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J Dent Res 83(8): 596-601, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

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;

* corresponding author, Theodora.Danciu{at}tch.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Physical forces play a critical role in the survival and proliferation of many cell types, including fibroblasts. Gingival fibroblasts are exposed to mechanical stress during mastication, orthodontic tooth movement, and wound healing following periodontal surgery. The aim of this study was to examine the effect of mechanical strain on human gingival fibroblasts (hGF). Cells were subjected to short-term (up to 60 min) and long-term (up to 48 hrs) 20% average elongation at 0.1 Hz. We monitored survival signaling by evaluating the phosphorylation status and localization of Forkhead box (FoxO) family members, which are mediators of apoptosis. We also examined strain-induced proliferation by measuring the level of proliferating cell nuclear antigen (PCNA). We observed that cyclic strain caused the phosphorylation and retention in the cytoplasm of FoxO family members. Moreover, mechanical strain resulted in increased ERK kinase phosphorylation and PCNA expression. In conclusion, cyclic strain delivers anti-apoptotic and proliferative stimuli to hGF.

KEY WORDS: gingival fibroblasts • stretch • FoxO • MAPK • PCNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gingival fibroblasts are essential for maintaining oral homeostasis. They participate in tissue repair and contribute to tissue remodeling following the application of physiological forces such as mastication. Mechanical stimulation of gingiva—as occurs, for example, during toothbrushing—promotes periodontal health by increasing capillary gingival circulation and fibroblast proliferation. During healing following various dental procedures such as periodontal surgery, tensile forces develop within the wound matrix which may be essential for fibroblast survival. Support for this hypothesis originates from studies in which fibroblasts in mechanically loaded collagen matrices showed little or no apoptosis compared with cells in mechanically unloaded conditions (Grinnell et al., 1999). How a mechanical stimulus is translated into cellular responses in gingival fibroblasts is, at present, unknown.

Mechanical forces are converted into signals that regulate metabolism in various cell types. Application of external forces to fibroblasts leads to activation of ion channels and growth factor receptors, which in turn activate several signaling pathways, including the MAPK pathway, resulting in changes in gene expression, cell proliferation, and protein synthesis (Silver et al., 2003). The ability of mechanical stimuli to promote cell survival has been attributed, at least in part, to the phosphoinositide 3'-(PI3)/Akt kinase cascade (Adam et al., 2003; Danciu et al., 2003). One mechanism by which Akt appears to regulate apoptosis is by phosphorylating and inactivating FoxO family members, including FoxO1 (also called FKHR1, forkhead in rhabdomyosarcoma) and FoxO4 (also known as AFX, ALL1 fused gene from chromosome X). Phosphorylated, and thus inactive, FoxO factors are predominantly localized in the cytoplasm. In the absence of survival factors, when Akt is inactive, unphosphorylated FoxO family members localize preferentially in the nucleus, where they bind to insulin response elements and/or Fas ligand promoter and activate transcription of target genes that induce apoptosis (Brunet et al., 1999).

The aim of this study was to explore the signal transduction pathways involved in the responses of human gingival fibroblasts to a controlled mechanical stimulus. By showing cytoplasmic translocation of FoxO1 as well as ERK phosphorylation and increased expression of PCNA, we conclude that cyclic stretch delivers anti-apoptotic and proliferative stimuli in hGF.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Tissue culture medium (Dulbecco’s modified Eagle’s medium, DMEM) was purchased form Life Technologies, Inc. (Rockville, MD, USA). The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): anti-FoxO1 polyclonal antibody (pAb; cross-reacts with FoxO4), anti-phospho-Ser256-FoxO1 pAb (cross-reacts with phospho-FoxO4), anti-p44/42 MAP Kinase pAb, and anti-phospho-p44/42 MAP Kinase (Thr202/Tyr204) pAb. Monoclonal anti-PCNA (Proliferating Cell Nuclear Antigen) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The reporter antibodies used were donkey anti-mouse or sheep anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL, USA). Fluorescein-conjugated donkey anti-rabbit IgG was purchased from Jackson ImmmunoResearch Laboratories, Inc. (West Grove, PA, USA). Enhanced chemiluminescence reagents were purchased from Pierce (Rockford, IL, USA).

