Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.
(1/175)
The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster (approximately 70 kDa, pI 5) and a lower molecular weight (approximately 35-45 kDa, pI 5.5) protein cluster were released from the oolemmal surface as a result of PI-PLC treatment. It is likely that these GPI-anchored egg surface proteins are required for sperm-egg binding and fusion. (+info)
The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in particular alpha3beta1, alpha4beta1 and alpha6beta1), MHC antigens and several unidentified molecules. The molecules associated with tetraspans are readily detected after immunoprecipitation performed in mild detergents such as Brij 97 or CHAPS. In this study we show that another classical mild detergent, digitonin, dissociated most of these associated molecules, including integrins, from the tetraspans CD9, CD37, CD53, CD63, CD82, Co-029, Talla-1 and NAG-2. In contrast, reciprocal immunoprecipitations from various cell lines demonstrated that two other tetraspans, CD81 and CD151, formed complexes with integrins not disrupted by digitonin. These complexes were CD81/alpha4beta1, CD151/alpha3beta1 and CD151/alpha6beta1. Furthermore, a new anti-CD151 monoclonal antibody (mAb), TS151r, was shown to have a restricted pattern of expression, inversely related to the sum of the levels of expression of alpha6beta1 and alpha3beta1. This mAb was unable to co-precipitate integrins in digitonin, suggesting that its epitope is blocked by the association with integrins. Indeed, the binding of TS151r to the cell surface was quantitatively diminished following alpha3beta1 overexpression. Altogether, these data suggest that, among tetraspans, CD81 interacts directly with the integrin alpha4beta1, and CD151 interacts directly with integrins alpha3beta1 and alpha6beta1. Because all tetraspan-tetraspan associations are disrupted by digitonin, it is likely that the other tetraspans interact indirectly with integrins, through interactions with CD81 or CD151. (+info)
CD9 is expressed on the cell surface of human granulosa cells and associated with integrin alpha6beta1.
(3/175)
The CD9 molecule is a 24-27 kDa cell surface glycoprotein which is reported to be involved in cell adhesion and migration. Recently, CD9 was shown to be associated with beta1-related integrins. We have previously found that integrin alpha6beta1 is expressed on human granulosa cells (GC) and regulates luteinization of GC in concert with its ligand laminin. In this study, we examined the expression of CD9 in human ovary and the relationship between CD9 and integrin alpha6beta1 in GC. By immunohistochemistry, CD9 was detected on GC in a small antral follicle of <1 mm in diameter. In growing follicles, CD9 was moderately expressed on both GC and theca interna cells (TI). The expression intensity of CD9 on GC increased in preovulatory follicles. In the early luteal phase, CD9 was expressed in both luteinizing GC and TI. The expression intensity on large luteal cells decreased in the mid-luteal phase. In the corpus luteum (CL) of pregnancy, CD9 continued to be expressed on large luteal cells, but not on small luteal cells. By flow cytometry, CD9 was detected on the cell surface in approximately 90% of the isolated GC from patients undergoing in vitro fertilization. The molecular weight of CD9 in the isolated GC was shown to be 26.5 kDa by Western blotting. CD9 mRNA was also detected in the isolated GC and CL by reverse transcription-polymerase chain reaction (RT-PCR). The proteins purified from GC by immunoaffinity chromatography using anti-integrin alpha6 monoclonal antibodies were shown by Western blotting to include CD9 as well as integrin beta1. These findings suggest that CD9 is a differentiation-related molecule of GC and TI and that it is associated with integrin alpha6beta1 on the cell surface of GC, suggesting that CD9 is implicated in the function of human GC in cooperation with integrin alpha6beta1. (+info)
The nonintegrin laminin binding protein (p67 LBP) is expressed on a subset of activated human T lymphocytes and, together with the integrin very late activation antigen-6, mediates avid cellular adherence to laminin.
(4/175)
A search for genes expressed in activated T cells revealed that the nonintegrin, 67-kDa laminin binding protein (p67 LBP) is expressed on the surface of a subset (10-15%) of activated peripheral blood T cells. Surface p67 LBP expression is detectable by FACS using the anti-p67 LBP mAb, MLuC5, within 6 h of T cell activation with phorbol dibutyrate and ionomycin, peaks 18-36 h postactivation, and persists for 7-10 days. The subset of T cells expressing p67 LBP is composed of mature, single-positive cells (85% CD4+8-, 15% CD4-8+) of memory cell phenotype (100% CD45 RO+/CD45 RA-). The p67 LBP+ T cells also express the integrin alpha6 chain (CD49f), which is known to associate with p67 LBP on tumor cells. In addition, the p67 LBP+ T cells express the integrin beta1, which associates with alpha6 in the laminin-specific integrin receptor very late activation Ag (VLA)-6 (alpha6beta1). Expression of an exogenous cDNA encoding the 37-kDa LBP precursor (p37 LBPP) confers p67 LBP surface expression on a p67 LBP-negative Jurkat T cell line (B2.7). Expression of p67 LBP induces B2.7 transfectants to adhere to laminin, but avid laminin binding depends on coexpression of VLA-6. Taken together, these data indicate that p67 LBP is an activation-induced surface structure on memory T cells that, together with VLA-6, mediates cellular adherence to laminin. (+info)
Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha6beta1: implications for murine fertilization.
