Tetraspanin CD9 is Regulated by miR-518f-5p and Functions in Breast Cell Migration and In Vivo Tumor Growth.

Breast cancer is the most commonly diagnosed and the second leading cause of cancer-related mortality among women worldwide. miR-518f-5p has been shown to modulate the expression of the metastasis suppressor CD9 in prostate cancer. However, the role of miR-518f-5p and CD9 in breast cancer is unknown. Therefore, this study aimed to elucidate the role of miR-518f-5p and the mechanisms responsible for decreased CD9 expression in breast cancer, as well as the role of CD9 in de novo tumor formation and metastasis. miR-518f-5p function was assessed using migration, adhesion, and proliferation assays. miR-518f-5p was overexpressed in breast cancer cell lines that displayed significantly lower CD9 expression as well as less endogenous CD9 3'UTR activity, as assessed using qPCR and dual luciferase assays. Transfection of miR-518f-5p significantly decreased CD9 protein expression and increased breast cell migration in vitro. Cd9 deletion in the MMTV/PyMT mouse model impaired tumor growth, but had no effect on tumor initiation or metastasis. Therefore, inhibition of miR-518f-5p may restore CD9 expression and aid in the treatment of breast cancer metastasis.

miR-518f-5p decreases tetraspanin CD9 protein levels and differentially affects non-tumourigenic prostate and prostate cancer cell migration and adhesion.

Tetraspanin CD9 is generally considered to be a metastasis suppressor, with decreased levels associated with progression and metastasis in many advanced stage cancers. Little is known about the cause of CD9 dysregulation in prostate cancer, however there are several miRNA-binding sites in the 3´UTR of the transcript suggesting it could be post-transcriptionally regulated. Using microarrays and luciferase assays in tumourigenic and non-tumourigenic prostate cell lines we identified miR-518f-5p as a regulator of the CD9 3'UTR gene expression, and decreased expression of endogenous CD9 in non-tumorigenic prostate RWPE1 and prostate cancer DU145 cells. This resulted in differential functional effects, in which RWPE1 cells showed increased migration and decreased adhesion to extracellular matrix substrates, whereas DU145 cells showed decreased migration and increased adhesion. Moreover, overexpression of miR-518f-5p significantly increased proliferation between 48h and 72h in normal RWPE1 cells, with no effect on tumourigenic DU145 cell proliferation. These results show that tetraspanin CD9 is regulated by miRNAs in prostate cell lines and that due to differential functional effects in non-tumourigenic versus tumourigenic prostate cells, miR-518f-5p may be an effective biomarker and/or therapeutic target for prostate cancer progression.

Characterization of the early molecular changes in the glomeruli of Cd151 -/- mice highlights induction of mindin and MMP-10.

In humans and FVB/N mice, loss of functional tetraspanin CD151 is associated with glomerular disease characterised by early onset proteinuria and ultrastructural thickening and splitting of the glomerular basement membrane (GBM). To gain insight into the molecular mechanisms associated with disease development, we characterised the glomerular gene expression profile at an early stage of disease progression in FVB/N Cd151 -/- mice compared to Cd151 +/+ controls. This study identified 72 up-regulated and 183 down-regulated genes in FVB/N Cd151 -/- compared to Cd151 +/+ glomeruli (p < 0.05). Further analysis highlighted induction of the matrix metalloprotease MMP-10 and the extracellular matrix protein mindin (encoded by Spon2) in the diseased FVB/N Cd151 -/- GBM that did not occur in the C57BL/6 diseased-resistant strain. Interestingly, mindin was also detected in urinary samples of FVB/N Cd151 -/- mice, underlining its potential value as a biomarker for glomerular diseases associated with GBM alterations. Gene set enrichment and pathway analysis of the microarray dataset showed enrichment in axon guidance and actin cytoskeleton signalling pathways as well as activation of inflammatory pathways. Given the known function of mindin, its early expression in the diseased GBM could represent a trigger of both further podocyte cytoskeletal changes and inflammation, thereby playing a key role in the mechanisms of disease progression.

CD151, a laminin receptor showing increased expression in asthmatic patients, contributes to airway hyperresponsiveness through calcium signaling.

