CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling - PubMed (original) (raw)

CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling

Chloé C Féral et al. J Cell Biol. 2007.

Abstract

Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis.

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Figures

Figure 1.

Figure 1.

CD98hc deficiency blocks Fn fibril assembly in vivo. (A) Fn assembly was analyzed by staining WT (left) and CD98hc-null (right) tumors with an anti-Fn antibody. Fn staining is in red, whereas the nuclei are in green. Staining of two independent tumors per genotype is shown. Although CD98hc-null tumors do not show Fn assembly, Fn fibrils can be seen outside the tumor region (arrows). (B) Neovascularization during tumor development. Sections of tumors (WT and CD98hc null) were harvested and stained to reveal blood vessels. Whole mounts of tissue samples were stained with fluorescent IB4 lectin and analyzed by laser-scanning confocal microscopy. Sections (z series) were merged, and the resulting images are shown. (C) Immunofluorescent staining of WT and CD98hc-null tumors for the endothelial cells marker PECAM-1 (red) and smooth muscle cells marker α-smooth muscle (SM) actin (green). CD98hc-null tumors exhibit poor endothelial cell and smooth muscle cell organization. Bars: (A and C) 50 μm; (B) 200 μm.

Figure 2.

Figure 2.

Characterization of CD98hc- conditional allele. (A) Conditional allele strategy. Representation of WT CD98hc locus and the targeted allele after CRE recombination (see Materials and methods). LoxP sites are depicted as triangles and Flp sites as ovals. (B) PCR analysis of genomic DNA after CRE recombination on MEFs. After the recombination occurred, CD98hc exons 1 and 2 were deleted. Expected sizes are 1.9 kb (floxed or WT allele; i.e., before CRE) and 382 bp (knockout allele; i.e., after CRE). (C) Flow cytometry analysis of cell surface expression of endogenous CD98 and of integrin subunits in CD98hc-deficient cells. WT cells are shown in dotted line and CRE cells in filled histogram. These FACS data show deletion of CD98hc in CRE cells (i.e., CD98hc null) and no change in integrin repertoire as a result of this deletion. Control is staining with irrelevant IgG.

Figure 3.

Figure 3.

Deletion of CD98hc blocks Fn assembly in vitro. (A) WT (left) and CD98hc-deficient (right) MEFs were incubated for 48 h on 4 μg/ml Fn-coated coverslips. Fixed cells were stained for Fn (green) and actin (red). Nuclei appear blue. Note the absence of Fn fibrils in the CD98hc-null cells. Bars, 25 μm. (B) Fn assembly was also evaluated biochemically by analyzing the DOC-soluble and -insoluble portions of the cell matrix in both WT and CD98hc-null MEFs. Cells were cultured in complete medium (i.e., containing Fn). CD98hc-null MEFs were impaired in their ability to incorporate Fn in both their DOC-insoluble and -soluble pools in the presence of exogenous Fn. Level of Fn in the conditioned medium was similar for both WT and CD98hc-null cells.

Figure 4.

Figure 4.

CD98hc-null cells' loss of assembly is not due to deficient Fn biosynthesis or reduced integrin affinity. (A) Fn biosynthesis was evaluated biochemically as described in Fig. 3. Levels of soluble Fn produced by WT and CD98hc-null cells (conditioned medium) were analyzed. Cells were cultured in Fn-depleted medium (Fn−). Both WT and CD98hc-null MEFs produced Fn, although only WT cells were able to assemble it into fibrils. Basal medium (Fn+) refers to complete Fn-containing medium. (B) WT (left) and CD98hc-deficient (right) MEFs were incubated for 48 h in the presence of 25 μg/ml exogenous soluble Fn. Fixed cells were stained for Fn (green), actin (red), and nucleus (blue). Addition of soluble Fn to CD98hc-null cell culture did not rescue Fn assembly. (C) Effect of activating anti-β1 mAb (9EG7) on α5β1 integrin binding to soluble Fn. Binding to the soluble cell binding domain of Fn (Fn 9–11) in the absence (−9EG7), and in the presence (+9EG7) of β1 activating antibody, are illustrated for WT (filled bars) and CD98hc-deficient (open bars) cells. Error bars indicate SEM. (D) WT (left) and CD98hc-deficient (right) MEFs were incubated for 48 h in the presence of 10 μg/ml activating β1 integrin mAb, 9EG7. Fixed cells were stained for Fn (green), actin (red), and nucleus (blue). Treatment of CD98hc-null cells with activating β1 mAb did not rescue Fn assembly in vitro. Note the presence of perinuclear intracellular staining for Fn in the CD98hc-null cells, consistent with Fn biosynthesis. Bars, 25 μm.

