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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02921

Journal of Cell Science 119, 1886-1895 (2006)
Published by The Company of Biologists 2006
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Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of {alpha}2ß1 integrin

Zhi-Gang Zhang1,2, Ingo Bothe1,2, Frank Hirche1, Manon Zweers1, Donald Gullberg3, Gabriele Pfitzer4, Thomas Krieg1,5, Beate Eckes1 and Monique Aumailley2,5,*

1 Department of Dermatology, Medical Faculty, University of Cologne, 50931 Cologne, Germany
2 Center for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany
4 Institute for Vegetative Physiology, Medical Faculty, University of Cologne, 50931 Cologne, Germany
3 Department of Biomedicine, Division of Physiology, University of Bergen, 5020 Bergen, Norway
5 Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany


Figure 1
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  		Fig. 1. Adhesion of wild-type and {alpha}2-deficient fibroblasts to extracellular matrix proteins. (A) Equal numbers of wild-type (white columns) and {alpha}2-deficient (black columns) fibroblasts were seeded on triplicate wells coated with optimal concentrations of collagen I (Col I, 40 µg/ml), collagen IV (Col IV, 10 µg/ml), laminin 1 (LM 1, 20 µg/ml) and laminin 5 (LM 5, 5 µg/ml) as indicated. After 30 minutes, adhesion of {alpha}2-deficient fibroblasts to collagen I is slightly lower than that of wild-type cells. The mean average of triplicate wells and s.d. are shown. (B,C) At the end of the adhesion assay, wild type (+/+) and integrin {alpha}2-deficient (-/-) fibroblasts adhering to collagen I were photographed under phase-contrast microscopy.

 

Figure 2
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  		Fig. 2. Adhesion of mouse fibroblasts to collagen I after siRNA downregulation of integrin {alpha}2 expression. Subconfluent wild-type mouse fibroblasts were transfected once (A) or twice (B) with 100 nM each siRNA2 and siRNA3 targeting integrin {alpha}2 subunit or an unrelated siRNA as described in the Materials and Methods. Graphs show adhesion assays (30 minutes) to increasing concentrations of collagen I performed 76 hours after a single (A) or two successive (B) transfections. Equal numbers of control (unrelated siRNA; open symbols) and specific siRNA-transfected ({alpha}2-specific; closed symbols) fibroblasts were seeded on the collagen coats. The mean average of adherent cells in triplicate wells and s.d. are shown. Equal aliquots of fibroblasts transfected with control (c) and specific siRNAs (si) were lysed for immunoblotting analysis of integrin {alpha}2 subunit expression. The blots shown to the right of the graphs correspond to the fibroblasts used for the adhesion assays (arrow indicates the position of integrin {alpha}2 subunit). Densitometry measurement of band intensity in the blots shown in A and B indicate that expression of the {alpha}2 subunit is downregulated to 26% and 19%, respectively, of the corresponding controls. Molecular size markers are indicated in kDa on the left of the blots.

 

Figure 3
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  		Fig. 3. The {alpha}2ß1 integrin is important for initial fibroblast adhesion to collagen I. Equal numbers of wild-type (open circles) and {alpha}2-deficient (closed circles) fibroblasts were seeded on triplicate wells coated with increasing concentrations of collagen I and for different periods of time as indicated. After 15 minutes, adhesion of {alpha}2-deficient fibroblasts to collagen I was markedly decreased compared with normal cells and partially rescued at longer incubation times. The means ± s.d. of triplicate wells are shown.

 

Figure 4
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  		Fig. 4. The {alpha}1ß1 integrin has a major role for fibroblast adhesion to collagen IV and laminin 1, and to a lesser extent to collagen I. Wild-type and {alpha}2-deficient mouse fibroblasts were seeded on wells coated with collagen I, collagen IV, laminin 1 and laminin 5, as indicated, in the absence (-) or the presence of function-blocking antibodies (20 µg/ml) against integrin {alpha}1, {alpha}2 and ß1 subunits. After 30 minutes of incubation, the extent of cell adhesion was determined as in Fig. 2. The results were expressed as a percentage of the control without antibodies set as 100%.

