of IGFBP2 expression in primary tumors, concomitant with the presence of mesenchymal biomarkers in HCC. Through in vitro and in vivo experimentation, we demonstrate that the overexpression of IGFBP2 expedites the progression of epithelial-mesenchymal transition (EMT) and facilitates the metastatic potential of HCC cells, chiefly mediated by the Wnt/ß-catenin signaling pathway. Notably, knockdown of IGFBP2 significantly decreased the expression of total and nuclear ß-catenin, N-cadherin and vimentin in the treatment of the specific activator of Wnt/ß-catenin CHIR-99021. Collectively, our findings identify IGFBP2 as a pivotal regulator within the HCC EMT axis, whereby its overexpression confers the distinctly aggressive clinical features characteristic of the disease.

Hsa_circ_0007401 regulates gemcitabine resistance of pancreatic cancer through the hsa-miR-6509-3p/fli1 axis.

Pancreatic cancer is a highly malignant cancer with a poor prognosis. Owing to the strong drug resistance of pancreatic cancer, adjuvant chemotherapy has failed to achieve good results in clinical practice. The expression profile data of circular RNA (circRNA) (GSE110580), microRNA (miRNA) (GSE79234), and messenger RNA (mRNA) (GSE140077, GES35141) were obtained from the gene expression omnibus database. The Cancer-Specific circRNA Database identified the structural pattern of circRNA, and the starBase and circBank databases together predicted the miRNA of circRNA. The mirDIP database predicts the target mRNAs of miRNAs and identifies the ceRNA network of circRNA-miRNA-mRNA via negative regulatory mechanisms. The final validation was performed using clinical data from the cancer treatment response gene signature database of patients treated with gemcitabine for pancreatic cancer of the cancer genome atlas. By differential expression analysis, 22 differential circRNAs (8 upregulated and 14 downregulated), 70 differential microRNAs (37 upregulated and 33 downregulated), and 256 differential messenger RNA (DEmRNA) (161 upregulated and 95 downregulated) were obtained. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses showed that DEmRNAs were associated with drug response, exogenous cellular stimulation, and the tumor necrosis factor signaling pathway. The screened downregulated differential circular RNA (hsa_circ_0007401), upregulated differential microRNA (hsa-miR-6509-3p), and downregulated DEmRNA (FLI1) were consistent with the negative regulation mechanism of the ceRNA network, and FLI1 was significantly downregulated in the data of gemcitabine-resistant pancreatic cancer patients in the cancer genome atlas (n = 26).

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation.

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6'-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-ß-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

Paeoniflorin-6'O-benzene sulfonate suppresses fibroblast-like synoviocytes proliferation and migration in rheumatoid arthritis through regulating GRK2-Gßγ interaction.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Enhanced G protein coupled receptor kinase 2 (GRK2) translocation and prostaglandin E4 receptor (EP4) desensitization play a critical role in fibroblast-like synoviocytes (FLS) dysfunction. Paeoniflorin-6'O-benzene sulfonate (CP-25) exerts a protective effect in arthritis in the RA animal models. To demonstrate the role of Gßγ in EP4 desensitization and the mechanisms of CP-25 that protects FLS in RA, RA-FLS and adjuvant-induced arthritis (AA-FLS) were isolated from synovium of RA patients and AA rats. RA-FLS, AA-FLS and MH7A were treated with CP-25, Gßγ agonist and antagonist. The cell membrane expression of EP4, GRK2, and Gßγ were detected using western blot analysis. Co-immunoprecipitation (Co-IP) and immunofluorescence were adopted to detect the interactions of GRK2-Gßγ, GRK2-EP4, and EP4-Gßγ. Cell Counting Kit-8 and Transwell assay were used to analyze the proliferation and migration of the FLS. An increased membrane expression of GRK2 and Gßγ, enhanced GRK2-Gßγ interaction and decreased EP4 membrane expression in the RA synovial tissue were identified. In vitro, prostaglandin E2 (PGE2) enhanced the proliferation and migration of FLS. CP-25 exhibited an inhibition effect similar to Gßγ inhibitor, which downregulated GRK2-EP4 interaction, blocked the translocation of GRK2, and reversed EP4 desensitization, leading to the suppression of the proliferation and migration induced by PGE2. These results elucidated that an enhanced GRK2-Gßγ interaction was involved in the EP4 desensitization and dysfunction. CP-25 regulated EP4-GRK2-Gßγ signaling and re-sensitized EP4 by inhibiting GRK2-Gßγ interaction. The regulation of EP4-Gßγ-GRK2 signaling may be a novel potential therapeutic target in RA.

Dominant negative TGF-ß receptor type II in T lymphocytes promotes anti-tumor immunity by modulating T cell subsets and enhancing CTL responses.

