Tetraspanin CD82: a suppressor of solid tumors and a modulator of membrane heterogeneity.

Tetraspanin CD82 suppresses the progression and metastasis of a wide range of solid malignant tumors. However, its roles in tumorigenesis and hematopoietic malignancy remain unclear. Ubiquitously expressed CD82 restrains cell migration and cell invasion by modulating both cell-matrix and cell-cell adhesiveness and confining outside-in pro-motility signaling. This restraint at least contributes to, if not determines, the metastasis-suppressive activity and, also likely, the physiological functions of CD82. As a modulator of cell membrane heterogeneity, CD82 alters microdomains, trafficking, and topography of the membrane by changing the membrane molecular landscape. The functional activities of membrane molecules and the cytoskeletal interaction of the cell membrane are subsequently altered, followed by changes in cellular functions. Given its pathological and physiological importance, CD82 is a promising candidate for clinically predicting and blocking tumor progression and metastasis and also an emerging model protein for mechanistically understanding cell membrane organization and heterogeneity.

Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases.

KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

The Role of CK7, S100A1, and CD82 (KAI1) Expression in the Differential Diagnosis of Chromophobe Renal Cell Carcinoma and Renal Oncocytoma.

Renal oncocytoma is a benign renal tumor originated from intercalated cells of collecting ducts like chromophobe renal cell carcinoma (RCC). The differential diagnosis of these 2 tumors is important because while they are histologically and cytologically similar, they show different biological behavior. For the differential diagnosis, several immunohistochemical markers have been investigated. But, differential diagnostic challenges remain and the identification of additional markers is needed. Cytokeratin 7 (CK7) is one of ductal-type keratins, which is expressed in tumors of breast, pancreas, lung, thyroid, ovary, endometrium, urinary bladder, and the kidney. S100A1 is the first defined member of the calcium-binding S100 protein family and it organizes several cellular functions including cell cycle progression and cell differentiation.CD82 is a tetraspanin membrane protein, which functions as a metastasis supressor. In this study, we immunohistochemically investigated the expressions of CK7, S100A1, and CD82 in 30 chromophobe RCC (23 classic and 7 eosinophilic variant) and 19 oncocytomas. When these markers were evaluated separately and together, their expressions in chromophobe RCC and renal oncocytoma show statistically significant difference (P<0.001). Similar statistically significant results were also seen between eosinophilic chromophobe RCC and oncocytoma (P<0.001). For both classic and eosinophilic-variant chromophobe RCCs, CK7+/S100A1-/CD82+ profile being the most common. In oncocytomas, the most frequently observed profile was CK7-/S100A1+/CD82-. Our results showed that the application of a panel consisting of CK7, S100A1, and CD82 may provide accurate categorization of the tumors in difficult cases.

The tetraspanin KAI1/CD82 is expressed by late-lineage oligodendrocyte precursors and may function to restrict precursor migration and promote oligodendrocyte differentiation and myelination.

In the adult mammalian brain, oligodendrocyte progenitors can differentiate into mature oligodendrocytes during remyelination. Mechanisms that regulate migration and differentiation of progenitors are of great importance in understanding normal development and demyelinating/remyelinating conditions. In a microarray analysis comparing adult and neonatal O4-positive (+) cells, we found that the tetraspanin KAI1/CD82 is far more highly expressed in adult O4(+) cells than in neonatal O4(+) cells (Lin et al., 2009). CD82 is a metastasis suppressor, and its expression is often downregulated or lost in the advanced stages of metastatic cancer. We hypothesized that CD82 could be a factor that restricts migration and promotes differentiation of maturing oligodendrocytes. Western blot analysis of isolated adult O4(+) cells confirms the elevated levels of CD82, which continues to be expressed as these become O1(+) in vitro. In the adult rat white matter, CD82 is coexpressed with CC1 and olig2 but not with NG2 or GFAP. Immature cells of the neonatal forebrain subventricular zone (SVZ) infected in vivo with a retrovirus that constitutively expresses CD82 do not remain immature but differentiate into either CC1(+) and MBP(+) myelinating oligodendrocytes in the white matter or zebrinII(+) astrocytes in the cortex. Their migration from the SVZ is severely restricted. In contrast, downregulation of CD82 in SVZ cells in vivo, using retroviral-expressed short hairpin RNAs (shRNAs), prevents their differentiation into myelinating oligodendrocytes. shRNA-expressing cells remained PDGF receptor alpha positive, olig2(+), or NG2(+) or became CC1(+) nonmyelinating oligodendrocytes or GFAP(+) astrocytes. CD82 thus appears to be a critical molecule in the regulation of oligodendrocyte progenitor migration and myelination.

