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CELLULAR AND MOLECULAR
Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina
Received July 18, 2006; accepted September 29, 2006.
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Abstract |
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from the cytosol to the membrane, and pretreatment with the phospholipase C (PLC) inhibitor, U73122
[GenBank]
[1-[6-[[(17
)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione], blocked ERK1/2 activation induced by CHA. Transfection of short interfering RNA targeting PKC blocked the CHA-induced ERK1/2 activation and the secretion of MMP-2. These results confirm the existence of functional adenosine A1 receptors in the trabecular meshwork cells. These receptors are coupled to the activation of ERK1/2 through Gi/o proteins and dependent upon the upstream activation of PLC and PKC. These studies provide evidence that adenosine A1 receptor agonists increase outflow facility through sequential activation of Gi/o > PLC > PKC > c-Raf > mitogen-activated protein kinase kinase > ERK1/2, leading to secretion of MMP-2.
; Fredholm et al., 2001). In the eye, adenosine A1 receptor agonist has been shown to lower intraocular pressure (IOP) in rabbits, mice, and monkeys (Crosson and Gray, 1994; Crosson, 1995; Tian et al., 1997; Avila et al., 2001; Crosson, 2001). This reduction in IOP results primarily from an increase in outflow facility and is dependent on the secretion and activation of matrix metalloproteinases (MMPs) from cells within the conventional outflow pathway (Tian et al., 1997; Crosson, 2001; Crosson et al., 2005). A principle source for MMPs in the conventional outflow path is the trabecular meshwork cells. Previous studies have provided evidence that in transformed trabecular meshwork cells, the activation of adenosine A1 receptors increases the secretion of MMPs via extracellular signal-regulated kinase (ERK) 1- and 2-dependent processes (Shearer and Crosson, 2002). However, the effect of A1 agonists on primary trabecular cells are not known nor are the signaling events linking adenosine A1 receptors to ERK1/2 activation.
The activation of mitogen-activated protein kinase cascades by G protein-coupled receptors can occur by several mechanisms. This activation may be dependent on or independent of protein kinase A, protein kinase C (PKC), Src tyrosine kinase, or Ras activation and involve cross-activation of receptor tyrosine kinases (Pearson et al., 2001; Roux and Blenis, 2004). Studies have shown that each adenosine receptor subtype can activate ERK1/2; however, the cellular events linking adenosine receptor activation to the stimulation of ERK1/2 have led to conflicting results (Schulte and Fredholm, 2003). These divergent results may reflect the use of artificial transfection systems to study adenosine receptor signaling, the fact that a number of pathway inhibitors also act as adenosine receptor antagonists, or the existence of multiple cellular compartments each with distinct activation pathways (Schulte and Fredholm, 2002; Schulte and Fredholm, 2003). The purpose of these studies was to investigate the cell signaling mechanisms linking adenosine A1 receptors to ERK1/2 activation in human trabecular meshwork cells. The present study provides evidence that PLC/PKC plays a primary role in adenosine A1-mediated activation of the ERK1/2 and associated secretion of MMP-2 from primary human trabecular cells.
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Materials and Methods |
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)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione (U-73343), and MRS-1191 were obtained from Sigma Chemical Co. (St. Louis, MO). Monoclonal antibodies to PKC isoforms (, , 
, 
, 
, 
, µ, 
, and 
) were purchased from BD Transduction Laboratories (San Diego, CA), and ERK antibodies were obtained from Cell Signaling Technologies (Danvers, MA). Transfection reagent, SilentFect, was obtained from Bio-Rad Laboratories (Hercules, CA), and siRNA duplexes targeting PKC and PKC were obtained from Dharmacon RNA Technologies (Dharmacon Inc., Lafayette, CO). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT), and all cell culture supplies were obtained from Cell Gro (Herndon, VA).
