Background & Aims
Oral cancer pain is more prevalent and severe than in any other cancer (1). This type of pain is attributed to high levels of proteases released into the cancer microenvironment (2). These proteases activate the protease-activated receptor 2 (PAR2) (2) and sensitize the Transient Receptor Potential channels: Ankyrin 1 (TRPA1) (3) and Vanilloid 1 (TRPV1) (4) to mediate pain (5). The cysteine protease cathepsin S, involved in oral cancer pain, is reported to sensitize TRPV4 (6). Whereas trypsin, the serine protease and canonical activator of PAR2, sensitizes TRPV1 (7) and TRPV4 (8). RNA sequencing of human oral cancer samples identified increased expression of two proteases that have not been explored in oral cancer pain; matrix metalloprotease (MMP1) and serine protease 23 (PRSS23) (9). The aim of this research is to study the role of MMP1 and PRSS23 in PAR2 activation by examining the ability of these proteases to trigger (1) calcium influx ([Ca2+]i) and (2) to sensitize TRP channels.
Methods
We studied the effect of proteases in PAR2-evoked [Ca2+]i using HEK293 PAR1 knock-out cells overexpressing PAR2 (HEK-PAR2). Cells were challenged with the proteases MMP1, PRSS23, or trypsin (positive control). [Ca2+]i was measured using the calcium-sensitive dye, FURA-2, AM. To confirm PAR2 cleavage by MMP1 and PRSS23, cells were pre-incubated with these proteases, followed by the addition of trypsin. To study the sensitization of TRP channels, we used T-Rex-293 cells overexpressing PAR2 and expressing either TRPA1, TRPV1, or TRPV4 (TRex-PAR2-TRP) using a tetracycline-inducible system (10). TRex-PAR2-TRP cells were treated with MMP1 or PRSS23, followed by the addition of trypsin or an agonist for TRPA1 (AITC), TRPV1(capsaicin), or TRPV4 (GSK1016790A). T-Rex-293 cells express PAR1; thus, to assess only PAR2-mediated responses, experiments were performed in the presence of the PAR1 antagonist SCH79797.
Results
The addition of MMP1 and PRSS23 to HEK-PAR2 cells did not elicit [Ca2+]i. By contrast, trypsin, evoked a rapid and transient increase in [Ca2+]i. PAR2 cleavage by MMP1 and PRSS23 was demonstrated by a decrease in trypsin evoked [Ca2+]i in cells pre-treated with these proteases.
Addition of MMP1 to TRex-PAR2-TRP cells did not elicit [Ca2+]i. TRex-PAR2-TRP cells, pre-treated with MMP1 and followed by trypsin, enhanced trypsin-evoked [Ca2+]i in the presence of TRPA1 but not TRPV1 or TRPV4. The TRPA1 antagonist AM-902, abolished trypsin-evoked [Ca2+]i in TRex-PAR2-TRPA1 cells, validating the involvement of TRPA1 in the enhanced trypsin-evoked [Ca2+]i. To determine if MMP1 directly activates TRPA1, TRex-PAR2-TRPA1 cells were pre-treated with MMP1 and stimulated with AITC. MMP1 elicited less AITC-mediated [Ca2+]i compared to control cells pre-treated with a vehicle, indicating that MMP1 does not directly activate TRPA1.
Conclusions
MMP1 and PRSS23 do not induce a change in [Ca2+]i in HEK-PAR2 or TRex-PAR2-TRP cells.
Pre-incubation with MMP1 increased trypsin-evoked [Ca2+]i in TRex-PAR2-TRPA1 cells, but not in TRex-PAR2-TRPV1 or -TRPV4 cells. Increased trypsin-evoked [Ca2+]i depended on the presence of TRPA1. MMP1 does not directly activate TRPA1, as AITC (TRPA1 agonist) stimulation of MMP1-treated TRex-PAR2-TRPA1 cells decreased AITC-mediated [Ca2+]i.
MMP1 alone does not sensitize TRPA1 and requires prior PAR2 activation to sensitize the channel. In this work, prior PAR2 activation was achieved by trypsin; however, the high level of proteases present in the oral cancer microenvironment could facilitate prior PAR2 activation. The mechanisms by which MMP1 enhances trypsin-evoked [Ca2+]i warrant further exploration.
References
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Presenting Author
Leticia Arbex
Poster Authors
Topics
- Mechanisms: Biological-Molecular and Cell Biology