Decreased expression of KAI1/CD82 metastasis suppressor gene is associated with loss of heterozygosity in melanoma cell lines.

KAI1/CD82, a metastasis suppressor gene of prostate cancer, is located on the human chromosome 11p11.2. Down-regulation of KAI1/CD82 during tumor progression and metastasis has been reported in several cancers, but the mechanism of this down-regulation remains unknown. The relationship between down-regulation of KAI1/CD82 mRNA expression and KAI1/CD82 gene alterations in human melanoma cell lines were investigated. The promoter methylation status was examined after a 331-bp GC-rich fragment of the promoter region was amplified in G361, SK-MEL-24 and SK-MEL-28 cell lines treated with bisulfite. In order to detect methylated CpGs in all three cell lines, 331-bp fragments were sequenced. To examine the restoration of KAI1/CD82 mRNA and protein expression, the cells were exposed to methylase inhibitor, 5-aza-2'-deoxycytidine (5-AzaC). Bisulfite-sequencing data showed no methylation in G361 and SK-MEL-24 cells, and slight methylation in SK-MEL-28 cells at CpG sites 23-26 in the promoter. Real-time PCR and flow cytometry analysis showed that 5-AzaC-treated cells restored KAI1/CD82 mRNA and protein expression in SK-MEL-24 and SK-MEL-28 cells, compared to the controls. The restoration of KAI1/CD82 mRNA and protein expression detected no significant difference between SK-MEL-24 and SK-MEL-28 cells. This means that 5-AzaC did not affect the methylated cells only. Loss of heterozygosity (LOH) at polymorphic microsatellite loci on the human chromosome 11 in the human melanoma cells was also examined. Microsatellite analysis showed LOH at D11S1344 in SK-MEL-24 and SK-MEL-28 cells, and G361 showed allelic imbalance. In conclusion, this study suggests that down-regulation of KAI1/CD82 mRNA expression in human melanoma cell lines is related to LOH or allelic imbalance, but not to methylation of the KAI1/CD82 gene region.

The expression of ubiquitin-conjugating enzyme E2C and KAI1 in ovarian carcinoma and their clinical significance.