 

Figure 5
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  		Fig. 5. The {alpha}2ß1 integrin is indispensable for keratinocyte adhesion to collagens. (A-C) Lysates of primary keratinocytes (K) and fibroblasts (F) from wild type (+/+) and {alpha}2-deficient (-/-) mice were immunoblotted with primary antibodies against the integrin {alpha}1 (A), {alpha}2 (B) and {alpha}11 (C) subunit as indicated. (D) Equal numbers of wild-type (white columns) and {alpha}2-deficient (black columns) keratinocytes were seeded on triplicate wells coated with collagen I (Col I), collagen IV (Col IV), laminin 1 (LM 1) and laminin 5 (LM 5) as described in Fig. 1A. Compared with wild-type cells, there is nearly no adhesion of {alpha}2-deficient keratinocytes to collagen I and collagen IV whereas adhesion of both strains of keratinocytes to laminin 1 and laminin 5 is similar. (E) Wild-type mouse keratinocytes were seeded on coats of collagen I (Col I), collagen (IV) and laminin 1 (LM 1) in the absence (- ab) or presence (+ ab) of monoclonal antibody against integrin {alpha}2 subunit. The antibody inhibits keratinocyte adhesion to collagens but not laminin 1.

 

Figure 6
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  		Fig. 6. The {alpha}2ß1 integrin is important for fibroblast force transmission within 3D collagen networks. (A) Equal numbers of wild type (WT) and integrin {alpha}2-deficient ({alpha}2-/-) fibroblasts were seeded within triplicate gels of collagen I. The time course of collagen gel contraction was monitored by photographing the gels at successive time intervals as indicated. Note that gel contraction is delayed for integrin {alpha}2-deficient fibroblasts. (B) The gel diameters were measured at successive time points and used to calculate the gel areas, which are plotted as a percentage of the original area at the onset of the experiment. Each point represents the mean ± s.d. of three independent experiments. (C) The isometric forces generated by wild-type (circles) and integrin {alpha}2-deficient fibroblasts (squares) embedded in tethered collagen lattices were monitored over time as indicated. Each data point represents the mean ± s.d. of four measurements.

 

Figure 7
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  		Fig. 7. The {alpha}2ß1 integrin plays a role in the distribution of focal adhesions in fibroblasts. Wild type (WT) and {alpha}2-deficient ({alpha}2-/-) fibroblasts were seeded on glass coverslips coated with collagen I (40 µg/ml). After 60 minutes of adhesion, adherent cells were fixed and double-stained for fibrillar actin (A,D) and vinculin (B,E). The cells were observed by confocal microscopy and images were captured using single channel excitation. The two images were superimposed (C,F) using Photoshop (Adobe). Numerous vinculin-positive focal adhesions are present on the entire basal surface of {alpha}2-deficient fibroblasts, but not of wild-type fibroblasts (asterisk). Inserts in C and F represent higher magnifications of focal adhesions at actin stress fibre termini (arrows).

 

Figure 8
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  		Fig. 8. Activation of Rho GTPases upon fibroblast adhesion to collagen I. Wild-type (WT) and {alpha}2-deficient (-/-) fibroblasts were serum-starved for 24 hours and subsequently seeded at the same cell density on collagen I. After 60 to 120 minutes of adhesion the cells were lysed and active, GTP-bound RhoA, Rac1 and Cdc42 were pulled down as described in the Materials and Methods. (A) Representative blots of total (lysate) and GTP-bound RhoA, Rac1 and Cdc42 (pull-down) after 90 (lane 1) and 120 (lane 2) minutes of adhesion to collagen for wild-type (WT) and {alpha}2-deficient (-/-) fibroblasts. (B) Band intensity of total and GTP-bound proteins were measured by densitometry on blots from different experiments and the relative amounts of active, GTP-bound proteins were normalized to those of the corresponding lysates (arbitrary units). The graphs show the results from different experiments performed for 60, 90 and 120 minutes as indicated below each set of columns. White columns, control fibroblasts; black columns, {alpha}2-deficient fibroblasts.

 

Figure 9
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  		Fig. 9. Reduction of wound area is normal in integrin-{alpha}2-null mice. Full-thickness wounds (6 mm in diameter) were created on the backs of {alpha}2-deficient (black bars) and wild-type mice (white bars). Wounds (n>=16 per time point and genotype) were photographed at the indicated time points and each wound area was calculated using Adobe Photoshop software. Results are expressed as means ± s.d.

 

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© The Company of Biologists Ltd 2006