Cell Culture. Human eyes without any evidence of disease were obtained from Life-Point Ocular Tissue Division (Charleston, SC). From these eyes, primary cultures of human trabecular meshwork cells were established from trabecular meshwork explants using a modification of the methods described previously by Shearer and Crosson (2002). In brief, small strips of trabecular meshwork tissues were dissected from human eyes and homogenized by means of a Teflon hand-held homogenizer in DMEM containing 15% FBS. The homogenized tissues were plated onto T-25 cell culture flasks and allowed to grow for 2 weeks in DMEM containing 15% FBS. The resulting colonies of cells were harvested and plated onto polypropylene cell culture plates in DMEM containing 10% FBS. These cells were allowed to grow to approximately 80% confluence. Studies involving these cells utilized second to fourth passage cells. In selected studies, the human trabecular meshwork (HTM)-3 cell line was used. The transformed HTM-3 cell line was maintained on polypropylene cell culture plates and grown in DMEM containing 10% heat-inactivated FBS (Pang et al., 1994). The cells were passaged at 3- to 4-day intervals and allowed to grow to approximately 80% confluence.
Extracellular Signal-Regulated Kinase Assay. Cells were cultured in serum-free medium for 16 h before the addition of any agent. Unless otherwise noted, cells were treated with CHA (1 µM) for 10 min. In experiments evaluating the adenosine A1 receptor antagonist CPT (1 µM), A3 receptor antagonist MRS-1191 (1 µM), PLC inhibitor U73122
[GenBank]
(1 µM), or its inactivated analog U-73343 (1 µM), Src tyrosine kinase inhibitor PP2 (1 µM), or the PI3 kinase inhibitor LY-294002 (1 µM), cells were pretreated for 30 min with the inhibitor before the addition of the agonist. In experiments evaluating pertussis toxin (100 ng/ml), cells were pretreated for 16 h before CHA treatment. At the end of the incubation periods, cells were rinsed with ice-cold phosphate-buffered saline and lysed by the addition of lysis buffer (50 mM Tris-HCl buffer, pH 8.0, containing 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 50 mM NaF, 1 mM/l Na3VO4, 5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 50 µg/ml aprotinin) for 20 min on ice. To determine the level of ERK1/2 activation (phosphorylation), equivalent amounts of protein (15 µg) were loaded onto 10% SDS-polyacrylamide gels, and proteins were separated according to mol. wt. using standard SDS-polyacrylamide gel electrophoresis protocols and transferred to a nitrocellulose membrane. The membranes were then probed with anti-phospho-ERK1/2 antibodies for 2 h at room temperature. Bands were visualized by the addition of anti-rabbit HRP-conjugated secondary antibodies (at 1:3000) and ECL reagents. Blots were then stripped by incubation in stripping buffer (62.5 mM Tris-HCl, pH 6.7, 100 mM -mercaptoethanol, and 2% SDS) for 30 min at 50°C, and total ERK levels (phosphorylated and nonphosphorylated forms) were determined by immunoblot techniques using polyclonal anti-ERK1/2 antibodies. Band densities were quantified with a BioRad Versa Doc Imaging System (Bio-Rad Laboratories). The level of phosphorylated ERK1/2 isoforms was normalized for differences in loading using the total ERK protein band intensities.
Protein Kinase C Assays. Cells were maintained in serum-free media for 16 h. Cells were then treated with vehicle or CHA (1 µM) for 10 min. To detect PKC isoforms in cell lysate and cytosolic and membrane fractions, cells were lysed in Tris-HCl buffer, pH 7.5, containing 1 mM EGTA, 2.5 mM EDTA, 5 mM dithiothreitol, 0.3 M sucrose, 1 mM Na3VO4, 20 mM NaF, and protease cocktail inhibitor, using G-20 syringes followed by centrifugation at 600g for 10 min at 4°C. To separate cytosolic and membrane fractions, the supernatant was again centrifuged at 100,000g for 45 min at 4°C. The supernatant served as the cytosolic fraction. The pellet was resuspended in lysis buffer containing 0.1% Triton X-100 and served as the membrane fraction. Equal amounts of protein were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk followed by incubation with anti-PKC isoform antibodies (1: 1000) for 16 h at 4°C with mild shaking. After washing, the membranes were incubated with secondary antibodies (HRP-conjugated anti-mouse IgG at 1:3000) for 1 h at 20°C followed by ECL exposure. Rat brain extract, Jurkat, and WI-38 were run as positive controls for PKC isoforms. Band densities were quantified by means of a Bio-Rad Versa Doc Imaging System (Bio-Rad Laboratories).
