Cathepsin S Signals via PAR2 and Generates a Novel Tethered Ligand Receptor Agonist

Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation

Cathepsin S Signals via PAR2 and Generates a Novel Tethered Ligand Receptor Agonist

Abstract Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation

Cathepsin S cleavage of the PAR2 N-terminus requires glycine⁴⁰.

<p>a) Luciferase luminescence was measured after cathepsin S (2 µM) treatment of HeLa cells expressing luciferase-PAR2 (bar 1), PAR2M1 (bar 2), PAR2M2 (bar 3), and PAR2M3 (bar 4). Luminescence of the transfected cells without cathepsin S treatment was subtracted from each of the measurements. b) Western blots were performed on supernatants of luciferase-PAR2 and PAR2 mutant-transfected HeLa cells following treatment with cathepsin S. Luciferase-PAR2, cathepsin S(−) (lane 1); luciferase-PAR2, cathepsin S (+) (lane 2); PAR2M1, cathepsin S (−) (Lane 3); PAR2M1 cathepsin S (+) (lane 4); PAR2M2, cathepsin S (−) (lane 5); PAR2M2, cathepsin S (+) (lane 6); PAR2M3, cathepsin S (−) (lane 7); PAR2M3, cathepsin S (+) (lane 8) and non-transfected HeLa cells, cathepsin S (+) (lane 9). Molecular weight markers on the left side of the blot are in kDa.</p

Cathepsin S fails to activate mutant PAR2 but KVDGTS retains activity on mutant PAR2.

<p>a) Calcium imaging was performed in HeLa cells transfected with native or mutant PAR2 receptors following treatment with cathepsin S. Cathepsin S (2 µM) was able to stimulate native PAR2 and PAR2M1 but not PAR2M2 or PAR2M3. b) In contrast, KVDGTS activated PAR2 and all three substitution mutants. Cathepsin S (2 µM), KVDGTS (100 µM).</p

Concentration-effect curves for PAR2 derived peptides.

<p>Calcium-dependent responses were determined in PAR2-transfected HeLa cells following incubation with the indicated peptides. The curves for activating peptides are similar in shape although that for KVDGTS is shifted to the right and has a lower peak calcium response as compared to SLIGRL and SLIGKVDGTS. The inverse hexapeptides, STGDVK and LRGILS were not active.</p

PAR2 N-terminal hexapeptides are capable of activating the receptor.

<p>a) Single-cell calcium imaging in HeLa cells transfected with PAR2 cDNA demonstrated a similar response to KVDGTS (thick solid line) and SLIGRL (thick dotted line). A weaker response was observed in response to IGKVDG (thin solid line). HeLa cells transfected with salmon sperm DNA and stimulated with KVDGTS did not respond (horizontal dotted line). b) Cathepsin S (solid line) and KVDGTS (100 µM) (dotted line) elicit similar calcium responses in NHEKs. KVDGTS (100 µM) and SLIGRL (10 µM), IGKVDG (100 µM) and cathepsin S (2 µM).</p

KVDGTS alters PAR2 response to cathepsin S.

<p>a) HeLa cells transfected with PAR2 cDNA were treated with KVDGTS and subsequently with cathepsin S after a 2 minute interval (dotted line). Pre-treatment with KVDGTS attenuates the response to cathepsin S. HeLa cells treated with cathepsin S failed to respond subsequently to KVDGTS (100 µM) (solid line). b) KVDGTS (100 µM) elicted calcium responses in NHEKs and the subsequent response to cathepsin S (2 µM) was attenuated. Treatment with cathepsin S abolishes the response to the subsequent addtion of KVDGTS. The second agent was delivered at the 300 second timepoint. KVDGTS (100 µM), cathepsin S (2 µM).</p