Transforming growth factor-ß (TGF-ß) is a multifunctional regulatory cytokine that maintains tolerance in the immune system by regulating the proliferation, differentiation and survival of lymphocytes. TGF-ß blockade therapy for cancer has achieved some results but shows limited efficacy and side effects because these drugs are not selective and act on various types of cells throughout the body. We demonstrate here that dominant negative TGF-ß receptor type II specifically targeting T cells decreases tumor load in tumor-bearing mice. In addition, the dominant negative TGF-ß receptor type II promotes the proliferation and differentiation of T cells and increases the expression of T-bet, which in turn promotes the secretion of granzyme A, granzyme B, perforin and IFN-γ secreted by T cells, and enhances the cytotoxicity and anti-tumor effects of T cells. Moreover, we also found that dominant negative TGF-ß receptor type II reduces the proportion of regulatory T cells (Tregs) in tumor tissue and spleen of tumor-bearing mice. Co-culture experiments with T cells and tumor cells revealed that dominant negative TGF-ß receptor type II inhibited tumor cell proliferation and increased apoptosis. Our results indicate that specifically inhibiting TGF-ß receptor type II in T cells increases anti-tumor immunity and has a strong therapeutic potential.

Insulin-Like Growth Factor Binding Protein-2 Promotes Proliferation and Predicts Poor Prognosis in Hepatocellular Carcinoma.

BACKGROUND: Insulin-like growth factor binding protein-2 (IGFBP2) levels are significantly increased in the plasma of hepatocellular carcinoma (HCC) patients. However, the correlation between IGFBP2 levels and clinical parameters and the exact role of IGFBP2 in HCC are unclear. In this study, we identified the role and potential molecular mechanisms of IGFBP2 in HCC. MATERIALS AND METHODS: ELISA assays were used to detect plasma IGFBP2 levels in HCC patients and healthy controls, and the correlations with patients' clinicopathological data were analyzed. The CCK8 assay was used to explore cell proliferation. Luciferase reporter, co-immunoprecipitation, and immunofluorescence assays were used to demonstrate the molecular mechanism of IGFBP2 in HCC. RESULTS: Plasma IGFBP2 levels were determined blindly in 37 HCC patients and 37 matched healthy controls. The mean plasma IGFBP2 concentrations in HCC patients were higher than in healthy controls, and IGFBP2 levels in HCC were positively correlated with the degree of differentiation, tumor size, metastasis, and portal venous invasion. Exogenous IGFBP2 activated integrin ß1 and thus induced the combination and colocalization of activated integrin ß1 and p-FAK, which promoted the phosphorylation of FAK, Erk, and Elk1, eventually inducing EGR1-mediated proliferation of the HCC cell lines HepG2 and HCCLM3. Meanwhile, neutralization of integrin ß1 inhibited IGFBP2-induced FAK, Erk, Elk1, and EGR1 activation. CONCLUSION: Taken together, these results indicated that exogenous IGFBP2 promoted the integrin ß1/FAK/Erk/Elk1/EGR1 pathway, which stimulated the proliferation of HCC cells. Plasma IGFBP2 could be a novel prognostic biomarker for HCC patients.

CP-25 inhibits PGE2-induced angiogenesis by down-regulating EP4/AC/cAMP/PKA-mediated GRK2 translocation.

G protein-coupled receptor kinase 2 (GRK2), a type of cytosolic enzyme, transiently translocates to the plasma membrane upon G protein-coupled receptors (GPCRs) activation, and it also binds to extracellular signal-regulated kinase (ERK) to inhibit the activation of ERK. GRK2 deficiency in endothelial cells (ECs) leads to increased pro-inflammatory signaling and promotes recruitment of leukocytes to activated ECs. However, the role of GRK2 in regulating angiogenesis remains unclear. Here, we show that GRK2 is a novel regulatory molecule on migration and tube formation of ECs, vessel sprouting ex vivo and angiogenesis in vivo. We identify that EP4/AC/cAMP/protein kinase A (PKA)-mediated GRK2 translocation to cells membrane decreases the binding of GRK2 and ERK1/2 to inhibit ERK1/2 activation, which promotes prostaglandin E2 (PGE2)-induced angiogenesis. GRK2 small interfering RNA (siRNA) inhibits the increase in PGE2-induced HUVECs migration and tube formation. In vivo, PGE2 increases ECs sprouting from normal murine aortic segments and angiogenesis in mice, but not from GRK2-deficient ones, on Matrigel. Further research found that Lys220 and Ser685 of GRK2 play an important role in angiogenesis by regulating GRK2 translocation. Paeoniflorin-6'-O-benzene sulfonate (CP-25), as a novel ester derivative of paeoniflorin (pae), has therapeutic potential for the treatment of adjuvant arthritis (AA) and collagen-induced arthritis (CIA), but the underlying mechanism of CP-25 on angiogenesis has not been elucidated. In our study, CP-25 inhibits the migration and tube formation of HUVECs, and angiogenesis in mice by down-regulating GRK2 translocation activation without affecting GRK2 total expression. Taken together, the present results revealed that CP-25 down-regulates EP4/AC/cAMP/PKA-mediated GRK2 translocation, restoring the inhibition of GRK2 for ERK1/2, thereby inhibiting PGE2-stimulated angiogenesis.

Development of Inflammatory Immune Response-Related Drugs Based on G Protein-Coupled Receptor Kinase 2.

G protein-coupled receptor kinase 2 (GRK2), as a vital Ser/Thr kinase, is an important regulatory protein in the inflammatory immune response (IIR) by maintaining the balance between the function of inflammatory immune cells and non-conventional inflammatory immune cells and regulating inflammatory immune cell infiltration, inflammatory cytokine secretion, and the signaling associated with endothelial function. However, the imbalance of GRK2 expression and activity plays an important role in the development of IIR-related diseases, such as hypertension, heart failure, Alzheimer's disease, type 2 diabetes mellitus, insulin resistance, rheumatoid arthritis, thyroid cancer, multiple sclerosis, and liver cancer. Small molecule GRK2 inhibitors, including balanol, Takeda inhibitors, paroxetine and derivatives, M119 and gallein, peptides, RNA aptamers, Raf kinase inhibitory protein, and microRNAs, that can directly inhibit GRK2 kinase activity have been identified by different strategies. This review discusses recent progress in one of the hallmark molecular abnormalities of GRK2 in IIR-related diseases and explores the soft regulation of IIR by innovative drugs reducing the excessive activity of GRK2 to basal levels, without damaging normal physiological function, to ameliorate inflammatory disorders.

Is macrophage polarization important in rheumatoid arthritis?

Macrophages are myeloid immune cells which are strategically positioned throughout the body, where they engulf and degrade debris, dead cells, and foreign substances, and coordinating the inflammatory processes. Macrophages can be divided into two extreme subsets, classical activation (M1), and alternatively activation (M2). The symptoms and signs of rheumatoid arthritis (RA) would exacerbate with the increase in pro-inflammatory cytokines, whereas anti-inflammatory cytokines will alleviate the symptoms and signs of RA. This review, mainly discusses the effects of Notch, JNK and ERK signaling pathways on the regulation of macrophage polarization, and the effects of pro-inflammatory factors and/or anti-inflammatory cytokines produced by polarized macrophages in RA. Also, we will make an attempt to find out the importance of macrophage polarization in RA treatment as a drug target.

Insulin-like growth factor-binding protein-3 inhibits IGF-1-induced proliferation of human hepatocellular carcinoma cells by controlling bFGF and PDGF autocrine/paracrine loops.

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) produced by hepatocellular carcinoma (HCC) cells are responsible for the growth of HCC cells. Accumulating evidence shows that insulin-like growth factor-binding protein-3 (IGFBP-3) suppresses HCC cell proliferation in both IGF-dependent and independent manners. It's unknown, however, whether treatment with exogenous IGFBP-3 inhibits bFGF and PDGF production in HCC cells. The present study demonstrates that IGFBP-3 suppressed IGF-1-induced bFGF and PDGF expression while it does not affect their expression in the absence of IGF-1. To delineate the underlying mechanism, western-blot and RT-PCR assays confirmed that the transcription factor early growth response protein 1 (EGR1) is involved in IGFBP-3 regulation of bFGF and PDGF. IGFBP-3 inhibition of type 1 insulin-like growth factor receptor (IGF1R), ERK and AKT activation is IGF-1-dependent. Furthermore, transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1, bFGF and PDGF expression. In conclusion, these findings suggest that IGFBP-3 suppresses transcription of EGR1 and its target genes bFGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation. It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation, suggesting that IGFBP-3 could be a target for the treatment of HCC.

GRK2 overexpression inhibits IGF1-induced proliferation and migration of human hepatocellular carcinoma cells by downregulating EGR1.

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that is involved in a variety of important signaling pathways and alternation of GRK2 protein level or activity causes diseases such as heart failure, rheumatoid arthritis, and obesity. However, the role and mechanism of GRK2 in hepatocellular carcinoma (HCC) progression is not fully investigated. In this study we found that GRK2 plays an inhibitory role in IGF1-induced HCC cell proliferation and migration. Overexpression of GRK2 causes a decrease in early growth response-1 (EGR1) expression, while knockdown of GRK2 leads to marked increase in EGR1 expression in the treatment of IGF1. Through co-immunoprecipitation and western blot assay, we confirmed that GRK2 can interact with insulin-like growth factor 1 receptor (IGF-1R) and inhibits IGF1-induced activation of IGF1R signaling pathway. Silencing EGR1 attenuates GRK2 overexpression-caused inhibition of cell proliferation, tumor colony number and migration activity, while overexpressing of EGR1 restores the anti-proliferative and migratory effect by GRK2 overexpression in HCCLM3 cells. Collectively, these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through downregulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.