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(Investigative Ophthalmology and Visual Science. 2007;48:3126-3137.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1375
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p38 MAP Kinase Pathway and Stromelysin Regulation in Trabecular Meshwork Cells

Mary J. Kelley, Anastasia Rose, Kaili Song, Barbara Lystrup, John W. Samples, and Ted S. Acott

From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Increased expression of stromelysin-1 (matrix metalloproteinase [MMP]-3) by the trabecular meshwork (TM) initiates extracellular matrix turnover and increases aqueous humor outflow facility. Tumor necrosis factor (TNF){alpha} and interleukin (IL)-1{alpha} are efficacious inducers of MMP-3 in TM. To facilitate understanding of the regulation of MMP-3, the authors investigated the involvement of p38 MAP kinase pathway proteins in this process.

METHODS. Western immunoblots were used to determine the effects of these cytokines and p38 MAP kinase pathway inhibitors on MMP-3 protein levels, p38 MAP kinase isoforms, and phosphorylation levels in human and porcine TM cells. The effects of a dominant-negative p38 MAP kinase construct on MMP-3 expression were evaluated. Morphologic changes in the cells were also examined.

RESULTS. Both cytokines increased MMP-3 levels. The p38 MAP kinase inhibitor SB202190 diminished MMP-3 induction by TNF{alpha} at all times and at 24 hours by IL-1{alpha} but potentiated the IL-1{alpha}–induced increase in MMP-3 at later times. MMP-3 induction by both cytokines was blocked by dominant-negative p38 MAP kinase constructs. Each cytokine increased phosphorylation of the p38 MAP kinase pathway components and altered TM cell morphology. The p38 inhibitor blocked only the morphologic changes produced by TNF{alpha}. Human and porcine TM cells expressed p38 {alpha}, ß, {delta}, and {gamma} isoforms, which migrate coincident with bands of specific phosphorylation.

CONCLUSIONS. The effects of p38 inhibitors and the dominant-negative construct on TNF{alpha} and IL-1{alpha} induction of MMP-3 demonstrate an essential role for p38 in this signaling process. Differences between p38 inhibitor effects on TNF{alpha} and IL-1{alpha} induction of MMP-3 suggest divergent p38 isoform use, as do the morphologic responses. The anomalous p38 inhibitor effect on IL-1{alpha} induction of MMP-3 and phosphorylation of p38 {delta}/{gamma} suggests complex interactions between p38 MAP kinase isoforms and their differential uses by TNF{alpha} and IL-1{alpha} in TM.

Mitogen-activated protein (MAP) kinases are a superfamily of serine/threonine protein kinases that function in transducing various extracellular signals to regulate a number of cellular functions.1 2 3 4 5 Several parallel MAP kinase pathways are known, with multiple isoforms of each. The most well-known of these pathways includes the extracellular signal-regulated kinases (Erks), the c-Jun N-terminal kinases (JNKs), and the p38 MAP kinases.1 2 3 6 Activation of these pathways by a vast array of stimuli produces an equally wide array of discrete and overlapping cellular responses. The pathways function as hierarchal protein phosphorylation cascades, though they do not generally exhibit the strong signal amplification seen in some kinase cascades.

In previous studies of the pathways in which the cytokines TNF and IL-1 stimulate matrix metalloproteinase (MMP)-3 production in TM, we found that protein kinase Cµ, Erk 1/2, and JNK 1/2 pathways were all necessary for signal transduction.7 8 9 TNF and IL-1 were shown to mediate the MMP-3 increase, which occurs in response to laser trabeculoplasty, a common treatment for glaucomatous elevated intraocular pressure.10 11 12 The extracellular matrix turnover initiated by the MMP-3 increase appears to be a critical component in the therapeutic efficacy of this glaucoma treatment.13 14

Because these cytokines often use the p38 MAP kinase pathway in ocular and nonocular systems, the potential involvement of this pathway in the TM was of interest.3 6 15 Previous studies with p38 MAP kinase inhibitors had implicated this kinase pathway in the IL-1{alpha} induction of MMP-3 in TM cells.16 Thus, we evaluated the responses of p38 pathway components to these cytokines and the effects of p38 inhibitors and dominant-negative mutations on TM cell MMP-3 expression. When complex responses to the p38 inhibitors were observed, p38 isoforms in the TM were evaluated. The p38 isoforms in the TM were previously unknown. Differential sensitivities of the various isoforms to p38 inhibitors have been reported.6 17 18 19 20 In addition, cross-talk and opposing actions have been reported for some p38 isoforms.21 22 23 24 25 26


