EMMPRIN promotes spheroid organization and metastatic formation: comparison between monolayers and spheroids of CT26 colon carcinoma cells.

Background: In vitro studies often use two-dimensional (2D) monolayers, but 3D cell organization, such as in spheroids, better mimics the complexity of solid tumors. To metastasize, cancer cells undergo the process of epithelial-to-mesenchymal transition (EMT) to become more invasive and pro-angiogenic, with expression of both epithelial and mesenchymal markers. Aims: We asked whether EMMPRIN/CD147 contributes to the formation of the 3D spheroid structure, and whether spheroids, which are often used to study proliferation and drug resistance, could better model the EMT process and the metastatic properties of cells, and improve our understanding of the role of EMMPRIN in them. Methods: We used the parental mouse CT26 colon carcinoma (CT26-WT) cells, and infected them with a lentivirus vector to knock down EMMPRIN expression (CT26-KD cells), or with an empty lentivirus vector (CT26-NC) that served as a negative control. In some cases, we repeated the experiments with the 4T1 or LLC cell lines. We compared the magnitude of change between CT26-KD and CT26-WT/NC cells in different metastatic properties in cells seeded as monolayers or as spheroids formed by the scaffold-free liquid overlay method. Results: We show that reduced EMMPRIN expression changed the morphology of cells and their spatial organization in both 2D and 3D models. The 3D models more clearly demonstrated how reduced EMMPRIN expression inhibited proliferation and the angiogenic potential, while it enhanced drug resistance, invasiveness, and EMT status, and moreover it enhanced cell dormancy and prevented CT26-KD cells from forming metastatic-like lesions when seeded on basement membrane extract (BME). Most interestingly, this approach enabled us to identify that EMMPRIN and miR-146a-5p form a negative feedback loop, thus identifying a key mechanism for EMMPRIN activities. These results underline EMMPRIN role as a gatekeeper that prevents dormancy, and suggest that EMMPRIN links EMT characteristics to the process of spheroid formation. Conclusions: Thus, 3D models can help identify mechanisms by which EMMPRIN facilitates tumor and metastasis progression, which might render EMMPRIN as a promising target for anti-metastatic tumor therapy.

Preconditioning-Induced Facilitation of Lactate Release from Astrocytes Is Essential for Brain Ischemic Tolerance.

A sublethal ischemic episode [termed preconditioning (PC)] protects neurons in the brain against a subsequent severe ischemic injury. This phenomenon is known as brain ischemic tolerance and has received much attention from researchers because of its robust neuroprotective effects. We have previously reported that PC activates astrocytes and subsequently upregulates P2X7 receptors, thereby leading to ischemic tolerance. However, the downstream signals of P2X7 receptors that are responsible for PC-induced ischemic tolerance remain unknown. Here, we show that PC-induced P2X7 receptor-mediated lactate release from astrocytes has an indispensable role in this event. Using a transient focal cerebral ischemia model caused by middle cerebral artery occlusion, extracellular lactate levels during severe ischemia were significantly increased in mice who experienced PC; this increase was dependent on P2X7 receptors. In addition, the intracerebroventricular injection of lactate protected against cerebral ischemic injury. In in vitro experiments, although stimulation of astrocytes with the P2X7 receptor agonist BzATP had no effect on the protein levels of monocarboxylate transporter (MCT) 1 and MCT4 (which are responsible for lactate release from astrocytes), BzATP induced the plasma membrane translocation of these MCTs via their chaperone CD147. Importantly, CD147 was increased in activated astrocytes after PC, and CD147-blocking antibody abolished the PC-induced facilitation of astrocytic lactate release and ischemic tolerance. Taken together, our findings suggest that astrocytes induce ischemic tolerance via P2X7 receptor-mediated lactate release.

MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Thai traditional medicines reduce CD147 levels in lung cells: Potential therapeutic candidates for cancers, inflammations, and COVID-19.