Ubiquitin-conjugating enzyme E2C (UBE2C) is considered to play an important role in the tumorigenesis of many cancers and promote cell cycle progression. Kangai 1 (KAI1) is considered as a suppressor gene of tumor metastasis. However, the clinicopathological significance and their each relationship of UBE2C and KAI1 in epithelial ovarian carcinoma (EOC) are not widely reported. The purpose of this study is to detect the expression of UBE2C and KAI1 in EOC and their clinical significance.The expression of UBE2C and KAI1 in 180 cases of EOC tissues, 60 cases of normal ovarian epithelial tissues, and 60 cases of ovarian benign tumor tissues were detected by immunohistochemistry. Patients data were also collected.Positive expression of UBE2C in EOC (38.9%) was significantly higher than that both in the normal group (0%) and benign tumors group (10.0%). Furthermore, the expression of UBE2C was positively associated with grades of differentiation, implants, lymph node metastasis (LNM), as well as the International Federation of Gynecology and Obstetrics (FIGO) stages. Positive expression of KAI1 in EOC (25.0%) was significantly lower than that both in the normal group (100%) and benign tumors group (75.0%). And the expression of KAI1 was inversely associated with grades of differentiation, implants, LNM, and FIGO stages. Kaplan-Meier survival analyses demonstrated that UBE2C positive expression for patients with EOC had unfavorably overall survival (OS) time when compared with negative UBE2C for patients. And KAI1 positive expression for patients had favorably OS time when compared with negative KAI1 for patients. Multivariate analysis showed that positive expression of UBE2C and KAI1, implants, and FIGO stages were considered as independently prognostic factors for OS in patients with EOC. Moreover, UBE2C expression was significantly higher in high grade serous adenocarcinoma (SA) when compared with low grade SA; and KAI1 expression was significantly lower in high grade SA when compared with low grade SA. High grade SA patients had higher rates of implants, LNM, and high FIGO stages when compared with low grade SA. High grade SA patients had unfavorably OS time when compared with low grade SA.UBE2C and KAI1 should be considered as potential biomarkers of EOC prognosis.

Molecular cloning and expression analysis of CD82 in pig.

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.

[Effect of metastasis suppressor gene KAI1 on the proliferation and invasive ability of cervical carcinoma cells].

OBJECTIVE: To explore the effect of metastasis suppressor gene KAI1 on the proliferation and invasive ability of cervical cancer cell line CaSki. METHODS: pCMV-KAI1 cDNA plasmid was transferred into cervical carcinoma cell line CaSki by liposome, which had low level of endogenous KAI1 expression. The expressions of KAI1 protein and mRNA were determined by immunohistochemistry and real-time fluorescence quantitative PCR (RT-PCR), the proliferation of KAI1-transfected CaSki cells was investigated by MTT assay and the invasive ability of these cells was evaluated by in vitro invasion assays. RESULTS: After the transfection of pCMV-KAI1 cDNA, the level of KAI1 mRNA and protein expression in CaSki cell were increased (P < 0.05), while the cell proliferation was suppresssed, and the migrative ability of passing through the membrane filte also decreased evidently (P < 0.05). CONCLUSION: The KAI1 metastasis suppressor gene suppressed the ability of proliferation and invasion of cervical cancer cell CaSki in vitro.

[Expression of metastasis suppressor gene KAI1 in cervical carcinoma and infections of HPV16 E6, E7 and HPV18 E6/E7].

OBJECTIVE: To investigate the expression of metastasis suppressor gene KAI1 in cervical carcinoma and the impact of human papillomavirus 16 E6, E7, 18 E6/E7 infection on the expression of KAI1. METHODS: The expressions of KAI1 protein in the formalin-fixed, paraffin-embedded specimens of 20 normal cervical epthelium, 15 cervical in situ carcinoma and 70 primary invasive cervical carcinoma were detected by immunohistochemistry SP. Polymerase chain reaction (PCR) tests were also undertaken to detect the HPV16 E6, E7 and HPV18 E6/E7 DNA. RESULTS: The expression of KAI1 protein was down-regulated in the invasive carcinoma and in situ carcinoma compared with the controls (P < 0.05). No significant difference was found in the expression of KAI1 protein between invasive carcinoma and in situ carcinoma. The infections of HPV16 E6, E7 and HPV18 E6/E7 were found in 67.1%, 54.3% and 12.9% of the invasive carcinoma, respectively. However, there was no correlation between the expression of KAI1 and the infections of HPV16 E6, E7 and HPV18 E6/E7. CONCLUSION: The expression of KAI1 protein is down-regulated in cervical carcinoma, which is not associated with the infection of HPV16 E6, E7 and 18 E6/E7.

KAI-1 protein expression in odontogenic cysts.

