Topographic Changes of Focal Adhesion Components and Modulation of p125FAK Activation in Stretched Human Periodontal Ligament Fibroblasts

Abstract

Mechanical stress has been shown in vitro to modulate integrin-β1-mediated activation of p125^FAK/FAK. To test the hypothesis whether this also applies to periodontal ligament fibroblasts (PDLs), we subjected human PDLs to mechanical stretch and analyzed stress-induced changes of p125^FAK activation by quantitative immunoprecipitation of p125^FAK and changes in the topography of molecules localizing in focal adhesions by indirect immunofluorescence. Generally, all components of focal contacts under study-including detection of phosphotyrosine, i.e., integrin-β1, p125^FAK, and paxillin-revealed a relative co-localization during stretch application. Under stretch, we observed a re-distribution of all components from the cell periphery to the cytoplasm following the main axes. Tyrosine phosphorylation of p125^FAK was monitored up to 72 hours under stretch. While the amount of p125^FAK remained essentially constant, the activation of p125^FAK was clearly modulated. Tyrosine phosphorylation of p125^FAK increased from 15 minutes up to 1 hour and declined after stretching periods of 24, 48, and 72 hours. The analysis of our data indicated a stretch-induced re-distribution of focal adhesion components and a modulation of p125^FAK activation, suggesting alterations in focal adhesions and their associated signal cascade.

Keywords

integrins p125FAK/FAK paxillin PDL fibroblasts stretching

Get full access to this article

View all access options for this article.

References

Aplin AE , Howe AK , Juliano RL (1999). Cell adhesion molecules, signal transduction and cell growth. Curr Opin Cell Biol 11:737-744.

Bellis SL , Perrotta JA , Curtis MS , Turner CE (1997). Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin. Biochem J 325:375-381.

Bolcato-Bellemin AL , Elkaim R. , Abehsera A. , Fausser JL , Haikel Y. , Tennenbaum H. (2000). Expression of mRNAs encoding for a and P integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts. J Dent Res 79:1712-1716.

Carvalho RS , Scott JE , Yen EH (1995). The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells. Arch Oral Biol 40:257-264.

Carvalho RS , Bumann A. , Schwarzer C. , Scott E. , Yen Ehk (1996). A molecular mechanism of integrin regulation from bone cells stimulated by orthodontic forces. Eur J Orthodont 18:227-235.

Chen HC ( 1995). Interaction of focal adhesion kinase with cytoskeletal protein talin. J Biol Chem 270:16995-16999.

Danker K. , Gabriel B. , Heidrich C. , Reutter W. (1998). Focal Adhesion Kinase ppl25FAK and the β1 integrin subunit are constitutively complexed in HaCaT cells. Exp Cell Res 239:326-331.

Galbraith CG , Sheetz MP (1998). Forces and/or adhesive contacts affect on cell function. Curr Opin Cell Biol 10:566-571.

Gates RE , King LE Jr , Hanks SK , Nanney LB (1994). Potential role for focal adhesion kinase in migrating and proliferating keratinocytes near epidermal wounds and in culture. Cell Growth Diff 5:891-899.

10.

Guan JL , Trevithick JE , Hynes RO (1991). Fibronectin/integrin interaction induces tyrosine phosphorylation of 120-kDa protein. Cell Regul 2:951-964.

11.

Howard PS , Kucich U. , Taliwal R. , Korostoff JM (1998). Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts. J Periodontal Res 33:500-508.

12.

Hynes RO (1992). Integrins: versatility modulation and signaling in cell adhesion. Cell 69:11-25.

13.

Juliano RL , Haskill S. (1993). Signal transduction from the extracellular matrix . J Cell Biol 120:577-585.

14.

Ni H. , Simonsen N. , Wilkins JA (1998). Integrin activation by Dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the β1-integrin chain. J Biol Chem 273:7981-7987.

15.

Ruhl M. , Johannsen M. , Atkinson J. , Manski D. , Sahin E. , Somasundaram R. , et al. (1999). Soluble collagen VI induces tyrosine phosphorylation of paxillin and focal adhesion kinase and activates the MAP kinase erk2 in fibroblasts. Exp Cell Res 250:548-557.

16.

Sai X. , Naruse K. , Sokabe M. (1999). Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts. J Cell Sci 112:1365-1373.

17.

Saito M. , Saito S. , Ngan PW , Shanfeld J. , Davidovitch Z. (1991). I1-1β and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro . Am J Orthod Dentofac Orthop 99:226-240.

18.

Schaller MD , Borgman CA , Cobb BS , Vines RR , Reynolds AB , Parsons JT (1992). ppl25FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci USA 89:5192-5196.

19.

Schlaepfer DD , Hauck CR , Sieg DJ ( 1999). Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71:435-478.

20.

Schwartz MA , Schaller MD , Ginsberg MH (1995). Integrins: emerging paradigms of signal transduction . Annu Rev Cell Dev Biol 11:549-599.

21.

Sieg DJ , Hauck CR , Ilic D. , Klingbeil CK , Schaefer E. , Damsky CH , et al. (2000). FAK integrates growth-factor and integrin signals to promote cell migration. Nature Cell Biol 2:249-257.

22.

Sokabe M. , Naruse K. , Sai S. , Yamasda T. , Kawakami K. , Inoue M. , et al. (1997). Mechanotransduction and intracellular signaling mechanisms of stretch-induced remodeling in endothelial cells. Heart Vessels 12:191-193.

23.

Yano Y. , Geibel J. , Sumpio BE (1996). Tyrosine phosphorylation of ppl25FAK and paxillin in aortic endothelial cells induced by mechanical strain. Am J Physiol 271:635-649.