Figure 5.

Figure 5.

CD98hc mediates cellular traction forces on the extracellular matrix. (A) WT and CD98hc-deficient MEFs were mixed with 200 μl of Fn-containing platelet-poor plasma, 200 μl of 28 mM CaCl2, and 5 U/ml human thrombin in Hepes-DME. Tubes were incubated for 2 h at 37°C. Depicted are digital images of WT and CD98hc-null clots. (B) Quantification of the percentage of clot contraction is presented (see Materials and methods). Values represent the mean ± SEM of triplicate determinations. The assay was repeated three times with similar results. (C) Decreased cellular traction forces in CD98-deficient MEFs. WT and CD98hc-deficient MEFs were plated on a 120-kD fragment of Fn-coated polyacrylamide sheets, in which fluorescent beads were embedded, as described in Materials and methods. Strain maps (green) overlaid with the brightfield images of WT (top) and CD98hc-null (bottom) cells plated on 120-kD Fn-coated polyacrylamide substrate (gray). CD98hc-deficient cells demonstrate reduced traction forces compared with WT cells. Bars, 10 μm.

Figure 6.

Figure 6.

CD98hc mediates adhesion-induced RhoA activation and matrix contraction to enable Fn matrix assembly. (A) RhoA activity was measured in an ELISA-based Rho assay in WT and CD98hc-deficient cells after plating on a 3D Fn matrix. The error bars represent SEM. The assay was repeated three times with similar results. Samples were also resolved by SDS-PAGE and immunoblotted with anti-RhoA antibody (total RhoA) to confirm that both WT and CD98hc-null MEFs express similar amounts of total RhoA (not depicted). (B) Activation of RhoA by LPA in WT and CD98hc-null MEFs. Adherent serum-starved CD98hc-null cells were treated with 1 μg/ml LPA or buffer, and RhoA activity was measured after 5 min. Values represent the mean and range of duplicate determinations. The assay was repeated twice with similar results. (C) Activation of RhoA bypasses the defect in matrix contraction in CD98hc-null cells. Clot contraction was measured 1 h after WT, CD98hc-null, and CD98hc-null MEFs were stimulated with LPA (see Materials and methods for details). Values represent the mean ± SEM of triplicate determinations. Depicted is one of two such experiments with identical results. (D) Activation of RhoA bypasses the defect in Fn matrix assembly in CD98hc-null cells. DOC-insoluble Fn produced by WT, CD98hc-null, and CD98hc-null MEFs treated with LPA was evaluated biochemically as described in Fig. 3. CD98hc-null MEFs stimulated with LPA were able to assemble Fn into fibrils as efficiently as WT cells. Depicted are the means of triplicate measurements. The assay was repeated two times with similar results.

Figure 7.

Figure 7.

CD98hc–integrin interaction mediates Fn assembly. (A) CD98hc-deficient MEFs reconstituted with each chimera depicted in B (C98T69E98, C69T98E98, or C98T98E69) were treated as described in Fig. 3 A. Fixed cells were stained for Fn (green), actin (red), and nucleus (blue). Only C98T98E69 rescued CD98hc-deficient cells' ability to assemble Fn fibrils. Bars, 25 μm. (B) Expression and schematic of the chimeras used in A. CD98hc protein is depicted in black and CD69 in gray. Each chimera is defined by its cytoplasmic (C), transmembrane (T), or extracellular (E) domain derived from either CD98hc (98) or CD69 (69). CD98hc extracellular domain is necessary and sufficient for amino acid transport, whereas the intracellular and transmembrane domains are required for interactions with integrins (Fenczik et al., 2001). Flow cytometry analysis (right) of cell surface expression of exogenous chimeras in CD98hc-deficient cells (filled histogram) is shown. Control staining (empty histogram) was performed with irrelevant IgG. (C) CD98–integrin association is required for contraction of the extracellular matrix. WT, CD98hc-deficient, and CD98hc-deficient MEFs reconstituted with C98T98E69 were mixed with 200 μl of Fn-containing platelet-poor plasma, 200 μl of 28 mM CaCl2, and 5 U/ml human thrombin in Hepes-DME. Tubes were incubated for 1 h at 37°C. Depicted are digital pictures of clots, as well as the calculated percentage of clot contraction (see Materials and methods). Values represent the mean ± SEM of triplicate determinations.

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