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Human recombinant TNF{alpha} and human and porcine recombinant IL-1{alpha} were obtained from R&D Systems (Minneapolis, MN). Primary MMP-3 antibodies were from TriplePoint Biologics (Forest Grove, OR). Phosphospecific antibodies to MAP kinase kinase 3 and 6 (MKK3/MKK6; Ser189/Thr207), p38 MAP kinase (Thr180/Tyr182), MAP kinase–activated protein kinase-2 (MAPKAPK-2; Thr334), and secondary antibodies conjugated with horseradish peroxidase were from Cell Signaling Technology (Beverley, MA). Phosphospecific ATF-2 (Ser383 or Thr71) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). p38 MAP kinase isoform-specific antibody to the {alpha} isoform was obtained from Calbiochem (San Diego, CA); to the ß isoform was obtained from Santa Cruz Biotechnology; to the {delta} and {gamma} isoforms was obtained from Upstate/Millipore (Charlottesville, VA). High- and low-glucose Dulbecco modified Eagle medium (DMEM), antibiotics, and antimycotics were from Invitrogen-Gibco (Grand Island, NY); fetal bovine serum (FBS) was from Hyclone (Logan, UT); chemiluminescence detection kits were from Pierce (SuperSignal; Rockford, IL); secondary antibodies (Alexa Fluor 680-conjugated) and assay kits (Picogreen DNA) were from Molecular Probes (Eugene, OR); secondary antibodies (IRDye 800-conjugated) were from Rockland (Gilbertsville, PA); and p38 MAP kinase inhibitors SB202190 and SB203580 were from Calbiochem. Porcine eyes were purchased from Carlton Packing Company (Carlton, OR), and human eyes were obtained from the Lions’ Eye Bank of Oregon (Portland, OR). The p38 kinase dead dominant-negative clone (T180A/Y183F)27 was a gracious gift of Roger Davis (University of Massachusetts Medical Center, Worcester, MA). Statistical significance when comparing groups subjected to different treatments used Student’s t-test or the Mann–Whitney U test.

Cell Culture
Human and porcine TM cells were isolated, and cultures were established as previously described7 8 28 29 30 31 and grown to confluence in medium-glucose (a 1:1 mix of high and low glucose) DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic mix. In the second passage, TM cells were plated into flasks or six-well plates. Confluent cells were maintained serum free for 48 hours before and during experiments. Cytokine effects were determined by treating cells with recombinant human TNF{alpha} (10 ng/mL), recombinant human IL-1{alpha} (10 ng/mL), or recombinant porcine IL-1{alpha} (10 ng/mL) for 5, 15, and 60 minutes or for 24, 48, or 72 hours. For p38 MAP kinase inhibitor studies, SB202190 (100 nM) or, in some cases, SB203580 (100 nM) was added to TM cells 1 hour before the addition of cytokines. Parallel controls with and without equivalent levels of vehicle (dimethyl sulfoxide) were included in these studies.

SDS-PAGE and Western Immunoblotting
At the end of the experiments, culture medium was removed, centrifuged at 2000g for 5 minutes, and snap frozen at –20°C. Cells were rapidly rinsed in phosphate-buffered saline (PBS), snap frozen in liquid nitrogen, and stored at –80°C. To obtain cellular proteins, thawed cells were extracted with 4°C modified radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors (2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 10 mM NaP4O7, 1 mM phenylmethylsulfonyl fluoride, 20 µg/mL leupeptin, 20 µg/mL aprotinin, 20 µg/mL pepstatin, and 50 mM Tris, pH 7.5). After incubation on ice, cells were scraped, extract was removed, and 6x SDS-PAGE sample buffer was added. Samples were then boiled, snap frozen, and stored at –80°C until they were subjected to standard SDS-PAGE on 8% separating gels.32 For MMP-3 assays, media aliquots were concentrated 5x to 8x with the use of spin columns (Centricon YM-10; Millipore, Bedford, MA). After 6x sample buffer was added, media aliquots were subjected to standard SDS/PAGE separations. After electrophoresis, proteins were electrophoretically transferred to polyvinylidene difluoride or nitrocellulose membranes. For some studies, membranes were blocked and extensively washed with 5% bovine serum albumin (BSA) in 1x TBST (10 mM Tris, 150 mM NaCl, 0.01% Tween-20, pH 7.5) before and after incubation in secondary antibody. To verify uniform total protein loading and transfer, blots were stained (Ponceau; Sigma-Aldrich, St. Louis, MO) after transfer and before the addition of the blocking agent. Detection was performed with the appropriate conjugated horseradish peroxidase secondary antibodies with chemiluminescent substrate (SuperSignal West Pico; Pierce, Rockford, IL). Exposed films from the chemiluminescent blots were scanned (ScanJet II CX/T; Hewlett-Packard, Palo Alto, CA), and relative band densities were determined (LabWorks software; UVP, Upland, CA).33 In other cases, blocking buffer (Odyssey; LI-COR Biosciences, Lincoln, NE) was used to block membranes before incubation with primary antibodies. Bands were detected with the appropriate secondary antibody (Alexa Fluor 680 or IRDye 800 conjugated; Molecular Probes). Blots were then scanned, and relative band densities were determined (Odyssey Infrared Imager; LI-COR Biosciences).

