Effects of Protease Activated Receptor 2 Agonists on Neuronal SH-SY5Y Cells Survival

Background: Protease-activated receptors (PARs) area family of G protein-coupled receptors that are activated by proteolytic cleavage by a diverse set of proteases (1). Trypsin has a high affinity for PAR2, but can also activate other PARs (2). Pharmacologically a peptide (SLIGRL-NH2, PAR2-AP) has been used to selectively activate PAR2. However, there is emerging evidence that proteases and PAR2-APact as biased agonists of PAR2 (3). PAR2 is expressed in many different cell and tissue types regulating many biological functions. PAR2 has been linked to both neuroprotective and neurodegenerative responses (4). Indeed, PAR2 may play both protective and pathogenic role in Alzheimer’s disease (5), and dysregulation of serine proteases has been suggested as an important contributor to central nervous system pathophysiology including Alzheimer’s disease, Parkinson’sdisease and stroke (6). However, the role of PAR2in neuronal cell function and survival remains unclear.

Aim: To elucidate the role of biased PAR2agonists in neurodegenerative and neuroprotective responses in differentiated and undifferentiated human SH-SY5Y neuroblastoma cells.

Methods: SH-SY5Y cells were differentiated using retinoic acid (10µM, 4 days). Differentiated and undifferentiated SH-SY5Y cells were exposed to saline (control), trypsin (0.1-100 nM) or PAR2-AP (0.1-100 µM) and cell viability assessed at 24 and 48 h using MTT assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 0.5 mg/mL HEPES-Hank’s Balanced Salt Solution). Data were expressed as percentage of control (100%) and analysed by ANOVA followed by a Tukey test (mean±S.E.M).

Results: In undifferentiated SH-SY5Y cells,trypsin (0.1-100 nM, 24, 48 h) had no effect on cell viability. In contrast, PAR2-AP significantly decreased cell viability (0.1 µM, 79±8%; 1 µM, 68±4%; 10 µM, 78±2%; 100 µM, 79±2%, n=4, p≤0.01) after 24 h. Surprisingly, no effect was observed after 48 h. In differentiated cells, trypsin and PAR2-AP (24 and 48 h) significantly increased cell viability (trypsin, 24 h; 1 nM, 129±8%; 10 nM, 131±7%; 48 h, 0.1 nM, 125±4%; 1 nM, 127±7%; 10 nM, 126±3%; 100 nM, 129±6%; PAR2-AP, 24 h, 10 µM, 120±7%; 100 µM, 120±4%; 48 h, 1 µM, 119±5%; 10 µM, 118±7%; 100 µM, 123±5%;n=4, p≤0.05).

Discussion and conclusion: In undifferentiated SH-SY5Y cells, trypsin and PAR2-AP had differing effects on cell viability, indicating that the PAR2 agonists are activating diverse signaling cascades in undifferentiated cells. In contrast, both PAR2 agonists promoted cell viability in differentiated SH-SY5Y cells, suggesting they activate similar signalling cascades. Furthermore, we also observed that PAR2-AP has opposing effects on the cell viability of undifferentiated and differentiated SH-SY5Y cells. This data indicates a role, not only for agonist-mediated bias in PAR2 signaling, but also a role for the differentiation state of the cell. Determining the molecular mechanisms underlying biased activation of PAR2and differentiation-dependent PAR2 signalling will further define the role of PAR2 and serine proteases in neuronal cell death and survival. Elucidating the fundamental mechanisms controlling both agonist- and differentiation-dependent PAR2 signaling may unearth new targets to regulate the viability of neuronal cells and lead to the development of new treatments for neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases.

References:

(1) Adams MN et al. (2011) Pharmacol Ther. 130, 248-282

(2) Xu et al. (1998) PNAS 95, 6642-6

(3) Ramachandran et al. (2009) Mol Pharmacol 76, 791-801

(4) Noorbakhsh F et al. (2005) J Immunol 174, 7320-7329

(5) Alkhami-Goli et al. (2007) J Immunol 179, 5493-503.

(6) Yoon H et al. (2013) J Neurochem 127, 283–298.