Cell Culture
Human gingival fibroblasts (hGF; HGF-1, CRL-2014, American Type Culture Collection, Manassas, VA, USA; isolated from a healthy volunteer) were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), sodium bicarbonate (1.5 g/L), glucose (4.5 g/L), penicillin (100 U/mL), and streptomycin (100 µg/mL; all reagents from Life Technologies, Inc.), in a humidified 5% CO2–95% air atmosphere at 37°C. All experiments were performed on cells between passages 5 and 6.

Mechanical Loading Apparatus and Cyclic Stretch Conditions
Cells were stretched as previously described (Nguyen et al., 2000). Briefly, 1 x 105 hGF/well were plated onto six-well culture plates with silicone elastomer bottoms coated with collagen type I (Bioflex, Flexcell, Hillsborough, NC, USA). Cells were grown to 80% confluence and were rendered quiescent by 24-hour incubation in serum-free DMEM. For immunofluorescence studies, cells were at 30% confluence. Cells were then subjected to continuous cycles of stretch-relaxation in the FX-3000 Flexercell Strain Unit (Flexcell, Hillsborough, NC, USA). Each cycle consisted of 5 sec of stretch and 5 sec of relaxation (0.1 Hz, 20% stretch). The stretch conditions included 25% maximum radial stretch at the membrane periphery. It should be noted that the degree of stretch deformation was not uniform throughout different regions of the membrane; however, since samples were harvested from each well and pooled from several wells, variations in response due to variability in the degree of stretch deformation were averaged.

Growth Factor and Serum Stimulation
hGF were rendered quiescent by 24-hour incubation in serum-free DMEM. Platelet-derived growth factor (PDGF BB; R&D Systems, Minneapolis, MN, USA) was then added to the culture medium at a final concentration of 10 ng/mL for 24 or 48 hrs. For serum experiments, FBS was added to the serum-free medium for a final concentration of 10%.

Immunoblotting
Cell monolayers were washed with PBS (phosphate-buffered saline) and detached with PBS/EDTA (2 mM disodium EDTA; ethylenediaminetetraacetic acid). Cells then underwent lysis in a buffer containing 1% Triton X-100, 10 mM Tris pH 7.6, 500 mM NaCl, 2mM PMSF, and 60 mM octylglucoside. For immunoblotting, proteins separated by SDS/PAGE (10% acrylamide; SDS/PAGE = sodium dodecyl sulfate/polyacrylamide gel electrophoresis) were transferred electrophoretically to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) in transfer buffer (20 mM Tris/150 mM glycine buffer, pH 8.3). Phospho-specific antibodies were diluted in 5% BLOTTO at 1:1000; all other antibodies were diluted in 5% milk powder (Nestlé Carnation, Frederick, MD, USA) PBS/Tween (0.5% Tween-20) at 1:1000. All immunoblotting experiments were repeated at least twice with similar results.

Immunofluorescence Staining
Cells were grown to 30% confluence on two-well chamber slides (Becton Dickinson, Franklin Lakes, NJ, USA) or on the stretch chambers described above. Cells were incubated with serum-free DMEM for 24 hrs prior to serum stimulation or stretch. Cells were fixed for 5 min in cold methanol. After 3 PBS washes, cells were incubated for 1 hr at room temperature in 10% donkey serum. Cells were then incubated in primary anti-FoxO1 pAb (1:50 dilution) or no primary antibody, in 10% donkey serum overnight at 4°C in a humidified chamber. Cells were then washed 5 times in PBS, then incubated for 1 hr at room temperature in the dark with fluorescein-conjugated donkey anti-rabbit secondary antibody (1:100). After 5 PBS washes, slides were mounted by means of Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). For the stretch/static conditions, the bottom of each silicone stretch chamber was cut and placed on a glass slide just prior to being mounted. Cells were then viewed under a Zeiss epifluorescence photo microscope. Photographs of representative fields of view are presented (Fig. 3Go).



