Tumor-derived endomucin promotes colorectal cancer proliferation and metastasis.

BACKGROUND: Endomucin (EMCN) is a type I transmembrane glycoprotein and a mucin-like component of the endothelial cell glycocalyx. The mechanism of EMCN action in colorectal cancer (CRC) remains unclear. AIMS: Our aim was to explore the role of EMCN in the progression of CRC. MATERIALS & METHODS: We examined EMCN expression in CRC tissues and normal para-carcinoma tissues. The function and mechanisms of EMCN were checked in CRC cell lines and in mouse xenograft. Additionally, we used co-immunoprecipitation and mass spectrometry to identify the potential EMCN-binding proteins. Functional annotation analysis showed where these genes were enriched. RESULTS: We found that EMCN was overexpressed in tumor tissues compared with that in normal para-carcinoma tissues. We also found that overexpression of EMCN induced CRC proliferation and metastasis both in vitro and in vivo. EMCN knockdown prevents epithelial-mesenchymal transition in vitro. We identified 178 potential EMCN-binding partners. Furthermore, functional annotation analysis indicated that these genes were considerably enriched in carcinogenic-related functions and pathways. Collectively, the identification of EMCN-binding partners enhanced our understanding of the mechanism of EMCN-mediated malignant phenotypes, and this research may provide valuable insights into the molecular mechanisms underlying CRC. CONCLUSION: Tumor-derived endomucin promotes colorectal cancer proliferation and metastasis. We identified 178 EMCN-binding proteins and initially screened three potential EMCN-interacting proteins: NALCN, and TPM2, ANKK1. Our study provides valuable insights into the molecular mechanisms underlying CRC development.

Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.

Endothelial Notch activation promotes neutrophil transmigration via downregulating endomucin to aggravate hepatic ischemia/reperfusion injury.

Inflammatory leukocytes infiltration is orchestrated by mechanisms involving chemokines, selectins, addressins and other adhesion molecules derived from endothelial cells (ECs), but how they respond to inflammatory cues and coordinate leukocyte transmigration remain elusive. In this study, using hepatic ischemia/reperfusion injury (HIRI) as a model, we identified that endothelial Notch activation was rapidly and dynamically induced in liver sinusoidal endothelial cells (LSECs) in acute inflammation. In mice with EC-specific Notch activation (NICeCA), HIRI induced exacerbated liver damage. Consistently, endothelial Notch activation enhanced neutrophil infiltration and tumor necrosis factor (TNF)-α expression in HIRI. Transcriptome analysis and further qRT-PCR as well as immunofluorescence indicated that endomucin (EMCN), a negative regulator of leukocyte adhesion, was downregulated in LSECs from NICeCA mice. EMCN was downregulated during HIRI in wild-type mice and in vitro cultured ECs insulted by hypoxia/re-oxygenation injury. Notch activation in ECs led to increased neutrophil adhesion and transendothelial migration, which was abrogated by EMCN overexpression in vitro. In mice deficient of RBPj, the integrative transcription factor of canonical Notch signaling, although overwhelming sinusoidal malformation aggravated HIRI, the expression of EMCN was upregulated; and pharmaceutical Notch blockade in vitro also upregulated EMCN and inhibited transendothelial migration of neutrophils. The Notch activation-exaggerated HIRI was compromised by blocking LFA-1, which mediated leukocyte adherence by associating with EMCN. Therefore, endothelial Notch signaling controls neutrophil transmigration via EMCN to modulate acute inflammation in HIRI.

Endomucin restores depleted endothelial glycocalyx in the retinas of streptozotocin-induced diabetic rats.

Endothelial glycocalyx plays a significant role in the development and progression of diabetic complications. Endomucin (EMCN) is an anti-inflammatory membrane glycoprotein that is mainly expressed in venous and capillary endothelial cells. However, the function of EMCN in diabetic retinopathy (DR) progression is still completely unknown. We first investigated the change of EMCN expression in the retina and human retinal microvascular endothelial cells. We then overexpressed EMCN in the retina with adeno-associated virus and induced DR with streptozotocin (STZ). We analyzed EMCN's effect on the integrity of endothelial glycocalyx under conditions of DR. Furthermore, we investigated EMCN's protective effect against inflammation and blood-retinal barrier (BRB) destruction. We found that EMCN is specifically expressed in retinal endothelial cells and that its levels are decreased during hyperglycemia in vitro and in vivo. Overexpression of EMCN can restore the retinal endothelial glycocalyx of STZ-induced diabetic rats. Furthermore, EMCN overexpression can decrease leukocyte-endothelial adhesion to ameliorate inflammation and stabilize the BRB to inhibit vessel leakage in rats with DR. EMCN may protect patients with diabetes from retinal vascular degeneration by restoring the endothelial glycocalyx. EMCN may thus represent a novel therapeutic strategy for DR because it targets endothelial glycocalyx degradation associated with this disease.-Niu, T., Zhao, M., Jiang, Y., Xing, X., Shi, X., Cheng, L., Jin, H., Liu, K. Endomucin restores depleted endothelial glycocalyx in the retinas of streptozotocin-induced diabetic rats.

Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

We have previously shown that knockdown of endomucin (EMCN), an integral membrane glycocalyx glycoprotein, prevents VEGF-induced proliferation, migration, and tube formation in vitro and angiogenesis in vivo. In the endothelium, VEGF mediates most of its angiogenic effects through VEGF receptor 2 (VEGFR2). To understand the role of EMCN, we examined the effect of EMCN depletion on VEGFR2 endocytosis and activation. Results showed that although VEGF stimulation promoted VEGFR2 internalization in control endothelial cells (ECs), loss of EMCN prevented VEGFR2 endocytosis. Cell surface analysis revealed a decrease in VEGFR2 following VEGF stimulation in control but not siRNA directed against EMCN-transfected ECs. EMCN depletion resulted in heightened phosphorylation following VEGF stimulation with an increase in total VEGFR2 protein. These results indicate that EMCN modulates VEGFR2 endocytosis and activity and point to EMCN as a potential therapeutic target.-LeBlanc, M. E., Saez-Torres, K. L., Cano, I., Hu, Z., Saint-Geniez, M., Ng, Y.-S., D'Amore, P. A. Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

Mutant p53s generate pro-invasive niches by influencing exosome podocalyxin levels.

Mutant p53s (mutp53) increase cancer invasiveness by upregulating Rab-coupling protein (RCP) and diacylglycerol kinase-α (DGKα)-dependent endosomal recycling. Here we report that mutp53-expressing tumour cells produce exosomes that mediate intercellular transfer of mutp53's invasive/migratory gain-of-function by increasing RCP-dependent integrin recycling in other tumour cells. This process depends on mutp53's ability to control production of the sialomucin, podocalyxin, and activity of the Rab35 GTPase which interacts with podocalyxin to influence its sorting to exosomes. Exosomes from mutp53-expressing tumour cells also influence integrin trafficking in normal fibroblasts to promote deposition of a highly pro-invasive extracellular matrix (ECM), and quantitative second harmonic generation microscopy indicates that this ECM displays a characteristic orthogonal morphology. The lung ECM of mice possessing mutp53-driven pancreatic adenocarcinomas also displays increased orthogonal characteristics which precedes metastasis, indicating that mutp53 can influence the microenvironment in distant organs in a way that can support invasive growth.

Organ-Specific Differences in Endothelial Permeability-Regulating Molecular Responses in Mouse and Human Sepsis.

In patients with sepsis-induced multi-organ dysfunction syndrome, diverging patterns of oedema formation and loss of function in organs such as lung and kidney suggest that endothelial permeability-regulating molecular responses are differentially regulated. This potential differential regulation has been insufficiently studied at the level of components of adherens and tight junctions. We hypothesized that such a regulation by endothelial cells in sepsis takes place in an organ-specific manner. We addressed our hypothesis by studying by quantitative real time polymerase chain reaction the expression of a predefined subset of EC permeability-related molecules (occludin, claudin-5, PV-1, CD-31, endomucin, Angiopoietin-1, Angiopoietin-2, Tie2, VEGFA, VEGFR1, VEGFR2, and VE-cadherin) in kidney and lung after systemic lipopolysacharide injection in mice, and in kidneys of patients who died of sepsis. We showed that baseline endothelial expression of permeability-related molecules differs in mouse kidney and lung. Moreover, we showed differential regulation of these molecules after lipopolysacharide injection in the two mouse organs. In lung we found a decrease in expression levels of molecules of the adherence and tight junctions complex and related signaling systems, compatible with increased permeability. In contrast, in kidney we found expression patterns of these molecules compatible with decreased permeability. Finally, we partially corroborated our findings in mouse kidney in human kidneys from septic patients. These findings may help to understand the clinical difference in the extent of oedema formation in kidney and lung in sepsis-associated organ failure.

Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions.

Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45(+) and NIMP-R14(+) cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues.

Induction of Sd(a)-sialomucin and sulfated H-sulfomucin in mouse small intestinal mucosa by infection with parasitic helminth.