The KAI-1 tumor suppressor gene is widely distributed in normal tissues and its down-regulation may be correlated with the invasive phenotype and metastases in several different epithelial tumors. The aim of the present study was an evaluation of KAI-1 expression in radicular cysts (RC), follicular cysts (FC), orthokeratinized keratocysts (OOKC), and parakeratinized keratocysts (POKC). Eighty-five odontogenic cysts, 28 RC, 22 FC, and 35 OKC (16 OOKC, 19 POKC) were selected. All the POKC were negative and only four of 16 of the OOKC were positive for KAI-1. On the contrary, all RC and FC cases were positive and immunoreactivity for KAI-1 was detected throughout all the layers of the cyst epithelium. The lack of KAI-1 expression in POKC could help to explain the differences in the clinical and pathologic behavior of OKC and, according to what has been reported for epithelial tumors, could be related to the increased aggressive behavior and invasiveness of OKC.

Gangliosides play an important role in the organization of CD82-enriched microdomains.

Four-transmembrane-domain proteins of the tetraspanin superfamily are the organizers of specific microdomains at the membrane [TERMs (tetraspanin-enriched microdomains)] that incorporate various transmembrane receptors and modulate their activities. The structural aspects of the organization of TERM are poorly understood. In the present study, we investigated the role of gangliosides in the assembly and stability of TERM. We demonstrated that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase [NB-DGJ (N-butyldeoxygalactonojirimycin) and PPMP (D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol.HCl)] resulted in specific weakening of the interactions involving tetraspanin CD82. Furthermore, ectopic expression of the plasma-membrane-bound sialidase Neu3 in mammary epithelial cells also affected stability of the complexes containing CD82: its association with tetraspanin CD151 was decreased, but the association with EGFR [EGF (epidermal growth factor) receptor] was enhanced. The destabilization of the CD82-containing complexes upon ganglioside depletion correlated with the re-distribution of the proteins within plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together, our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains. Furthermore, these results demonstrate that the association between different tetraspanins in TERM is controlled by distinct mechanisms and identify Neu3 as a first physiological regulator of the integrity of these microdomains.

A viral microRNA downregulates metastasis suppressor CD82 and induces cell invasion and angiogenesis by activating the c-Met signaling.

Kaposi's sarcoma (KS) as the most common AIDS-associated malignancy is etiologically caused by KS-associated herpesvirus (KSHV). KS is a highly disseminated and vascularized tumor. KSHV encodes 12 pre-microRNAs that yield 25 mature microRNAs (miRNAs), but their roles in KSHV-induced tumor metastasis and angiogenesis remain largely unclear. KSHV-encoded miR-K12-6 (miR-K6) can generate two mature miRNAs, miR-K6-5p and miR-K6-3p. Recently, we have shown that miR-K6-3p induced cell migration and angiogenesis via directly targeting SH3 domain binding glutamate-rich protein (SH3BGR). Here, by using mass spectrometry, bioinformatics analysis and luciferase reporter assay, we showed that miR-K6-5p directly targeted the coding sequence of CD82 molecule (CD82), a metastasis suppressor. Ectopic expression of miR-K6-5p specifically inhibited the expression of endogenous CD82 and strongly promoted endothelial cells invasion and angiogenesis. Overexpression of CD82 significantly inhibited cell invasion and angiogenesis induced by miR-K6-5p. Mechanistically, CD82 directly interacted with c-Met to inhibit its activation. MiR-K6-5p directly repressed CD82, relieving its inhibition on c-Met activation and inducing cell invasion and angiogenesis. Lack of miR-K6 abrogated KSHV suppression of CD82 resulting in compromised KSHV activation of c-Met pathway, and KSHV induction of cell invasion and angiogenesis. In conclusion, our data show that by reducing CD82, KSHV miR-K6-5p expedites cell invasion and angiogenesis by activating the c-Met pathway. Our findings illustrate that KSHV miRNAs may be critical for the dissemination and angiogenesis of KSHV-induced malignant tumors.

