Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line and mechanisms involved in adherence of these cells to laminin and kalinin.
ABSTRACT Keratinocytes attach to an underlying basement membrane by adhesion junctions called hemidesmosomes. We have characterized a cell line, RAC-11P/SD, established from a murine mammary tumor, which differentiates into squamous epithelium and forms well defined hemidesmosomes. These hemidesmosomes contain the integrin alpha 6 beta 4 as well as the hemidesmosomal plaque proteins BP230 and HD1 and are associated with a matrix containing kalinin and laminin. We examined how these cells adhere to laminin and to kalinin present in matrices as well as immunopurified kalinin. We show that adhesion to laminin is energy dependent but does not require an intact actin-containing cytoskeleton. The affinity for kalinin proved to be greater and binding to kalinin was still observed when cells had been treated with deoxyglucose and azide to inhibit metabolic energy. Binding to laminin (or fragment E8), but not to kalinin was partially blocked by a monoclonal antibody specific for the integrin alpha 6 subunit, and only in the initial phase of adhesion. The antibody efficiently blocked adhesion to laminin of cells treated with the microfilament disrupting drug cytochalasin B, but only partially blocked the adhesion of cytochalasin B-treated cells to kalinin, while adherence of cells treated with deoxyglucose and azide to kalinin was blocked completely. The integrin alpha 6 beta 4 is redistributed to the basal surface during adhesion and then is organized into ring-like structures when cells are bound to laminin and localized into hemidesmosomes in cells adhered to kalinin. We suggest that anti-alpha 6 hinders the binding of the alpha 6 beta 4 integrins to its ligands laminin and kalinin, but cannot prevent adhesion when clustering of the integrin has become complete. In addition, there is evidence that adhesion to kalinin is mediated by a second receptor, which associates with the actin-containing cytoskeleton. The presence of such a second receptor is suggested because the cells can spread on kalinin, but not when they have been treated with cytochalasin B. On laminin spreading does not occur, irrespective of whether cells have been treated with cytochalasin B or not. The integrin alpha 3 beta 1, which has been identified as a receptor for kalinin but not for laminin, is strongly expressed in RAC-11P/SD cells and it seems likely that this integrin is responsible for spreading of cells on kalinin.
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ABSTRACT: Therapeutic approaches aimed at targeting tumor surface markers using monoclonal antibodies provide a powerful strategy in cancer treatment. Here we report selection of single variable domains (VHH) of llama heavy chain antibodies, using a VHH-phage-display library. A reverse proteomic approach was used to identify the cognate proteins recognized by enriched VHH on HeLa cells. One of these VHH bound the integrin alpha 3 beta 1 (VLA-3) and was further characterized. Most interestingly, this VHH could inhibit VLA-3 mediated cell-matrix adhesion. Our approach provides a fast and efficient method to screen for novel cell surface markers on normal and tumor cells that may find diagnostic or therapeutic application in disease management or treatment.Molecular Immunology 05/2009; 46(10):2022-8. DOI:10.1016/j.molimm.2009.03.002 · 3.00 Impact Factor
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ABSTRACT: Laminin 5/laminin 332 (LN332) is an adhesion substrate for epithelial cells. After secretion of LN332, a regulated cleavage occurs at the carboxy-terminus of its alpha3 subunit, which releases a tandem of two globular modules named LG4/5. We show that the presence of the LG4/5 domain in precursor LN332 decreases its integrin-mediated cell adhesion properties in comparison with mature LN332. Whereas cell adhesion to the recombinant LG4/5 fragment relies solely on the heparan sulfate proteoglycan (HSPG) receptor syndecan-1, we reveal that both syndecan-1 and the alpha3beta1 integrin bind to precursor LN332. We further demonstrate that syndecan-1 mediated cell adhesion to the LG4/5 fragment and pre-LN332 allows the formation of fascin-containing protrusions, depending on the GTPases Rac and Cdc42 activation. Reducing syndecan-1 expression in normal keratinocytes prevents cell protrusions on pre-LN332 with subsequent failure of the peripheral localization of the alpha3beta1 integrin. We finally show that cell migration on pre-LN332 requires syndecan-1. Therefore, the LG4/5 domain in precursor LN332 appears to trigger intracellular signaling events, which participate in keratinocyte motility.Journal of Cellular Physiology 01/2008; 214(1):238-49. DOI:10.1002/jcp.21184 · 3.87 Impact Factor
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ABSTRACT: An increased expression of the integrin alpha6beta4 is correlated with a poor prognosis in patients with squamous cell carcinomas. However, little is known about the role of alpha6beta4 in the early stages of tumor development. We have isolated cells from mouse skin (mouse tumor-initiating cells [mTICs]) that are deficient in both p53 and Smad4 and carry conditional alleles of the beta4 gene (Itgb4). The mTICs display many features of multipotent epidermal stem cells and produce well-differentiated tumors after subcutaneous injection into nude mice. Deletion of Itgb4 led to enhanced tumor growth, indicating that alpha6beta4 mediates a tumor-suppressive effect. Reconstitution experiments with beta4-chimeras showed that this effect is not dependent on ligation of alpha6beta4 to laminin-5, but on the recruitment by this integrin of the cytoskeletal linker protein plectin to the plasma membrane. Depletion of plectin, like that of beta4, led to increased tumor growth. In contrast, when mTICs had been further transformed with oncogenic Ras, alpha6beta4 stimulated tumor growth, as previously observed in human squamous neoplasms. Expression of different effector-loop mutants of Ras(V12) suggests that this effect depends on a strong activation of the Erk pathway. Together, these data show that depending on the mutations involved, alpha6beta4 can either mediate an adhesion-independent tumor-suppressive effect or act as a tumor promotor.Molecular Biology of the Cell 12/2007; 18(11):4210-21. DOI:10.1091/mbc.E06-08-0720 · 4.55 Impact Factor