BACKGROUND: Airway smooth muscle (ASM) contraction underpins airway constriction; however, underlying mechanisms for airway hyperresponsiveness (AHR) remain incompletely defined. CD151, a 4-transmembrane glycoprotein that associates with laminin-binding integrins, is highly expressed in the human lung. The role of CD151 in ASM function and its relationship to asthma have yet to be elucidated. OBJECTIVE: We sought to ascertain whether CD151 expression is clinically relevant to asthma and whether CD151 expression affects AHR. METHODS: Using immunohistochemical analysis, we determined the expression of CD151 in human bronchial biopsy specimens from patients with varying asthma severities and studied the mechanism of action of CD151 in the regulation of ASM contraction and bronchial caliber in vitro, ex vivo, and in vivo. RESULTS: The number of CD151+ ASM cells is significantly greater in patients with moderate asthma compared with those in healthy nonasthmatic subjects. From loss- and gain-of-function studies, we reveal that CD151 is required for and enhances G protein-coupled receptor (GPCR)-induced peak intracellular calcium release, the primary determinant of excitation-contraction coupling. We show that the localization of CD151 can also be perinuclear/cytoplasmic and offer an explanation for a novel functional role for CD151 in supporting protein kinase C (PKC) translocation to the cell membrane in GPCR-mediated ASM contraction at this site. Importantly, CD151-/- mice are refractory to airway hyperreactivity in response to allergen challenge. CONCLUSIONS: We identify a role for CD151 in human ASM contraction. We implicate CD151 as a determinant of AHR in vivo, likely through regulation of GPCR-induced calcium and PKC signaling. These observations have significant implications in understanding the mechanism for AHR and the efficacy of new and emerging therapeutics.

Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors.

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

FMS-like tyrosine kinase 3 (FLT3) inhibitors: Molecular docking and experimental studies.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) occur in 25% of acute lymphoid and 30% of acute myeloid leukaemia cases. Therefore, FLT3 is a potential therapeutic target for small molecule kinase inhibitors. In this study, protein-ligand interactions between FLT3 and kinase inhibitors (CEP701, PKC412, sunitinib, imatinib and dasatinib) were obtained through homology modelling and molecular docking. A cellular system for experimental testing of the inhibitors was also established by expressing wildtype and internal tandem duplication mutant FLT3 (FLT3-WT and FLT3-ITD) in FDC-P1 cells. Imatinib and dasatinib could not be docked into any of the FLT3 models, consistent with their lack of activity in the experimental assays. CEP701, PKC412 and sunitinib interacted with the ATP-binding pocket of FLT3, forming H-bonds with Cys694 and Glu692. Based on the EC50 values in the cell proliferation assay, CEP701 was the most potent inhibitor; sunitinib and PKC412 were ranked second and third, respectively. Sunitinib was the most selective inhibitor, followed by PKC421 and CEP701. The potency of sunitinib and to a lesser extent CEP701 in inhibition of FLT3 autophosphorylation was lower than the cell proliferation inhibition, indicating that inhibition of FLT3 downstream proteins may contribute to the cellular effects. It was shown in this study that the docking procedure was able to differentiate FLT3 inhibitors from ineffective compounds. Additionally, interaction with the phosphate binding region in the ATP-binding pocket increased potency at the cost of selectivity. These findings can be applied in designing highly effective and selective inhibitors for FLT3 and other related kinases.

A complementary role for tetraspanin superfamily member CD151 and ADP purinergic P2Y12 receptor in platelets.

