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First published online 17 July 2007
doi: 10.1242/jcs.03476

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Mechanical force modulates global gene expression and -catenin signaling in colon cancer cells

Christopher L. Avvisato^1,2,*, Xiang Yang^2,*, Salim Shah², Becky Hoxter², Weiqun Li², Richard Gaynor³, Richard Pestell², Aydin Tozeren⁴ and Stephen W. Byers^2,

¹ Department of Biomedical Engineering, The Catholic University of America, Washington, DC, USA
² Departments of Oncology, Biochemistry, Molecular and Cellular Biology, Georgetown University School of Medicine and Lombardi Comprehensive Cancer Center, Washington, DC 20007, USA
³ Simmons Cancer Center, University of Texas Southwestern, Dallas, TX, USA
⁴ School of Engineering, Drexel University, Philadelphia, PA, USA

View larger version (39K): [in this window] [in a new window]   Fig. 1. Shear flow activates p38, Rac1 and NFB in colon cancer cells. (A) SW480 cells were exposed to shear stress (15 dyn/cm2) for 0 to 60 minutes. MAPK pathway activity was measured by western blot of phospho-ERK, -JNK and -p38. (B) HT29 cells exposed to shear stress (15 dyn/cm2) for 0 to 60 minutes. (C) Rac1 activity in SW480 cells exposed to shear stress. GTPS treatment increased the sensitivity of the GST-PBD pull-down assay. SW480 cells were exposed to shear stress (15 dyn/cm2) for 0, 2, 5 and 20 minutes. Lysates were treated with GTPS and GST-PBD pull-down was performed, followed by Rac1 western blot. (D) SW480 cells were exposed to shear stress for 0 to 12 hours and lysates were collected. Total RNA was purified and DKK1 mRNA concentration was analyzed by real-time qPCR. Cells were treated with the JNK inhibitor SB600125 and the p38 inhibitor SP202190 for 1 hour prior to initiation of shear. Cells were collected and DKK1 mRNA was analyzed by real-time qPCR. Inset in D, western blot analysis of DKK1 expression in SW480 cells exposed to shear stress for 0 to 12 hours. (E) As D, using HT29 cells. All experiments were performed in triplicate with consistent and repeatable results. (F) SW480 cells were transiently transfected with 1 µg/ml of NFB reporter and either 1 µg/ml of DN IKK or DN IKK and exposed to 15 dyn/cm2 of shear stress for 12 hours. Asterisk indicates significant difference in comparison with control (Student's t-test, P<0.01).

View larger version (32K): [in this window] [in a new window]   Fig. 2. Shear flow negatively regulates -catenin signaling and cell cycle. (A) SW480 cells transiently transfected with TOPFLASH reporter were exposed to shear stress (0-35 dyn/cm2) for 12 hours. Inset in A, western blot of -catenin in SW480 cell lysates before and after shear flow. (B) SW480 cells transiently transfected with cyclin D1 reporter gene (with or without TCF sites) were exposed to shear stress (0-35 dyn/cm2) for 12 hours. (C) SW480 cells plated at low (800,000 cells) and high (6 million cells) density were transiently transfected with TOPFLASH and exposed to shear stress of 15 dyn/cm2 for 12 hours. (D) SW480 cells transiently transfected with TOPFLASH were exposed to shear stress of 15 dyn/cm2 for 0 to 24 hours. (E) SW480 cells transiently transfected with TOPFLASH were exposed to shear stress (15 dyn/cm2) for 12 hours and incubated in static culture for 0 to 25 hours. (F) A1N4 cells, transiently transfected with TOPFLASH, FOPFLASH or pcDNA, and -catenin were exposed to shear stress of 15 dyn/cm2 for 12 hours. (G,H) Control cells (G) or cells exposed to shear flow for 24 hours (H) were analyzed by FACS. Experiments were performed in triplicate with consistent and repeatable results.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Shear flow modulation of -catenin signaling is ECM substrate- and integrin-dependent. (A) SW480 cells in static culture were transfected with TOPFLASH. (B) SW480 cells were transfected with TOPFLASH, incubated on glass, laminin or fibronectin, and exposed to 15 dyn/cm2 of shear stress for 12 hours. (C,D) SW480 cells transiently transfected with TOPFLASH were treated with 5, 6, 1 (10 µg/ml) or 4 (40 µg/ml) anti-integrin antibody for 2 hours. Cells were then plated on laminin and exposed to 15 dyn/cm2 of shear stress (C) or static (D) conditions for 12 hours. (E,F) Shear flow negatively regulates -catenin signaling through PI 3-kinase and Rac1. (E) SW480 cells were transiently transfected with the indicated plasmids and exposed to 15 dyn/cm2 of shear stress for 12 hours. (F) SW480 cells were transiently transfected with the indicated plasmids and exposed to 15 dyn/cm2 of shear stress for 12 hours. All experiments were performed in triplicate with consistent and repeatable results. Asterisk indicates significant difference in comparison with control (Student's t-test, P<0.01).

View larger version (76K): [in this window] [in a new window]   Fig. 4. Shear flow negatively regulates the level and localization of activated -catenin. Localization of cyclin D1 in control cells (A) and cells exposed to shear stress (B). Localization of -catenin in control cells (C) and cells exposed to shear stress (D). Localization of dephosphorylated activated -catenin in control cells (E) and cells exposed to shear stress (F). Lanes 1-4 of the inset in F show a western blot of dephosphorylated -catenin in the cytoplasmic (lanes 1, 3) and NP-40 pools (lanes 2, 4) before (lanes 1, 2) and after (lanes 3, 4) shear stress. The numbers above the lanes indicate the densitometric ratio of activated -catenin (static:shear).

View larger version (31K): [in this window] [in a new window]   Fig. 5. (A,B) Localization of -catenin phosphorylated on residues 33, 37 and 41 in control cells (A) and in cells exposed to shear stress (B). (C,D) Localization of -catenin phosphorylated on residues 41 and 45 in control cells (C) and in cells exposed to shear stress (D). (E,F) Cells were transfected with -catenin mutated on residues 33 and 37 and with TOPFLASH (E) or with a –163 cyclin D1 reporter (F) and exposed to shear stress for 16 hours. (G) HCT116 cells, which express -catenin missing serine 45, were transfected with TOPFLASH and exposed to shear stress for 12 hours.