Transformation of chicken embryo fibroblasts by v-src uncouples beta1 integrin-mediated outside-in but not inside-out signaling

Abstract

Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.

FIG. 1

Cells were allowed to spread and then were treated for 6 h with CSAT monoclonal antibody at 30 μg/ml. CSAT is a function-blocking anti-chicken β1 antibody. (A) Normal CEF. (B) Normal CEF treated with CSAT. (C) v-src-transformed CEF. (D) v-src-transformed CEF treated with CSAT.

FIG. 2

Adhesion assays for normal and v-src-transformed CEF obtiained by using the spinning disk assay. (A) Mean adhesion strength for normal CEF and v-src-transformed CEF [CEF(SRA)] for adhesion to fibronectin (160 ng/cm²) for 15 min in the absence or presence of CSAT antibody (∗; <2 dynes/cm²). Error bars indicate the standard deviation (n = 3). (B) Mean adhesion strength for normal CEF (white bars) and v-src-transformed CEF (black bars) for cells were allowed to adhere to fibronectin for 6 or 48 h before analysis by using the spinning disk. Error bars indicate the standard deviation (n = 3).

FIG. 3

Chemical cross-linking of fibronectin-bound α5β1. Level of substrate-bound β1 and α5 integrins after 1 h of attachment of cells to fibronectin was detected by Western blot of β1 and α5 integrins. X-link, recovered integrin subunits after cleavage of the cross-linker; Supernatant, extract of non-cross-linked integrins; CEF, normal cells; SRA, v-src-transformed cells.

FIG. 4

Chemical cross-linking inhibits proteolytic removal of fibronectin. v-src-transformed CEF were plated on fibronectin-coated or chemically cross-linked fibronectin-coated cover glasses. (A) Cells plated for 48 h on cross-linked fibronectin and stained with HFN7.1 monoclonal antibody showing diffuse staining of the initial human fibronectin. (B) Same field as panel A but stained with ethidium homodimer. (C) Cells plated on normal fibronectin for 48 h and stained with HFN7.1 showing that most of the fibronectin has been removed by the cells. (D) Same field as panel C but stained with ethidium homodimer. (E and F) Quantification of residual fibronectin levels after 48 h by using HFN7.1 antibody (E) or rabbit anti-fibronectin (F). Fn, fibronectin only; X-Fn, cross-linked fibronectin only; Fn-C, fibronectin plus transformed cells; X-Fn-C, cross-linked fibronectin plus transformed cells. (G and H) Modified enzyme-linked immunosorbent assay showing the removal of normal (●) or cross-linked (○) fibronectin by transformed cells as a function of fibronectin density by using HFN7.1 antibody (G) and rabbit anti-fibronectin antibody (H). Error bars indicate the standard deviation (n = 3).

FIG. 5

Effects of fibronectin cross-linking and hyaluronidase on the morphology of v-src transformed CEF. (A) Plated for 12 h in the absence of serum on cover glasses coated with fibronectin. (B) Plated for 12 h in the absence of serum on cover glasses coated with glutaraldehyde cross-linked fibronectin. (C) Plated for 48 h on fibronectin. (D) Plated for 48 h of glutaraldehyde cross-linked fibronectin. (E) Plated for 7 days on glutaraldehyde cross-linked fibronectin in the presence of hyaluronidase and phalloidin stained. (F) Corresponding phase micrograph (i.e., for panel E). (G) Plated for 7 days on fibronectin in the presence of hyaluronidase and phalloidin stained. (H) Corresponding phase micrograph (i.e., for panel G).

FIG. 6

v-src-transformed cells can assemble a fibronectin matrix. v-src-transformed CEF were incubated for 48 h on coated cover glasses. (A) Cross-linked fibronectin stained with anti-chicken fibronectin monoclonal antibody B3D6. (B) Same field as panel A but stained with ethidium homodimer. (C) Non-cross-linked fibronectin stained with B3D6. (D) Same field as panel C but stained with ethidium homodimer.

FIG. 7

Mean fluorescence index (calculated as geometric mean integrin – geometric mean control/geometric mean control) as determined by flow cytometry for expression of β1 and β3 integrin after 48 h on normal fibronectin (black bars) or glutaraldehyde cross-linked fibronectin (white bars).

FIG. 8

Specific induction of FAK phosphorylation by α5β1-mediated adhesion to fibronectin. Cells were serum starved overnight, trypsinized, neutralized with soybean trypsin inhibitor, and plated on fibronectin-coated plates at densities of 0, 50, 100, 150, and 350 ng/cm² for 60 min at room temperature (without phosphatase inhibitors). (A) Normal CEF extracts were analyzed by for phosphorylation of FAK(Y397), FAK(Y407), FAK(Y577), FAK(Y861), FAK(Y925), and total FAK protein. (B) Quantification of relative phosphorylation of Y397 in CEF; phosphorylated pY397/total FAK for each point. (C) Similar quantification of relative phosphorylation of Y861. (D) v-src-transformed CEF extracts were analyzed for specific phosphorylation of FAK(Y397), FAK(Y407), FAK(Y577), FAK(Y861), FAK(Y925), and total FAK. (E) Quantification of relative phosphorylation of Y397, Y861, and Y925 normalized for total FAK in each extract. (F) Similar quantification of Y407 and Y577. Error bars indicate the standard deviations (n = 3). Symbols: ▪, pY397; ▾, pY407; ▪, pY577; ○, pY861; ▾, pY925.

FIG. 9

Adhesion-dependent control of cell growth rate in the absence of serum. Cells were plated on substrates for 48 h in serum-free medium. BrdU was added from 48 to 72 h, the cells were stained with anti-BrdU, and the proportion of BrdU positive nuclei was counted. In experiment 1, normal CEF were plated on polylysine (1-CEF PL) or fibronectin (1-CEF-Fn). In experiment 2, normal CEF were plated on normal (2-CEF-Fn) or cross-linked (2-CEF-X-Fn) fibronectin, and v-src-transformed CEF were plated on normal (2-src-Fn) or cross-linked (2-src-X-Fn) fibronectin. Error bars indicate the standard deviations (n = 3).