Plasmid Constructs
To generate the hMMP3p-SEAP reporter construct, a 2.3-kb DNA fragment containing the human MMP-3 promoter (GenBank accession no. U435111) was amplified from human genomic DNA by PCR. The promoter was then subcloned into MluI/BglII restriction sites upstream of the secreted alkaline phosphatase (SEAP) gene in SEAP-Basic reporter vector (Clontech, Palo Alto, CA). Correct insertion and sequence of the MMP-3 promoter in the hMMP-3 promoter-SEAP construct was confirmed by DNA sequencing. The vector was amplified in Escherichia coli cells (One-Shot; Invitrogen, Carlsbad, CA) and subsequently extracted (EndoFree Plasmid Maxi Kit; Qiagen, Valencia, CA). The dominant-negative p38 plasmid was a double mutant (T180A and Y182F) in a pCMV5 vector.27 The control plasmid was pcDNA 3.1 from Invitrogen-Gibco.

Cotransfection of TM Cells and Chemiluminescent SEAP Assay
Porcine TM cells were seeded in 12-well plates at a density of 9 x 105 cells per plate and maintained in DMEM with 10% FBS and 1% antibiotic/antimycotic mix overnight. Cells were washed twice with serum-free DMEM before cotransfection. TM cells in each well were cotransfected with 0.6 µg total DNA (either 0.2 µg hMMP3 promoter-SEAP reporter vector or 0.2 µg SEAP-Basic and either 0.4 µg dominant-negative p38 plasmid construct or 0.4 µg pcDNA 3 plasmid) using the transfection reagent (Transfectam; Promega, Madison, WI) according to the manufacturer’s instructions.

After 2 hours of incubation at 37°C, cells in each well were overlaid with 2 mL DMEM with 10% FBS and 1% antibiotic/antimycotic mix and were allowed to recover overnight. Transfected TM cells were washed twice with, and then incubated in, serum-free medium for 48 hours before treatment with human TNF{alpha} (20 ng/mL) or porcine IL-1{alpha} (10 ng/mL). After 72, 96, and 120 hours of treatment, aliquots of conditioned medium were collected and stored at –20°C. Promoter activity was determined by chemiluminescent SEAP activity assay with the use of detection kits (Great EscAPe SEAP Chemiluminescence Detection; Clontech) according to the manufacturer’s directions. Two complete independent experiments were conducted in triplicate with each sample. Additional controls to evaluate transfection efficiency and for general expression and transfection effects included cotransfection of pcDNA3-GFP (green fluorescence protein) constructs with the reporter or the dominant-negative constructs. Fluorescence was assessed using an inverted fluorescence-equipped culture microscope. Neither the dominant-negative nor the reporter constructs affected GFP expression (data not shown).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of TNF{alpha}, IL-1{alpha}, and p38 MAP Kinase Inhibitor on MMP-3 Levels
MMP-3 levels in the culture media were elevated with increasing incubation times for porcine TM cells treated with TNF{alpha} or IL-1{alpha} (Figs. 1A 1B ; solid bars). Pretreatment of cells with the p38 MAP kinase inhibitor SB202190 reduced but did not completely block the TNF{alpha}-induction of MMP-3 at 24, 48, and 72 hours. The inhibitor slightly reduced the MMP-3 response of IL-1{alpha} at 24 hours, but this was not statistically significant. Surprisingly, it actually potentiated the MMP-3 increases produced by IL-1{alpha} at 48 and 72 hours (Fig. 1B) . Similar responses were also seen with a second p38 MAP kinase inhibitor, SB203580 (data not shown).


Figure 1
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  		FIGURE 1. Effects of TNF{alpha}, IL-1{alpha}, and p38 MAP kinase inhibitor on MMP-3 levels. Medium MMP-3 was analyzed by Western immunoblot after 24-, 48-, or 72-hour treatment of porcine TM cells with TNF{alpha} (A) or IL-1{alpha} (B), with or without the p38 MAP kinase inhibitor SB202190 (SB), as indicated. Mean MMP-3 relative band density and SEMs are shown, with n = 4 to 6. t-Test significance is as indicated above the lines that connect the pairs, which were compared. Corresponding lanes from one representative immunoblot are shown below each bar. Control and control + SB lanes and values for the respective bars are from the 24-hour treatments. Both types of control were also conducted for 48 and 72 hours in parallel with these treatments but are not shown because, in all cases, they were virtually identical with the 24-hour controls.

