Mechanical Stretch Stimulates Protein Kinase B/Akt Phosphorylation in Epidermal Cells via Angiotensin II Type 1 Receptor and Epidermal Growth Factor Receptor

Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis. Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis. The application of mechanical forces is a ubiquitous challenge to skin cells. Both differentiation processes and proliferation can be induced by different qualities of mechanical stimulation. In vitro studies have demonstrated that mechanical pressure gives rise to differentiation processes (1Bernd A. Ramirez-Bosca A. Görmar F.E. Bereiter-Hahn J. Holzmann H. Eur. J. Dermatol. 1992; 2: 450-451Google Scholar, 2Görmar F.E. Bernd A. Bereiter-Hahn J. Holzmann H. Bernd A. Bereiter-Hahn J. Hevert F. Holzmann H. Cell and Tissue Culture Models in Dermatological Research. Springer, Heidelberg, Germany1993: 87-97Google Scholar), while mechanical stretch supports proliferation in human epidermal cells (3Takei T. Rivas-Gotz C. Delling C.A. Koo J.T. Mills I. McCarthy T.L. Centrella M. Sumpio B.E. J. Cell. Physiol. 1997; 173: 64-72Crossref PubMed Scopus (83) Google Scholar, 4Kippenberger S. Bernd A. Loitsch S. Guschel M. Müller J. Bereiter-Hahn J. Kaufmann R. J. Investig. Dermatol. 2000; 114: 408-412Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Kippenberger S. Loitsch S. Müller J. Guschel M. Ramirez-Bosca A. Kaufmann R. Bernd A. Pigm. Cell Res. 2000; 4: 278-280Crossref Scopus (27) Google Scholar). These findings fit the in vivo situation where mechanical pressure provokes horny skin formation, and mechanical stretch, as present in abdominal stretching during pregnancy, induces skin enlargement. Previous in vitro studies on human skin keratinocytes have shown that mechanical stretch induces proliferation-associated signaling cascades of the mitogen-activated protein kinase pathway (4Kippenberger S. Bernd A. Loitsch S. Guschel M. Müller J. Bereiter-Hahn J. Kaufmann R. J. Investig. Dermatol. 2000; 114: 408-412Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Particularly the activation of extracellular signal-regulated kinase 1/2 may have functional aspects in stretch-mediated proliferation. As initial events in mechanotransduction, surface receptors of the integrin family are thought to recognize mechanical energy and transduce it into biological signals (6Ingber D. Curr. Opin. Cell Biol. 1991; 3: 841-848Crossref PubMed Scopus (591) Google Scholar, 7Ingber D. Annu. Rev. Physiol. 1997; 59: 575-599Crossref PubMed Scopus (1347) Google Scholar). This assumption is supported by the finding that blocking of β1 integrins abrogates stretch-induced activation of extracellular signal-regulated kinase 1/2 (4Kippenberger S. Bernd A. Loitsch S. Guschel M. Müller J. Bereiter-Hahn J. Kaufmann R. J. Investig. Dermatol. 2000; 114: 408-412Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In addition to increased proliferation, it could be that induction of antiapoptotic signaling pathways may contribute to the increase in cell number in response to mechanical stretch. In particular, the antiapoptotic kinase PKB 1The abbreviations used are: PKB, protein kinase B; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide 3-OH kinase; HEK, human embryonic kidney; AT1-R, angiotensin II type 1 receptor; GPCR, G protein-coupled receptor; PBS, phosphate-buffered saline; TGFα, transforming growth factor α./Akt is a candidate signaling molecule shown to play a key role in suppression of apoptotic cell death (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1318) Google Scholar, 9Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (985) Google Scholar). Prototypically, PKB/Akt activation is transduced by cell surface receptors in response to insulin and mitogens such as epidermal growth factor (10Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4471) Google Scholar, 11Pearl L.H. Barford D. Curr. Opin. Struct. Biol. 2002; 12: 761-767Crossref PubMed Scopus (65) Google Scholar, 12Okano J. Gaslightwala I. Birnbaum M.J. Rustgi A.K. Nakagawa H. J. Biol. Chem. 2000; 275: 30934-30942Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). In addition, recent advances have shown that integrins, namely the β1 and β4 subsets, stimulate PKB/Akt phosphorylation and therefore may contribute to antiapoptosis (13Fornaro M. Steger C.A. Bennett A.M. Wu J.J. Languino L.R. Mol. Biol. Cell. 2000; 11: 2235-2249Crossref PubMed Scopus (40) Google Scholar, 14Tang K. Nie D. Cai Y. Honn K.V. Biochem. Biophys. Res. Commun. 1999; 264: 127-132Crossref PubMed Scopus (38) Google Scholar). Proximal from surface receptors, the phosphoinositide 3-OH kinase (PI3K) conveys activation of PKB/Akt via phosphoinositide-dependent kinases. It has been demonstrated that phosphoinositide-dependent kinase-1 phosphorylates PKB/Akt at threonine 308 (15Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar), whereas the mechanism of the serine 473 phosphorylation is still under debate (16Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 17Attwell S. Roskelley C. Dedhar S. Oncogene. 