ETHNOPHARMACOLOGICAL RELEVANCE: The cluster of differentiation 147 (CD147) is identified as the signaling protein relevant importantly in various cancers, inflammations, and coronavirus disease 2019 (COVID-19) via interacting with extracellular cyclophilin A (CypA). The reduction of CD147 levels inhibits the progression of CD147-associated diseases. Thai traditional medicines (TTMs): Keaw-hom (KH), Um-ma-ruek-ka-wa-tee (UM), Chan-ta-lee-la (CT), and Ha-rak (HR) have been used as anti-pyretic and anti-respiratory syndromes caused from various conditions including cancers, inflammations, and infections. Thus, these medicines would play a crucial role in the reduction of CD147 levels. AIM OF THE STUDY: This article aimed to investigate the effects of KH, UM, CT, and HR for reducing the CD147 levels through in vitro study. Additionally, in silico study was employed to screen the active compounds reflexing the reduction of CD147 levels. MATERIALS AND METHODS: The immunofluorescent technique was used to evaluate the reduction of CD147 levels in human lung epithelial cells (BEAS-2B) stimulated with CypA for eight extracts of KH, UM, CT, and HR obtained from water decoction (D) and 70% ethanol maceration (M) including, KHD, UMD, CTD, HRD, KHM, UMM, CTM, and HRM. RESULTS: UM extracts showed the most efficiency for reduction of CD147 levels in the cytoplasm and perinuclear of BEAS-2B cells stimulated with CypA. Phenolic compounds composing polyphenols, polyphenol sugars, and flavonoids were identified as the major chemical components of UMD and UMM. Further, molecular docking calculations identified polyphenol sugars as CypA inhibitors. CONCLUSIONS: UMD and UMM are potential for reduction of CD147 levels which provide a useful information for further development of UM as potential therapeutic candidates for CD147-associated diseases such as cancers, inflammations, and COVID-19.

NOS3 prevents MMP-9, and MMP-13 induced extracellular matrix proteolytic degradation through specific microRNA-targeted expression of extracellular matrix metalloproteinase inducer in hypertension-related atherosclerosis.

BACKGROUND: Endothelial nitric oxide synthase (NOS3) elicits atheroprotection by preventing extracellular matrix (ECM) proteolytic degradation through inhibition of extracellular matrix metalloproteinase inducer (EMMPRIN) and collagenase MMP-13 by still unknown mechanisms. METHODS: C57BL/6 mice lacking ApoE , NOS3, and/or MMP13 were fed with a high-fat diet for 6 weeks. Entire aortas were extracted and frozen to analyze protein and nucleic acid expression. Atherosclerotic plaques were detected by ultrasound imaging, Oil Red O (ORO) staining, and Western Blot. RNA-seq and RT-qPCR were performed to evaluate EMMPRIN, MMP-9, and EMMPRIN-targeting miRNAs. Mouse aortic endothelial cells (MAEC) were incubated to assess the role of active MMP-13 over MMP-9. One-way ANOVA or Kruskal-Wallis tests were performed to determine statistical differences. RESULTS: Lack of NOS3 in ApoE null mice fed with a high-fat diet increased severe plaque accumulation, vessel wall widening, and high mortality, along with EMMPRIN-induced expression by upregulation of miRNAs 46a-5p and 486-5p. However, knocking out MMP-13 in ApoE/NOS3 -deficient mice was sufficient to prevent mortality (66.6 vs. 26.6%), plaque progression (23.1 vs. 8.8%), and MMP-9 expression, as confirmed in murine aortic endothelial cell (MAEC) cultures, in which MMP-9 was upregulated by incubation with active recombinant MMP-13, suggesting MMP-9 as a new target of MMP-13 in atherosclerosis. CONCLUSION: We describe a novel mechanism by which the absence of NOS3 may worsen atherosclerosis through EMMPRIN-induced ECM proteolytic degradation by targeting the expression of miRNAs 146a-5p and 485-5p. Focusing on NOS3 regulation of ECM degradation could be a promising approach in the management of atherosclerosis.

Basigin mediation of Plasmodium falciparum red blood cell invasion does not require its transmembrane domain or interaction with monocarboxylate transporter 1.

Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from its transmembrane domain-mediated interactions, including with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin-MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

Intervention with extracellular matrix metalloproteinase inducer in osteoclasts attenuates periodontitis-induced bone resorption.

Extracellular matrix metalloproteinase inducer (EMMPRIN) plays critical roles in the regulation of inflammation and bone metabolism. The roles of EMMPRIN signaling in osteoclasts are worthy of deep study. The present study aimed to investigate bone resorption in periodontitis through the intervention of EMMPRIN signaling. The distribution of EMMPRIN in human periodontitis was observed. RANKL-induced osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) were treated with EMMPRIN inhibitor in vitro. Rats with ligation-induced periodontitis were treated with EMMPRIN inhibitor and harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, and double immunofluorescence analysis. Positive expressions of EMMPRIN could be found in the CD68+-infiltrating cells. Downregulated EMMPRIN restrained osteoclast differentiation of BMMs in vitro, which also inhibited MMP-9 expression (*P < 0.05). In vivo, EMMPRIN inhibitor restrained ligation-induced bone resorption by decreasing tartrate-resistant acid phosphatase-positive osteoclasts. Both EMMPRIN-positive and MMP-9-positive osteoclasts were less common in the EMMPRIN inhibitor groups than in the control groups. Intervention of EMMPRIN signaling in osteoclasts could probably provide a potential therapeutic target for attenuating ligation-induced bone resorption.

High-Mannose-Type Glycan of Basigin in Endothelial Cells Is Essential for the Opening of the Blood-Brain Barrier Induced by Hypoxia, Cyclophilin A, or Tumor Necrosis Factor-α.

Pathologic opening of the blood-brain barrier accelerates the progression of various neural diseases. Basigin, as an essential molecule for the opening of the blood-brain barrier, is a highly glycosylated transmembrane molecule specified in barrier-forming endothelial cells. This study analyzed the involvement of basigin in the regulation of the blood-brain barrier focusing on its glycosylation forms. First, basigin was found to be expressed as cell surface molecules with complex-type glycan as well as those with high-mannose-type glycan in barrier-forming endothelial cells. Monolayers of endothelial cells with suppressed expression of basigin with high-mannose-type glycan were then prepared and exposed to pathologic stimuli. These monolayers retained their barrier-forming properties even in the presence of pathologic stimuli, although their expression of basigin with complex-type glycan was maintained. In vivo, the blood-brain barrier in mice pretreated intravenously with endoglycosidase H was protected from opening under pathologic stimuli. Pathologically opened blood-brain barrier in streptozotocin-injected mice was successfully closed by intravenous injection of endoglycosidase H. These results show that high-mannose-type glycan of the basigin molecule is essential for the opening of the blood-brain barrier and therefore a specific target for protection as well as restoration of pathologic opening of the blood-brain barrier.

Basigin Deficiency Induces Spontaneous Polycystic Kidney in Mice.

BACKGROUND: Polycystic kidney disease is the most common hereditary kidney disorder with early and frequent hypertension symptoms. The mechanisms of cyst progression in polycystic kidney disease remain incompletely understood. METHODS: Bsg (basigin) heterozygous and homozygous knockout mice were generated using cas9 system, and Bsg overexpression was achieved by adeno-associated virus serotype 9 injection. Renal morphology was investigated through histological and imaging analysis. Molecular analysis was performed through transcriptomic profiling and biochemical approaches. RESULTS: Bsg-deficient mice exhibited significantly elevated arterial blood pressure. Further investigation demonstrated that Bsg deficiency triggers spontaneous cystic formation in mouse kidneys, which shares similar cyst pathological features and common transcriptional regulatory pathways with human polycystic kidney disease. Moreover, Bsg disruption promoted polycystin-1 ubiquitination and degradation, leading to activation of polycystic kidney disease associated cAMP and AMPK signaling pathways in Bsg knockout mouse kidneys. Finally, adeno-associated virus serotype 9 mediated Bsg reexpression reversed cystic progression in Bsg knockout mice in vivo, and Bsg overexpression inhibited the expansion of Madin-Darby canine kidney cysts in vitro. CONCLUSIONS: Our findings show that Bsg deficiency leads to an early-onset spontaneous polycystic kidney phenotype, suggesting that dysregulated Bsg signaling may be a contributing factor in cystogenesis.

