Membrane-associated RING-CH-1 Research Articles

Curcumin Inhibits the Growth of Hepatocellular Carcinoma via the MARCH1-mediated Modulation of JAK2/STAT3 Signaling.

Curcumin has been reported to have anti-hepatocellular carcinoma (HCC) effects, but the underlying mechanism is not well known. To investigate whether membrane-associated RING-CH 1 (MARCH1) is involved in the curcumin-induced growth suppression in HCC and its underlying molecular mechanism. A few recent patents for curcumin for cancer are also reviewed in this article. The effect of curcumin on growth inhibition of HCC cells was analyzed through in vitro and in vivo experiments, and the expression levels of MARCH1, Bcl-2, VEGF, cyclin B1, cyclin D1, and JAK2/STAT3 signaling molecules were measured in HCC cells and the xenograft tumors in nude mice. Cell transfection with MARCH1 siRNAs or expression plasmid was used to explore the role of MARCH1 in the curcumin-induced growth inhibition of HCC cells. Curcumin inhibited cell proliferation, promoted apoptosis, and arrested the cell cycle at the G2/M phase in HCC cells with the decrease of Bcl-2, VEGF, cyclin B1, and cyclin D1 expression as well as JAK2 and STAT3 phosphorylation, resulting in the growth suppression of HCC cells. MARCH1 is highly expressed in HCC cells, and its expression was downregulated after curcumin treatment in a dose-dependent manner. The knockdown of MARCH1 by siRNA decreased the phosphorylation levels of JAK2 and STAT3 and inhibited the growth of HCC cells. In contrast, opposite results were observed when HCC cells overexpressed MARCH1. A xenograft tumor model in nude mice also showed that curcumin downregulated MARCH1 expression and decelerated the growth of transplanted HCC with the downregulation of JAK2/STAT3 signaling and functional molecules. The ADC value of MRI analysis showed that curcumin slowed down the progression of HCC. Our results demonstrated that curcumin may inhibit the activation of JAK2/STAT3 signaling pathway by downregulating MARCH1 expression, resulting in the growth suppression of HCC. MARCH1 may be a novel target of curcumin in HCC treatment.

MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation.

Persistent antigenic signaling leads to T cell exhaustion, a dysfunctional state arising in many chronic infections and cancers. Little is known concerning mechanisms limiting exhaustion in immune-stimulatory diseases such as asthma. We report that membrane-associated RING-CH1 (MARCH1), the ubiquitin ligase that mediates surface turnover of MHC class II (MHCII) and CD86 in professional APCs, plays an essential role in restraining an exhaustion-like program of effector CD4+ T cells in a mouse model of asthma. Mice lacking MARCH1 or the ubiquitin acceptor sites of MHCII and CD86 exhibited increased MHCII and CD86 surface expression on lung APCs, and this increase promoted enhanced expression of immune-inhibitory receptors by effector CD4+ T cells and inhibited their proliferation. Remarkably, ablation of MARCH1 in mice with established asthma reduced airway infiltration of eosinophils and Th2 cells. Thus, MARCH1 controls an exhaustion-like program of effector CD4+ T cells during allergic airway inflammation and may serve as a therapeutic target for asthma.

5‐FU inhibits migration and invasion of CRC cells through PI3K/AKT pathway regulated by MARCH1

Colorectal cancer is a major health problem with a significant impact on the patients' quality of life. 5‐Fluorouracil is the most common chemotherapy drug used for this type of cancer. While its molecular mechanism is the inhibition of DNA synthesis via the inhibition of thymine nucleotide synthetase, its complete anticancer mechanism is not clear. Membrane‐associated RING‐CH‐1 (MARCH1) is an E3 ubiquitin ligase that plays an important role in antigen presentation. However, MARCH1 has not been studied in the context of colorectal cancer. In this study, we demonstrated that MARCH1 is highly expressed in colorectal cancer tissues and cell lines. Furthermore, migration and invasion of colorectal tumor cells were inhibited via transfection with small interfering RNAs to suppress the expression of MARCH1. The western blot analysis showed that MARCH1 regulates epithelial–mesenchymal transition and the PI3K/AKT pathway. Moreover, 5‐fluorouracil inhibited the proliferation, migration, and invasion of tumor cells, via the targeting of MARCH1 and the consequent downregulation of the PI3K/AKT pathway, impacting the progression of epithelial–mesenchymal transition. In conclusion, our study shows that MARCH1 may play a role as an oncogene in colorectal cancer and may represent a new target molecule of 5‐fluorouracil.

MARCH1 encourages tumour progression of hepatocellular carcinoma via regulation of PI3K-AKT-β-catenin pathways.

Membrane‐associated RING‐CH‐1 (MARCH1) is a membrane‐anchored E3 ubiquitin ligase that is involved in a variety of cellular processes. MARCH1 was aberrantly expressed as a tumour promoter in ovarian cancer, but the signalling about the molecular mechanism has not yet been fully illuminated. Here, we first determined that MARCH1 was obviously highly expressed in human hepatocellular carcinoma samples and cells. In addition, our findings demonstrated that the proliferation, migration and invasion of hepatocellular carcinoma were suppressed, but the apoptosis was increased, as a result of MARCH1 knockdown by either siRNA targeting MARCH1 or pirarubicin treatment. Conversely, the proliferation, migration and invasion of hepatocellular carcinoma were obviously accelerated, and the apoptosis was decreased, by transfecting the MARCH1 plasmid to make MARCH1 overexpressed. Moreover, in vivo, the results exhibited a significant inhibition of the growth of hepatocellular carcinoma in nude mice, which were given an intra‐tumour injection of siRNA targeting MARCH1. Furthermore, our study concluded that MARCH1 functions as a tumour promoter, and its role was up‐regulated the PI3K‐AKT‐β‐catenin pathways both in vitro and in vivo. In summary, our work determined that MARCH1 has an important role in the development and progression of hepatocellular carcinoma and may be used as a novel potential molecular therapeutic target in the future treatment of hepatocellular carcinoma.

The E3 ubiquitin ligase MARCH1 regulates glucose-tolerance and lipid storage in a sex-specific manner.

Type 2 diabetes is typified by insulin-resistance in adipose tissue, skeletal muscle, and liver, leading to chronic hyperglycemia. Additionally, obesity and type 2 diabetes are characterized by chronic low-grade inflammation. Membrane-associated RING-CH-1 (MARCH1) is an E3 ubiquitin ligase best known for suppression of antigen presentation by dendritic and B cells. MARCH1 was recently found to negatively regulate the cell surface levels of the insulin receptor via ubiquitination. This, in turn, impaired insulin sensitivity in mouse models. Here, we report that MARCH1-deficient (knockout; KO) female mice exhibit excessive weight gain and excessive visceral adiposity when reared on standard chow diet, without increased inflammatory cell infiltration of adipose tissue. By contrast, male MARCH1 KO mice had similar weight gain and visceral adiposity to wildtype (WT) male mice. MARCH1 KO mice of both sexes were more glucose tolerant than WT mice. The levels of insulin receptor were generally higher in insulin-responsive tissues (especially the liver) from female MARCH1 KO mice compared to males, with the potential to account in part for the differences between male and female MARCH1 KO mice. We also explored a potential role for MARCH1 in human type 2 diabetes risk through genetic association testing in publicly-available datasets, and found evidence suggestive of association. Collectively, our data indicate an additional link between immune function and diabetes, specifically implicating MARCH1 as a regulator of lipid metabolism and glucose tolerance, whose function is modified by sex-specific factors.

Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress antitumor immune responses, promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of antitumor immunity. Specifically, MDSCs in patients with melanoma and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells markedly delayed tumor growth and endowed antitumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, whereas transcriptome analysis revealed substantial differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation, thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase that mediates the lysosomal degradation of MHC II in M-MDSCs attenuated their suppressive function, and resulted in markedly decreased tumor volume followed by development of a robust antitumor immunity. Collectively, these findings depict autophagy as a molecular target of MDSC-mediated suppression of antitumor immunity.

MARCH1-mediated ubiquitination of MHC II impacts the MHC I antigen presentation pathway.

Major histocompatibility complex class II (MHC II) expression and turn-over are regulated via its ubiquitination by the membrane associated RING-CH 1 (MARCH1) E3 ligase. Unexpectedly, we show that MHC II ubiquitination also impacts MHC I. Lack of MARCH1 in B cells and dendritic cells (DCs) resulted in a significant reduction in surface MHC I expression. This decrease was not directly caused by changes in MARCH1 ubiquitination of MHC I but indirectly by altered MHC II trafficking in the absence of its ubiquitination. Deletion of MHC II in March1-/- cells restored normal MHC I surface expression and replacement of wild type MHC II by a variant that could not be ubiquitinated caused a reduction in MHC I expression. Furthermore, these cells displayed inefficient presentation of peptide and protein antigen via MHC I to CD8+ T cells. In summary, we describe an unexpected intersection between MHC I and MHC II such that the surface expression of both molecules are indirectly and directly regulated by MARCH1 ubiquitination, respectively.

Molecular mechanism and cellular function of MHCII ubiquitination.

The major histocompatibility complex class II (MHCII) is ubiquitinated via the evolutionarily conserved lysine in the cytoplasmic tail of the β chain in dendritic cells (DCs) and B cells. The ubiquitination is mediated by the membrane-associated RING-CH1 (MARCH1) ubiquitin ligase although it can be also mediated by the homologous ligase MARCH8 in model cell lines. The ubiquitination promotes MHCII endocytosis and lysosomal sorting that results in a reduction in the level of MHCII at cell surface. Functionally, MHCII ubiquitination serves as a means by which DCs suppress MHCII expression and reduce antigen presentation in response to the immune regulatory cytokine interleukin-10 (IL-10) and regulatory T cells. Recently, additional roles of MHCII ubiquitination have emerged. MHCII ubiquitination promoted DC production of inflammatory cytokines in response to the Toll-like receptor ligands. It also potentiated DC ability to activate antigen-specific naive CD4(+) T cells while limiting the amount of antigens presented at cell surface. Similarly, MHCII ubiquitination promoted DC activation of CD4(+) thymocytes supporting regulatory T-cell development independent of its effect of limiting antigen presentation. Thus, ubiquitination appears to confer MHCII a function independent of presenting antigens by a mechanism yet to be identified.