2000; 19: 3811-3815Crossref PubMed Scopus (204) Google Scholar). Studies regarding the impact of mechanical stimuli on PKB/Akt have been mainly carried out in endothelium-derived cells as hemodynamic changes caused by coronary circulation are known to contribute to pathophysiological processes of the blood vessel system (18Dimmeler S. Assmus B. Hermann C. Haendeler J. Zeiher A.M. Circ. Res. 1998; 83: 334-341Crossref PubMed Scopus (377) Google Scholar, 19Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3080) Google Scholar, 20Fisslthaler B. Popp R. Michaelis U.R. Kiss L. Fleming I. Busse R. Hypertension. 2001; 38: 1427-1432Crossref PubMed Scopus (69) Google Scholar, 21Liu X.M. Ensenat D. Wang H. Schafer A.I. Durante W. FEBS Lett. 2003; 541: 52-56Crossref PubMed Scopus (73) Google Scholar). In this context, vascular smooth muscle cells may also respond to mechanical stretch by activation of PKB/Akt and therefore may contribute to the onset of proliferative disease of the vascular system (22Sedding D.G. Seay U. Fink L. Heil M. Kummer W. Tillmanns H. Braun-Dullaeus R.C. Circulation. 2003; 108: 616-622Crossref PubMed Scopus (71) Google Scholar). It is hypothesized that mechanical stress induced by therapeutic interventions such as stent implantation and balloon catheters contributes to the frequently observed restenosis (23Zhou R.H. Lee T.S. Tsou T.C. Rannou F. Li Y.S. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 2015-2020Crossref PubMed Scopus (50) Google Scholar). In the present approach, it was found that PKB/Akt activation in response to mechanical stretch is also present in a keratinocyte model. Moreover the signaling events involved in the activation of PKB/Akt were identified. Transactivation of epidermal growth factor receptor (EGFR) by pertussis-sensitive GPCRs demonstrates a novel mechanism of PKB/Akt activation triggered by mechanical stretch. Antibodies and Reagents—The following antibodies were used. Phosphospecific antibodies raised against PKB/Akt (serine 473 and threonine 308) and an antibody against total PKB/Akt were from Cell Signaling Technology Inc. Antibodies raised against the activated form of the EGFR and total EGFR were purchased from BD Transduction Laboratories and Santa Cruz Biotechnology Inc. Detection of caspase 8 and 3 degradation was performed using antibodies from Cell Signaling Technology Inc. The PI3K inhibitors wortmannin and LY-294002 and the specific inhibitors of the epidermal growth factor receptor tyrosine kinase, AG1478 and PD168393, were purchased from Calbiochem-Novabiochem. Pertussis toxin from Bordetella pertussis to inactivate the α subunits in the Gi and Go subfamilies of heterotrimeric G proteins was purchased from Calbiochem-Novabiochem. Losartan to block the angiotensin II type 1 receptor (AT1-R) was a kind gift from Merck Sharp and Dohme. The endothelin receptor antagonists BQ-123 and BQ-788 were purchased from Calbiochem-Novabiochem. The apoptosis inducer staurosporine was purchased from Roche Applied Science. Cell Culture—The spontaneously immortalized human keratinocyte cell line (HaCaT) (a generous gift from Professor Fusenig, German Cancer Research Institute, Heidelberg, Germany) was cultured in carbonate buffered Hanks' medium with 5% fetal calf serum and 1% penicillin-streptomycin at 37 °C in a 5% CO2 atmosphere. The human epidermoid carcinoma cell line A-431 (American Type Culture Collection) and the human epithelial kidney line HEK-293 were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. All experiments were done in agreement with the local ethics commission. Mechanical Stimulation—Stretching of in vitro cultured cells was performed as described previously (4Kippenberger S. Bernd A. Loitsch S. Guschel M. Müller J. Bereiter-Hahn J. Kaufmann R. J. Investig. Dermatol. 2000; 114: 408-412Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Kippenberger S. Loitsch S. Müller J. Guschel M. Ramirez-Bosca A. Kaufmann R. Bernd A. Pigm. Cell Res. 2000; 4: 278-280Crossref Scopus (27) Google Scholar). Briefly, silicone elastomer MED-4011 (Armando Medizin Technik, Düsseldorf, Germany) was stirred, poured into preformed Teflon matrices, and allowed to polymerize. To allow cell attachment, silicone dishes were treated with 5.7% KOH in methanol for 5 min to neutralize the polymerization-derived HCl. After washing with double distilled water, silicone dishes were coated with 2% arginine for 2 h to facilitate cell attachment (24Karasek M.A. Curr. Probl. Dermatol. 