Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V

1996 ◽  
Vol 109 (7) ◽  
pp. 1865-1874 ◽  
Author(s):  
F. Ruggiero ◽  
J. Comte ◽  
C. Cabanas ◽  
R. Garrone
Keyword(s):  
Cell Adhesion ◽  
Cell Line ◽  
Binding Sites ◽  
Triple Helix ◽  
Helical Conformation ◽  
Epithelial Cell Line ◽  
Ht1080 Cell ◽  
Native Collagen ◽  
Beta 1 Integrin ◽  
Hbl100 Cell

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50–200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

scholarly journals Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell-cell adhesion.

1996 ◽  
Vol 135 (6) ◽  
pp. 1643-1654 ◽  
Author(s):  
S Islam ◽  
T E Carey ◽  
G T Wolf ◽  
M J Wheelock ◽  
K R Johnson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Squamous Cell ◽  
Epithelial Cell Line ◽  
Squamous Epithelial Cell ◽  
Squamous Carcinoma Cells ◽  
E Cadherin ◽  
Cell Cell

E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent, homotypic cell-cell adhesion and plays an important role in maintaining the normal phenotype of epithelial cells. Disruption of E-cadherin activity in epithelial cells correlates with formation of metastatic tumors. Decreased adhesive function may be implemented in a number of ways including: (a) decreased expression of E-cadherin; (b) mutations in the gene encoding E-cadherin; or (c) mutations in the genes that encode the catenins, proteins that link the cadherins to the cytoskeleton and are essential for cadherin mediated cell-cell adhesion. In this study, we explored the possibility that inappropriate expression of a nonepithelial cadherin by an epithelial cell might also result in disruption of cell-cell adhesion. We showed that a squamous cell carcinoma-derived cell line expressed N-cadherin and displayed a scattered fibroblastic phenotype along with decreased expression of E- and P-cadherin. Transfection of this cell line with antisense N-cadherin resulted in reversion to a normal-appearing squamous epithelial cell with increased E- and P-cadherin expression. In addition, transfection of a normal-appearing squamous epithelial cell line with N-cadherin resulted in downregulation of both E- and P-cadherin and a scattered fibroblastic phenotype. In all cases, the levels of expression of N-cadherin and E-cadherin were inversely related to one another. In addition, we showed that some squamous cell carcinomas expressed N-cadherin in situ and those tumors expressing N-cadherin were invasive. These studies led us to propose a novel mechanism for tumorigenesis in squamous epithelial cells; i.e., inadvertent expression of a nonepithelial cadherin.

Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin

1994 ◽  
Vol 267 (2) ◽  
pp. G235-G244 ◽  
Author(s):  
P. Singh ◽  
S. Narayan ◽  
R. B. Adiga
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Tyrosine Kinases ◽  
Intestinal Cell ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Intact Cells ◽  
Intestinal Cell Line ◽  
Endogenous Proteins

Intracellular mechanisms that mediate mitogenic effects of gastrin remain largely unknown. The present studies were designed to examine if protein tyrosine kinases (PTKs) mediate growth effects of gastrin on a rat intestinal epithelial cell line (IEC-6 cells). Gastrin (< 10 nM) was mitogenic for IEC-6 cells. PTK activity of cell membranes was stimulated in response to 0.01-10.0 nM and 0.05-10.0 microM gastrin in a double biphasic manner. Cells labeled with H3(32)PO4 were stimulated with gastrin and cellular proteins immunoprecipitated with phosphotyrosine antibodies. Endogenous proteins were phosphorylated in a dose- (100% effective dose = 0.1-1.0 nM) and time-dependent manner; at > 10 nM gastrin, the second peak of response was not measured in intact cells. Thus the growth and phosphorylation response of intact cells to gastrin was similar. Both high [dissociation constant (Kd) = 1 nM]- and low (Kd = approximately 0.1 microM)-affinity gastrin binding sites are present on IEC-6 cells. The results of the present study suggest that occupancy of both high- and low-affinity gastrin-binding sites can potentially activate membrane-associated PTKs. However, in intact cells, occupancy of low-affinity sites apparently attenuates kinase activity resulting in reduced protein phosphorylation. Eight protein bands [with relative molecular weight (M(r)) of 32-145 kDa] were tyrosine phosphorylated in intact cells in response to 0.1-1.0 nM gastrin, including two pp60 src-like proteins (with M(r) of 54 and 62 kDa). Thus the growth response pattern of a target cell to gastrin may depend on the stimulation of kinases and other factors (phosphatases?) that phosphorylate and/or dephosphorylate several proteins including c-src-like proteins in a dose-dependent manner.