 
Parallel studies with human TM cells produced generally similar results (data not shown). However, in human TM cells, the p38 MAP kinase inhibitor significantly reduced IL-1{alpha} induction of MMP-3 at 24 hours by approximately 75%. The degree of potentiation of the IL-1{alpha} induction of MMP-3 by the p38 inhibitor at 72 hours was also greater for human TM cells than for porcine TM cells (data not shown).

Effects of Dominant-Negative p38 MAP Kinase on TNF-{alpha}– or IL-1{alpha}–Induced MMP-3 Promoter Activity
As an alternative method to evaluate the involvement of the p38 MAP kinase pathway in regulating MMP-3 expression by TM cells, a SEAP reporter plasmid driven by a 2.3-kb MMP-3 promoter fragment (hMMP-3 Promoter-SEAP) was cotransfected into TM cells with a dominant-negative p38 construct (DN-p38). In response to stimulation by TNF{alpha} (Fig. 2A) or IL-1{alpha} (Fig. 2B) , cells transfected with the hMMP-3 promoter-SEAP construct but no DN-p38 produced high levels of SEAP compared with untreated, similarly transfected cells or with cells transfected with the control SEAP plasmid without the MMP-3 promoter (Figs. 2A 2B) . Cotransfection with hMMP-3 promoter-SEAP and DN-p38 reduced TNF{alpha} and IL-1{alpha} induction of SEAP reporter activity (Figs. 2A 2B) .


Figure 2
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  		FIGURE 2. Effects of dominant-negative p38 MAP kinase on TNF{alpha} and IL-1{alpha} stimulation of MMP-3 promoter activity. Porcine TM cells were cotransfected with the SEAP reporter vector without a promoter (Basic-SEAP) or with the human MMP-3 promoter (hMMP-3 Promoter-SEAP) and with pcDNA or with the dominant-negative p38 (DN-p38) construct. SEAP activity in the media was assessed after TNF{alpha} (A) or IL-1{alpha} (B) treatment, as indicated. Mean SEAP activity is shown with SEM, with n = 6. t-Test significance is as indicated above the lines that connect the pairs, which were compared.

 
Phosphorylation of MKK3, MKK6, or Both
MKK3 and MKK6, the normal upstream kinases of p38 MAP kinases,3 6 34 were phosphorylated in response to treatment with TNF{alpha} or IL-1{alpha} (Figs. 3A 3B) . TM cells incubated with TNF{alpha} (Fig. 3A) achieved peak levels by 5 minutes, whereas the IL-1{alpha} response (Fig. 3B) was slightly slower and was maximized at 15 minutes. Phosphorylation in response to TNF{alpha} declined by 60 minutes and was modestly but significantly elevated at 24 hours. With IL-1{alpha} treatment, a slight decrease in phosphorylation was seen at 1 hour, but at 24 hours, total phosphorylation was approximately the same as at 15 minutes. Although this antibody did not differentiate between MKK6 and MKK3, their sizes were slightly different. In some lanes, MKK6 could be seen to migrate slightly slower than MKK3.


Figure 3
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  		FIGURE 3. Effects of TNF{alpha} and IL-1{alpha} on MKK3/6 phosphorylation levels. MKK3 and MKK6 phosphorylation on S189/S207 was determined by Western immunoblot of porcine TM cell lysates after 5, 15, and 60 minutes and 24 hours of treatment with (A) TNF{alpha} or (B) IL-1{alpha}. Mean band density and SEMs are shown, and n is as indicated. Paired t-test significance is shown above the lines between the pairs of samples that were compared. The 15-minute and 24-hour controls are shown. Bands from representative immunoblots are shown below each figure.

 
Phosphorylation of p38 MAP Kinase
Phosphorylation of the 38-kDa band of p38 MAP kinase followed a generally similar temporal pattern. TNF{alpha} (Fig. 4A) produced maximum phosphorylation at 5 minutes, declined at 15 minutes, and declined further, while remaining slightly above controls, at 60 minutes and 24 hours. IL-1{alpha} responses were slower but also more sustained (Fig. 4B) . Phosphorylation was slightly elevated at 5 minutes, reached a maximum at 15 minutes, was slightly down at 1 hour, but remained elevated above control at 24 hours. After some treatments, additional bands of phospho-p38 immunostaining were observed.


Figure 4
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  		FIGURE 4. Effects of TNF{alpha} and IL-1{alpha} on p38 MAP kinase phosphorylation. Phosphorylation levels of the 38-kDa p38 MAP kinase band determined for porcine TM cell extracts on Western immunoblots probed with phosphospecific antibodies to Thr180/Tyr182. TM cells were treated with TNF{alpha} (A) or IL-1{alpha} (B) for the indicated times. Mean relative band densities and SEMs are shown with the n and t-test significance, as indicated. Corresponding lanes from representative immunoblots are shown below each bar. Control lanes and values for control bars are from 15-minute treatments, but 5- and 60-minute and 24-hour control treatments were included in experiments and were virtually identical.