CD147/Basigin Is Involved in the Development of Malignant Tumors and T-Cell-Mediated Immunological Disorders via Regulation of Glycolysis.

CD147/Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, is a multifunctional molecule with various binding partners. CD147 binds to monocarboxylate transporters (MCTs) and supports their expression on plasma membranes. MTC-1 and MCT-4 export the lactic acid that is converted from pyruvate in glycolysis to maintain the intracellular pH level and a stable metabolic state. Under physiological conditions, cellular energy production is induced by mitochondrial oxidative phosphorylation. Glycolysis usually occurs under anaerobic conditions, whereas cancer cells depend on glycolysis under aerobic conditions. T cells also require glycolysis for differentiation, proliferation, and activation. Human malignant melanoma cells expressed higher levels of MCT-1 and MCT-4, co-localized with CD147 on the plasma membrane, and showed an increased glycolysis rate compared to normal human melanocytes. CD147 silencing by siRNA abrogated MCT-1 and MCT-4 membrane expression and disrupted glycolysis, inhibiting cancer cell activity. Furthermore, CD147 is involved in psoriasis. MCT-1 was absent on CD4+ T cells in CD147-deficient mice. The naïve CD4+ T cells from CD147-deficient mice exhibited a low capacity to differentiate into Th17 cells. Imiquimod-induced skin inflammation was significantly milder in the CD147-deficient mice than in the wild-type mice. Overall, CD147/Basigin is involved in the development of malignant tumors and T-cell-mediated immunological disorders via glycolysis regulation.

Extracellular matrix metalloproteinases inducer gene polymorphism and reduced serum matrix metalloprotease-2 activity in preeclampsia patients.

Preeclampsia increases the risk of pregnancy-related complications, nevertheless a successful spiral vessel remodeling, and trophoblast invasion reduces disorders of pregnancy. Matrix metalloproteinase-2 (MMP-2) clears the path for trophoblast invasion, and activation of MMP-2 largely depends on extracellular matrix metalloproteinases inducer (EMMPRIN) protein. This study aimed to investigate EMMPRIN gene polymorphism and MMP-2 activity in preeclampsia patients. Archival whole blood and serum samples of 74 preeclampsia and 66 normotensive pregnant women age-matched were used in this case-control study. Genomic DNA was extracted from the whole blood samples and EMMPRIN gene amplified with specific primers following fragments sequence mutation analysis. Serum MMP-2 activity was determined using enzyme-linked immunosorbent assay (ELISA) and socio-demographic data of participants retrieved from the database. Age of preeclampsia patients (32.78 ± 6.39) years and body mass index (BMI) (33.09 ± 7.27) kg/m2 compared with the normotensive counterparts (32.33 ± 5.56) years and (32.33 ± 5.56) kg/m2,respectively, were not statistically significant (P > 0.05). Serum matrix metalloprotease-2 (MMP-2) activity was significantly reduced in preeclampsia group (16.34 ± 7.07) compared with the normotensives (25.63 ± 4.56) (P < 0.001), and rs424243T/G variant (55.6%) was overrepresented among the cases compared with the normotensives (16.7%). The single-nucleotide polymorphism T/G was found to be associated with preeclampsia (odds ratio [OR] = 7.63; 95% confidence interval [CI] = 3.95-14.75; P < 0.0001). Decreased activity of MMP-2 and rs424243T/G SNP of EMMPRIN gene was reported in preeclampsia. These preliminary data warrant a further investigation into the relationship between EMMPRIN gene polymorphism and MMP-2 activity in preeclampsia.