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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. 3AGo. 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. 3AGo, 3BGo. 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. 4AGo. Immunohistochemistry was then performed in a manner identical to that described for Figs. 3AGo, 3BGo. The right panel illustrates that the immunofluorescent signal is distributed evenly throughout the cytoplasm. (E) Negative control for Figs. 3Go and 4Go. 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. 3Go and 4Go. This Fig. illustrates very faint cytoplasmic straining and demonstrates the specificity of the FoxO1 antibody used in the immunofluorescent studies. Magnification: 80X.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stretch Affects Alignment and Cellular Morphology of Human Gingival Fibroblasts.
hGF exposed to 24 hrs of cyclic stretch assumed a stellate morphology with marked cytoplasmic contraction (Fig. 1Go). Cells were plated onto the stretch and static chambers at the same density 24 hrs prior to stretch initiation. In this study, the direction of the stretch was perpendicular to the cell-culture surface. In studies lasting up to 48 hrs, there was no evidence of cell detachment, suggesting that cells were viable during the experimental period.



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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.

 
FoxO1 is Activated in Response to Stretch.
We have previously shown that stretch activates the anti-apoptotic kinase Akt in osteoblasts (Danciu et al., 2003). Factors (such as growth factors) that activate the PI3K signal transduction pathway also cause phosphorylation and nuclear exclusion of FoxO family members such as FoxO1 and FoxO4 (Arden and Biggs, 2002). As a positive control for phosphorylation and thus inactivation of FoxO1 and FoxO4, we used serum. Serum stimulation after 24 hrs of serum withdrawal caused a rapid phosphorylation of FoxO1 and FoxO4 (Fig. 2AGo). Stretch also caused the rapid (within 5 min) phosphorylation of FoxO1 and FoxO4 (Fig. 2BGo) that was sustained for at least 60 min. Fig. 2CGo represents densitometry data of phosphorylated FoxO1 normalized to total FoxO1 from immunoblots (as shown in Fig. 2BGo) from two independent experiments. To explore further the effects of stretch and serum on FoxO family members, we used immunofluorescence to determine their cellular localization. Twenty-four hrs of serum withdrawal caused nuclear accumulation of FoxO1 consistent with the initiation of apoptosis in the absence of growth factors (Fig. 3AGo). In contrast, in the presence of serum, most FoxO1 was localized in the cytoplasm (Fig. 3BGo).



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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).

 
At 24 hrs, cyclic stretch caused nuclear exclusion of FoxO1, even in the absence of serum, suggesting that mechanical stimulation delivers a strong anti-apoptotic stimulus in these cells (Fig. 3DGo). In contrast, static cultures showed nuclear accumulation of FoxO1 (Fig. 3CGo), suggesting that, in the absence of serum or stretch, FoxO1 is localized to the nucleus, where it can interact with apoptosis-promoting genes. Minimal background staining occurred in the absence of primary antibody (Fig. 3EGo).

Stretch Activates Proliferative Pathways in Human Gingival Fibroblasts
Various investigators have demonstrated that stretch activates ERK in cardiac fibroblasts (MacKenna et al., 1998). In this study, we extend the observation to include gingival fibroblasts. ERK1 and ERK2 become rapidly phosphorylated (within 1 min) in response to stretch and sustain this response for the entire study period (60 min; Fig. 4AGo). In contrast, stretch has no effect on total ERK1 and 2 (Fig. 4AGo).



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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).

 
ERK phosphorylation is an indication of kinase activation. Since the ERK pathway is activated by a variety of mitogens, we assessed cellular proliferation by measuring PCNA protein level. PCNA protein is increased at 24 hrs of stretching and returns to the static level by 36 and 48 hrs (Fig. 4BGo). This implies that stretch after 36 hrs is not a sufficiently strong mitogenic stimulus to override the effect of the serum-free condition. We compared the effects of PDGF, a potent growth factor which has been shown to induce PCNA mRNA in fibroblasts (Miura et al., 1994), with those of stretch. Fig. 4CGo represents densitometry data of PCNA normalized to ß-actin from immunoblots (as shown in Fig. 4BGo) from two independent experiments. The effect of PDGF on PCNA levels at 24 hrs was similar to that of stretch (Fig. 4DGo). In contrast to stretch, PDGF alone can modulate PCNA expression in the absence of serum, even at 48 hrs (Fig. 4DGo). Since we did not observe any changes in ß-actin expression with stretch, we decided to use it as a normalization marker.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ability of fibroblasts to perceive substrate deformation and to respond to it influences the adaptation of these cells to mechanical forces. Cells in periodontal tissues are subjected to constant forces during normal masticatory function as well as during wound contraction. Moreover, previous reports suggest that the release of mechanical loading triggers an apoptotic response in fibroblasts (Grinnell et al., 1999). In trying to understand the mechanism whereby a mechanical stimulus protects gingival fibroblasts from apoptosis, we subjected these cells to mechanical forces. The cells used in this study have been isolated from a healthy volunteer and thus, we believe, accurately reflect the responses of human gingival fibroblasts to mechanical stimuli. We determined that stretch phosphorylates and thereby inactivates the FoxO family members (FoxO1, FoxO3a, and FoxO4), which play a crucial role in cellular death. Furthermore, stretch results in ERK phosphorylation and an increase in the expression of PCNA, leading us to conclude that stretch stimulates cellular proliferation. To our knowledge, this is the first report of FoxO1 and FoxO4 inactivation in gingival fibroblasts in response to a mechanical stimulus. Our findings are consistent with those of Horiuchi et al.(2002), who demonstrated in vivo that mechanically stimulated fibroblasts show increased PCNA expression.

Signaling through the Akt pathway regulates apoptosis by ultimately modulating the expression of a defined subset of genes involved in cell death. Under conditions during which Akt is activated, FoxO family members are inactivated by phosphorylation and retained in the cytoplasm. When Akt is not active, these factors are not phosphorylated and are localized to the nucleus (Biggs et al., 1999; Brunet et al., 1999), where they can interact with various genes involved in apoptosis. One such target is the gene encoding for the cytokine FasL. Binding of FasL to its surface receptor Fas triggers a cascade of events leading to apoptosis (Datta et al., 1999). In this study, we have demonstrated that mechanical stimulation of human gingival fibroblasts results in FoxO phosphorylation and cytoplasmic localization in a manner similar to serum, which is a potent growth stimulus. This finding is consistent with that of Grinnell et al.(1999), who demonstrated that the release of mechanical tension triggers fibroblast apoptosis.

Mitogen-activated protein kinases become activated upon phosphorylation and mediate extracellular signals which regulate cell growth, differentiation, survival, and death (reviewed in Davis, 1993). The MAPK superfamily is a widely distributed group of enzymes that can be divided into several subfamilies: (1) the extracellular-regulated kinases (ERKs); (2) the c-Jun N-terminal kinases; and (3) the p38 MAPKs cascade (reviewed in Ruwhof and van der Laarse, 2000). The best-characterized ERKs are the 44-kDa MAPK (ERK1), the 42-kDa MAPK (ERK2), and the 63-kDa MAPK (ERK3) (Boulton et al., 1991). In addition to growth factors, mechanical stress has also been reported to activate ERK1 and ERK2 in various cell types, including fibroblasts (MacKenna et al., 1998) and cardiac myocytes (reviewed in Ruwhof and van der Laarse, 2000), resulting in diverse biological responses such as proliferation and differentiation (Matsuda et al., 1998; Sanchez-Esteban et al., 2003). In this study, we assessed cellular proliferation by measuring the level of PCNA expression.