(5/175)
CD9 is a tetraspan protein that associates with several beta1 integrins, including alpha6beta1. Because alpha6beta1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin beta), we first asked whether CD9 is present on murine eggs and whether it functions in sperm-egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm-egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin beta disintegrin domain. (Both ligands bind to the egg via alpha6beta1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin alpha6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, alpha6A and CD9 both are expressed on the apical epithelial surface at the uterine-oviduct junction. These findings correlate with the observation that fertilin beta "knockout" sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin beta(-/-) male mice. Our results suggest that high-avidity binding between fertilin beta (ADAM 2) and alpha6beta1 requires cooperation between alpha6beta1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm-egg interactions. (+info)
Integrins mediate a neuronal survival signal for oligodendrocytes.
(6/175)
Target-dependent survival of newly differentiated cells is an important part of neural development. In the case of myelin-forming oligodendrocytes, it matches the number of oligodendrocytes to the available axons [1]. In addition to growth factors, an axonal signal regulates this survival: when axons are transected, oligodendrocytes die and, conversely, when the number of axons is increased by genetic manipulation, oligodendrocyte numbers increase [2] [3]. Newly formed oligodendrocytes that fail to contact axons undergo apoptosis, and co-culture experiments that model axon-glial interactions in vitro reveal a neuronal survival effect not present in neuron-conditioned medium [4] [5], suggesting that the signal is non-diffusible and present on the surface of axons. The nature of these neuronal signals is unknown, as are the mechanisms by which they interact with growth-factor-mediated survival signals. As integrins can regulate survival in other cell types [6] [7] [8], we determined whether integrins are involved in the neuronal survival effect. We found that the laminin receptor alpha6beta1 integrin, which is expressed on oligodendrocytes, enhances the sensitivity of oligodendrocytes to the survival effect of growth factors. On the basis of this interaction between integrin and growth-factor-mediated signalling, we propose a simple model by which signals from axons and other cell types might interact to regulate oligodendrocyte cell numbers. (+info)
Upregulation of integrin alpha6/beta1 and chemokine receptor CCR1 by interleukin-12 promotes the migration of human type 1 helper T cells.
(7/175)
CD4(+) T helper 1 (Th1) cells and Th2 cells are distinguished based on the pattern of cytokines they are able to produce. Selectin ligands and chemokine receptors are differentially expressed in Th1 and Th2 cells, providing a basis for tissue-specific recruitment of helper T-cell subsets. However, the modes and mechanisms regulating tissue-specific localization of Th1 and Th2 cells are still largely unknown. Here, we show the preferential expression on Th1 cells of the integrin alpha6/beta1, which is distinctly regulated by the Th1-inducing cytokines interleukin-12 (IL-12) and interferon-alfa (IFN-alpha). The pattern of integrin alpha6/beta1 regulation closely mirrors that of the chemokine receptor CCR1. Analysis of signal transducer and activator of transcription 4 (Stat4) activation by IL-12 and IFN-alpha shows distinct signaling kinetics by these cytokines, correlating with the pattern of CCR1 and integrin alpha6/beta1 expression. Unlike IFN-alpha, the ability of IL-12 to generate prolonged intracellular signals appears to be critical for inducing integrin alpha6/beta1 upregulation in Th1 cells. The expression and upregulation of CCR1 and alpha6/beta1 integrin promotes the migration of Th1 cells. These findings suggest that the exquisite regulation of integrin alpha6/beta1 and CCR1 may play an important role in tissue-specific localization of Th1 cells. (+info)
Embryonic neurons adapt to the inhibitory proteoglycan aggrecan by increasing integrin expression.
(8/175)
The primary mediators of cell migration during development, wound healing and metastasis, are receptors of the integrin family. In the developing and regenerating nervous system, chondroitin sulfate proteoglycans (CSPGs) inhibit the integrin-dependent migration of neuronal growth cones. Here we report that embryonic sensory neurons cultured on the growth-promoting molecule laminin in combination with the inhibitory CSPG aggrecan rapidly adapt to inhibition. Adaptation is associated with a two- to threefold increase in the levels of RNA and surface protein for two laminin receptors, integrin alpha6beta1 and alpha3beta1, indicating that integrin expression is regulated by aggrecan. Increased integrin expression is associated both with increases in neuronal cell adhesion/outgrowth and with decreases in the ability of aggrecan to inhibit cell adhesion. Directly increasing integrin expression by adenoviral infection is sufficient to eliminate the inhibitory effects of aggrecan, indicating that upregulation of integrin receptors may promote neuronal regeneration in the presence of inhibitory matrix components. (+info)