Hypertonic induction of the cell adhesion molecule beta 1-integrin in MDCK cells

1997 ◽  
Vol 273 (3) ◽  
pp. C902-C908 ◽  
Author(s):  
D. Sheikh-Hamad ◽  
W. N. Suki ◽  
W. Zhao
Keyword(s):  
Cell Adhesion ◽  
Osmotic Stress ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Organic Osmolytes ◽  
Heparin Binding ◽  
Renal Epithelial Cell ◽  
Beta 1 Integrin

Cells of many organisms adapt to osmotic stress by accumulating compatible organic osmolytes. In Madin-Darby canine kidney (MDCK) cells, a renal epithelial cell line widely used as a culture model for the study of osmotic regulation in mammals, extracellular hypertonicity induces genes responsible for the accumulation of organic osmolytes. We have recently cloned from these cells a partial cDNA corresponding to a member of the transmembrane 4 superfamily (tetraspan) family, CD9 antigen, and demonstrated its induction by hypertonicity [D. Sheikh-Hamad, J. D. Ferraris, J. Dragolovich, H. G. Preuss, M. B. Burg, and A. Garcia-Perez. Am. J. Physiol. 270 (Cell Physiol. 39): C253-C258, 1996]. An association between CD9 and potential regulatory proteins, such as the heparin-binding epidermal growth factor-like protein and the cell adhesion molecule beta 1-integrin, has been reported. Here, using beta 1-integrin-specific monoclonal antibodies and an antisense oligonucleotide probe, we demonstrate expression and hypertonic induction of beta 1-integrin in MDCK cells. Induction of the mRNA and protein occurs in 2 h and is maximal at 6 h, consistent with a regulatory role in the adaptation to osmotic stress. In addition, we show that accumulation of organic osmolytes markedly attenuates the hypertonic induction of the mRNA, a feature shared with genes involved in hyperosmotic stress response. Finally, we demonstrate that CD9 and beta 1-integrin are expressed in association at the cell membrane. Our findings suggest the existence of a cluster of integral membrane proteins that includes but may not be limited to CD9 and the adhesion molecule beta 1-integrin, which may play a role in the adaptation of kidney cells to osmotic stress, possibly at the regulatory level.

Ultrastructure of NPC/HK1 cells heterotransplanted in athymic nude mice

1982 ◽  
Vol 40 ◽  
pp. 236-237
Author(s):  
E.C. Chew ◽  
C.L. Li ◽  
D.P. Huang ◽  
H.C. Ho ◽  
L.S. Mak ◽  
...  
Keyword(s):  
Cell Line ◽  
Nude Mice ◽  
Biopsy Specimen ◽  
Epithelial Cell Line ◽  
Present Communication ◽  
Recurrent Tumour ◽  
Athymic Nude Mice ◽  
Cell Line Establishment ◽  
Fine Structures ◽  
Well Differentiated

An epithelial cell line, NPC/HK1, has recently been established from a biopsy specimen of a recurrent tumour of the nasopharynx which was histologically diagnosed as a moderately to well differentiated squamous cell carcinoma. A definite decrease in the amount of tonofilaments and desmosomes in the NPC/HK1 cells during the cell line establishment was observed. The present communication reports on the fine structures of the NPC/HK1 cells heterotraneplanted in athymic nude mice.

An Established Epithelial Cell Line (NPC/HK1) from a Nasopharyngeal Carcinoma - II. A Sem Study

1982 ◽  
Vol 40 ◽  
pp. 224-225
Author(s):  
Li C.L. ◽  
Chew E.C. ◽  
Huang D.P. ◽  
Ho H.C. ◽  
Mak L.S. ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Epithelial Cell ◽  
Surface Morphology ◽  
Cell Line ◽  
Epithelial Cell Line ◽  
Present Communication ◽  
Cells In Culture ◽  
Sem Study ◽  
Well Differentiated

An epithelial cell line, NPC/HK1, has recently been successfully established from a nasopharyngeal carcinoma of the moderately to well differentiated squamous type. The present communication reports on the surface morphology of the NPC/HK1 cells in culture.

A New Human Breast Tumor Cell Line

1975 ◽  
Vol 33 ◽  
pp. 388-389
Author(s):  
R.E. Nordquist ◽  
R.M. Wasik ◽  
P.J. Riggs ◽  
P.L. Munson ◽  
F.B. Schafer
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Calf Serum ◽  
Tumor Cell Line ◽  
Electron Microscopic Examination ◽  
Epithelial Cell Line ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Excised Tissue ◽  
Caucasian Female

An infiltrating ductal cell carcinoma was removed from the breast of a postmenopausal Caucasian female. The excised tissue was divided into three parts; one part for electron microscopy, one part for tissue culture and the remainder frozen for immunological studies.The tissue for culture was minced finely with sterile razor blades and cultured in Falcon flasks containing Eagel's MEM supplemented with 10% heat denatured fetal calf serum. The tissue for electron microscopy was fixed in 6.25% glutaraldehyde in 0.1 M PO4 buffer plus 5% sucrose and postfixed in 1% OsO4 in the same buffer. The fixed tissue was dehydrated in graded ethanol and embedded in Spurr.The tissue which was cultured began to grow out after approximately six weeks and became a continuous epithelial cell line which was designated BOT-2 (Breast Original Tumor). Electron microscopic examination revealed that these cells had epithelial characteristics, i.e. the presence of tonofilaments and well formed desmosomes.

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