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Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype

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Abstract

Cyclophilin A (CypA) was originally identified as a cytosolic protein possessing peptidyl–prolyl isomerase activity. CypA has been shown to play a pivotal role in the immune response, but little is known about other molecular mechanisms of CypA-mediated biologic events. In our present study, we demonstrate that knockdown CypA expression using RNAi in U2OS cells resulted in disruption of the F-actin structure, as well as decreased anchorage-independent growth, proliferation, and migration. Wild-type U2OS cells treated with cyclosporine A (CsA), a peptidyl–prolyl isomerase inhibitor, displayed the same phenotype as knockdown CypA cells, suggesting that the isomerase activity of CypA is required to maintain a normal phenotype. In vitro and in vivo binding assays revealed that CypA binds to N-WASP, which functions in the nucleation of actin via the Arp2/3 complex. Pulse-chase labeling study indicated an enhanced degradation of N-WASP in cell lacking CypA, suggesting that CypA is required for stabilizing N-WASP to form a N-WASP/Arp2/3 complex for the nucleation/initiation of F-actin polymerization.

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Acknowledgments

We thank Dr. Matthew J. Schibler of the UCLA Brain Research Institute for assistance with confocal microscopy. We are also grateful to Uma Dandekar, Assistant Director, Sequencing Core and Martin Phillips, Manager, Biochemistry Instrumentation Facility for their help with this study. This work was supported by a grant CA066746 to RC. Colonya C. Calhoun was supported by the NIH Training Grant #T32 DE07296.

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Authors and Affiliations

  1. Dental Research Institute, UCLA School of Dentistry, Los Angeles, CA, 90095, USA

    Colonya C. Calhoun, Ying-Chun Lu, Jun Song & Robert Chiu

  2. Department of Surgery/Oncology, UCLA School of Medicine, Los Angeles, CA, 90095, USA

    Robert Chiu

  3. Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, 90095, USA

    Robert Chiu

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  1. Colonya C. Calhoun
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  2. Ying-Chun Lu
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Correspondence to Robert Chiu.

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Calhoun, C.C., Lu, YC., Song, J. et al. Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype. Mol Cell Biochem 320, 35–43 (2009). https://doi.org/10.1007/s11010-008-9896-0

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  • DOI: https://doi.org/10.1007/s11010-008-9896-0

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