Loss of tetraspanin-7 expression reduces pancreatic ß-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

BACKGROUND: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate ß-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic ß-cell function is not yet fully understood. METHODS: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. ß-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. RESULTS: Tspan7 is expressed in insulin-containing granules of pancreatic ß-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- ß-cells, but ß-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent ß-cells, exocytotic Ca2+ sensitivity was reduced in a human ß-cell line (EndoC-ßH1) following Tspan7 KD. CONCLUSION: Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in ß-cells. Its function is more significant in human ß-cells than their rodent counterparts.

Tetraspanin-7 regulation of L-type voltage-dependent calcium channels controls pancreatic ß-cell insulin secretion.

KEY POINTS: Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca2+ ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how ß-cell TSPAN-7 regulates Ca2+ handling and hormone secretion. We find that TSPAN-7 reduces ß-cell glucose-stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls ß-cell function through a direct interaction with L-type voltage-dependent Ca2+ channels (CaV 1.2 and CaV 1.3), which reduces channel Ca2+ conductance. TSPAN-7 slows activation of CaV 1.2 and accelerates recovery from voltage-dependent inactivation; TSPAN-7 also slows CaV 1.3 inactivation kinetics. These findings strongly implicate TSPAN-7 as a key regulator in determining the set-point of glucose-stimulated Ca2+ influx and insulin secretion. ABSTRACT: Glucose-stimulated insulin secretion (GSIS) is regulated by calcium (Ca2+ ) entry into pancreatic ß-cells through voltage-dependent Ca2+ (CaV ) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling, and thus they may also modulate GSIS. TSPAN-7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN-7 is highly expressed in ß- and α-cells; however, the function of islet TSPAN-7 has not been determined. Here, we show that TSPAN-7 knockdown (KD) increases glucose-stimulated Ca2+ influx into mouse and human ß-cells. Additionally, mouse ß-cell Ca2+ oscillation frequency was accelerated by TSPAN-7 KD. Because TSPAN-7 KD also enhanced Ca2+ entry when membrane potential was clamped with depolarization, the effect of TSPAN-7 on CaV channel activity was examined. TSPAN-7 KD enhanced L-type CaV currents in mouse and human ß-cells. Conversely, heterologous expression of TSPAN-7 with CaV 1.2 and CaV 1.3 L-type CaV channels decreased CaV currents and reduced Ca2+ influx through both channels. This was presumably the result of a direct interaction of TSPAN-7 and L-type CaV channels because TSPAN-7 coimmunoprecipitated with both CaV 1.2 and CaV 1.3 from primary human ß-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human ß-cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN-7 modulation of ß-cell L-type CaV channels is a key determinant of ß-cell glucose-stimulated Ca2+ entry and thus the set-point of GSIS.

Tetraspanin 7 and its closest paralog tetraspanin 6: membrane organizers with key functions in brain development, viral infection, innate immunity, diabetes and cancer.

Tetraspanin (TSPAN) protein family forms a family of transmembrane proteins that act as organizers/scaffold for other proteins. TSPANs are primarily present on plasma membranes although they are also found in other biological membranes. They are organized in tetraspanin-enriched microdomains (TEMs), which allow spatiotemporal tuning of protein functions through the control of their membrane localization. TSPAN6 and TSPAN7 are close paralogs expressed in different tissues, TSPAN7 being highly expressed in the brain. Their functions only started to be unveiled in the late 2000's and are still poorly understood. Here, we introduce how TSPAN7 was first highlighted has a protein mutated in some forms of X-linked mental retardation, which was later proposed to be caused by defects in neuronal morphogenesis and synaptic transmission. We then discuss the impacts TSPAN7 has on cell morphology of dendritic cells and osteoclasts, through rearrangement of actin cytoskeleton and how TSPAN7 was shown to be a target of autoantibody in patients suffering from type 1 diabetes. Finally, we are addressing the double edge sword that is TSPAN7 in cancer. In the second part of this review, we address the known roles of TSPAN6 and how this protein was shown to participate in synaptic transmission and in amyloid precursor protein secretion, which may contribute to Alzheimer's disease pathology. We conclude this review by discussing the anti-inflammatory effect of TSPAN6.

Cytoplasmic ends of tetraspanin 7 harbour epitopes recognised by autoantibodies in type 1 diabetes.

AIMS/HYPOTHESIS: The beta cell protein tetraspanin 7 is a target of autoantibodies in individuals with type 1 diabetes. The aim of this study was to identify autoantibody epitope-containing regions and key residues for autoantibody binding. METHODS: Autoantibody epitope regions were identified by immunoprecipitation of luciferase-tagged single or multiple tetraspanin 7 domains using tetraspanin 7 antibody-positive sera. Subsequently, amino acids (AAs) relevant for autoantibody binding were identified by single AA mutations. RESULTS: In tetraspanin 7 antibody-positive sera, antibody binding was most frequent to tetraspanin 7 proteins that contained the NH2-terminal cytoplasmic domain 1 (C1; up to 39%) or COOH-terminal C3 (up to 22%). Binding to C3 was more frequent when the domain was expressed along with the flanking transmembrane domain, suggesting that conformation is likely to be important. Binding to external domains was not observed. Single AA mutations of C3 identified residues Y246, E247 and R239 as critical for COOH-terminal binding of 9/10, 10/10 and 8/10 sera tested, respectively. Mutation of cysteines adjacent to the transmembrane domain at either residues C235 or C236 resulted in both decreased (8/178 and 15/178 individuals, respectively; >twofold decrease) and increased (30/178 and 13/178 individuals, respectively; >twofold increase) binding in participant sera vs wild-type protein. CONCLUSIONS/INTERPRETATION: We hypothesise that conformation and, potentially, modification of protein terminal ends of tetraspanin 7 may be important for autoantibody binding in type 1 diabetes.

