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Differential uPA Expression by TGF-ß1 in Gingival Fibroblasts

¹ Faculty of Odontology and
² Laboratory of Cell Biology, Institute of Nutrition and Food Technology (INTA), University of Chile, Olivos 943, Casilla 1903, Santiago, Chile

View larger version (56K): [in a new window]   Figure 1. Characterization of healthy and granulation-tissue fibroblasts. (A) At passage 4, human gingival fibroblasts obtained from healthy and granulation-tissue gingiva were stained with Alexa Fluorphalloidin or antibodies recognizing -SMA or vimentin. Arrowheads indicate -SMA-negative cells. Arrows indicate -SMA-positive cells. Bar = 20 µm. (B) The average of -SMA expression was obtained after we counted positively stained cells in cell cultures derived from HAG, GT-W, HMG, and GT-PD patients, and analyzed them by Student’s t test.

View larger version (33K): [in a new window]   Figure 2. Regulation of uPA production by TGF-ß1 and EGF in healthy and granulation-tissue fibroblasts. (A) Serum-free cultures of gingival fibroblasts from healthy and granulation-tissue (80,000 cells) were stimulated with increasing concentrations of TGF-ß1 or EGF for 48 hrs in DMEM without serum. Conditioned medium, normalized after the quantification of total protein amounts in the cell lysate at the end of the experiment, was analyzed through casein zymography. (B) uPA protein levels were determined through Western blot analysis of the concentrated conditioned medium of 500,000 cells stimulated with 10 ng/mL TGF-ß1 or 10 ng/mL EGF. (C) uPA-derived proteolytic activity was determined in conditioned medium of cells stimulated with 10 ng/mL TGF-ß1 or 10 ng/mL EGF through a radial diffusion assay. (D) PAI-1 production was identified through Western blotting of concentrated conditioned medium of cells stimulated with 10 ng/mL TGF-ß1 or 10 ng/mL EGF. (E) TGF-ß1-stimulated uPA production, determined through casein zymography, in each individual was normalized against non-stimulated uPA production (indicated as 1 in the graph) and compared among the 4 groups under study. Bars indicate averages ± standard deviations. Statistical analysis was performed with Student’s t test (n=33). (F) We performed Pearson correlation analysis, comparing TGF-ß1-stimulated uPA production through casein zymography and -SMA expression.

View larger version (28K): [in a new window]   Figure 3. Signal transduction pathways involved in TGF-ß1-stimulated uPA production. (A) Healthy and granulation-tissue gingival fibroblasts (80,000 cells) were cultured in the presence of specific MAPK inhibitors: 40 µM PD98059, 10 µM SB203580, or 10 µM SP600125 in serum-free DMEM. After 15 min, cell cultures were treated with 10 ng/mL TGF-ß1 for 48 hrs. Secreted uPA activity was evaluated by casein zymography. (B) Dose-response analysis of the effects of the JNK inhibitor SP600125 on TGF-ß1-stimulated uPA production determined through casein zymography in 3 independent experiments. (C) Gingival granulation-tissue fibroblasts (300,000 cells) were incubated with TGF-ß1 (10 ng/mL) in DMEM without serum for different periods of time, as indicated. The level of activated JNK (pJNK) was determined by Western blotting with specific antibodies.

View larger version (95K): [in a new window]   Figure 4. Immunolocalization of -SMA expression and uPA production in sections of gingival wounds and healthy gingiva. (A,C,E) -SMA expression. (B,D,F) uPA production. (A–D) Acute gingival wounds. (E,F) Healthy gingiva. GT = granulation tissue. HT = healthy tissue. BV = blood vessel. (G) Negative control section. Arrows indicate -SMA staining; arrowheads indicate uPA staining. Bar = 50 µm.