Cluster of differentiation 147 (CD147) as potential membrane protein biomarker for bladder cancer cells.

Studies reveal that alterations in membrane protein (MP) patterns are associated with underlying drug resistance to chemotherapy. Therefore, the tryptic-digested MPs from the bladder cancer cell line were subjected to global proteomics using LC-MS/MS to identify the highly expressed potential MPs in bladder cancer cells. Our findings revealed the identification of MP biomarkers, CD147, and caveolin-1. Immunocytochemistry analysis confirmed the presence of CD147 on the cell membrane, while caveolin-1 showed positive signals without apparent staining on the membrane, suggesting its existence in multiple locations. Western blot analysis confirmed the higher expression of CD147 in non-invasive (RT 112) and metastatic (UM-UC-13) bladder cancer cells compared to invasive bladder cancer cells (5637 and J82), suggesting its potential as an MP biomarker for both of the former subtypes. The identified MPs could be used as drug therapy targets aimed at improving drug sensitivity and enhancing treatment outcomes in bladder cancer patients. SIGNIFICANCE: Identification of the membrane proteins associated with bladder cancer recurrence is crucial to understanding the mechanisms underlying the drug resistance to chemotherapy.

S-nitrosylation of EMMPRIN influences the migration of HSCs and MMP activity in liver fibrosis.

The mechanism of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of liver fibrosis has not been clarified. This study aims to investigate the role of EMMPRIN S-nitrosylation (SNO) in the regulation of hepatic stellate cell (HSC) migration and matrix metalloproteinase (MMP) activities in liver fibrosis. The results from the tissue microarrays and rat/mouse liver tissues suggest that EMMPRIN mRNA and protein levels in the fibrotic livers are lower than those in the corresponding normal control livers, but higher SNO level of EMMPRIN in fibrotic liver area was shown by immunohistochemistry, immunofluorescence staining, and biotin-switch assay conversely in vivo. Primary EMMPRIN comes from hepatocytes and liver sinus epithelial cells (LSECs) rather than quiescent HSCs. To mimic the uptake of extrinsic EMMPRIN, supernatants from mouse primary hepatocytes/293 cells transfected with EMMPRIN wild-type plasmids (WT) and EMMPRIN SNO site (cysteine 87) mutation plasmids (MUT) were collected and added to JS-1/LX2 cell medium. The MUT EMMPRIN diminishes SNO successfully, enhances the activities of MMP2 and MMP9, and subsequently increases HSC migration. In conclusion, SNO of EMMPRIN influences HSC migration and MMP activities in liver fibrosis. This finding may shed light on the possible regulatory mechanism of MMPs in ECM accumulation in liver fibrosis.

Basigin is released in extracellular vesicles derived from the renal tubular epithelium in response to albuminuria.

AIM: Irrespective of the cause, albumin/proteinuria induces tubulointerstitial damage and accelerates the progression of kidney diseases. Our series of studies demonstrated that proteinuria, an independent prognostic factor for chronic kidney disease (CKD), is correlated with urinary basigin/CD147 (Bsg) levels. We examined the morphology and origin of Bsg in the tubular lumen through the effects of filtered glucose and protein solutes on the tubules. METHODS: Diabetic kidney disease (DKD) patients (N = 50) were treated with spironolactone 25 mg for 4 weeks or by conservative treatment. The associations between urinary Bsg values and clinical indicators were examined. Primary-cultured proximal tubular epithelial cells (PTECs) from human adult kidneys were exposed to high glucose or bovine serum albumin (BSA). RESULTS: In patients with early phase DKD, urinary Bsg levels were closely correlated with proteinuria but not HbA1c. Full-length Bsg on extracellular vesicles (EVs) was investigated primarily in urine collected from DKD patients. EVs obtained from the urine of DKD patients included Bsg and SGLT2 proteins. Notably, spironolactone treatment concomitantly suppressed the release of Bsg-bearing EVs in correlation with decreased albuminuria. Exposure of PTECs to BSA (but not high glucose) enhanced the storage of supernatant Bsg in EVs despite the absence of exposure-specific changes in Bsg transcription. CONCLUSION: Proteinuria induces the release of Bsg-bearing EVs derived from PTECs into the tubular lumen.