Mucin is a major component of mucus on gastrointestinal mucosa. Mucin alteration in the host is considered to be the principal event for expulsion of intestinal helminths. However, it is unclear what mucin alterations are induced by various helminth infections. In this study, the alterations of mouse small intestinal mucin after infection with two nematodes, Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which parasitize the jejunal epithelium, and a cestode, Vampirolepis nana, which parasitizes the ileal epithelium, were examined biochemically and histologically using two anti-mucin monoclonal antibodies (mAbs), HCM31 and PGM34, which recognize Sd(a) antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, and sulphated H type 2 antigen, Fucα1-2Galß1-4GlcNAc(6SO3H)ß-, respectively. The goblet cell mucins that reacted with HCM31 increased conspicuously on the jejunal mucosa concurrently with expulsion of N. brasiliensis. Increased levels of HCM31-reactive mucins were observed in the jejunal mucosa after H. polygyrus infection, despite the ongoing parasitism. Goblet cell mucins that reacted with PGM34 increased on the ileal mucosa during V. nana parasitism. Small intestinal goblet cells reacting with the two mAbs were not observed in non-infected mice, although sialomucins and sulfomucins were abundantly present. Additionally, the number of ileal goblet cells that reacted with the two mAbs was increased at the time of expulsion of heterophyid trematode. These results indicate that the type of specific acidic mucins expressed after infection varies among species of intestinal helminth, and, furthermore, that the relationship with worm expulsion is also different.

A multigene assay identifying distinct prognostic subtypes of clear cell renal cell carcinoma with differential response to tyrosine kinase inhibition.

Patients with clear cell renal cell carcinoma (ccRCC) have divergent survival outcomes and therapeutic responses, which may be determined by underlying molecular diversity. We aimed to develop a practical molecular assay that can identify subtypes with differential prognosis and response to targeted therapy. Whole-genome expression analysis of formalin-fixed paraffin-embedded (FFPE) material from 55 ccRCC patients was performed and two molecular subtypes with differential clinical outcomes were identified by hierarchical clustering. An eight-gene quantitative polymerase chain reaction assay for classification into two subtypes was developed for FFPE material. The primary objective was to assess assay performance by correlating ccRCC prognostic subtypes to cancer-specific survival (CSS) and, for patients receiving targeted therapy, radiologic response. In three validation cohorts, patients could be distinguished into prognostic subtypes with differential CSS (Singapore General Hospital FFPE cohort: n = 224; p = 1.48 × 10(-8); the Cancer Genome Atlas RNA-Sequencing cohort: n = 419; p = 3.06 × 10(-7); Van Andel Research Institute microarray cohort: n=174; p=0.00743). For 48 patients receiving tyrosine kinase inhibitor (TKI) treatment, the prognostic classification was associated with radiologic response to treatment (p = 5.96 × 10(-4)) and prolonged survival on TKI treatment (p=0.019). The multigene assay can classify ccRCCs into clinical prognostic subtypes, which may be predictive of response in patients receiving TKI therapy.

Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro.

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.

Erythropoietin modulation of podocalyxin and a proposed erythroblast niche.

Epo's erythropoietic capacity is ascribed largely to its antiapoptotic actions. In part via gene profiling of bone marrow erythroblasts, Epo is now shown to selectively down-modulate the adhesion/migration factors chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4) and to up-modulate growth differentiation factor-3 (Gdf3), oncostatin-M (OncoM), and podocalyxin like-1 (PODXL). For PODXL, Epo dose-dependent expression of this CD34-related sialomucin was discovered in Kit(+)CD71(high) proerythroblasts and was sustained at subsequent Kit(-)CD71(high) and Ter119(+) stages. In vivo, Epo markedly induced PODXL expression in these progenitors and in marrow-resident reticulocytes. This was further associated with a rapid release of PODXL(+) reticulocytes to blood. As studied in erythroblasts expressing minimal Epo receptor (EpoR) alleles, efficient PODXL induction proved dependence on an EpoR-PY343 Stat5 binding site. Moreover, in mice expressing an EpoR-HM F343 allele, compromised Epo-induced PODXL expression correlated with abnormal anucleated red cell representation in marrow. By modulating this select set of cell-surface adhesion molecules and chemokines, Epo is proposed to mobilize erythroblasts from a hypothesized stromal niche and possibly promote reticulocyte egress to blood.

Nepmucin, a novel HEV sialomucin, mediates L-selectin-dependent lymphocyte rolling and promotes lymphocyte adhesion under flow.

Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function-associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains.