Blockade of alpha6-integrin reveals diversity in homing patterns among human, baboon, and murine cells.
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ABSTRACT:
SUBMITTER: Bonig H
PROVIDER: S-EPMC2866506 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Integrins play a major role in cell adhesion and migration. Previous work reported that a cleaved form of integrin alpha6 (alpha6p) was detected in invasive human prostate cancer tissue, absent in normal prostate tissue and was produced by urokinase-type Plasminogen Activator (uPA) in a plasmin-independent manner. Using site-directed mutagenesis we identified amino acid residues R594 and R595, located in the "stalk" region of integrin alpha6, as essential for cleavage. The cleavage site is located on the extracellular region of the protein between the beta-barrel domain and the thigh domain. Prostate cancer cells (PC3N) were stably transfected to overexpress the cleavable, wild-type (PC3N-alpha6-WT) or the non-cleavable form of integrin alpha6 (PC3N-alpha6-RR). The number of cells invading laminin 111- and laminin 332-coated filters by PC3N-alpha6-WT cells increased by threefold as compared to PC3N-alpha6-RR cells. Plasminogen activator inhibitor-1 (PAI-1) reduced the invasion of PC3N-alpha6-WT cells by approximately 42% through laminin 332-coated filters and plasmin inhibitor aprotinin had no significant effect. Linear cell migration increased production of integrin alpha6p in the PC3N-alpha6-WT cells and not in the PC3N-alpha6-RR cells and 32% of the PC3N-alpha6-WT cells migrated on laminin 111 in the linear migration assay as compared to the 5% PC3N-alpha6-RR cells. These data taken together suggest that the uPA-mediated cell surface cleavage of the alpha6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin.
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Project description:In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.
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Project description:The physiological importance of CD151 tetraspanin is known from somatic cells and its outside-in signalling through integrins was described. In male germ cells, two tetraspanins, CD9 and CD81, are involved in sperm-egg membrane fusion, and similarly to integrins, they occupy characteristic regions. We report here on a newly discovered presence of CD151 in sperm, and present its expression and distribution during spermatogenesis and sperm transition during the acrosome reaction. We traced CD151 gene and protein expression in testicular cell subpopulations, with strong enrichment in spermatogonia and spermatids. The testicular and epididymal localization pattern is designated to the sperm head primary fusion site called the equatorial segment and when compared to the acrosome vesicle status, CD151 was located into the inner acrosomal membrane overlying the nucleus. Moreover, we show CD151 interaction with ?6 integrin subunit, which forms a dimer with ?4 as a part of cis-protein interactions within sperm prior to gamete fusion. We used mammalian species with distinct sperm morphology and sperm maturation such as mouse and bull and compared the results with human. In conclusion, the delivered findings characterise CD151 as a novel sperm tetraspanin network member and provide knowledge on its physiology in male germ cells.
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Project description:Inflammatory bowel diseases (IBD) in humans are characterized by chronic inflammation and gastrointestinal tissue damage, caused by a combination of genetic and environmental factors. It has been largely documented that IBD frequently lead to colorectal cancers (CRC). The identification of causative factors of IBD is thus essential to understand CRC progression and develop therapeutical approaches. Models have been described in which molecular alterations are combined with inflammatory treatments in order to recapitulate IBD-associated CRC. Here, we describe a mouse line, α6fl/fl Villin-Cre, in which inactivation of the gene encoding the integrin alpha-6 subunit (ITGA6) specifically in the intestinal mucosa results into chronic inflammation and intestinal carcinogenesis. In these mice, the loss of integrin alpha-6 beta-4, a receptor mediating the attachment of epithelial cells to laminins, leads to epithelial detachment, hyperplasia, chronic inflammation, rectal prolapses, and ultimately adenocarcinomas. Alterations of differentiation affecting mucus secreting (goblet) cells as well as changes in expression of essential intestinal transcription factors were detected. Thus alpha-6 beta-4 integrin is a key factor for the maintenance of intestinal integrity and its loss may represent a risk factor for tumor progression associated with IBD.
2016-07-07 | GSE37749 | GEO
Project description:Functional heterologous expression of naturally-expressed and apparently functional mammalian ?6*-nicotinic acetylcholine receptors (nAChRs; where '*' indicates presence of additional subunits) has been difficult. Here we wanted to investigate the role of N-terminal domain (NTD) residues of human (h) nAChR ?6 subunit in the functional expression of h?6*-nAChRs. To this end, instead of adopting random mutagenesis as a tool, we used 15 NTD rare variations (i.e., Ser43Pro, Asn46Lys, Asp57Asn, Arg87Cys, Asp92Glu, Arg96His, Glu101Lys, Ala112Val, Ser156Arg, Asn171Lys, Ala184Asp, Asp199Tyr, Asn203Thr, Ile226Thr and Ser233Cys) in nAChR h?6 subunit to probe for their effect on the functional expression of h?6*-nAChRs.N-terminal ?-helix (Asp57); complementary face/inner ?-fold (Arg87 or Asp92) and principal face/outer ?-fold (Ser156 or Asn171) residues in the h?6 subunit are crucial for functional expression of the h?6*-nAChRs as variations in these residues reduce or abrogate the function of h?6h?2*-, h?6h?4- and h?6h?4h?3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal ?-helix) in h?6 subunit reduce h?6h?2*-nAChRs function those at residues Arg96 (?2-?3 loop), Asp199 (loop F) or Ser233 (?10-strand) increase h?6h?2*-nAChR function. Similarly substitution of NTD ?-helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in h?6 subunit increase the function of h?6h?4-nAChRs. All other variations in h?6 subunit do not affect the function of h?6h?2*- and h?6h?4*-nAChRs. Incorporation of nAChR h?3 subunits always increase the function of wild-type or variant h?6h?4-nAChRs except for those of h?6(D57N, S156R, R87C or N171K)h?4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in h?6 subunit drive the h?6h?4h?3-nAChRs into a nonfunctional state as at spontaneously open h?6(D57N, S156R or N171K)h?4h?3V9'S-nAChRs (V9'S; transmembrane II 9' valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of h?6h?4- and/or h?6h?4h?3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in h?6 subunit.Hence investigating functional consequences of natural variations in nAChR h?6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of h?6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in h?6 subunit that compromise h?6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.
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