Thrombin-induced endothelin-1 synthesis and secretion in retinal pigment epithelial cells is rho kinase dependent

Abstract

Purpose: The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood–retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1.

Methods: Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels.

Results: Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion.

Conclusions: Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.

FIG. 1.

Secreted endothelin-1 (ET-1) in mature ARPE-19 (A) and primary human retinal pigment epithelium (hRPE) (B) cells measured by radio-immunoassay (RIA). Cells were treated with the indicated agonists and/or inhibitors for 24 h in serum-free Dulbecco's modified Eagle's medium/F-12 medium. Immunoreactive ET-1 (ir-ET-1) released in the medium was extracted and measured by RIA. In mature RPE (mRPE) cells, thrombin significantly increased ir-ET-1 secretion versus control, an effect that was concentration dependent. A similar effect was observed in primary hRPE cells. Hirudin (30 nM) when preincubated with thrombin (10 nM) inhibited ET-1 secretion as opposed to thrombin (10 nM) alone. Peptidergic protease-activated receptor-1 (pPAR-1), but not pPAR-4 (both at 50 μM), significantly increased ET-1 secretion, suggesting that thrombin-mediated effects on ET-1 secretion involves the PAR-1. Data are represented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using analysis of variance and Student-Newman Keuls (SNK) test. *Significance versus control (P < 0.05) (n = at least 6 per treatment).

FIG. 2.

Time-dependent increase in ir-ET-1 secretion in mRPE cells after thrombin (10 nM) stimulation. Mature ARPE-19 (mRPE) cells were treated with thrombin (10 nM) for 1, 4, 8, 16, and 24 h. The medium was collected and assayed for ir-ET-1 content as previously described. Thrombin stimulated ir-ET-1 secretion in a time-dependent manner. A significant increase in ir-ET-1 was observed at the end of 4, 8, 16, and 24 h compared to control. Secretion of ir-ET-1 reached a plateau after 16 h. Data are represented as mean ± SEM. Statistical comparisons were performed by t-test. Asterisk (*), double asterisk (**), pound (#), and double pound symbols (##) denote significance versus controls at 4, 8, 16, and 24 h, respectively (P < 0.001) (n = 9).

FIG. 3.

Intracellular [Ca²⁺]_i measurements in mRPE cells. Representative [Ca²⁺]_i trends in response to thrombin (10 nM), pPAR-1 (50 μM), and pPAR-4 (50 μM) in mRPE cells. Thrombin-mediated rise in mean [Ca²⁺]_i mobilization was concentration dependent and was attenuated by thrombin neutralization agent Hirudin or the PLC inhibitor U73122 (see Tables 1 and 2). Thrombin was more potent in mobilizing [Ca²⁺]_i in mRPE cells than pPAR-1 or pPAR-4 (compare y-axes scales and Tables 1 and 2). Arrows represent time of addition of compound to cells in buffer.

FIG. 4.

RhoA pull-down assay in ARPE-19 cell lysates after thrombin treatment. Thrombin-activated RhoA in ARPE-19 cells at 5, 10, and 30 min compared to unstimulated cells (A, top panel). Total RhoA in cell lysates remain unchanged (A, lower panel). (B) The scanning densitometric data plotted as a ratio of active RhoA/total RhoA at the indicated periods. Data are represented as mean SEM. Statistical comparisons were performed by t-test. Asterisk (*) and double asterisk (**) denote significance versus control at 20 and 30 minutes respectively.

FIG. 5.

Measurement of preproET-1 (ppET-1) mRNA by quantitative polymerase chain reaction (PCR) in mature ARPE-19 and primary hRPE cells. Quantitative RT-PCR was performed using the SYBR-green PCR core reagents. Quantitation of ppET-1 transcripts was done by the comparative C_T method (see the Materials and Methods section) with β-actin cDNA as the external control. Thrombin-induced ppET-1 mRNA reached a maximal level at 1 h and returned to basal values at all time points tested thereafter (A). Thrombin-induced rise in ppET-1 mRNA was completely inhibited by Y27632, a ROCK1/2 inhibitor, but not the PLC inhibitor-U73122 (B). Similar effects were observed in primary hRPE cells (C). Data are represented as mean ± SEM. Statistical comparisons were performed by t-test. Asterisks (*) denote significance versus control, and pound (#) denotes significance versus thrombin, 1 h (P < 0.05).

FIG. 6.

Secreted ET-1 in mature ARPE-19 (mRPE) measured by RIA. Cells were treated with the indicated agonists (thrombin or pPAR-1) or were pretreated with the indicated inhibitors for 20–30 min followed by the agonist for 24 h in serum-free Dulbecco's modified Eagle's medium/F-12 medium. ir-ET-1 released in the medium was extracted and measured by RIA. Thrombin-mediated increase in ET-1 secretion was partially blocked by the PLC inhibitor, U73122, and completely inhibited by ROCK1/2 inhibitor, Y27632. The pan-PKC inhibitor (Ro 31-8425) had no effect on thrombin-induced ET-1 release. pPAR-1 (SFLLR)-mediated ET-1 secretion was also completely inhibited by Y27632. Data are represented as mean ± SEM. Statistical comparisons were performed using analysis of variance and student newman keuls (SNK) test. Asterisks (*) denote significance versus control, double asterisk (**) denotes significance versus thrombin 10 nM, and pound (#) denotes significance versus pPAR-1 50 μM (P < 0.05) (n = at least 6 per treatment).

FIG. 7.

Thrombin-induced ET-1 secretion in RPE is rho/ROCK1/2 dependent. Thrombin-mediated PAR-1 activation can simultaneously activate the G_q/11 and G_12/13-dependent pathways, which in turn activates PLC-dependent IP₃/DAG (Inositol 1,4,5-triphosphate/diacylglycerol) production and Rho activation, respectively. IP₃-dependent [Ca²⁺]_i elevation along with DAG can activate protein kinase C (PKC) that may influence ET-1 synthesis in some cells. We demonstrate that the predominant effect of thrombin on ET-1 production in ARPE-19 cells was via the Rho/ROCK1/2-dependent pathway. ROCK1/2 may increase ppET-1 mRNA synthesis by activating the GATA family of transcription factors (i.e., GATA-2/4) or by regulating the AP-1 family of transcription factors known to activate ppET-1 transcription. The physiological function of ET-1 secreted by the RPE is presently unknown. Considering some of the known actions of ET-1, it may mediate tissue repair by acting on its receptors in an autocrine manner or cause further damage to the neural retina by acting on its receptors in the inner retina.