Expression of KITENIN, a KAI1/CD82 binding protein and metastasis enhancer, in bladder cancer cell lines: relationship to KAI1/CD82 levels and invasive behaviour.

KITENIN is a newly identified binding partner of the KAI1/CD82 metastasis suppressor. Recent studies using a mouse model of colon cancer, have suggested that KITENIN might be a metastasis enhancer whose functions are modulated by an interaction with KAI1/CD82. To begin exploration of the possible importance of KITENIN to human cancer, we examined KITENIN mRNA (by RT-PCR) and protein expression (by Western blotting) in a large series of bladder cancer cell lines, and then compared these levels to the expression of KAI1/CD82 and of previously determined in vitro invasive behaviour of these same cancer cell lines. We report that KITENIN was uniformly expressed in all cancer cell lines, but those lines in which KAI1/CD82 was not detected, had a higher in vitro invasive ability and altered actin organisation (as determined by fluorescence microscopy), than those lines in which KAI1/CD82 was present. Our data suggest that the relationship between KITENIN and KAI1/CD82 may be an important determinant of tumour cell behaviour.

CD82/KAI1 Maintains the Dormancy of Long-Term Hematopoietic Stem Cells through Interaction with DARC-Expressing Macrophages.

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 in niche-mediated LT-HSC maintenance. We found that CD82/KAI1 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82(-/-) mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-ß1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC(+) BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche.

Evaluation of tumor malignancy in esophageal squamous cell carcinoma using different characteristic factors.

BACKGROUND: We have been investigating various molecules correlated with the malignancy of esophageal squamous cell carcinoma and, in the present study, we examined the correlation of four of them (KAI1, FAK, EphA2, Ki-67 labeling index) with the prognosis of affected patients. Furthermore, the use of biopsy samples was studied to evaluate whether the grade of tumor malignancy can be determined before treatment in a clinical setting. MATERIALS AND METHODS: Tissue specimens that had been surgically removed from 91 patients with thoracic esophageal cancer and 247 biopsy samples were examined. The malignancy index (MI) was defined in terms of the KAI1, FAK and EphA2 scores and the Ki-67 labeling index, and the reliability and utility of the correlation between MI and prognosis was evaluated. RESULTS: The mean 5-year survival rate of patients with MI=0 was 100%, while that of patients with MI=1, 2 and 3 was 70%, 48% and 10%, respectively. Patients with MI=4 all died, with the exception of one who has been observed for 3 years. The rate of concordance between the biopsy samples and surgical specimens was 79.4% for KAI1, 88.2% for FAK and 73.5% for EphA2, and the rates of concordance for 1, 2, 3, 4, 5, 6, 7 and 8 biopsy samples were 66.7%, 64.1%, 74.5%, 90.7%, 91.7%, 83.3%, 100% and 100%, respectively. CONCLUSION: It may be feasible to evaluate the malignancy of tumor cells and to predict patient outcome by using multiple marker molecules. It is anticipated that such data will accelerate the development of "tailor-made" therapy.

[Expression of KAI1 gene in human laryngeal carcinoma and its clinical significance].