 
Phosphorylation and Inhibition of MAPKAPK-2
One downstream target of p38 MAP kinase is MAPKAPK-2.3 35 Phosphorylation of MAPKAPK-2, which appeared as a doublet around 47 kDa, increased at 5 minutes, reached a maximum at 15 minutes, and declined thereafter (Figs. 5A 5B) . After TNF{alpha} treatment, phosphorylation levels were only slightly, and not significantly, elevated at later time points. After IL-1{alpha} treatment, they were lower but significantly elevated at 1 and 24 hours. Treatment with the p38 inhibitor SB202190 effectively blocked the TNF{alpha} and IL-1{alpha} stimulation of MAPKAPK-2 phosphorylation.


Figure 5
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  		FIGURE 5. Effects of TNF{alpha}, IL-1{alpha}, and p38 MAP kinase inhibitor SB202190 on MAPKAPK-2 phosphorylation. Western immunoblots of porcine TM cell lysates were probed with a phosphospecific MAPKAPK-2 antibody to T334. TM cells were treated with TNF{alpha} (A) or IL-1{alpha} (B) for 5 minutes, 15 minutes, 60 minutes, or 24 hours, with or without 1-hour pretreatment with the p38 MAP kinase inhibitor SB202190 (SB), as indicated below each figure. A typical immunoblot is shown below each figure. Mean relative band densities and SEMs are shown, with n and t-test significance as indicated above the lines connecting the groups compared.

 
Phosphorylation of Transcriptional Activator ATF-2
Another potential downstream substrate for p38 MAP kinase is the transcription factor ATF-2.36 37 ATF-2 phosphorylation increased several fold by 15 minutes after TNF{alpha} or IL-1{alpha} treatment and, at this time point, was not affected by SB202190 (Fig. 6) . By 60 minutes or 24 hours, phosphorylation had decreased but remained greater than in controls, particularly after IL-1{alpha} treatment. At these times, SB202190 did not reduce ATF-2 phosphorylation. In fact, at 60 minutes and at 24 hours, SB202190 increased ATF-2 phosphorylation above the level produced by TNF{alpha} or IL-1{alpha} alone (Figs. 6A 6B) .


Figure 6
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  		FIGURE 6. Effects of TNF{alpha}, IL-1{alpha}, and p38 MAP kinase inhibitor SB202190 on ATF-2 phosphorylation. Western immunoblots of porcine TM cell lysates were probed with a phosphospecific ATF-2 antibody to T69/71. TM cells were treated with TNF{alpha} (A) or IL-1{alpha} (B) for 5 minutes, 15 minutes, 60 minutes, or 24 hours, with or without 1 hour pretreatment with the p38 MAP kinase inhibitor SB202190 (SB), as indicated below each figure. A typical immunoblot is shown below each figure. Mean relative band densities and SEMs are shown, with n and t-test significance as indicated above the lines connecting the groups compared.

 
Effects of Inhibitor Treatment on TM Cell Shape
Twenty-four hours after TNF{alpha} treatment, TM cells look "dendritic" (Fig. 7C) . Although they appear to have retained at least some of their extracellular matrix attachment, much of the cell body was retracted, and their normal flat shape was lost. Pretreatment with the p38 MAP kinase inhibitor SB202190 before TNF{alpha} treatment (Fig. 7D) blocked this response, and cells resembled the untreated control (Fig. 7A) . SB202190 alone added to TM cultures had no effect (Fig. 7B) . IL-1{alpha} produced an even stronger dendritic appearance (Fig. 7E) . Pretreatment with SB202190 (Fig. 7F) did not block, and perhaps even accentuated, this very strong IL-1{alpha} effect. Changes in TM cell shape in response to either cytokine were transient. These changes were apparent by 12 hours, reached a maximum around 18 to 24 hours, and were diminished by 36 to 48 hours (data not shown).


Figure 7
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  		FIGURE 7. Morphologic effects of TNF{alpha}, IL-1{alpha}, and SB202190 on TM cells. Phase-contrast microscopic images of porcine TM cells after 24-hour treatment. (A) Control. (B) SB202190 (SB). (C) TNF{alpha}. (D) TNF{alpha} and SB. (E) IL-1{alpha}. (F) IL-1{alpha} and SB.

