Protease-activated receptor 2 activates CRAC-mediated Ca2+ influx to cause prostate smooth muscle contraction

Abstract

Protease activated receptor 2 (PAR2) is a G-protein coupled receptor that contributes to prostate fibrosis and lower urinary tract symptoms (LUTS). In addition to fibrosis, aberrant smooth muscle tone in the prostate has been hypothesized to play a role. We therefore examined PAR2 expression in primary human prostate smooth muscle cells (PSMC) and studied the downstream signaling effects of PAR2 activation. Signaling pathways involved in the process were assessed using the PAR2 activating peptide SLIGKV-NH2. We show that PAR2 is expressed in PSMC and that PAR2 activation mediates a biphasic elevation in intracellular Ca²⁺ and phosphorylation of myosin light chain 20 (MLC20), causing cellular contraction as assessed in a gel contraction assay. Intracellular Ca²⁺ flux was inhibited by a phosphoinositide hydrolysis inhibitor, U73122, showing a requirement for phospholipase C β (PLCβ) activation. PSMC expressed mRNA for L-type voltage dependent Ca²⁺ channels (VDCC) as well as Ca²⁺ release activated channels (CRAC), a hitherto unreported finding. Secondary intracellular Ca²⁺ oscillations were abrogated only by BTP2, the CRAC channel inhibitor, but not by nifedipine, an inhibitor of VDCC. These data suggest that, PAR2 activation and subsequent Ca²⁺ entry through CRAC channels are important mechanisms in prostate smooth muscle contraction.

Keywords: G-protein coupled receptor; PAR2; calcium channels; phospholipase; prostate; smooth muscle.

Figure 1

Expression of PAR2 in mice and human prostate smooth muscle cells. (A) Expression of PAR2 in smooth muscle cells in mouse prostate by immunofluorescence. Arrows in first row indicate representative cells with colocalization and in the second row cells where there si absence of colocalization and (B) in primary smooth muscle cells (PSMC) derived from human prostate by RT‐ PCR. WPMY‐1 and RWPE‐1 are myofibroblast and epithelial cell lines, respectively, derived from human prostate that are known to express PAR2 and were used as positive controls in the RT‐PCR reaction. Scale bar represents 50 µm

Figure 2

PAR2 causes contraction of smooth muscle cells from human prostate. (A and B) Decrease in diameter of collagen hydrogels after PAR2 activation with SLIGKV (80 µM). (C) Representative western blot and (D) densitometry showing time dependent increase in level of phosphorylated MLC20 in PSMC after PAR2 is activated. Data represent mean ± SEM of at least three independent experiments. Diameter of collagen hydrogels were measured in ImageJ (version 1.50i) and significance analyzed in Prism (version 7.04) with one‐way ANOVA followed by Tukey's multiple comparison test. *P < 0.05, **P < 0.01

Figure 3

Inhibiting PLCβ prevents PAR2‐mediated contraction in PSMC and Ca²⁺ flux. (A) Representative images of collagen hydrogels and (B) bar graph demonstrating significantly reduced diameter of collagen hydrogel gels upon U73122 (10 µM) pretreatment compared to control. (C and D) Increased Ca²⁺ flux in PSMC after PAR2 stimulation with 80 µM SLIGKV (solid line), that is significantly decreased when cells are pretreated with U73122 (dashed line). Data represent mean ± SEM of three independent experiments. Diameter of collagen hydrogels were measured in ImageJ and significance analyzed by a T‐test. [Ca²⁺]_i was monitored using Fura‐2AM fluorescence and represented as the 340/380 nm ratio. Baseline levels of [Ca²⁺]_i was recorded for 60 seconds and then recorded until 600 seconds after delivery of various reagents (represented with solid arrow). Peak increase of 340/380 ratio from baseline levels after addition of reagents was analyzed in Prism (version 7.04) with one‐way ANOVA followed by Tukey's multiple comparison test. *P < 0.05, ***P < 0.001, ****P < 0.0001

Figure 4

PAR2 activation causes a biphasic Ca²⁺ flux. (A and B) When Ca²⁺ is present in the external bath solution, PAR2 stimulation causes oscillatory calcium flux in PSMC (dashed line). BAPTA‐AM (10 µM) pretreatment abrogates Ca²⁺ flux with PAR2 activation (solid line). (C and D) In the absence of Ca²⁺ in the external solution, PAR2 activation causes initial Ca²⁺ flux in cells, but subsequent fluxes are abrogated (dashed line). BAPTA‐AM abolishes PAR2‐mediated Ca²⁺ flux in PSMC (solid line). Data represent mean ± SEM of three independent experiments. [Ca²⁺]_i was monitored using Fura‐2AM fluorescence and represented as the 340/380 nm ratio. Baseline levels of [Ca²⁺]_i was recorded for 60 seconds and then SLIGKV (80 µM) was delivered to cells and recorded until 600 seconds. Peak increase of 340/380 ratio from baseline levels after addition of SLIGKV was analyzed in Prism (version 7.04) with unpaired two tailed Student's t test. ***P < 0.001, ****P < 0.0001

Figure 5

CRAC channels are involved in contraction of prostate smooth muscle cells. (A) qRT‐PCR analysis to determine expression of various CRAC and L‐type voltage channels in PSMC. (B and C) collagen hydrogels showing significantly reduced contractility upon BTP2 (12 µM) pretreatment but not after nifedipine (10 µM) pretreatment. (D and E) BTP2 (12 µM) pretreatment reduced the amplitude of the secondary Ca²⁺ oscillations in PSMC (grey line), resulting in significantly reduced area of secondary oscillations. Data represent mean ± SEM of three independent experiments. qRT‐PCR samples of respective Ca²⁺ channel mRNA relative to the expression of GAPDH as a housekeeping gene. Diameter of collagen hydrogels were measured in ImageJ (version 1.50i) and significance analyzed in Prism (version 7.04) with one‐way ANOVA followed by Tukey's multiple comparison test. [Ca2+]_i was monitored using Fura‐2AM fluorescence and represented as the 340/380 nm ratio. Area of the secondary oscillations was determined by area under the curve analysis performed in Prism (version 7.04) with unpaired two tailed Student's t test. *P < 0.05, **P < 0.01

Figure 6

Schematic representation of signaling pathway activated by PAR2 to cause smooth muscle contraction in prostate. Stimulation of PAR2 activates PLCβ/IP₃ signal cascade to release Ca²⁺ from sarcoplasmic reticulum (SR) in smooth muscle cells of the prostate. The released Ca²⁺ initiates smooth muscle contraction via MLC kinase activation and increased phosphorylation of MLC20. Furthermore, Ca²⁺ released from the SR also activates STIM channels on the SR membrane, that activate the Orai CRAC channels, causing an influx of extracellular Ca²⁺ that sustains smooth muscle contraction by a mechanism that may involve MLC20 phosphorylation