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J. Biol. Chem., Vol. 282, Issue 36, 26089-26100, September 7, 2007

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Trypsin IV or Mesotrypsin and p23 Cleave Protease-activated Receptors 1 and 2 to Induce Inflammation and Hyperalgesia^*

Wolfgang Knecht¹, Graeme S. Cottrell¹, Silvia Amadesi¹, Johanna Mohlin, Anita Skåregärde, Karin Gedda, Anders Peterson, Kevin Chapman^¶, Morley D. Hollenberg^¶, Nathalie Vergnolle^¶||**, and Nigel W. Bunnett²

From the Molecular Pharmacology and Lead Generation, AstraZeneca Research and Development, Mölndal 431 83, Sweden, the Departments of Surgery and Physiology, University of California, San Francisco, California 94143-0660, the ^¶Department of Pharmacology and Therapeutics, and Department of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada, ^||INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-310300, France, and the **Université Toulouse III Paul Sabatier, Toulouse F-31000, France

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR₁, PAR₂, and PAR₄ at sites that would expose the tethered ligand (PAR₁ = PAR₄ > PAR₂). Trypsin IV increased [Ca²⁺]_i in transfected cells expressing human PAR₁ and PAR₂ with similar potencies (PAR₁, 0.5 µM; PAR₂, 0.6 µM). p23 also cleaved fragments of PAR₁ and PAR₂ and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca²⁺]_i in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR ^–/–₁(trypsin IV) and PAR ^–/–₂ (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR ^–/–₂ mice but maintained in PAR ^–/–₁ mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR₁- and PAR₂-dependent inflammation and PAR₂-dependent hyperalgesia.

Received for publication, May 9, 2007 , and in revised form, July 5, 2007.

^* This work was supported by National Institutes of Health Grants DK57480, DK43207, and DK39957 (to N. W. B.), by a Proteinases and Inflammation Network group grant from the Canadian Institutes of Health Research (to M. D. H. and N. V.), and by INSERM-Avenir and the "Fondation BettencourtSchueller" (to N. V.). 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.

This article is dedicated to the memory of our colleague Anita Skåregärde, who died during the completion of this work.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Tables S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: University of California San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936; E-mail: nigel.bunnett{at}ucsf.edu.

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