 
TM Cell Isoforms and Additional Phosphorylation of p38 MAP Kinase
A number of p38 MAP kinase isoforms and mRNA splice variants that exhibit different sensitivities to SB202190 have recently been identified.6 To explain the anomalous effect of this p38 inhibitor on the MMP-3 level changes in response to IL-1{alpha}, we determined which p38 MAP kinase isoforms were present in the TM (Figs. 8A) . Porcine and human TM cells express {alpha}, ß, {delta}, and {gamma} isoforms of p38. The {alpha} isoform migrates at approximately 38 kDa, and the {delta} and {gamma} isoforms migrate at approximately 42 to 43 kDa. The ß isoform migrates as an intermediate band at approximately 40 kDa. No significant differences were seen between porcine and human TM cell isoform profiles or sizes. In Figure 8A , lanes probed with phosphospecific p38 antibody are shown to the right of each lane of porcine TM cell extract probed with the indicated isoform-specific antibodies to correlate migration positions of the phosphorylation bands with the isoform bands.


Figure 8
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  		FIGURE 8. Isoforms of p38 MAP kinase and phosphorylation at 15 minutes and 24 hours. (A) Western immunoblots of human TM (HTM) and porcine TM (PTM) cell extracts were probed with p38 MAP kinase {alpha}, ß, {delta}, and {gamma} isoform–specific antibodies and with a phospho-p38 antibody, as indicated below each lane. PTM cells had been treated with IL-1{alpha} for 15 minutes or 24 hours before protein extractions. (B) Immunoblot of lysate from porcine TM cells treated for 15 minutes with vehicle (Control), TNF{alpha}, or IL-1{alpha} and probed with phosphospecific p38 antibody. (C) Immunoblot of lysates from porcine TM cells treated for 24 hours with vehicle (Control), TNF{alpha}, or IL-1{alpha}, with and without SB202190 (SB). The blot was probed with phosphospecific p38 MAP kinase antibodies. The approximate apparent molecular weight based on standards for each band is as indicated (arrowheads). Representative gels are shown from three experiments.

 
Analysis of p38 phosphorylation after 15 minutes (Fig. 8B) or 24 hours (Fig. 8C) showed that the predominant band of phosphorylation migrated at approximately 38 kDa, coincident with the p38 {alpha} isoform. This is the band that was analyzed earlier (Fig. 4) . The second band of phosphorylated p38 was the 42- or 43-kDa band, which migrated coincident with the {delta} and {gamma} p38 isoforms. After some treatments, such as 15-minute IL-1{alpha} (Fig. 8B) , one or more light bands of phosphorylation were also noticeable migrating between the 38- and the 42-kDa bands, roughly coincident with the ß p38 isoform.

The amount of phosphorylation of the 38-kDa band was similar for TNF{alpha} and IL-1{alpha}, though the time courses were different. However, IL-1{alpha} was much more effective at producing phosphorylation of the 42-kDa band than was TNF{alpha} (Figs. 8B 8C) . Surprisingly, at 24 hours, SB202190 actually increased the 42-kDa band of p38 MAP kinase phosphorylation in the presence of IL-1{alpha}, though it did not affect phosphorylation levels alone or with TNF{alpha} (Fig. 8C) .


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Previous studies showed that the protein kinase Cµ, Erk, and JNK pathways were all necessary for the transduction of signals between TNF{alpha} or IL-1{alpha} and MMP-3 production in the TM.7 8 9 16 The ability of the p38 MAP kinase inhibitor SB202190 and of the dominant-negative p38 MAP kinase mutant to interfere in the MMP-3 elevation, which occurs in response to TNF{alpha} or IL-1{alpha}, provides strong support for a similar requirement for p38 MAP kinase in this signal transduction process. The phosphorylation of MKK3/MKK6 and p38 MAP kinase in response to these cytokines provides additional support for involvement of this pathway. A previous study analyzing the effects of various pathway inhibitors on the IL-1{alpha} induction of MMP-3 in human TM cells further supports this multiple pathway hypothesis.16

A diagram depicting a simplified model for TNF and IL-1 signal transduction in the TM shows the most common components of these pathways (Fig. 9) . At the cell surface, either TNF or IL-1 binds its specific receptor, which transduces the signal through associated scaffold/adaptor proteins and kinase cascades.2 38 39 40 In this diagram, the question marks indicate one or more possible coupling proteins that mediate activation of the first tier of the MAP kinase or the protein kinase C pathways. The three primary MAP kinase pathways—Erk, JNK, and p38—are approximately parallel cascades composed of related but different kinases acting at each level. The MEK or MKK level kinases produce a dual phosphorylation of the MAP kinases in their activation loop on a Thr and a Tyr separated by one amino acid (as indicated in Fig. 9 ).2 6 17 38 This activates the MAP kinases, which then phosphorylate intermediary kinases such as MAPKAPK-2 or directly phosphorylate transcription factors or other cellular proteins.4 6 41 Because MMP-3 has AP-1 (Jun/Fos) and Ets (Ets/Elk) enhancer sites in its core promoter, members of these transcription factor families are clearly important (Song K, et al. IOVS 2005;46:ARVO E-Abstract 1356).42 43 44 45 46 47 Additional upstream enhancer and repressor sites are present in the MMP-3 promoter, but the transcription factors involved in these cytokine responses are not yet known (Song K, et al. IOVS 2005;46:ARVO E-Abstract 1356).48 49 A variety of posttranscriptional regulatory effects on MMP-3 levels are also possible50 51 52 but have not been investigated in the TM.