Tetraspanin 7 autoantibodies predict progressive decline of beta cell function in individuals with LADA.

AIMS/HYPOTHESIS: The aim of this work was to investigate whether tetraspanin 7 autoantibodies (TSPAN7A) are valuable in predicting poor beta cell function in individuals with latent autoimmune diabetes in adults (LADA). METHODS: The cross-sectional study involved participants with LADA (n = 173), type 1 diabetes (n = 158), type 2 diabetes (n = 204) and healthy control participants (n = 170). The longitudinal study involved 53 participants with LADA, with a 3-year follow-up. In both cohorts, TSPAN7A in the sera were measured by a luciferase immunoprecipitation system assay, and physical and clinical characteristics were recorded. RESULTS: The prevalence of TSPAN7A in LADA, type 1 diabetes, type 2 diabetes and healthy control participants was 21.4% (37/173), 26% (41/158), 0.5% (1/204) and 1.2% (2/170), respectively. Importantly, measurement of TSPAN7A significantly increased the number of individuals with LADA found to be positive for multiple antibodies (32.4% vs 22%; p < 0.001). Further logistic regression analysis demonstrated that positivity for TSPAN7A (OR 2.87, p = 0.034), disease duration (OR 1.83, p = 0.019) and GAD antibody titre (OR 2.67, p = 0.009) were risk factors for beta cell function in LADA, while BMI (OR 0.34, p = 0.001) was a protective factor. In the prospective study in individuals with LADA, the median annual decrease in rates of fasting C-peptide and 2 h postprandial C-peptide in individuals who were positive for TSPAN7A was significantly higher when compared with the decrease in those who were negative for TSPAN7A (34.6% vs 7.9%, p = 0.043 and 33.2% vs 11%, p = 0.041, respectively). CONCLUSIONS/INTERPRETATION: TSPAN7A are valid islet autoantibodies for use in East Asian populations with autoimmune diabetes and can discriminate individuals with LADA who have lower beta cell function after disease progression.

Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts.

Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules.

Tetraspanin 7 autoantibodies in type 1 diabetes.

AIMS/HYPOTHESIS: Autoantibodies to pancreatic beta cell proteins are markers of asymptomatic type 1 diabetes. The aim was to determine whether autoantibodies to the beta cell protein tetraspanin 7 would improve the ability to identify autoimmunity against pancreatic beta cells. METHODS: Full length and external domain fragments of tetraspanin 7 were expressed as luciferase-tagged fusion proteins and used in immunoprecipitation assays to measure autoantibodies in samples from 363 patients with type 1 diabetes at onset of disease, 503 beta cell autoantibody negative first-degree relatives of patients, and 212 relatives with autoantibodies to insulin, glutamic acid decarboxylase, insulinoma antigen 2 or zinc transporter 8. RESULTS: Antibody binding was observed against the full length and external domains of tetraspanin 7, and was strongest against the full length protein. Autoantibodies that could be inhibited by untagged tetraspanin 7 were detected in 5 (1%) of 503 autoantibody negative relatives, 3 (3.2%) of 94 autoantibody negative patients, 95 (35.3%) of 269 autoantibody positive patients, 1 (1%) of 98 single autoantibody positive relatives and 25 (21.9%) of 114 multiple autoantibody positive relatives. Progression to diabetes did not differ between multiple autoantibody positive relatives with and without tetraspanin 7 autoantibodies. CONCLUSIONS/INTERPRETATION: Tetraspanin 7 is an autoantigen in type 1 diabetes. Tetraspanin 7 autoantibodies are a marker of type 1 diabetes, but provide minor additional value to existing autoantibodies in identifying beta cell autoimmunity.

Tetraspanin 7 (TSPAN7) expression is upregulated in multiple myeloma patients and inhibits myeloma tumour development in vivo.

BACKGROUND: Increased expression of the tetraspanin TSPAN7 has been observed in a number of cancers; however, it is unclear how TSPAN7 plays a role in cancer progression. METHODS: We investigated the expression of TSPAN7 in the haematological malignancy multiple myleoma (MM) and assessed the consequences of TSPAN7 expression in the adhesion, migration and growth of MM plasma cells (PC) in vitro and in bone marrow (BM) homing and tumour growth in vivo. Finally, we characterised the association of TSPAN7 with cell surface partner molecules in vitro. RESULTS: TSPAN7 was found to be highly expressed at the RNA and protein level in CD138(+) MM PC from approximately 50% of MM patients. TSPAN7 overexpression in the murine myeloma cell line 5TGM1 significantly reduced tumour burden in 5TGM1/KaLwRij mice 4 weeks after intravenous adminstration of 5TGM1 cells. While TSPAN7 overexpression did not affect cell proliferation in vitro, TSPAN7 increased 5TGM1 cell adhesion to BM stromal cells and transendothelial migration. In addition, TSPAN7 was found to associate with the molecular chaperone calnexin on the cell surface. CONCLUSION: These results suggest that elevated TSPAN7 may be associated with better outcomes for up to 50% of MM patients.