title: Regulation of tension-induced mechanotranscriptional signals by the microtubule network in fibroblasts.
abstract: Mechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through beta1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured fibroblasts at focal adhesions. Force enhanced recruitment of alpha-tubulin and the plus end microtubule-binding protein cytoplasmic linker protein-170 (CLIP-170) at focal adhesions. Immunoprecipitation studies demonstrated no direct binding of tubulin to actin or filamin A, but CLIP-170 interacted with tubulin, filamin A, and beta-actin. The association of CLIP-170 with beta-actin was enhanced by force. Force activated the p38 mitogen-activated protein kinase, increased filamin A expression, and induced the relocation of p38 and filamin A to focal adhesions. Disruption of microtubules with nocodazole, independent of force application, enhanced filamin A expression and Sp1-mediated filamin A promoter activity, while stabilization of microtubules with Taxol inhibited force induction of both filamin A mRNA and protein. We conclude that in response to tensile forces applied through beta1 integrins and actin the microtubule network modulates mechanotranscriptional coupling of filamin A.
authors.affiliation: Canadian Institutes of Health Research (CIHR) Group in Matrix Dynamics, Institute of Dental Research, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors.forename: Mario
authors.initials: M
authors.lastname: D'Addario
authors.forename: Pamela D
authors.initials: PD
authors.lastname: Arora
authors.forename: Richard P
authors.initials: RP
authors.lastname: Ellen
authors.forename: Christopher A G
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D003285:Contractile Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D064448:Filamins; D022001:Focal Adhesions / Q000378:metabolism; D005881:Gingiva / Q000166:cytology; D006801:Humans; D015151:Immunoblotting; D008280:Magnetics; D008840:Microfilament Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009363:Neoplasm Proteins; D017239:Paclitaxel / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013314:Stress, Mechanical; D014158:Transcription, Genetic; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D000199:Actins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014404:Tubulin; C076744:cytoplasmic linker protein 170; D012313:RNA; D003094:Collagen; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D017239:Paclitaxel