OBJECTIVE: To explore the mRNA expression of KAI1 gene in laryngeal squamous-cell carcinoma and its clinical significance. METHODS: Fresh laryngeal cancer samples taken from 40 laryngeal carcinoma cases and normal control laryngeal tissues from 9 subjects were examined with semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Moderate, low and negative expression rates of KAI1 gene mRNA in nine normal laryngeal tissues were 33.3% (3/9), 33.3% (3/9) and 33.3% (3/9), respectively. The high, moderate, low and negative expression rates of KAI1 mRNA in 25 laryngeal cancers without lymph node metastasis were 40.0% (10/25), 28.0% (7/25), 20.0% (5/25) and 12.0% (3/25), respectively. The moderate, low and negative expression rates of KAI1 mRNA in 15 laryngeal cancers with lymph node metastasis were 20.0% (3/15), 26.7% (4/15) and 53.3% (8/15), respectively. The KAI1 mRNA expression in the laryngeal cancers without lymph node metastasis was higher than that in normal laryngeal tissues (P < 0.05). The KAI1 mRNA expression in the laryngeal cancers with lymph node metastasis was lower than that in the laryngeal cancers without lymph node metastasis (P < 0.05). The high, moderate and low expression rates of KAI1 mRNA in 10 highly differentiated laryngeal cancers were 50.0% (5/10), 30.0% (3/10) and 20.0% (2/10), respectively. The high, moderate, low and negative expression rates of KAI1 mRNA in 12 low differentiation laryngeal cancers were 8.3% (1/12), 16.7% (2/12), 16.7% (2/12) and 58.3% (7/12), respectively. The differences of KAI1 mRNA expression between high and low differentiation laryngeal cancers were statistically significant (P < 0.05). CONCLUSION: The decrease of KAI1 mRNA expression may be related to lymph node metastasis and low differentiation of laryngeal squamous-cell carcinoma.

Pharmacological targeting of the ß-amyloid precursor protein intracellular domain.

Amyloid precursor protein (APP) intracellular domain (AICD) is a product of APP processing with transcriptional modulation activity, whose overexpression causes various Alzheimer's disease (AD)-related dysfunctions. Here we report that 1-(3',4'-dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid) (CHF5074), a compound that favorably affects neurodegeneration, neuroinflammation and memory deficit in transgenic mouse models of AD, interacts with the AICD and impairs its nuclear activity. In neuroglioma-APPswe cells, CHF5074 shifted APP cleavage from Aß42 to the less toxic Aß38 peptide without affecting APP-C-terminal fragment, nor APP levels. As revealed by photoaffinity labeling, CHF5074 does not interact with γ-secretase, but binds to the AICD and lowers its nuclear translocation. In vivo treatment with CHF5074 reduced AICD occupancy as well as histone H3 acetylation levels and transcriptional output of the AICD-target gene KAI1. The data provide new mechanistic insights on this compound, which is under clinical investigation for AD treatment/prevention, as well as on the contribution of the AICD to AD pathology.

The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density.

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell-matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82's contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.

CD82 expression alters with human endometrial cycles and affects the uterine endometrial receptivity in vitro.

Embryo implantation is a process that requires both temporal and spatial synchronization of the uterine endometrium and the embryo, and the endometrium becomes receptive to the embryo during the window of implantation. Although the expression patterns of many implantation-related molecules change dynamically during this process, the impact of CD82 on endometrial receptivity has not been elucidated. By immunohistochemical staining, we found that CD82 levels rose from the proliferative phase to the secretory phase in human endometrium. Specifically, the highest level appeared in mid- and late-secretory phases. Consistently, RL95-2 cells, representative of high-receptive endometrial epithelium, expressed higher levels of CD82 than did HEC-1A cells, which are representative of low-receptive endometrial epithelium, as detected by reverse transcription-polymerase chain reaction, Western blot and immunofluorescence. Furthermore, progesterone up-regulated the expression of CD82 in both epithelial cell lines. Down-regulation of CD82 in RL95-2 cells by either CD82 siRNA transfection or treatment with a CD82 antibody significantly decreased the adhesion of human embryonic JAR cells to RL95-2 cell monolayers (P < 0.01) and inhibited the phosphorylation of focal adhesion kinase (FAK). In contrast, up-regulation of CD82 in HEC-1A cells by CD82 cDNA transfection promoted embryonic JAR cell adhesion to HEC-1A monolayers (P < 0.05) and activated the phosphorylation of FAK. In conclusion, the expression of CD82 increases in endometrial tissues during the window of embryo implantation, CD82 expression affects endometrial receptivity of the uterine epithelial cells in vitro, and the FAK signaling pathway may be involved in this phenomenon. The correlation between CD82 and endometrial receptivity suggests that CD82 may serve as a potential marker of endometrial function.