Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.

Abstract

Abstract. We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts, mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates, mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibrol~last fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.T HE interaction of cells with solid substrates is important for their anchorage, proliferation, migration, and differentiation. Cells can attach, spread, and migrate on a variety of extracellular glycoproteins including fibronectin, laminin, vitronectin, and collagen. These interactions occur through specific cell surface receptors. Many of these cell adhesion receptors belong to a large superfamily of related cell surface complexes called the integrins or the cytoadhesins (for reviews see 3,11,26,30,33,37,52). The integrins are noncovalent, heterodimeric glycoprotein complexes that consist of a 140,000-180,000-D alpha subunit and a 105,000-125,000-D beta subunit. The alpha subunit often consists of a small, transmembrane polypeptide of 20,000 D disulfide bonded to a larger, extracellular polypeptide ...

Cancer–Stromal Interactions in Scirrhous Gastric Carcinoma

“…The upregulation of α2-, α3-, and α5-integrin by TGFβ under hypoxic conditions may be one of the mechanisms responsible for the high metastatic potential of scirrhous gastric cancer cells. The adhesion polypeptides, Tyr-Ile-Gly-Ser-Arg (YIGSR) and Arg-Gly-Asp (RGD), are the cell binding domain of the extra-cellular matrix (ECM) [42,43], and these peptides interfere with cell binding by integrins [44]. The adhesion polypeptides, YIGSR and RGD inhibit the adhesion ability of β1-integrin on gastric cancer cells.…”

Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells

“…It has been shown that mesenchymal matrix proteins such as fibronectin and collagen-I can induce taxol resistance in a 2D culture system by a β1-integrin mechanism that depends on Akt/PKB (Aoudjit and Vuori, 2001). Therefore, we tested whether the 3D matrix-induced changes in resistance or sensitivity to taxol treatment could be prevented or reversed in the presence of the β1-integrin function-blocking antibody mAb13 (Akiyama et al, 1989). In addition, to test whether the effects observed were dependent on the matrix configuration (3D matrix) versus the matrix composition (2D matrix), we tested the taxol responses in the presence or absence of β1-integrin inhibition using 2D plastic control as a contrasting substrate.…”

“…StartingBlock™ T20 (TBS) Blocking Buffer was purchased from Pierce (Rockford, IL). Functional blocking anti β1-integrin antibody, mAb13 (Akiyama et al, 1989), was kindly provided by Kenneth M. Yamada (NIH/NIDCR/CDBRB, Bethesda MD). Purified Rat IgG used as non-specific antibody control was from Sigma Aldrich.…”

αV integrin promotes in vitro and in vivo survival of cells in metastatic melanoma

“…Cells were collected by centrifugation, exposed to saturating concentration of antibodies against ␣V integrin (L230), ␣1 (Pharmingen, San Diego, CA), ␣2 integrin (12F1), 28 ␣4 integrin (Pharmingen), ␣5 integrin (mAb 16), 29 ␤1 integrin (R-322, rabbit polyclonal), 30 ␤3 integrin (Southern Biotech, Birmingham, U.K.) or ␣V␤5 integrin (PIF6) 31 in 1% FCS/PBS for 30 min at 4°C. For labeling, cells were incubated with rabbit antimouse (1:20 dilution), rabbit antirat (1:100 dilution) or swine antirabbit (1:20 dilution) IgG coupled to fluorescein (all from Dako, Glostrup, Denmark) for 30 min at 4°C, washed twice with PBS and suspended in the same buffer.…”