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J. Biol. Chem., Vol. 283, Issue 45, 30471-30481, November 7, 2008

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The Human Cathelicidin LL-37 Modulates the Activities of the P2X₇ Receptor in a Structure-dependent Manner^*

Linda Tomasinsig¹, Cinzia Pizzirani¹, Barbara Skerlavaj, Patrizia Pellegatti, Sara Gulinelli, Alessandro Tossi^¶, Francesco Di Virgilio, and Margherita Zanetti^||2

From the Department of Biomedical Sciences and Technology, University of Udine, 33100 Udine, Italy, Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Interdisciplinary Center for the Study of Inflammation, University of Ferrara, 44100 Ferrara, Italy, ^¶Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, 34127 Trieste, Italy, and ^||National Laboratory of the Interuniversity Consortium for Biotechnology, Area Science Park, 34012 Trieste, Italy

Extracellular ATP, released at sites of inflammation or tissue damage, activates the P2X₇ receptor, which in turn triggers a range of responses also including cell proliferation. In this study the ability of the human cathelicidin LL-37 to stimulate fibroblast growth was inhibited by commonly used P2X₇ blockers. We investigated the structural requirements of the growth-promoting activity of LL-37 and found that it did not depend on helix sense (the all-D analog was active) but did require a strong helix-forming propensity in aqueous solution (a scrambled analog and primate LL-37 orthologs devoid of this property were inactive). The involvement of P2X₇ was analyzed using P2X₇-expressing HEK293 cells. LL-37 induced proliferation of these cells, triggered Ca²⁺ influx, promoted ethidium bromide uptake, and synergized with benzoyl ATP to enhance the pore and channel functions of P2X₇. The activity of LL-37 had an absolute requirement for P2X₇ expression as it was blocked by the P2X₇ inhibitor KN-62, was absent in cells lacking P2X₇, and was restored by P2X₇ transfection. Of particular interest, LL-37 led to pore-forming activity in cells expressing a truncated P2X₇ receptor unable to generate the non-selective pore typical of the full-length receptor. Our results indicate that P2X₇ is involved in the proliferative cell response to LL-37 and that the structural/aggregational properties of LL-37 determine its capacity to modulate the activation state of P2X₇.

Received for publication, March 19, 2008 , and in revised form, August 8, 2008.

^* This work was supported by the Italian Ministry of Education, University and Research (Progetti di Ricerca di Interesse Nazionale 2005 Grants 2005068150_001 and 2005051341_004), Regione Friuli-Venezia Giulia (Grant art.23 L.R. 26/2005), the Italian Association for Cancer Research, Telethon of Italy (Grant GGP06070), the Italian Space Agency (Agenzia Spaziale Italiana-Osteoporosi e Atrofia Muscolare), the Commission of European Communities (7th Framework Program HEALTH-F2-2007-202231), and institutional funds from the University of Ferrara. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biomedical Sciences and Technology, University of Udine, P.le Kolbe 4, I-33100 Udine, Italy. E-mail: mzanetti{at}mail.dstb.uniud.it.

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