Mechanical Strain Delivers Anti-apoptotic and Proliferative Signals to Gingival Fibroblasts

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Mechanical Strain Delivers Anti-apoptotic and Proliferative Signals to Gingival Fibroblasts

T.E. Danciu¹, E. Gagari², R.M. Adam¹, P.D. Damoulis², and M.R. Freeman¹

¹ Enders Research Laboratories, Rm 1150.2, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA, and Department of Surgery, Harvard Medical School, Boston; and
² Tufts School of Dental Medicine, Boston, MA, USA;

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Figure 1. hGF change orientation in response to stretch. 1 x 10⁵ cells per well were plated onto six-well culture plates and subjected to stretch for 24 hrs (left panel) with the use of an FX-3000 Flexercell Strain Unit (0.1 Hz, 20% stretch) or static conditions (right panel). Magnification: 80X. Bar represents 140 µm.

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Figure 2. Phosphorylation of FoxO family members in response to serum and stretch in hGF. (A) hGF were cultured in serum-free medium for 24 hrs prior to stimulation. Cells were then stimulated with 10% FBS for the times indicated (in min). A 20-µg quantity of total cellular proteins was separated by SDS-PAGE, transferred to nitrocellulose, and probed with phospho-specific FoxO1/FoxO4 antibody (upper panels) or actin (lower panel). The upper panels represent different film exposure times. (B) hGF were cultured in serum-free medium for 24 hrs prior to stimulation. Cells were then subjected to stretch for the times indicated (in min). A 20-µg quantity of total cellular proteins was separated by SDS-PAGE, transferred to nitrocellulose, and probed with phospho-specific FoxO1/FoxO4 antibody (upper panel) or total FoxO1/FoxO4 antibody (lower panel). (C) Densitometric analysis of phosphorylated FoxO1 normalized to FoxO1 for two independent stretch experiments (represented as fold induction over static controls) (N = 2; 1 min – 1.49 ± 0.09; 5 min – 1.82 ± 0.014; 10 min – 2.37 ± 0.05; 30 min – 3.65 ± 0.28; 60 min – 4.50 ± 0.41).

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Figure 3. Cytoplasmic enrichment of FoxO1 in response to serum and stretch in hGF. (A) hGF were cultured in serum-free medium on glass immunohistochemistry chambers for 24 hrs. Cells were then fixed as described in MATERIALS & METHODS and stained with anti-FoxO1 antibody; mounting medium contained DAPI for identification of nuclei. a: DAPI nuclear staining. b: FoxO1 staining. c: superimposed image illustrating that the most intense FoxO1 staining corresponds to the nuclear area. (B) hGF were cultured in 10% FBS-containing medium on glass immunohistochemistry chambers for 24 hrs. Cells were then fixed and stained as in Fig. 3A

. The superimposed image (c) indicates that FoxO1 is evenly distributed in the cytoplasm. (C) hGF were cultured in serum-free medium for 24 hrs and kept under static conditions for an additional 24 hrs. The bottom areas of the silicone stretch chambers were then cut and placed on glass slides prior to being mounted. Immunohistochemistry was then performed in a manner identical to that described in Figs. 3A

, 3B

. The right panel illustrates that most of the immunofluorescent signal is localized to the nucleus. (D) hGF were cultured in serum-free medium for 24 hrs prior to being stretched for an additional 24 hrs. The chambers were treated as in Fig. 4A

. Immunohistochemistry was then performed in a manner identical to that described for Figs. 3A

, 3B

. The right panel illustrates that the immunofluorescent signal is distributed evenly throughout the cytoplasm. (E) Negative control for Figs. 3

and 4

. Cells were cultured in serum-containing medium for 24 hrs, fixed, and processed as indicated in MATERIALS & METHODS in the absence of a primary antibody. The image represents superimposed images of DAPI and FoxO1 taken with the same settings as in Figs. 3

and 4

. This Fig. illustrates very faint cytoplasmic straining and demonstrates the specificity of the FoxO1 antibody used in the immunofluorescent studies. Magnification: 80X.

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Figure 4. Stretch-induced ERK phosphorylation in hGF. (A) hGF were cultured in serum-free conditions for 24 hrs prior to being stretched for the indicated times (in min). A 20-µg quantity of total cellular proteins was separated by SDS-PAGE, transferred to nitrocellulose, and probed with phospho-specific ERK1,2 antibody (upper panel) or total ERK1,2 antibody (lower panel). + = positive control, MC3T3-E1 mouse osteoblast cell line [ERK1, 44 kDa; ERK2, 42 kDa].

Stretch and serum cause increased PCNA expression in hGF. (B) hGF were stretched in serum-free medium for the times indicated (in hrs). A 20-µg quantity of total cellular proteins was separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-PCNA antibody (upper panel) or actin antibody (lower panel). PC, positive control; hGF cultured in 10% FBS-containing medium for 24 hrs. (C) Densitometric analysis of PCNA normalized to ß-actin for two independent stretch experiments (represented as fold induction over static condition at each time point; N = 2; 24 hrs, 1.98 ± 0.233; 48 hrs, 1.16 ± 0.01). (D) hGF were cultured in serum-free medium for 24 hrs prior to stimulation with 10 ng/mL PDGF for 24 or 48 hrs as indicated. A 20-µg quantity of total cellular proteins was separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-PCNA antibody (upper panel) or actin antibody (lower panel).

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