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Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin

a role in TGFß-dependent matrix deposition

¹ Department of Biochemistry, The Holland Laboratory, American Red Cross, Rockville, MD 20855, USA
² Department of Biochemistry and Molecular Biology, The George Washington University, Washington, DC 20037, USA
^* Author for correspondence (e-mail: belkina{at}usa.redcross.org )

View larger version (70K): [in a new window]   Fig. 1. Cell-surface tTG promotes Fn assembly independent of its crosslinking activity. Populations of Swiss 3T3 transfectants (vector, tTG[1], tTG[2], tTGC277S[1] and tTGC277S[2]) were analyzed for integrin-tTG association and Fn biosynthesis and assembly. (A) Association of transfected tTG or its catalytically inactive mutant tTG(C277S) with endogenous 5ß1 integrin. 5ß1 integrin and tTG were immunoprecipitated from cell lysates with mAb BMA5 and polyclonal anti-tTG antibody, respectively. The resulting immunoprecipitates were blotted for tTG. (B) Biosynthesis of Fn in the transfectants. Fn was immunoprecipitated from 35S-labeled cell lysates and analyzed by SDS-PAGE and autoradiography. (C,D) tTG and tTG(C277S) stimulate incorporation of [125I]Fn into the deoxycholate-insoluble fraction. (C) Deoxycholate-insoluble [125I]Fn in the matrix of cells grown for 24 hours with 10 nM [125I]Fn was analyzed by reducing SDS-PAGE and autoradiography. Arrow points to Fn monomer; arrowhead marks tTG-crosslinked Fn polymers. Vector, tTG[1], tTG[2], tTGC277S[1] and tTGC277S[2] transfectants are marked as 1, 2, 3, 4 and 5, respectively (B,C). (D) 125I-labeled bands corresponding to Fn monomer and polymer were cut out of the gels and counted. The results are representative of three independent experiments (mean±s.e.m.). (E) tTG and tTG(C277S) promote Fn fibrillogenesis. Cells grown for 24 hours in the presence of 50 nM exogenous Fn were stained with anti-Fn antibody. Bar, 50 µm.

View larger version (53K): [in a new window]   Fig. 2. Expression of surface tTG induces the binding of 42 kDa Fn fragment and Fn to cells. (A) Analysis of binding of [125I]42 kDa fragment to vector, tTG[2] and tTGC277S[2] transfectants in suspension. (B) Anti-tTG antibody suppresses the binding of [125I]42 kDa fragment to tTG[2] transfectants in suspension. (C) Binding of [125I]Fn to vector and tTG[2] transfectants in suspension. (D) The lack of binding of [125I]42 kDa fragment to monolayers of vector, tTG[2] and tTGC277S[2] transfectants. Adherent cells plated for 2 or 72 hours were analyzed for binding [125I]42 kDa fragment. (B,C) Where indicated, 20 µg/ml anti-tTG antibody, blocking anti-5ß1 integrin mAb BMA5, control nonimmune IgG or 5 µM unlabeled 42 kDa fragment was used during incubation of cells with 125I-labeled proteins. Nonspecific binding in the presence of excess unlabeled 42 kDa fragment was subtracted. The results are representative of three independent experiments (mean±s.e.m.).

View larger version (60K): [in a new window]   Fig. 3. Stimulation of Fn assembly is mediated by interaction of surface tTG with the gelatin-binding domain of Fn. (A,B) tTG-stimulated increase in [125I]Fn incorporation into the deoxycholate-insoluble fraction is inhibited by the 42 kDa Fn fragment and anti-tTG antibody. (A) Deoxycholate-insoluble [125I]Fn in the matrix of Swiss 3T3 transfectants grown for 24 hours with 50 nM [125I]Fn was analyzed by reducing SDS-PAGE and autoradiography. (B) Quantitation of the [125I]Fn incorporation into the deoxycholate-insoluble fraction. 125I-labeled bands corresponding to Fn monomer and polymer were cut out of the gels and counted. The results are representative of three independent experiments (mean±s.e.m.). (C) tTG-mediated increase in Fn fibril formation by tTG[2] and tTGC277S[2] transfectants is blocked by the 42 kDa fragment and anti-tTG antibody. Cells grown for 24 hours with 50 nM exogenous Fn were stained with anti-Fn antibody. Bar, 50 µm. (A-C) Where indicated, cells were grown in the presence of 20 µg/ml anti-tTG antibody, blocking anti-5ß1 integrin mAb BMA5, control nonimmune IgG or 2 µM 42 kDa or 110 kDa fragments of Fn.

View larger version (29K): [in a new window]   Fig. 4. Differential effects of 29 kDa and 42 kDa Fn fragments of Fn assembly. Incorporation of [125I]Fn into the deoxycholate-insoluble fraction by Swiss 3T3 vector (A) or tTG[2] (B) transfectants was analyzed in the presence of indicated concentrations of the unlabeled 29 kDa fragment (), 42 kDa fragment () or a combination of the two Fn fragments ([UNK]). Data is presented as a percentage of [125I]Fn incorporated in the absence of the unlabeled Fn fragments for each of the two cell lines. The results are representative of three independent experiments (mean±s.e.m.).