P2Y12 receptor is required for sustained activation of integrin αIIbß3, irreversible platelet aggregation and thrombus stabilisation. Tetraspanin superfamily member CD151 associates with integrin αIIbß3 and plays critical roles in regulation of thrombus growth and stability in vivo. The possible functional relationship between P2Y12 and CD151 in a molecular cluster in platelets may affect thrombus formation. Hence our aim was to investigate the physical and functional requirements for this association in platelets. Our investigations reveal a specific and constitutive association between CD151 and P2Y12 receptor in human platelets shown by immunoprecipitation/western blot studies and by flow cytometry. Specifically, the prominent association involves CD151 with P2Y12 oligomers, and to a lesser extent P2Y12 monomers. This association is not altered by platelet aggregation induced by different agonists. There is also a distinct complex of tetraspanin CD151 with ADP purinergic receptor P2Y12 but not P2Y1. P2Y12 oligomer interaction with CD151 is selective as compared to other tetraspanins. To investigate the functional relationship between these receptors in platelets we used wild-type or CD151 knockout (KO) mice treated with either PBS or 50 mg/kg clopidogrel. CD151 KO mice treated with clopidogrel exhibited synergy in delayed kinetics of clot retraction, in PAR-4 and collagen-mediated platelet aggregation, platelet spreading on fibrinogen and without restricting cAMP inhibition. Clopidogrel treated CD151 KO arterioles showed smaller and less stable thrombi with increased tendency to embolise ex vivo and in vivo. These studies demonstrate a complementary role between CD151 and P2Y12 receptor in platelets in regulating thrombus growth and stability.

Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model.

BACKGROUND: Tetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with ß1 and ß4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems. METHODS: Here we investigated the effect of Cd151 deletion on mammary tumorigenesis by crossing Cd151-deficient mice with a spontaneously metastasising transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV). RESULTS: Cd151 deletion did not affect the normal development and differentiation of the mammary gland. While there was a trend towards delayed tumor onset in Cd151-/- PyMT mice compared to Cd151+/+ PyMT littermate controls, this result was only approaching significance (Log-rank test P-value =0.0536). Interestingly, Cd151 deletion resulted in significantly reduced numbers and size of primary tumors but did not appear to affect the number or size of metastases in the MMTV/PyMT mice. Intriguingly, no differences in the expression of markers of cell proliferation, apoptosis and blood vessel density was observed in the primary tumors. CONCLUSION: The findings from this study provide additional evidence that CD151 acts to enhance tumor formation initiated by a range of oncogenes and strongly support its relevance as a potential therapeutic target to delay breast cancer progression.

Differences in growth promotion, drug response and intracellular protein trafficking of FLT3 mutants.

OBJECTIVES: Mutant forms FMS-like tyrosine kinase-3 (FLT3), are reported in 25% of childhood acute lymphoid leukemia (ALL) and 30% of acute myeloid leukemia (AML) patients. In this study, drug response, growth promoting, and protein trafficking of FLT3 wild-type was compared with two active mutants (Internal Tandem Duplication (ITD)) and D835Y. MATERIALS AND METHODS: FLT3 was expressed on factor-dependent cells (FDC-P1) using retroviral transduction. The inhibitory effects of CEP701, imatinib, dasatinib, PKC412 and sunitinib were studied on cell proliferation and FLT3 tyrosine phosphorylation. Total expression and proportion of intracellular and surface FLT3 was also determined. RESULTS: FDC-P1 cells became factor-independent after expression of human FLT3 mutants (ITD and D835Y). FDC-P1 cells expressing FLT3-ITD grow 3 to 4 times faster than those expressing FLT3-D835Y. FD-FLT3-ITD cells were three times more resistant to sunitinib than the FD-FLT3-WT cells. The Geo means for surface FLT3 expression in FD-FLT3-ITD and -D835Y were 65 and 70% less than the FD-FLT3-WT cells. About 40% of expressed FLT3 was detected as intracellular in FD-FLT3-D835Y cell compared to 4 and 4.5% in FD-FLT3-WT and -ITD cells. CONCLUSION: Retention of D835Y FLT3 mutant protein may cause altered signaling, endoplasmic reticulum stress and activation of apoptotic signaling pathways leading to lower proliferation rate in FD-FLT3-D835Y than the FLT3-WT and ITD mutant., these may also also contribute, along with the preferential affinity, to the increased sensitivity of D835Y of CEP701 and PKC412. Studying these genetic variations can help determining the prognosis and designing a therapeutic plan for the patients with FLT3 mutations.

Knockout of the tetraspanin Cd9 in the TRAMP model of de novo prostate cancer increases spontaneous metastases in an organ-specific manner.