siRNA. Primary HTM cells were grown to 60 to 70% confluence, and transfection of duplex siRNA was preformed using a modification of the methods developed by Elbashir et al. (2001). In brief, cells were transfected with 10 or 100 nM siRNA using SilentFect (Bio-Rad Laboratories) following the protocol provided by the manufacturer. SilentFect was removed by placing the cells in fresh medium containing 10% FBS 4 h after transfection. Cells were then allowed to grow for 24 to 72 h following transfection. Preliminary studies demonstrated that cells transfected with siRNA concentration of 10 nM, in the presence of the lipid carrier SilentFect, produced maximal knockdown (80–90%) of the target PKC with little or no toxicity. This level of protein knockdown was maintained for up to 72 h. As a result, all subsequent studies were carried out in cells 48 h following transfection of siRNA at a concentration of 10 nM. PKC siRNA duplexes were synthesized by Dharmacon RNA Technologies. The sequences of PKC siRNAs were as follows: 5'-AAA GGC UGA GGU UGC UGA UTT-3' and 5'-AUC AGC AAC CUC AGC CUU UTT-3'. For PKC knockdown, we used the four pooled siRNAs developed by Dharmacon RNA Technologies (product no. MU-004653). All siRNAs were annealed and desalted in the 2'-hydroxyl form as provided by the manufacturer.
Cell Transfection and Expression of Dominant Negative Ras (N17 Ras). The HTM-3 cell line was grown to approximately 60% confluence and then cotransfected with either N17 Ras (gift from Dr. Larry Feig, Tufts University, Medford, MA) and hemoagglutinin-tagged ERK1 (HA-ERK1) (a gift from Dr. Melanie Cobb, University of Texas Southwestern, Dallas, TX) or a Lac-Z control vector and HA-ERK1. Transcription of each transcript was directed under a cytomegalovirus promoter. Transfections were performed using the Lipofectamine-Plus transfection reagents according to the manufacturer's recommendations using 1 µg/ml each plasmid. Cells were allowed to grow normally for 24 h following transfection and then were serum-deprived for 16 h as before. Cells were then treated for 10 min with 100 nM CHA and harvested as before. HA-ERK1 was then immunoprecipitated from the total cell lysate (200 µg) with a rabbit polyclonal HA-tagged antibody (BD Biosciences Clontech, Palo Alto, CA) for 1 h, followed by 30-min incubation with protein G conjugated to agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Samples were centrifuged, washed three times with lysis buffer, and the proteins were analyzed for total and phosphorylated HA-ERK1 by immunoblotting as before. Total cell lysates were also analyzed for N17Ras by immunoblotting to insure proper expression.
MMP-2 Assay. Human trabecular meshwork cells were starved in serum-free medium for 16 h. Cells were treated with vehicle or CHA for 2 h, and the media were collected and stored at –80°C until analyzed by Western blotting. Media were concentrated using concentrators (Amicon Ultra-10 centrifugal filter devices; 10-kDa cut-off; Millipore, Billerica, MA) and adjusted to a final concentration ratio of 10:1. Equivalent volumes of media (40 µl) were loaded onto 10% SDS-polyacrylamide gels followed by transfer to a nitrocellulose membrane. The membranes were then probed with anti-MMP-2 antibodies overnight at 4°C. Bands were visualized by the addition of anti-mouse HRP-conjugated secondary antibodies (at 1:3000) and ECL reagents. The band intensities were quantified by densitometry. Purified pro-MMP-2 was run in parallel as a positive control to identify the MMP-2 band.
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). To determine whether these signaling events are normally present in nontransformed trabecular cells, second and third passage trabecular meshwork cells derived from human explants were treated with the adenosine A1 receptor agonist CHA. As shown in Fig. 1, the addition of the adenosine A1 agonist CHA induced a dose-related increase in ERK1/2 in both primary human trabecular meshwork cells and the HTM-3 cell line. Regression analysis of concentration-response curves yielded EC50s and response maximums of 18 nM and 319% for primary human trabecular meshwork cells and 7.9 nM and 291% for the HTM-3 cell line. The addition of the A3 agonist, IB-MECA, produced only limited increases (10 to 27%) in ERK1/2 activation at concentrations up to 10–6 M (Fig. 1).