Expression profiles of MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps.

Objective: Metalloproteinases (MMPs) are implicated in tissue remodeling in chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to evaluate the expression profiles of MMP-9 and the extracellular matrix metalloproteinase inducer (EMMPRIN) in nasal polyps compared to healthy mucosa. Methods: Tissue samples from 37 CRSwNP patients undergoing functional endoscopic sinus surgery and mucosal specimens from 12 healthy controls were obtained intra-operatively. MMP-9 and EMMPRIN mRNA levels were assessed by reverse transcription-polymerase chain reaction and their protein expression by Western blot analysis. Results: MMP-9 mRNA expression levels were significantly elevated in CRSwNP compared to controls (p < 0.05). MMP-9 protein levels presented an increasing trend but with no statistical significance (p > 0.05). No statistically significant difference in EMMPRIN mRNA and protein levels was identified. Conclusions: Upregulation of MMP-9 in nasal polyps is evident and highlights its role in the pathogenesis of CRSwNP. The lack of concordance between MMP-9 mRNA and protein levels may be attributed to post-translational gene expression regulation. Although EMMPRIN expression was not significantly different between the two groups, its role remains to be elucidated. MMP-9 may be a valuable biomarker and treatment target in CRSwNP.

MMP-2 Regulation of Emmprin on Tumour Cells and CD73 on Fibroblasts During Tumour-Stromal Interaction.

BACKGROUND/AIM: We previously found that binding between CD73 and extracellular matrix metalloproteinase (MMP) inducer (emmprin) and suppression of CD73 in both tumour cells and fibroblasts suppressed MMP-2 production when co-cultured. However, the importance of CD73 expression in either fibroblasts or cancer cells for cancer invasion remains unknown. In this study, we used siRNA to separately down-regulate CD73 in individual cells, and then performed a 3D co-culture to investigate tumour invasion. MATERIALS AND METHODS: siRNA was used for suppression of CD73 in either fibroblasts (ST353i, HDF) or tumour cells (FU-EPS-1, A431, CRL-2095). Immunoblotting was performed for detecting MMP-2 production after CD73 suppression. 3D-co-cultures were performed for examining tumour invasion. RESULTS: CD73 suppression revealed that CD73 expression on fibroblasts and emmprin on tumour cells were important in regulating MMP-2 production, suggesting that emmprin on tumour cells does not bind CD73 at the cis-manner, but rather at the trans-manner to CD73 present on fibroblasts. CD73 suppression also reduced MMP-2 production at the transcription level and reduced tumour invasion. CONCLUSION: CD73 on fibroblasts acts as a receptor for emmprin, which forms a complex that increases MMP-2 production, possibly resulting in increased invasiveness.

The retina-specific basigin isoform does not induce IL-6 expression in mouse monocytes.