View larger version (48K): [in a new window]   Fig. 5. Visualization of binding sites for the 42 kDa Fn fragment on the cell surface. Swiss 3T3 vector (A,C) or tTG[2] (A,B,D) transfectants were preincubated in suspension for 1 hour with 25 µg/ml biotinylated 42 kDa fragment, washed and plated on Fn-coated (A) or untreated glass coverslips (B-D). Staining of fixed nonpermeabilized cells is shown. (A) Cell-surface binding sites for the 42 kDa fragment were visualized by double-staining with avidin-rhodamine and anti-ß1 integrin mAb HMß1-1 followed by fluorescein-labeled goat anti-hamster IgG. (B) Codistribution of surface-bound 42 kDa fragment with ß1 integrins, surface tTG and Fn fibrils. Cells were double-stained with avidin-rhodamine and anti-ß1 integrin mAb HMß1-1 or anti-tTG mAbs CUB7402/TG100, followed by secondary fluorescein-labeled IgG (middle and right panels). Alternatively, cells were double-stained with anti-tTG mAbs CUB7402/TG100 and anti-Fn antibody, followed by fluorecein-labeled anti-mouse IgG and Alexa-Fluor 350-labeled anti-rabbit IgG (left panels). (C,D) Cells were stained with avidin-rhodamine to visualize surface-bound 42 kDa fragment and then either stained for Fn (C) or double-stained for Fn and surface tTG (D). (A,B,) Yellow color in merged images shows codistribution of the exogenous 42 kDa fragment with ß1 integrins and surface tTG at focal adhesions (A) or cell-matrix contacts on the dorsal surface (B). (B,D) Arrows point to clusters of surface tTG colocalized with initiation sites of Fn fibril growth (B) or large clusters of Fn fibrils (D). Arrowheads (B) mark codistribution of surface-bound 42 kDa fragment with ß1 integrins and tTG at cell-matrix contacts. Bar, 20 µm (A,B) or 50 µm (C,D).

View larger version (30K): [in a new window]   Fig. 6. Stimulation of Fn assembly by surface tTG is separate from its effects on cell spreading. Vector and tTG[2] transfectants were plated on vitronectin or laminin-coated surfaces in serum-free medium. Three hours later the cells were supplemented with 20 µg/ml of unlabeled (A,C) or 125I-labeled (B) Fn in Fn-depleted 10% BCS and incubated for the following 6 hours. (A) tTG-mediated stimulation of Fn fibril formation on the dorsal cell surfaces. Cells were fixed and stained with anti-Fn antibody. Bar, 50 µm. (B) Quantitation of [125I]Fn incorporation into the deoxycholate-insoluble pool. The results are representative of three independent experiments (mean±s.e.m.). (C) Quantitation of the extent of cell spreading. The average areas on the substrates was determined for 120 sparsely plated cells in each population (mean±s.e.m.).

View larger version (58K): [in a new window]   Fig. 7. TGFß increases tTG biosynthesis and its association with ß1 integrins in WI-38 fibroblasts. (A) Effects of TGFß on biosynthesis of Fn, ß1 and ß3 integrins and tTG. ß1, ß3 integrins, tTG, Fn and actin were immunoprecipitated from 35S-labeled lysates (1.8x108 cpm per sample). (B) Effects of TGFß on Fn secretion. Fn was immunoprecipitated from 35S-labeled media (0.2x108 cpm per sample). 35S-labeled cell extracts (A) and secreted proteins in the media (B) of untreated and TGFß-treated cells reflect equal amounts of material taken for immunoprecipitation. (C) Association of tTG with ß1 but not with ß3 integrins is increased in TGFß-treated cells. ß1, ß3 integrins and tTG were immunoprecipitated from untreated or TGFß-stimulated cells and the immune complexes were blotted for tTG (lower panels) or ß1 and ß3 integrins (upper panels).

View larger version (30K): [in a new window]   Fig. 8. Binding of Fn and 42 kDa Fn fragment to the surfaces of WI-38 fibroblasts is stimulated by TGFß. (A) Binding of [125I]42 kDa fragment to untreated or TGFß-treated cells in suspension. (B) Binding of [125I]Fn to untreated or TGFß-treated cells in suspension. Nonspecific binding in the presence of excess unlabeled 42 kDa fragment or Fn was subtracted. Where indicated, cells were incubated with 20 µg/ml anti-tTG antibody. The results are representative of three independent experiments (mean±s.e.m.).

View larger version (47K): [in a new window]   Fig. 9. Surface tTG contributes to TGFß-dependent increase in Fn assembly by WI-38 fibroblasts. (A,B) 42 kDa Fn fragment and anti-tTG antibody inhibit TGFß-stimulated increase of [125I]Fn incorporation into the deoxycholate-insoluble fraction. (A) Deoxycholate-insoluble [125I]Fn in the matrix of cells grown for 36 hours with or without TGFß in the presence of 50 nM [125I]Fn was analyzed by reducing SDS-PAGE and autoradiography. (B) Quantitation of incorporation of [125I]Fn into the deoxycholate-insoluble fraction. 125I-labeled bands corresponding to Fn monomer and polymer were cut out of the gels and counted. The results are representative of three independent experiments (mean±s.e.m.). (C) TGFß-mediated enhancement of Fn fibril formation is inhibited by the 42 kDa Fn fragment and anti-tTG antibody. Cells grown for 36 hours with 50 nM exogenous Fn and either 2 µM 42 kDa fragment, 20 µg/ml anti-tTG antibody or without these treatments were stained with anti-Fn antibody. Bar, 50 µm. (A-C) Where indicated, cells were grown with 20 µg/ml anti-tTG antibody, blocking anti-5ß1 integrin mAb BMA5, control nonimmune IgG or 2 µM 42 kDa or 110 kDa Fn fragments.