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To investigate whether CHA-induced ERK1/2 activation in trabecular meshwork was dependent on Ras activation, HTM-3 cells were transfected with dominant negative Ras transcript (N17Ras). The limited transfection of N17 Ras in primary human trabecular meshwork cells prevented this assessment in these cells. Overexpression of N17Ras inhibited the activation of coexpressed HA-tagged ERK1 in CHA-treated cells by 31%. However, N17Ras overexpression did not significantly alter basal levels of ERK phosphorylation compared with the control vector (Fig. 4).
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The inhibition of CHA-induced ERK1/2 activation by the PLC inhibitor indicated that this process may involve the activation of PKC. Western blot analysis of different PKC isoforms in cytosolic and membrane fractions of human trabecular meshwork cells is shown in Fig. 6. PKC, , , , , and µ were detected in human trabecular meshwork cells. The PKC isoform showed the highest level of immunoreactivity in these cells compared with other PKC isoforms. PKC, , and isoforms exhibited moderate levels of expression, whereas PKC and µ isoforms exhibited low levels of expression. PKC and were not detected. As shown in Fig. 6, the addition of CHA (1 µM) induced a significant translocation of PKC to the membrane fraction (126%) compared with control preparations. The addition of CHA also induced a small increase in PKC association with the membrane fraction (39%); however, the addition of CHA did not produce any detectable change in the distribution of PKC, µ, , and isoforms (data not shown).
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To examine the involvement of PKC and PKC in CHA-induced ERK1/2 activation of primary human trabecular cells, cells were treated with siRNA targeting either PKC or PKC. As shown in Fig. 7, transfection of primary human trabecular cells with PKC siRNA resulted in over 80% knockdown of PKC. However, PKC was not affected by this treatment. Likewise, expression of other PKC isoforms was not altered by this treatment with siRNA targeting PKC (data not shown). In human trabecular cells transfected with siRNA targeting PKC, CHA-induced ERK1/2 activation was completely inhibited (Fig. 7). However, these responses were not significantly altered when cells were incubated with transfection reagents alone. In cells treated with PKC siRNAs, the expression of PKC was reduced by over 80%, but no detectable changes in PKC or other PKC isoforms were measured. In human trabecular cells transfected with siRNA targeting PKC, CHA-induced ERK1/2 activation was not significantly altered compared with cells treated with the transfection reagents alone.
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To assess whether the functional responses linked to CHA-induced ERK1/2 activation were also blocked by inhibiting PKC, primary human trabecular meshwork cells were transfected with siRNA targeting PKC before CHA administration. Cells were then treated with CHA (1 µM) for 2 h, and the media were collected and analyzed for MMP-2. As shown in Fig. 8, the CHA-induced secretion of MMP-2 was completely inhibited in siRNA-transfected cells. However, these responses were not inhibited when cells were incubated with transfection reagent alone.
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Discussion |
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). Experiments in our laboratory have shown that adenosine levels in the aqueous humor are positively correlated to IOP (Daines et al., 2003). In rabbits, mice, and primates, the activation of adenosine A1 receptors lowers IOP (Tian et al., 1997; Avila et al., 2001; Crosson, 2001). This decrease in IOP results primarily from an increase in outflow facility and is dependent on the secretion and activation of matrix metalloproteinases from cells within the outflow pathway (Crosson et al., 2005). Trabecular meshwork cells are thought to play a central role in the regulation of outflow resistance and resulting IOP. Previous studies have shown that the administration of adenosine A1 agonists to the trabecular meshwork cell line, HTM-3, stimulate the secretion of MMPs via the activation of the ERK1/2 pathway (Shearer and Crosson, 2002). Adenosine A3 receptors also have been identified in the trabecular meshwork and the activity of these receptors seems to be important in the regulation of intraocular pressure. However, the ocular hypotensive responses associated with adenosine A1 and A3 receptors seem to operate via different cellular mechanisms. In vivo studies evaluating A1 receptors have shown that A1 agonists lower intraocular pressure, whereas studies evaluating A3 receptors have shown that adenosine A3 antagonists lower intraocular pressure (Crosson, 1995; Avila et al., 2001; Fleischhauer et al., 2003).