Examining a potential link between antigen-presentation and metabolic dysregulation (HUM7P.289)

Abstract Antigen-presentation is induced following interaction of an antigen-presenting cell (APC) with stimuli, including microbial ligands and cytokines, whereupon Major Histocompatibility Complex II (MHC-II) and co-stimulatory molecule, CD86, surface expression is significantly upregulated. At the surface of the APC, the immune modulators provide signals critical for T cell activation and induction of the appropriate adaptive immune response. As key regulators of the T cell-APC conversation, it is imperative to gain a thorough understanding of the underlying mechanisms regulating expression and function of antigen-presenting molecules, and the biological consequences of dysregulation. Indeed, our lab has demonstrated that Membrane-Associated RING-CH1 (MARCH1), a highly-conserved E3 ubiquitin ligase identified as a potent regulator of both MHCII and CD86 in APCs, is stringently regulated at multiple steps, indicating a need for precise control of antigen-presentation. Interestingly, MARCH1-deficient and hemizygous mice show signs of mild hyperglycemia, an increase in the visceral adipose tissue (VAT), and increased inflammation of the VAT, all characteristic of insulin-resistance and type II diabetes progression. Collectively, the data support the hypothesis that antigen-presentation is tightly regulated to ensure a rapid adaptive immune response, and suggests a novel role for components of this pathway in the maintenance of insulin sensitivity in healthy adipose tissues.

MARCH1-mediated MHCII ubiquitination promotes dendritic cell selection of natural regulatory T cells

Membrane-associated RING-CH1 (MARCH1) is an E3 ubiquitin ligase that mediates ubiquitination of MHCII in dendritic cells (DCs). MARCH1-mediated MHCII ubiquitination in DCs is known to regulate MHCII surface expression, thereby controlling DC-mediated T cell activation in vitro. However, its role at steady state or in vivo is not clearly understood. Here, we show that MARCH1 deficiency resulted in a substantial reduction in the number of thymus-derived regulatory T cells (T reg cells) in mice. A specific ablation of MHCII ubiquitination also significantly reduced the number of thymic T reg cells. Indeed, DCs deficient in MARCH1 or MHCII ubiquitination both failed to generate antigen-specific T reg cells in vivo and in vitro, although both exhibited an increased capacity for antigen presentation in parallel with the increased surface MHCII. Thus, MARCH1-mediated MHCII ubiquitination in DCs is required for proper production of naturally occurring T reg cells, suggesting a role in balancing immunogenic and regulatory T cell development.

Cell-type specific regulation of MARCH1 E3 ubiquitin ligase by the anti-inflammatory cytokine IL-10

Membrane-associated RING-CH-1 (MARCH1) is an E3 ubiquitin ligase which targets MHC-II, CD86 and various other molecules for degradation. It is one of the most efficient post-translational regulators of antigen presentation. MARCH1 is expressed in resting immature dendritic cells and B cells but can be induced in other cell types. While activation of most immune cells results in a reduction in MARCH1 gene expression, its anti-inflammatory properties are highlighted by its induction in response to IL-10. Here, we review what is known about the regulation of MARCH1 gene expression in response to IL-10 by various immune cells. We speculate on the role of MARCH1 ininfection, its differential expression pattern and the promise that this E3 ubiquitin ligase holds to influence immune responses and mitigate immune pathologies.

Ubiquitin-mediated Regulation of CD86 Protein Expression by the Ubiquitin Ligase Membrane-associated RING-CH-1 (MARCH1)

The activation of naïve T cells requires antigen presentation by dendritic cells (DCs), and the process of antigen presentation is regulated over the course of DC maturation. One key aspect of this regulation is the cell surface up-regulation upon DC maturation of peptide·MHC-II complexes and the costimulatory molecule CD86. It is now clear that these critical induction events involve changes in ubiquitin-dependent trafficking of MHC-II and CD86 by the E3 ligase membrane-associated RING-CH-1 (MARCH1). Although ubiquitin-dependent trafficking of MHC-II has been well characterized, much less is known regarding the post-transcriptional regulation of CD86 expression. Here, we examined the physical and functional interaction between CD86 and MARCH1. We observed that CD86 is rapidly endocytosed in the presence of MARCH1 followed by lysosome-dependent degradation. Furthermore, we found that the association between CD86 and MARCH1 was conferred primarily by the transmembrane domains of the respective proteins. In contrast to MHC-II, which has a single, conserved ubiquitin acceptor site in the cytosolic domain, we found that multiple lysine residues in the cytosolic tail of CD86 could support ubiquitination consistent with the relative lack of sequence conservation across species within the CD86 cytosolic domain. These findings suggest that MARCH1 recruits multiple substrates via transmembrane domain-mediated interactions to permit substrate ubiquitination in the face of diverse cytosolic domain sequences.