Figure 9
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  		FIGURE 9. Hypothetical model for TNF{alpha} and IL-1{alpha} signal transduction of MMP-3 response in TM cells. TNF and IL-1 signal through their respective cell surface receptors to cytoplasmic adaptor complexes, including TNF receptor associated factor(s) (TRAFs). Protein kinase Cµ and three MAP kinase pathways are all necessary to initiate MMP-3 transcription, expression and secretion.

 
In the case of TNF{alpha}, the involvement of the p38 MAP kinase pathway in regulating MMP-3 fits this model without additional considerations. With IL-1{alpha}, however, additional complexity in the p38 MAP kinase signaling seems apparent. With porcine TM cells, for example, SB202190 does not significantly reduce MMP-3 at 24 hours and actually potentiates, rather than inhibits, it at 48 and 72 hours. In human TM at 24 hours, we observe a strong inhibition of IL-1{alpha} induction of MMP-3 by SB202190, which is in agreement with a previous report.16 However, at 48 or 72 hours, SB202190 also potentiates the MMP-3 response to IL-1{alpha}.

A partial explanation for the anomalous pattern observed with IL-1{alpha} appears to reside in differential use of the p38 MAP kinase isoforms. There are four known isoforms of p38 MAPK and a number of mRNA splicing variants.6 The {alpha} and ß isoforms of p38 are inhibited by the small molecule pyridinyl imidazole inhibitors, such as SB202190 and SB203580, which serve as competitive inhibitors that bind in the adenosine triphosphate (ATP)–binding pocket of the active site. The p38{gamma} and p38{delta} isoforms do not respond to these inhibitors because of differences in the three-dimensional structure of their ATP-binding pocket.6 17 18 19 20 Using isoform-specific antibodies, we showed that TM cells produce all four isoforms of p38 in easily detectable levels (Fig. 8) . Phosphorylation bands are seen that migrate coincident with the {alpha} isoform and with the {delta}/{gamma} isoforms. Given that the {delta} and {gamma} isoforms are of approximately the same size, their contribution to 42- to 43-kDa phosphorylation cannot be differentiated. The degree of phosphorylation of the p38 ß isoform is also very low under these conditions. In human and porcine cells, the TNF{alpha} response to SB202190 suggests a strong reliance on the p38{alpha}/ß isoforms because MMP-3 is effectively reduced by this inhibitor at all times. The IL-1{alpha} response to the same inhibitor at 24 hours suggests a similar reliance, at least in human TM, on p38{alpha}/ß. In porcine TM at all times and in human TM cells at 48 or 72 hours, however, SB202190 cannot inhibit MMP-3 induction by IL-1{alpha}, suggesting that p38 {delta} and {gamma} rather than p38 {alpha} and ß are of greater importance in these instances.

The ability of the dominant-negative p38 MAP kinase to block MMP-3 transcription induced by TNF{alpha} or IL-1{alpha} at all time points argues strongly for a critical role for p38 MAP kinase in this transduction process. The dominant-negative p38 mutant will interact with any of the upstream signaling kinases (i.e., MKK3/MKK6) but cannot be activated because of the mutations in the critical amino acids (T180A and Y182F).34 Therefore, a dominant-negative mutant of any of the p38 isoforms will bind and occupy the activated upstream kinases. Mutant p38 will not, however, pass the signal on because it cannot be activated. High levels of a dominant-negative/kinase dead form of any of the p38 isoforms should thus block signal transduction by all these isoforms. Consequently, the p38 Map kinase pathway does appear to be necessary for TNF{alpha} and IL-1{alpha} induction of MMP-3 in TM cells.

The potentiation of MMP-3 expression at 48 and 72 hours, produced by the p38 inhibitor with IL-1{alpha} but not with TNF{alpha}, indicates that the IL-1{alpha} signal transduction pathway includes additional complexity. From Figure 8B , it is apparent that IL-1{alpha} triggers more phosphorylation of p38 {delta} or {gamma} isoforms than does TNF{alpha}, though both produce strong p38 {alpha} phosphorylation. In addition, the inhibitor SB202190 actually increases the p38 {delta}/{gamma} phosphorylation after IL-1{alpha} treatment (Fig. 8C) . The implication is that active p38 {alpha}/ß can reduce p38 {delta}/{gamma} phosphorylation. If this does occur, it could be through an indirect feedback effect such as p38 {alpha}/ß activating a phosphoprotein phosphatase. From the literature, phosphoprotein phosphatase 2C (PP2C) would be a possible candidate. Inhibition of PP2C by okadaic acid has been shown to increase MKK3 and p38 {gamma} activity.23 If active p38 {alpha} does increase PP2C activity, then SB202190 inhibition of p38 {alpha} could increase MKK3 or MKK6 and p38 {delta}/{gamma} activity. This would increase MMP-3 induction at later time points. The other MAP kinases, including the Erks, JNKs, p38 {alpha}, and p38 ß, but not p38 {delta} or p38 {gamma}, are normally dephosphorylated by a family of dual-specificity phosphatases.6 23 Thus, our studies are compatible with some type of negative feedback effect of p38 {alpha}/ß on PP2C or another phosphatase, but this explanation is clearly speculative.