Extracellular signal-regulated kinase is a member of the mitogen-activated protein kinase family of serine/threonine kinases and is involved in the transduction of externally derived signals regulating cell growth, division, differentiation, motility, gene expression, and apoptosis. Activity of the ERK1/2 pathway is determined by stimulation of diverse classes of receptors (e.g., receptor tyrosine kinases, G protein-coupled receptors, and integrin) and inactivation by dual-specific phosphatases (Pearson et al., 2001; Alonso et al., 2004; Roux and Blenis, 2004; Ducruet et al., 2005). Each adenosine receptor subtype has been shown to activate ERK1/2 (Schulte and Fredholm, 2003); however, studies examining the cellular events coupling these receptors to the proximal kinase, Raf, in ERK activation have led to conflicting results. Studies on adenosine receptors have concluded that ERK activation may be PKC dependent or independent, Ras dependent or independent, or Src dependent or PI3 kinase dependent (Schulte and Fredholm, 2003). Our results demonstrated that the administration of CHA to primary human trabecular meshwork cells produced a rapid dose-dependent increase in ERK1/2 phosphorylation. This response was completely inhibited by pretreatment with the adenosine A1 receptor antagonist, CPT, or pertussis toxin but not altered by pretreatment with the adenosine A3 antagonist, MRS1191. In addition, the adenosine A3 agonist, IB-MECA, at concentrations up to 10–6 M produces little or no stimulation of ERK1/2 in primary human trabecular cells. Taken together, these results provide evidence that CHA-induced ERK1/2 activation in human trabecular cells is mediated through the activation of adenosine A1 receptors coupled to Gi/o proteins. However, the limited inhibition (31%) of ERK1 produced by transfection with the dominant negative N17 Ras indicates that in these cells, the high-affinity binding of RasGTP to Raf is not the primary event coupling adenosine A1 receptors to ERK1/2 activation. This limited Ras-dependent activation may reflect the ability of adenosine A1 receptors to cross-activate receptor tyrosine kinases (i.e., EGF receptors) via membrane shedding of epidermal-like growth factors. Previous studies have demonstrated that various G protein-coupled receptors can stimulate receptor tyrosine kinases and ERK via the activation of MMPs and release of endogenously expressed ligands (Tegeder and Geisslinger, 2004; Shah et al., 2005). Studies by this laboratory found that the tyrosine kinases inhibitor, genistein, reduces CHA-induced ERK1/2 activation (data not shown). Although these studies support the idea that the indirect activation of receptor tyrosine kinases contribute to adenosine stimulation of ERK1/2, they are complicated by the fact that genistein can also act as an adenosine receptor antagonist (Schulte and Fredholm, 2002). Hence, understanding the potential role of cross-activation in this response will require additional investigation.
In other systems, Ras-independent activation of ERK1/2 by G protein-coupled receptors has been shown to involve the upstream activation of Src family kinases, PI3 kinase or PKC (Takeda et al., 1999). To evaluate the role of Src kinases and PI3 kinase, human trabecular meshwork cells were pretreated with the Src family of tyrosine kinase inhibitor (PP2) or PI3 kinase inhibitor (LY-294002). In each case, CHA-induced ERK1/2 activation was not significantly altered by the presence of these inhibitors, indicating that adenosine A1-mediated activation of ERK1/2 is not mediated by Src tyrosine kinase or PI3K-dependent pathways.
