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Heparan Sulfate Interacting Protein (HIP/L29) Negatively Regulates Growth Responses to Basic Fibroblast Growth Factor in Gingival Fibroblasts

¹ Department of Biological Sciences, University of Delaware, Newark, DE 19716; and
² Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104;

View larger version (26K): [in a new window]   Figure 1. bFGF stimulates gingival fibroblast proliferation. (A) Cells were plated in 24-well plates at an initial density of 3500 cells/well and allowed to attach overnight in MEM containing 10% (v/v) FBS. The following day, cells were rinsed twice with PBS, and the media were changed to 0.2% (v/v) FBS with the addition of either 10 ng/mL bFGF or 10 µg/mL heparin or both compounds. In the control experiment, cells were cultured in MEM with 0.2% (v/v) FBS. Cell number was counted by means of a hemocytometer at days 0, 3, and 9 after treatments as described in MATERIALS & METHODS. Data are presented as the mean ± standard deviation (SD) of 6 experiments. (B) Cells were plated in 24-well plates as in (A). Subsequently, cells were rinsed twice with PBS and exposed to the indicated concentrations of bFGF in media containing 0.2% (v/v) FBS only. In the control experiment, cells were cultured in MEM with 0.2% (v/v) FBS. Cell number was counted on days 0, 3, and 9. Data are presented as the means ± SD of 6 experiments. *p < 0.05, **p < 0.001, ***p < 0.0001 relative to day 9 control.

View larger version (24K): [in a new window]   Figure 2. HIP/L29 antagonizes bFGF-induced proliferation. (A) Cells were plated as in Fig. 1. HIP/L29 (5 µg/mL) and bFGF (50 ng/mL) were added to the medium with or without 10-9 g/mL CsA. In the control experiment, cells were cultured in MEM with 0.2% (v/v) FBS. On days 0, 3, 9, and 13, cells were trypsinized and counted. Data are presented as ± the SD of 6 experiments. *p < 0.05, **p < 0.001, ***p < 0.0001 relative to bFGF only or bFGF + CsA at day 13. (B) Cells were plated as above. The following day, cells were rinsed twice with PBS and the medium was changed to 0.2% (v/v) FBS. Cells were exposed to HIP/L29 at the indicated concentrations in the presence of 50 ng/mL bFGF. In the control experiment, gingival fibroblasts were cultured in MEM with 0.2% (v/v) FBS. Cell number was counted on days 0, 3, and 9. Data are presented as the means ± SD of 6 experiments. *p < 0.05 relative to bFGF alone at day 9.

View larger version (22K): [in a new window]   Figure 3. Effect of HIP/L29 peptide on bFGF-induced proliferation. Gingival fibroblasts were plated in 24-well plates at an initial density of 3500 cells/well and allowed to attach as above. Subsequently, cells were rinsed twice with PBS and the medium was changed to 0.2% (v/v) FBS. HIP/L29 peptide (CRPKAKAKAKAKDQTK) (A) and BSA-conjugated peptide (AKAK-BSA) (B) were added to medium at the indicated concentrations in the presence of 50 ng/mL bFGF. On the indicated days, cells were trypsinized and counted. While peptide conjugated to BSA was very effective in reducing cell growth (bottom panel), unconjugated peptide had little effect (top panel). Data are presented as the means ± SD of 6 experiments. ***p < 0.0001 relative to corresponding day of group receiving bFGF only.

View larger version (27K): [in a new window]   Figure 4. Western blot analysis of MAPK in human gingival fibroblast. Cells were plated in 24-well plates at an initial density of 30,000 cells/well and allowed to attach overnight in MEM containing 10% (v/v) FBS. Subsequently, cells were rinsed twice with PBS, serum-starved for 24 hrs, treated as indicated, and then subjected to lysis on the plates. Antibodies were used for the detection of MAPK and phosphorylated MAPK as described in MATERIALS & METHODS. (A) Effects of bFGF and HIP on the phosphorylation of Erk-1 (p44) and Erk-2 (p42). (B) We verified equal loading by re-probing with antibody recognizing both phosphorylated and non-phosphorylated forms of p44 and p42.