Purpose: Basigin gene products are positioned on adjacent cell types in the neural retina and are thought to compose a lactate metabolon important for photoreceptor cell function. The Ig0 domain of basigin isoform 1 (basigin-1) is highly conserved throughout evolution, which suggests a conserved function. It has been suggested that the Ig0 domain has proinflammatory properties, and it is hypothesized to interact with basigin isoform 2 (basigin-2) for cell adhesion and lactate metabolon formation. Therefore, the purpose of the present study was to determine whether the Ig0 domain of basigin-1 binds to basigin-2 and whether the region of the domain used for binding is also used to stimulate interleukin-6 (IL-6) expression. Methods: Binding was assessed using recombinant proteins corresponding to the Ig0 domain of basigin-1 and endogenously expressed basigin-2 from mouse neural retina and brain protein lysates. The proinflammatory properties of the Ig0 domain were analyzed with exposure of the recombinant proteins to the mouse monocyte RAW 264.7 cell line and subsequent measurement of the IL-6 concentration in the culture medium via enzyme-linked immunosorbent assay (ELISA). Results: The data indicate that the Ig0 domain interacts with basigin-2 through a region within the amino half of the domain and that the Ig0 domain does not stimulate the expression of IL-6 in mouse cells in vitro. Conclusions: The Ig0 domain of basigin-1 binds to basigin-2 in vitro. In addition, contrary to previous reports, there was no evidence that the Ig0 domain potentiates IL-6 expression in a mouse monocyte cell line in vitro. However, it is possible that the Ig0 domain stimulates the expression of proinflammatory cytokines other than IL-6, or that the potential involvement of the Ig0 domain of basigin-1 in the acute inflammatory response is dependent on species.

Cyclophilin A Inhibitors Suppress Proliferation and Induce Apoptosis of MKN45 Gastric Cancer Stem-like Cells by Regulating CypA/CD147-Mediated Signaling Pathway.

Gastric cancer stem cells (GCSCs) are a subgroup of gastric cancer (GC) cells with high self-renewal and multi-lineage differentiation abilities that lead to tumor initiation, metastasis, drug resistance, and tumor relapse. Therefore, the eradication of GCSCs can contribute to the effective treatment of advanced or metastatic GC. In our previous study, compound 9 (C9), a novel derivative of nargenicin A1, was identified as a potential natural anticancer agent that specifically targeted cyclophilin A (CypA). However, its therapeutic effect and molecular mechanisms of action on GCSC growth have not been assessed. In this study, we investigated the effects of natural CypA inhibitors, including C9 and cyclosporin A (CsA), on the growth of MKN45-derived GCSCs. Compound 9 and CsA effectively suppressed cell proliferation by inducing cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade in MKN45 GCSCs. In addition, C9 and CsA potently inhibited tumor growth in the MKN45 GCSC-grafted chick embryo chorioallantoic membrane (CAM) model. Furthermore, the two compounds significantly decreased the protein expression of key GCSC markers including CD133, CD44, integrin α6, Sox2, Oct4, and Nanog. Notably, the anticancer activities of C9 and CsA in MKN45 GCSCs were associated with the regulation of CypA/CD147-mediated AKT and mitogen-activated protein kinase (MAPK) signaling pathways. Collectively, our findings suggest that the natural CypA inhibitors C9 and CsA could be novel anticancer agents used to combat GCSCs by targeting the CypA/CD147 axis.

Surfaceome Profiling Identifies Basigin-Chaperoned Protein Clients.

The surface proteome or "surfaceome" is a critical mediator of cellular biology, facilitating cell-to-cell interactions and communication with extracellular biomolecules. Constituents of the surfaceome can serve as biomarkers for changing cell states and as targets for pharmacological intervention. While some pathways of cell surface trafficking are well characterized to allow prediction of surface localization, some non-canonical trafficking mechanisms do not. Basigin (Bsg), a cell surface glycoprotein, has been shown to chaperone protein clients to the cell surface. However, understanding which proteins are served by Bsg is not always straightforward. To accelerate such identification, we applied a surfaceome proximity labeling method that is integrated with quantitative mass spectrometry-based proteomics to discern changes in the surfaceome of hepatic stellate cells that occur in response to the genetic loss of Bsg. Using this strategy, we observed that the loss of Bsg leads to corresponding reductions in the cell surface expression of monocarboxylate transporters MCT1 and MCT4. We also found that these relationships were unique to Bsg and not found in neuroplastin (Nptn), a related family member. These results establish the utility of the surfaceome proximity labeling method to determine clients of cell surface chaperone proteins.