摘要:

Examination of rat hepatic cell lines has revealed a correlation between the differentiated state of the cells and the gap junctional proteins, or connexins, they express. The cell lines RLC (Gershenson et al, 1970) and FTO.2B (Killary et al, 1984) were examined and compared to primary adult hepatocytes for expression of fetal and adult hepatic antigens under various tissue culture conditions. Maximal expression of fetal antigens was observed in cells grown in serum-supplemented medium, on either tissue culture plastic or type IV collagen. Maximal expression of adult specific antigens was seen in cells grown in a hormonally defined medium containing heparin, on type I or type IV collagen. The cell line RLC strongly expressed fetal antigens, while FTO.2B expressed both fetal and adult antigens.These cell lines and another poorly differentiated hepatic cell line, WB-F344 (Tsao et al., 1984) were used to assess the developmental profile of mRNAs encoding isoforms of gap junctions: connexins 26, 32, and 43. The cell lines each transcribed mRNAs of all three connexins, as determined by transcriptional elongation analysis. By contrast, only certain of the connexin mRNAs could be detected in specific cell lines by Northern analysis: RLC expressed only connexin 43 mRNA; WB-F344 expressed connexin 26 and 43 mRNAs; and FTO.2B, the most differentiated cell line, expressed connexin 32 and 43 mRNAs. Selection among the connexin mRNAs appears to occur post-transcriptionally. Culture of the cell lines in hormonally defined medium vs. serum supplemented medium did not affect the patterns of connexin mRNA abundance. When the cell lines were cultured in hormonally defined medium containing heparin, however, the level of connexin mRNAs did vary: Connexin 26 mRNA increased in WB-F344 cells, and connexins 32 and 43 mRNAs increased in FTO.2B, but connexin 43 mRNA decreased in WB-F344 and RLC. The abundance of connexin mRNAs also varied when the cell lines were analyzed at different cell densities: connexin 43 mRNA increased with cell density in RLC and WB-F344, and connexin 26 mRNA peaked at an intermediate density and fell at higher cell densities in WB-F344. The differences in connexin mRNA expression among cell lines characteristic of different stages of hepatic differentiation, and the differences in regulation of connexin mRNAs in the hepatic cell lines, suggest distinct biological roles of the highly homologous proteins. Moreover, connexin gene expression may be a marker of hepatic development: as hepatocytes differentiate the proportions of connexin 43 then 26 mRNAs decrease while that of connexin 32 mRNA increases.

DOI:10.3109/15419069609010768

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these,α5β1,αIIbβ3, andαvβ3, the structural requirements for binding to fibronectin differ. In the present study. we report that two additional integrins,αvβ6. andαvβ5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments.αvβ5, likeαvβ3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study.αvβ5 only minimally supported adhesion to intact fibronectin. but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat. alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated byαvβ5, as well as adhesion mediated byαvβ6.αvβ5 andαvβ6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.

DOI:10.3109/15419069609010769

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The lymphocyte integrinα4β1 is the receptor for the Hep II domain and CS-1 site in fibronectin (Fn) as well as for VCAM-1. We recently showed that upon activation with anti-β1 mAb TS2/16,α4β1 also recognizes the RGD Fn sequence. To determine the physiological role of these multiple interactions, we have now studied some intracellular events induced by“resting”and activatedα4β1 binding to its different ligands. Analyses of actin and tubulin reorganization upon adhesion of B lymphoid cells to Fn fragments or VCAM-1 showed that VCAM-1, a 38 kDa fragment (Hep II+CS-1), and the CS-1 synthetic peptide induced formation of transient cytoplasmic projections; however, cells attached to a 58 kDa (Hep II) or 80 kDa (RGD) fragments remained rounded. Using transfilter assays, we showed that VCAM-1, 38 kDa and CS-1 also induced dose-dependent B cell migration mediated byα4β1. Furthermore, these three ligands, but not the 80 kDa fragment or a synthetic peptide (H1) containing a sequence from Hep II shown to bindα4β1, induced tyrosine phosphorylation of a 110 kDa protein. Activation ofα4β1 with TS2/16 inhibited the cytoplasmic protrusions and cell migration but did not affect the pattern of phosphorylation. Our results indicate that the variousα4β1 ligands induce different cellular responses. Most importantly they show thatα4β1 interaction with CS-1 is sufficient to trigger intracellular events in B cells. Furthermore, they suggest a regulation by the activation form of the receptor as well as by the ligand in events involving lymphocyte adhesion and migration.

DOI:10.3109/15419069609010770

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Laminins are a family of multifunctional basement membrane glycoproteins. Over the last years, many laminin isoforms have been characterized, which were shown to be composed of distinct combinations of variantαβandγchains. Some of these isoforms show remark-able tissue specificity, which suggests functional involvement in local processes. In this study the previously described mAb 4C7. which recognize epithelial basement membranes as well as endothelial basement membranes in lymphoid follicles, was identified as an anti-laminin-5 antibody. Using a set of mAbs against various variant laminin chains we established that specifically theγ2chain of laminin-5 was confined to the endothelial basement membranes of vessels in lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid tissue. Additionally. the expression of the known integrin receptors of laminin-5 was also examined. Theα6β4 integrin-receptor for laminin was found to be colocalized with the laminin-5γ2chain on the abluminal surface of endothelial cells, whereas theα3 integrin chain could not be detected in lymphoid follicles. This finding suggests that theα6β4 integrin (and not theα3β1 integrin) serves as a laminin-5 receptor on endothelial cells in the follicular compartment of lymphoid tissue. Furthermore,α6β4 was also found in the same punctuated pattern on FDCs as laminin-5. The function of the laminin-α6β4 complex in this particular localisation is still obscure, but a role in the maintainance of the follicular compartment via hemidesmosome-like attachment sites is postulated.