PCNA functions as a DNA sliding clamp for DNA polymerase {delta} and is an essential component for eukaryotic chromosomal DNA replication (Tsurimoto, 1999). Horiuchi et al.(2002) have demonstrated an increase in PCNA-positive fibroblasts in vivo in response to another mechanical stimulus: toothbrushing. Our findings suggest that the increase in proliferation as assessed by PCNA expression in stretched hGF may be mediated by ERK kinases.

In summary, in this study we have demonstrated that human gingival fibroblasts respond to mechanical stimulation by activating FoxO family members, the ERK kinase pathway, and by increasing the expression of PCNA, which is a marker for cellular proliferation. These findings warrant further studies aimed at elucidating how a mechanical stimulus is interpreted by hGF and the precise role of each resulting in multiple biological responses such as proliferation and differentiation.


   ACKNOWLEDGMENTS
 
This work was supported by F32-DE14163 (T.E.D.) and by NIH R01-DK57691 and P50 DK65298 (M.R.F.) This paper is based on an abstract presented at the 32nd Annual Meeting and Exhibition of the American Association for Dental Research, March 12–15, 2003, San Antonio, TX, USA (J Dent Res 82[Spec Iss A]:1508, www.dentalresearch.org).

Received September 9, 2003; Last revision May 25, 2004; Accepted May 25, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adam RM, Roth JA, Cheng HL, Rice DC, Khoury J, Bauer SB, et al. (2003). Signaling through PI3K/Akt mediates stretch and PDGF-BB-dependent DNA synthesis in bladder smooth muscle cells. J Urol 169:2388–2393.[ISI][Medline]

Arden KC, Biggs WH 3rd (2002). Regulation of the FoxO family of transcription factors by phosphatidylinositol-3 kinase-activated signaling. Arch Biochem Biophys 403:292–298.[ISI][Medline]

Biggs WH 3rd, Meisenhelder J, Hunter T, Cavenee WK, Arden KC (1999). Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96:7421–7426.[Abstract/Free Full Text]

Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, et al. (1991). ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65:663–675.[ISI][Medline]

Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868.[ISI][Medline]

Danciu TE, Adam RM, Naruse K, Freeman MR, Hauschka PV (2003). Calcium regulates the PI3K-Akt pathway in stretched osteoblasts. FEBS Lett 536:193–197.[ISI][Medline]

Datta SR, Brunet A, Greenberg ME (1999). Cellular survival: a play in three Akts. Genes Dev 13:2905–2927.[Free Full Text]

Davis RJ (1993). The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553–14556.[Free Full Text]

Grinnell F, Zhu M, Carlson MA, Abrams JM (1999). Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue. Exp Cell Res 248:608–619.[ISI][Medline]

Horiuchi M, Yamamoto T, Tomofuji T, Ishikawa A, Morita M, Watanabe T (2002). Toothbrushing promotes gingival fibroblast proliferation more effectively than removal of dental plaque. J Clin Periodontol 29:791–795.[ISI][Medline]

MacKenna DA, Dolfi F, Vuori K, Ruoslahti E (1998). Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts. J Clin Invest 101:301–310.[ISI][Medline]

Matsuda N, Morita N, Matsuda K, Watanabe M (1998). Proliferation and differentiation of human osteoblastic cells associated with differential activation of MAP kinases in response to epidermal growth factor, hypoxia, and mechanical stress in vitro. Biochem Biophys Res Commun 249:350–354.[ISI][Medline]

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Sanchez-Esteban J, Wang Y, Gruppuso PA, Rubin LP (2003). Mechanical stretch induces fetal type II cell differentiation via an EGFR-ERK signaling pathway. Am J Respir Cell Mol Biol 30(1):76–83.

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