Prostate cancer is an extremely heterogeneous disease; patients that do progress to late-stage metastatic prostate cancer have limited treatment options, mostly palliative. Molecules involved in the metastatic cascade may prove beneficial in stratifying patients to assign appropriate treatment modalities and may also prove to be therapeutic antimetastatic targets. The tetraspanin group of molecules are integral membrane proteins that associate with motility-related proteins such as integrins. Clinical studies have mostly shown that reduced expression levels of the tetraspanin CD9 are correlated with tumour progression in a range of cancers. Furthermore, functional studies have shown CD9 to be involved in cell motility and adhesion and that it may influence metastasis. The effects of endogenous CD9 on prostate cancer initiation and progression were analysed by crossing a Cd9-/- (KO) murine model with a model of de novo developing and spontaneously metastasising prostate cancer, namely the transgenic adenocarcinoma of mouse prostate model. Our study demonstrates for the first time that ablation of Cd9 had no detectable effect on de novo primary tumour onset, but did significantly increase metastasis to the liver but not the lungs.

Therapeutic targeting of c-KIT in cancer.

INTRODUCTION: Mutated forms of the receptor tyrosine kinase c-KIT are "drivers" in several cancers and are attractive targets for therapy. While benefits have been obtained from use of inhibitors of KIT kinase activity such as imatinib, especially in gastrointestinal stromal tumours (GIST), primary resistance occurs with certain oncogenic mutations. Furthermore, resistance frequently develops due to secondary mutations. Approaches to addressing both of these issues as well as combination therapies to optimise use of KIT kinase inhibitors are discussed. AREAS COVERED: This review covers the occurrence of oncogenic KIT mutations in different cancers and the molecular basis of their action. The action of KIT kinase inhibitors, especially imatinib, sunitinib, dasatinib and PKC412, on different primary and secondary mutants is discussed. Outcomes of clinical trials in GIST, acute myeloid leukaemia (AML), systemic mastocytosis and melanoma and their implications for future directions are considered. EXPERT OPINION: Analysis of KIT mutations in individual patients is an essential prerequisite to the use of kinase inhibitors for therapy, and monitoring for development of secondary mutations that confer drug resistance is necessary. However, it is unlikely that KIT inhibitors alone can lead to cure. KIT mutations alone do not seem to be sufficient for transformation; thus identification and co-targeting of synergistic oncogenic pathways should lead to improved outcomes.

Genetic ablation of the tetraspanin CD151 reduces spontaneous metastatic spread of prostate cancer in the TRAMP model.

Tetraspanins are integral membrane proteins that associate with motility-related molecules such as integrins. Experimental studies have indicated that they may be important regulators of tumor invasion and metastasis, and high expression of the tetraspanin CD151 has been linked to poor prognosis in a number of cancers. Here, we show for the first time that genetic ablation of CD151 inhibits spontaneous metastasis in a transgenic mouse model of de novo tumorigenesis. To evaluate the effects of CD151 on de novo prostate cancer initiation and metastasis, a Cd151(-/-) (KO) murine model was crossed with the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. Mice were analyzed for initiation of prostate tumor by palpation and primary tumors were analyzed by immunohistochemistry. Liver and lungs were examined for incidence and size of spontaneous metastatic lesions by histopathology. Knocking-out Cd151 had no significant effect on prostate cancer initiation or on expression of markers of proliferation, apoptosis, or angiogenesis in primary tumors. However, it did significantly decrease metastasis in a site-specific fashion, notably to the lungs but not the liver. Thus, CD151 acts principally as promoter of metastasis in this model. Prostate cancer is the second highest cause of cancer-related deaths in men in most Western countries, with the majority of deaths attributed to late-stage metastatic disease. CD151 may prove to be a valuable prognostic marker for treatment stratification and is a possible antimetastatic target.

Expression of biologically active human colony stimulating factor-1 in Pichia pastoris.