Inhibition of the CHA-induced ERK1/2 activation by U73122
[GenBank]
indicates that adenosine A1 receptors are coupled to ERK activation by the sequential activation of PLC and PKC. Preliminary studies found that pretreatment with the non-selective PKC inhibitor, chelerythrine, also inhibited CHA-induced ERK1/2 activation (data not shown). However, these studies are again complicated by the study, demonstrating that chelerythrine also acts as an adenosine receptor antagonist (Schulte and Fredholm, 2002). Hence, to investigate the involvement of PKC as a mediator of adenosine receptor function, we used alternative biochemical assays and siRNA. The PKC family is comprised of at least 11 isoforms (, , , , , 
, µ, , , , and 
, which are encoded by different genes (Nishizuka, 1988; Walsh et al., 1994). As shown in Fig. 6, the PKC isoform exhibited the highest level of immunoreactivity in human trabecular meshwork cells and was located predominantly in the cytosol under resting conditions. The PKC, , and isoforms exhibited moderate levels of expression. PKC and µ isoforms exhibited low levels of expression, whereas PKC and were not detected. These results are similar to previous reports in human and porcine trabecular meshwork cells (Thieme et al., 1999; Alexander and Acott, 2001). Because most inactive PKC isoforms reside within the cytosol and translocate to the membrane (or other subcellular sites) upon stimulation, the cytosolic and membrane-associated levels of each PKC isoform were determined in the presence and absence of CHA. Treatment of human trabecular meshwork cells with CHA induced almost a complete translocation of PKC isoform from the cytosol to the membrane fraction. In contrast, CHA produced little or no change in the cellular distribution of other PKC isoforms. These data support the idea that the activation of adenosine A1 receptors in these cells is primarily coupled to the activation of PKC.
To determine whether the CHA-induced ERK1/2 activation, as well as ERK-dependent secretion of MMP-2, requires the activation of PKC, we used siRNA duplexes to knockdown endogenous expression of this isoform. Our results demonstrate that siRNA technologies can selectively reduce the expression of individual PKC isoforms in primary human trabecular meshwork cells. In cells treated with siRNA targeting PKC, the CHA-induced ERK1/2 activation was eliminated. However, transfection with siRNA targeting PKC did not significantly alter ERK1/2 induced by the adenosine A1 agonist CHA. In addition, the functional response to A1 receptor activation (i.e., MMP-2 secretion) in these cells is also blocked by siRNA targeting PKC.
Extracellular matrix material within the outflow pathway is thought to contribute to the resistance of aqueous humor outflow, and abnormal accumulations of these materials have been associated with elevation in IOP in glaucomatous individuals (Gabelt and Kaufman, 2005). Studies have shown that the activation of ocular adenosine A1 receptors in rabbits, mice, and monkeys lowers IOP. This reduction in IOP results, in large part, by the secretion and activation of MMPs from cells within the conventional outflow pathway. Although studies have linked the secretion of MMP induced by adenosine agonists and various cytokines to the activation of ERK1/2 (Shearer and Crosson, 2001, 2002; Alexander and Acott, 2003), there is little understanding of upstream signaling that mediates ERK activation. This study provides evidence that in trabecular meshwork cells adenosine A1 receptor-mediated increases in MMP-2 secretion results from the sequential activation of PLC, PKC, and ERK1/2 and that Ras activation plays little or no role in this process.
In summary, stimulation of adenosine A1 receptors in primary trabecular meshwork cells activates ERK1/2. This activation is coupled to Gi/o proteins and is primarily dependent upon the upstream activation of PLC and PKC. Taken together, these results demonstrate the existence of functional adenosine A1 receptors in human trabecular meshwork cells. In vivo, the activation of these receptors seems to modulate outflow facility and lower IOP through the sequential activation of Gi/o > PLC > PKC > c-Raf > mitogen-activated protein kinase kinase > ERK1/2, leading to secretion of MMP-2.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IOP, intraocular pressure; MMP, matrix metalloproteinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PLC, phospholipase C; CHA, N6-cyclohexyladenosine; CPT, 8-cyclopentyl-1,3-dimethylxanthine; LY-294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PP2, 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D] pyrimidin-4-amine; U73122
[GenBank]
, 1-[6-[[(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione; U-73343, 1-[6-[((17)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione; MRS-1191, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; HTM, human trabecular meshwork; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; HA-ERK1, hemoagglutinin-tagged ERK1; PI3, phosphoinositide kinase-3.
1 Current affiliation: Alcon Laboratories, Fort Worth, Texas. 
Address correspondence to: Dr. Craig E. Crosson, Medical University of South Carolina, 167 Ashley Avenue, Charleston, SC 29425. E-mail: crossonc{at}musc.edu
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