1980; 10: 143-158Crossref PubMed Google Scholar) and afterward rinsed with phosphate-buffered saline (PBS). Subsequently, the dishes were incubated with fetal calf serum for 2 h. After withdrawal of the serum, cells were plated in flexible silicone chambers and incubated for 24 h under regular conditions. Prior to the application of mechanical stretch, the cells were held for 24 h under serum-free conditions. Then the silicone chambers were extended to 10% for 5 min. After stretch stimulation, cells were either fixed for immunocytochemistry, or protein samples were prepared at the indicated time intervals. Transfection—HEK-293 cells seeded in silicone dishes were transiently transfected with kinase-defective mutants of EGFR (K721A) using Lipofectamine 2000 reagent (Invitrogen). The construct was a kind gift from Josef Yarden, Weizman Institute of Science, Rehovot, Israel (25Bao J. Alroy I. Waterman H. Schejter E.D. Brodie C. Gruenberg J. Yarden Y. J. Biol. Chem. 2000; 275: 26178-26186Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Because of a point mutation at position 721, Lys is replaced by Ala to inhibit the cytoplasmic kinase activity. After transfection, cells were allowed to synthesize proteins under regular conditions for 16 h. Before the application of mechanical stretch the cells were serum-starved for another 16 h. Immunoblotting—For detection of PKB/Akt and caspases 3 and 8, cells were lysed in 100 μl of SDS sample buffer (62.5 mm Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mm for 5 and on detection of EGFR, cells were into buffer mm (pH mm 1 mm 1 mm 1% mm 1 mm 1 mm 1 1 mm and of the was protein and using serum of protein were with SDS sample buffer and on Then proteins were to a The membrane was in blocking buffer saline (pH 5% for at 3 h at °C by with the antibody in saline (pH and 5% serum The antibodies were using and with the detection system cells were fixed with 5% for min at The silicone dishes were into in and incubated with the EGFR antibody in with 1% serum for min at 37 After with PBS, the silicone were incubated with in for min at the silicone were rinsed with PBS, on and with a Mechanical of cells were seeded into flexible chambers and into a stretching as described previously (4Kippenberger S. Bernd A. Loitsch S. Guschel M. Müller J. Bereiter-Hahn J. Kaufmann R. J. Investig. Dermatol. 2000; 114: 408-412Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). were to 10% for 5 min and were into SDS sample buffer and were for PKB/Akt phosphorylation by As shown in mechanical stretch induced a phosphorylation of PKB/Akt at both phosphorylation sites 308 and serine The phosphorylation and min in the epithelial cell of PI3K PKB/Akt in to role of PI3K was to the pathways the activation of PKB/Akt phosphorylation by mechanical stretch. is a of for the key role of PI3K in the activation of PKB/Akt in different cell (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1318) Google Scholar, A. P. S. J. J. 1997; PubMed Scopus Google Scholar). In study the of PI3K was by selective inhibitors and It was found that a for 1 h with LY-294002 the stretch-induced phosphorylation of PKB/Akt and To this cells were treated with wortmannin at and 100 In the of the phosphorylation of PKB/Akt was suppressed in a the stretch-induced phosphorylation of PKB/Akt and min stretch was by of wortmannin These findings phosphorylation of PKB/Akt in response to mechanical stretch. Mechanical Transactivation of Pertussis and Losartan EGFR Transactivation and of of the pathways to activation of PKB/Akt is the of growth to receptor tyrosine Nature. 1995; PubMed Scopus Google Scholar, T.F. K. A. Kaplan D.R. Tsichlis P.N. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google Scholar). In the present the of this signaling for was demonstrated a phosphorylation of EGFR in response to mechanical stretch and These were by The of EGFR in response to mechanical stretch indicated functional activation Furthermore it was whether the phosphorylation of EGFR in response to stretch is on cells were with pertussis toxin known to inactivate by the of α subunits in the Gi and Go subfamilies of heterotrimeric G In could that with pertussis toxin the stretch-induced phosphorylation of EGFR This finding gives for a of GPCRs to the transactivation of EGFR in response to mechanical stretch. of EGFR tyrosine kinase by with 1 EGFR transactivation by mechanical stretch. The functional EGFR, and PKB/Akt was by an of PKB/Akt by pertussis toxin To the different inhibitors against were from a vascular smooth muscle cell that angiotensin and endothelin receptors in the activation of extracellular signal-regulated kinase 1/2 in response to stretch H. S. T. Y. T. Biol. 2002; PubMed Scopus Google Scholar). In an of AT1-R, suppressed PKB/Akt phosphorylation in against the endothelin receptor and and These that and cascades in the phosphorylation of PKB/Akt in of by pertussis toxin abrogates stretch-induced phosphorylation of cells were for h with different of pertussis toxin and and for 5 min. protein was and and for PKB/Akt phosphorylation. from cells in the of pertussis toxin as a positive The pertussis of abrogates stretch-mediated activation of cells were with to block the or with 5 or both receptor antagonists specific for the type or type B were for 5 and total protein was prepared at the indicated time and for PKB/Akt phosphorylation. The EGFR and of EGFR PKB/Akt in to shown in 3 demonstrated phosphorylation of the EGFR in response to mechanical stretch. In the following it was investigated whether phosphorylation of EGFR has functional for PKB/Akt phosphorylation cells were treated with a specific of the epidermal growth factor receptor tyrosine kinase, and The treated with the phosphorylation of PKB/Akt In phosphorylation of PKB/Akt was observed in cells treated with 50 AG1478 To different of AG1478 were In the of mechanical stretch AG1478 and 100 on PKB/Akt phosphorylation at both phosphorylation sites at AG1478 was the phosphorylation of PKB/Akt the of of AG1478 on stretch-induced phosphorylation of After stretching the cells for 5 min the protein was and for PKB/Akt phosphorylation. the stretch a phosphorylation of PKB/Akt at both phosphorylation sites It was found that the stretch-induced phosphorylation of PKB/Akt was suppressed by AG1478 in a the phosphorylation of PKB/Akt was this was To findings another specific EGFR was of PKB/Akt in response to mechanical stretch was abrogated in the of In addition to expression of EGFR (K721A) was It was found that of in HEK-293 cells phosphorylation of the functional EGFR and PKB/Akt in the of mechanical stretch of EGFR by AG1478 or activation of cells were for 1 h with the indicated inhibitors and stimulated with for 5 min. different and were to the cells with 50 different and 100 were to the cells with 100 The of the EGFR tyrosine kinase by and dominant-negative expression abrogates stretch-mediated activation of serum-starved cells were with for 1 for 5 and and for PKB/Akt phosphorylation. HEK-293 cells were transiently transfected with mutants of EGFR (K721A) or for After proteins were and min and for PKB/Akt phosphorylation. The Functional of EGFR by AG1478 and of and that EGFR inhibitors AG1478 and abrogated phosphorylation of PKB/Akt in response to mechanical stretch in cells. To the functional of both inhibitors was cells were with of AG1478 or and and stimulated with 50 as an EGFR a of PKB/Akt phosphorylation in a cells were with a fixed of both inhibitors and stimulated with of and 100 of PKB/Akt phosphorylation was the functional of EGFR by both inhibitors Mechanical of 8 and the present study that mechanical stretch of the key in apoptosis. this it could be that mechanical stretch is to cells against apoptosis. This was using a of mechanical stretch and a known inducer of apoptosis. in that staurosporine for 16 h to a increase of caspase 8 and 3 2 and staurosporine and mechanical stretch were at the caspase 3 were the of caspase 8 was 5 and addition of staurosporine 1 and h to a in caspase These findings that mechanical stretch protects cells against the onset of apoptosis. in cell by the application of and forces are to expression and cell In particular, cell stretch is to be a cell proliferation. the of an for the application of mechanical stretch to increase skin to skin for J. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore is that mechanical stimuli contribute to Y. Cancer Res. 1992; Google Scholar). caused by increased and the of a functional circulation 1998; 4: PubMed Scopus Google Scholar). The increase of gives rise to stretch of the It that stretching of the proliferation of cells and therefore contributes to growth of The signaling pathways mechanical stretch to cell proliferation and are still In the present study that mechanical stretch to human epithelial cells the antiapoptotic kinase This supports the assumption that the increase in cell number in response to mechanical stretch at in to a suppression of apoptosis. of PKB/Akt in was to wortmannin and LY-294002 have shown that the of PI3K for PKB/Akt activation is by a and the of PKB/Akt (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1318) Google Scholar). in the present study a of receptor tyrosine EGFR the to was It was demonstrated that mechanical stretch induces EGFR formation and phosphorylation of the receptor. 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J. 2003; PubMed Scopus Google Scholar). to this the of EGFR, is for the induction of studies provided initial that activation of PKB/Akt signaling by mechanical stretch contributes to suppression of apoptosis. These findings contribute to the of the known induced by mechanical stretch. the for and be also for are to for of the