Colony Stimulating Factor-1 (CSF-1) is involved in proliferation, differentiation, and survival of the mononuclear lineage, in development of the female reproductive system and mammary glands during pregnancy and lactation. It is also implicated in the biology of breast cancer and promotion of its metastasis to bones. Therefore, CSF-1 is required for many applications in cellular and molecular biology studies. Commercial products, usually expressed in prokaryotic systems, are costly, with the likelihood of endotoxin contamination and also lack posttranslational modifications. These considerations provide the rationale to express growth factors in eukaryotic systems. In this study, the biologically active and soluble fragment (residues 33-182) of human (CSF-1) was cloned from K562 cell line and expressed in Pichia pastoris. The expression level of the active CSF-1 was about 100 µg/ml of the P. pastoris culture medium. Protein analysis revealed that the expressed CSF-1 appears in three bands with apparent molecular weight of 30, 26 and 20 kDa constituting 44%, 25% and 13% of all proteins in the culture medium, respectively. The expressed protein was partially purified and concentrated (10x) by ultrafiltration, then filter sterilized. The product was confirmed to be biologically active by stimulation of its receptor (FMS) autophosphorylation in THP-1 cells and also growth promotion of factor dependent FDC-P1 cells expressing human wild-type FMS (FD-FMS-WT). Therefore, P. pastoris is a highly efficient and cost-effective expression system for production of endotoxin-free CSF-1 for research and potentially for therapeutic applications.

The migration and invasion of human prostate cancer cell lines involves CD151 expression.

The molecular mechanisms underlying prostate cancer metastasis remain poorly understood. The tetraspanin family member CD151 has been reported as an 'adaptor' between integrins and signal pathways. The role of CD151 in prostate cancer metastasis in vitro was investigated in this study. LNCap cells were transfected with wild-type CD151 cDNA, mutated CD151 cDNA and vector cDNA. The mutant (QRD194-196 to INF) CD151 cDNA was created using QuickChange 2 site directed Mutagenesis kit (Stratagene). siRNAs were also used to knock down the CD151 expression in the prostate cancer cell line PC3. Proliferation, migration and invasion properties were measured after gene transfection and gene knock-down. There was no difference in proliferation of untransfected or control transfected LNCap cells vs. CD151 transfected LNCap cells (P>0.05). There was greater motility of CD151-transfected vs. control cells, when transferring through migration chambers with or without matrigel-coated membranes (P<0.01, P<0.01). Fewer numbers of mutant-transfected cells were found on the membranes for both migration and invasion studies (P<0.01, P<0.01). CD151 knock-down PC3 cells showed decreased motility (P<0.01), but no change in proliferation (P>0.05). Our data show that CD151 does not change the proliferative properties of prostate cancer cells, but does promote migration and invasion, and suggest that CD151 plays a specific role in promoting prostate cancer cell motility.

Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

Colony stimulating factor-1 receptor as a target for small molecule inhibitors.

Imatinib, dasatinib, sunitinib, CEP-701, and PKC-412, ATP-competitive small molecule inhibitors of type III receptor tyrosine kinases c-KIT and/or FLT3, were evaluated for binding to the closely related receptor, FMS, by docking into models of inactive and active conformations of the FMS kinase domain. To confirm the docking predictions, the drugs were tested for their activity and selectivity in inhibiting cell proliferation and FMS phosphorylation upon stimulation by the FMS ligand, CSF-1. All five drugs inhibited FMS activity. Imatinib, dasatinib and CEP-701 represent three different types of interactions determining drug potency and selectivity.

Tetraspanins CD37 and CD151 differentially regulate Ag presentation and T-cell co-stimulation by DC.

A major question in immunology is how DC can display limited amounts of individual peptide-MHC complexes and still induce cross-linking of T-cell receptors to initiate cellular responses. One suggested mechanism is that MHC exists at the cell surface in high avidity multimers, and tetraspanin proteins, known to laterally associate with both MHC classes I and II, promote MHC multimerisation. To validate this theory, we tested the ability of DC deficient in either one of two typical tetraspanin molecules: CD37 or CD151 to present peptide to Ag-specific T cells. Surprisingly, although they exhibited no developmental or maturation defects, DC lacking either CD37 or CD151 expression were hyper-stimulatory to T cells. We demonstrate that CD37 and CD151 control DC-mediated T-cell activation by two different mechanisms: CD151 regulates co-stimulation whereas CD37 regulates peptide/MHC presentation. The implications of these results on the model suggesting that tetraspanins promote MHC multimerisation are discussed.

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains.

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.

Tetraspanin CD151 regulates glycosylation of (alpha)3(beta)1 integrin.

The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.