β1 integrin signaling in asymmetric migration of keratinocytes under mechanical stretch in a co-cultured wound repair model
Tóm tắt
Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin. But the mechanisms of how mechanical stimuli regulate the migration of keratinocytes have been poorly understood. Human immortalized keratinocyte HaCaT cells were co-cultured with skin fibroblasts on PDMS membranes and transferred to the static stretch device developed in-house for additional 6 day culture under mechanical stretch to mimic surface tension in skin. To detect the expression of proteins on different position at different time points and the effect of β1 integrin mechanotransduction on HaCaT migration, Immunofluorescence, Reverse transcription-polymerase chain reaction, Flow cytometry, Western blotting assays were applied. Mechanical receptor of β1 integrin that recognizes its ligand of collagen I was found to be strongly associated with migration of HaCaT cells since the knockdown of β1 integrin via RNA silence eliminated the key protein expression dynamically. Here the expression of vinculin was lower but that of Cdc42 was higher for the cells at outward edge than those at inward edge, respectively, supporting that the migration capability of keratinocytes is inversely correlated with the formation of focal adhesion complexes but positively related to the lamellipodia formation. This asymmetric expression feature was further confirmed by high or low expression of PI3K for outward- or inward-migrating cells. And ERK1/2 phosphorylation was up-regulated by mechanical stretch. We reported here, a novel mechanotransduction signaling pathways were β1 integrin-dependent pattern of keratinocytes migration under static stretch in an in vitro co-culture model. These results provided an insight into underlying molecular mechanisms of keratinocyte migration under mechanical stimuli.
Tài liệu tham khảo
Singer AJ, Clark RAF. Mechanisms of disease—cutaneous wound healing. N Engl J Med. 1999;341:738–46.
Kirsner RS, Eaglstein WH. The wound healing process. Dermatol Clin. 1993;11:629–40.
Stadelmann WK, Digenis AG, Tobin GR. Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg. 1998;176:26–38.
Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453:314–21.
Bartkova J, Gron B, Dabelsteen E, Bartek J. Cell-cycle regulatory proteins in human wound healing. Arch Oral Biol. 2003;48:125–32.
Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol. 2007;5:9–18.
Bandyopadhyay B, Fan J, Guan S, Li Y, Chen M, Woodley DT, Li W. A “traffic control” role for TGF beta3: orchestrating dermal and epidermal cell motility during wound healing. J Cell Biol. 2006;172:1093–105.
Nickoloff BJ, Mitra RS, Riser BL, Dixit VM, Varani J. Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferative factors. Am J Pathol. 1988;132:543–51.
El-Ghalbzouri A, Gibbs S, Lamme E, Van Blitterswijk CA, Ponec M. Effect of fibroblasts on epidermal regeneration. Br J Dermatol. 2002;147:230–43.
Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002;3:349–63.
Urschel JD, Scott PG, Williams HT. The effect of mechanical stress on soft and hard tissue repair; a review. Br J Plast Surg. 1988;41:182–6.
Chiquet M. Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol. 1999;18:417–26.
Argenta LC, Morykwas MJ. Vacuum-assisted closure: a new method for wound control and treatment: clinical experience. Ann Plast Surg. 1997;38:563–76.
Morykwas MJ, Simpson J, Punger K, Argenta A, Kremers L, Argenta J. Vacuum-assisted closure: state of basic research and physiologic foundation. Plast Reconstr Surg. 2006;117:121S–6S.
Orgill DP, Manders EK, Sumpio BE, Lee RC, Attinger CE, Gurtner GC, Ehrlich HP. The mechanisms of action of vacuum assisted closure: more to learn. Surgery. 2009;146:40–51.
Lü DY, Liu XF, Gao YX, Huo B, Kang YY, Chen J, Sun SJ, Chen L, Luo XD, Long M. Asymmetric migration of human keratinocytes under mechanical stretch and co-cultured fibroblasts in a wound repair model. PLoS ONE. 2013;8:e74563.
Humphries JD, Byron A, Humphries MJ. Integrin ligands at a glance. J Cell Sci. 2006;119:3901–3.
Kong F, Li ZH, Parks WM, Dumbauld DW, García AJ, Mould AP, Humphries MJ, Zhu C. Cyclic mechanical reinforcement of integrin-ligand interactions. Mol Cell. 2013;49:1060–8.
Brakebusch C, Fassler R. β1 integrin function in vivo: adhesion, migration and more. Cancer Metast Rev. 2005;24:403–11.
Grose R, Hutter C, Bloch W, Thorey I, Watt FM, Fässler R, Brakebusch C, Werner S. A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair. Development. 2002;129:2303–15.
Lal H, Verma SK, Smith M, Guleria RS, Lu G, Foster DM, Dostal DE. Stretch-induced MAP kinase activation in cardiac myocytes: differential regulation through β1-integrin and focal adhesion kinase. J Mol Cell Cardiol. 2007;43:137–47.
Jalali S, del Pozo MA, Chen K, Miao H, Li Y, Schwartz MA, Shyy JY, Chien S. Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands. Proc Natl Acad Sci USA. 2001;98:1042–6.
Li J, Zhao ZH, Wang J, Chen GP, Yang JY, Luo SJ. The role of extracellular matrix, integrins, and cytoskeleton in mechanotransduction of centrifugal loading. Mol Cell Biochem. 2008;309:41–8.
Miyamoto S, Teramoto H, Gutkind JS, Yamada KM. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol. 1996;135:1633–42.
Matsubayashi Y, Ebisuya M, Honjoh S, Nishida E. ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing. Curr Biol. 2004;14:731–5.
Jeong HW, Kim IS. TGF-beta1 enhances betaig-h3-mediated keratinocyte cell migration through the alpha3beta1 integrin and PI3K. J Cell Biochem. 2004;92:770–80.
Katsumi A, Naoe T, Matsushita T, Kaibuchi K, Schwartz MA. Integrin activation and matrix binding mediate cellular responses to mechanical stretch. J Biol Chem. 2005;280:16546–9.
Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature. 1997;390:632–6.
Choma DP, Pumiglia K, DiPersio CM. Integrin alpha 3 beta 1 directs the stabilization of a polarized lamellipodium in epithelial cells through activation of Rac1. J Cell Sci. 2004;117:3947–59.
Ciobanasu C, Faivre B, Le Clainche C. Integrating actin dynamics, mechanotransduction and integrin activation: the multiple functions of actin binding proteins in focal adhesions. Eur J Cell Biol. 2013;92(10–11):339–48.
Jockusch BM, Bubeck P, Giehl K, Kroemker M, Moschner J, Rothkegel M, Rüdiger M, Schlüter K, Stanke G, Winkler J. The molecular architecture of focal adhesions. Annu Rev Cell Dev Biol. 1995;11:379–416.
Albuquerque ML, Flozak AS. Lamellipodial motility in wounded endothelial cells exposed to physiologic flow is associated with different patterns of beta1-integrin and vinculin localization. J Cell Physiol. 2003;195:50–60.
Askari JA, Tynan CJ, Webb SE, Martin-Fernandez ML, Ballestrem C, Humphries MJ. Focal adhesions are sites of integrin extension. J Cell Biol. 2010;188:891–903.
Nelson WJ. Adaptation of core mechanisms to generate cell polarity. Nature. 2003;422:766–74.
Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back. Science. 2003;302:1704–9.
Lämmermann T, Renkawitz J, Wu X, Hirsch K, Brakebusch C, Sixt M. Cdc42-dependent leading edge coordination is essential for interstitial dendritic cell migration. Blood. 2009;113:5703–10.
Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol. 2010;1:329–41.
Giancotti FG, Ruoslahti E. Integrin signaling. Science. 1999;285:1028–32.
Arulmoli J, Pathak MM, McDonnell LP, Nourse JL, Tombola F, Earthman JC, Flanagan LA. Static stretch affects neural stem cell differentiation in an extracellular matrix-dependent manner. Sci Rep. 2015;5:8499.
Jiménez C, Portela RA, Mellado M, Rodríguez-Frade JM, Collard J, Serrano A, Martínez-A C, Avila J, Carrera AC. Role of the PI3K regulatory subunit in the control of actin organization and cell migration. J Cell Biol. 2000;151:249–62.
Plotkin LI, Mathov I, Aguirre JI, Parfitt AM, Manolagas SC, Bellido T. Mechanical stimulation prevents osteocyte apoptosis: requirement of integrins, Src kinases, and ERKs. Am J Physiol Cell Physiol. 2005;289:C633–43.
Van Aelst L, D’SouzaSchorey C. Rho GTPases and signaling networks. Genes Dev. 1997;11:2295–322.
Schoenwaelder SM, Burridge K. Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol. 1999;11:274–86.
Watson A, Morris VL, Chan BM. Coordinated integrin and growth factor regulation of primary keratinocyte migration mediated through extracellular signal regulated kinase and phosphoinositide 3-kinase. Arch Dermatol Res. 2009;4:307–17.
Löffek S, Hurskainen T, Jackow J, Sigloch FC, Schilling O, Tasanen K, Bruckner-Tuderman L, Franzke CW. Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling. PLoS ONE. 2014;2:e87263.
Hsu CC, Chow SE, Chen CP, Tsai WC, Wang JS, Yu SY, Lee SC. Negative pressure accelerated monolayer keratinocyte healing involves Cdc42 mediated cell podia formation. J Dermatol Sci. 2013;3:196–203.
Raja, Sivamani K, Garcia MS, Isseroff RR. Wound re-epithelialization: modulating keratinocyte migration in wound healing. Front Biosci. 2007;12:2849–68.
Smola H, Thiekotter G, Fusenig NE. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction. J Cell Biol. 1993;122:417–29.
Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot L, Tarone G, Defilippi P. Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J. 1998;17:6622–32.
Schultz G, Rotatori DS, Clark W. EGF and TGF-alpha in wound healing and repair. J Cell Biochem. 1991;45:346–52.
Li Y, Fan JH, Chen M, Li W, Woodley DT. Transforming growth factor-alpha: a major human serum factor that promotes human keratinocyte migration. J Invest Dermatol. 2006;126:2096–105.