DOI:10.3109/15419069609010771

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

We report that cranin (dystroglycan) can become recruited to focal adhesions of cultured rat REF 52 fibroblasts and human aortic smooth muscle cells. Within mature focal adhesions, cranin was present within the plaque region defined byβ1 integrin, vinculin and phosphotyrosine staining, but occupied a larger domain corresponding to, the terminal segments of stress fibers that was more precisely co-extensive with the cytoskeletal proteins alpha-actinin, utrophin and aciculin. When REF 52 fibroblasts were plated on different substrata in the absence of protein synthesis and secretion in serum-free medium, focal clusters of cranin readily formed within 2 hours on matrix proteins that bind cranin directly (laminin or agrin) which were maintained as the focal adhesions became mature. In contrast, cranin failed to become targeted to cell-substratum attachment sites, either at early or later times. when cells were plated on a variety of other substrata that elicit formation of focal adhesions but do not bind cranin directly (fibronectin, vitronectin, collagen type IV, or anti-βintegrin antibody TS2/16). These data strongly suggest that targeting of cranin to focal adhesions was dependent upon the presence of an extracellular ligand capable of binding cranin directly. How-ever, some cultured nonmuscle cell lines (e.g., human umbilical vein endothelial cells, NIH 3T3 and CHO cells) failed to localize cranin to focal adhesions, even when plated on laminin. Cranin was also enriched at cell-cell adherens-type junctions of human normal breast MCF-10 epithelial cells, and at growth cones of E17 rat hippocampal axons. That cranin can become targeted to sites of cell-cell and cell-substratum contact in diverse cell types supports the hypothesis that cranin may be involved in mediating or regulating cell adhesion. The absence of muscle-specific and synapse-specific proteins within fibroblasts and epithelial cells provides a different context for thinking about cranin (dystroglycan) that may aid in discerning general principles of its structure and function.

DOI:10.3109/15419069609010772

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

CD9 is a member of the tetraspan (TM4) family of proteins and is abundantly expressed in the epidermis. As CD9 forms complexes withβ1integrins and the integrins are known to regulate keratinocyte behaviour, we investigated CD9 expression and function in human epidermal keratinocytes. CD9 was present in all the living layers of the epidermis, whereas theβ1integrins were largely confined to the basal layer; the same relative distribution was found in stratified cultures of keratinocytes. There was extensive co-localisation of CD9 andβ1integrins on microvilli and at cell-cell borders of basal keratinocytes; however, in contrast to the integrins, CD9 was not found in focal adhesions. CD9 was detected inβ1integrin immunoprecipitates and also in immunoprecipitates of CD44 and syndecan, but not of cadherins. CD9 was associated withα3β1but notα5β1; small amounts of CD9 also co-immunoprecipitated with antibodies toα2β1, andα6β4. Antibodies to CD9 did not affect the proportion of keratinocytes that adhered to laminin 1, type IV collagen and fibronectin, but did inhibit motility of keratinocytes on tissue culture plastic. Like antibodies to theβ1integrin subunit, anti-CD9 inhibited suspension-induced terminal differentiation. These results suggest that CD9 may play a role in regulating keratinocyte motility and differentiation.

DOI:10.3109/15419069609010773

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Loss of expression of specific integrins is a feature of poorly differentiated oral squamous cell carcinomas (SCCs) and cell lines derived from them. In order to test whether there is a direct link between reducedα6β4integrin expression and abnormal keratinocyte growth and differentiation we‘repaired' an SCC line. H376, by transfection of theβ4integrin subunit. We analysed five independentβ4transfectant clones and compared them with four empty vector control clones and with the parental cell line. Elevated cell surface expression ofα6β4was not correlated with changes in anchorage dependent or independent growth and was not sufficient to induce expression of the terminal differentiation marker, involucrin. Introduction of theβ4integrin subunit did not have a major effect on cell adhesion to laminin 1 or 5 and did not result in formation of stable anchoring contacts. We conclude that loss ofα6β4is not directly responsible for the abnormal behaviour of the H376 cell line.

DOI:10.3109/15419069609010774

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins inE. coll. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5μg/well compared to plasma-derived vitronectin which reached at 0.2μg/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.

DOI:10.3109/15419069609010775

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine spongeGeodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5′-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.

DOI:10.3109/15419069609010776

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Cultured epithelial cells interact massively, rapidly and stereospecifically with the {011} faces of calcium (R,R)-tartrate tetrahydrate crystals. It was suggested that the massive rapid adhesion represents an exaggerated and isolated form of the first initial events in the attachment of cultured cells to conventional tissue culture surfaces (Hanein, et al., Cells and Materials, 5, 197–210: 1995). Attachment is however not followed by normal cell spreading and development of focal adhesions, but results in massive cell death. In this study, the fate of the crystal-bound cells was characterized by electron microscopy, flow cytometry and microscopic morphometry and was found to display the characteristics of physiological cell death. We show that the direct interaction with the highly homogenous and repetitive {011} faces per se does not trigger the transduction of lethal transmembrane signals. We suggest that the excessive direct interactions between the cell membrane and the crystal. by impairing cell motion, prevent the evolution of RGD-dependent cell adhesion. This implies that the deprivation of proper extracellular matrix (ECM)-receptor contacts of substrate-attached epithelial cells eventually triggers physiological cell death.

DOI:10.3109/15419069609010777

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor