Stretch-induced mitogen-activated protein kinase activation in lung fibroblasts is independent of receptor tyrosine kinases.
Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cgamma1 (PLCgamma1) and activation of the small G-protein Ras. Human lung fibroblasts (LFs) were seeded on matrix-coated silicone membranes and exposed to equibiaxial 10 to 40% static stretch or 20% contraction. LFs were stimulated with EGF, FGF2, or PDGF-BB or exposed to stretch in the presence of inhibitors of EGFR (AG1478), FGFR (PD173074), and PDGFR (AG1296). Phospho-MAPK, phospho-RTK, and phospho-PLCgamma1 levels were measured by Western blotting. Active GTP-Ras was quantified by immunoblotting after pull-down with a glutathione S-transferase-Raf-RBD construct. Normalized p-ERK1/2, p-JNK, and p-p38 levels increased after stretch but not contraction. Ligands to RTKs broadly stimulated MAPKs, with the responses to EGF and PDGF most similar to stretch in terms of magnitude and rank order of MAPK responses. Stretching cells failed to elicit measurable activation of EGFR, FGFR (FRS2alpha phosphorylation), or PDGFR. Potent inhibitors of the kinase activity of each receptor failed to attenuate stretch-induced MAPK activation. PLCgamma1 and Ras, prominent effectors downstream of RTKs, were not activated by stretch. Our findings demonstrate that MAPKs are potently activated by stretch in lung fibroblasts, but, in contrast to stress responses observed in other cell types, RTKs are not necessary for stretch-induced MAPK activation in LFs.
Boudreault
Francis
F
Department of Environmental Health, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, USA.
Tschumperlin
Daniel J
DJ
D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008168:Lung / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D009119:Muscle Contraction; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D013314:Stress, Mechanical; D013997:Time Factors
D017468:Receptors, Fibroblast Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor
Oncogene. 2004 Oct 18;23(48):7957-68
15489913
Physiol Rev. 2001 Apr;81(2):807-69
11274345
FASEB J. 2006 May;20(7):811-27
16675838
J Appl Physiol (1985). 2004 Oct;97(4):1567-74; discussion 1549
15208290
Am J Med Genet C Semin Med Genet. 2007 May 15;145C(2):125-38
17436297
Adv Protein Chem. 2004;68:1-27
15500857
J Biol Chem. 2006 Mar 31;281(13):8917-26
16418172
Cytokine Growth Factor Rev. 2005 Apr;16(2):139-49
15863030
J Cell Sci. 2005 Jun 15;118(Pt 12):2557-66
15928048
Exp Cell Res. 2003 Mar 10;284(1):78-88
12648467
Physiol Rev. 2000 Oct;80(4):1291-335
11015615
N Engl J Med. 2000 May 4;342(18):1301-8
10793162
Anat Rec. 1978 Dec;192(4):467-79
736269
J Biol Chem. 1996 Mar 22;271(12):7066-71
8636139
J Cell Physiol. 2004 Mar;198(3):417-27
14755547
Nature. 1999 Dec 23-30;402(6764):884-8
10622253
J Appl Physiol (1985). 1999 Jun;86(6):2026-33
10368370
Endocr Rev. 2001 Apr;22(2):153-83
11294822
Am J Respir Crit Care Med. 2000 Aug;162(2 Pt 1):357-62
10934053
Am J Physiol. 1995 Aug;269(2 Pt 1):L178-84
7653578
Physiol Rev. 2001 Jan;81(1):153-208
11152757
Arterioscler Thromb Vasc Biol. 2007 Aug;27(8):1744-51
17541019
Endocr Relat Cancer. 2000 Sep;7(3):165-97
11021964
Am J Physiol. 1993 Oct;265(4 Pt 1):L403-9
8238375
J Appl Physiol (1985). 2008 Mar;104(3):766-72
18187612
Science. 1996 Nov 15;274(5290):1194-7
8895468
J Cell Biol. 1996 Feb;132(4):741-52
8647902
Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L786-98
11557582
Exp Lung Res. 2000 Mar;26(2):105-19
10742925
Pediatr Res. 2008 Feb;63(2):124-30
18091360
FASEB J. 1998 Sep;12(12):1135-42
9737716
Am J Respir Cell Mol Biol. 2008 Apr;38(4):407-13
17975175
Am J Physiol Renal Physiol. 2007 Mar;292(3):F1028-34
17107942
Annu Rev Biochem. 2001;70:281-312
11395409
Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L883-91
11943650
Mech Dev. 2008 Mar-Apr;125(3-4):314-24
18082381
Nature. 2001 Mar 1;410(6824):37-40
11242034
J Cell Sci. 1998 Nov;111 ( Pt 22):3379-87
9788879
Dev Cell. 2006 Jan;10(1):11-20
16399074
Cell. 2006 Dec 1;127(5):1015-26
17129785
J Biol Chem. 2005 Jan 28;280(4):3060-7
15545271
Am J Physiol Heart Circ Physiol. 2000 Feb;278(2):H521-9
10666084
Am J Respir Cell Mol Biol. 2004 Jan;30(1):76-83
12829451
Ciba Found Symp. 1990;150:6-14; discussion 14-22
2164910
J Invest Dermatol. 2004 Dec;123(6):1012-9
15610507
J Appl Physiol (1985). 2004 Nov;97(5):1992-8
15475557
Biomech Model Mechanobiol. 2006 Mar;5(1):1-16
16489478
J Appl Physiol (1985). 2000 Dec;89(6):2391-400
11090594
J Cell Biol. 1993 Nov;123(3):741-7
8227136
J Cell Physiol. 2008 Dec;217(3):759-68
18668519
Oncogene. 2000 Nov 20;19(49):5548-57
11114734
Cell Signal. 2008 Oct;20(10):1830-8
18644433
Mol Cell Biol. 2004 Jun;24(12):5172-83
15169883
J Biol Chem. 1996 Oct 25;271(43):26981-8
8900184