The small amount of p38 ß phosphorylation that we observed in TM could also be more important than it appears. Furthermore, the phosphorylation of MKK4 is increased by these treatments, and the phosphorylation state of MKK7 is constitutively moderate and unaffected by these treatments.9 Both have been shown to phosphorylate p38 {delta}/{gamma} in some systems.6 The relative efficacy of the individual MKKs at phosphorylating the p38 isoforms is unknown. Similarly, the relative efficacy of the p38 isoforms at activating the several MMP-3 transcriptional activator proteins is also unknown. Hence, no simple mechanism completely explains all the observations in a quantitative manner.

It seems probable that MAPKAPK-2 is not directly involved in signaling the MMP-3 increase because its phosphorylation is blocked by the inhibition of p38 {alpha}/ß in response to both cytokines. Given that p38 {delta} and {gamma} are not blocked by SB202190 or similar inhibitors, these isoforms must not be critical to this phosphorylation. MAPKAPK-2 is thus downstream from p38 {alpha}/ß but not upstream from MMP-3 production or from the cytoskeletal shape change. This pathway must bifurcate somewhere near this point, with MAPKAPK-2 activation leading to some other process triggered by these cytokines. Although we thought that MAPKAPK-2 might be related to the cell shape changes observed with the cytokine treatments, the shape changes appear to be regulated more like MMP-3 induction. Both cytokines produce strong changes in cell shape. In separate studies, we have observed microfilament disruption associated with the shape changes produced by these treatments (data not shown). The fact that SB202190 can mostly block the shape changes produced by TNF{alpha} but appears to accentuate the changes produced by IL-1{alpha} suggests a combination of p38 isoform use more similar to that observed in MMP-3 production. The time courses of the MMP-3 and cell shape changes suggest a parallel rather than a causal relationship between them.

The p38 {alpha}/ß isoforms also appear not to be directly responsible for ATF-2 phosphorylation because SB202190 does not inhibit this process. Previously, we showed that ATF-2 phosphorylation after cytokine treatment is blocked by a JNK inhibitor, which also blocks MMP-3 increases.9 However, the increase in ATF-2 phosphorylation at 60 minutes and 24 hours with SB202190, after either TNF{alpha} or IL-1{alpha} treatment, may imply involvement in the MMP-3 increases. That SB202190 also enhances the TNF{alpha} effect on ATF-2 phosphorylation makes the nature of this involvement less clear. The {delta} and {gamma} isoforms of p38 are more effective than the {alpha} and ß isoforms at activating some transcription factors.22 53 54 55 However, the participation of p38 MAP kinase in activating other transcriptional activators has not been clearly established in the TM.

The effects of TNF and IL-1 on TM cells and their signal transduction have several levels of significance. These cytokines have been shown to mediate the increase in MMP-3 that is produced by laser trabeculoplasty, a common treatment to reduce the intraocular pressure elevation associated with primary open-angle glaucoma.12 The addition of MMPs or their elevation by IL-1{alpha} increases aqueous humor outflow facility in perfused anterior segment organ culture.56 It seems, then, that an important component of the effectiveness of laser trabeculoplasty in restoring glaucomatous intraocular pressures to normal is the extracellular matrix turnover initiated by these MMPs. At another level, studies showing an association between chronic elevation of IL-1 and glaucoma have been presented.57 Signal transduction there, where the stress is chronic, appears to be permanently perturbed, resulting in pathology.15 No simple relationship between these long-term and our short-term studies has been established.


   Acknowledgements
 
The authors thank Genevieve Long, PhD, for editorial assistance.


   Footnotes
 
Supported by Grants EY003279, EY008247, and EY010572 from the National Institutes of Health; an unrestricted grant to Casey Eye Institute from Research to Prevent Blindness (New York, NY); and grants from the Glaucoma Research Foundation (San Francisco, CA), Alcon Labs (Fort Worth, TX), and the Kettering Family Foundation (Denver, CO).

Submitted for publication November 14, 2006; revised January 25, 2007; accepted April 5, 2007.

Disclosure: M.J. Kelley, None; A. Rose, None; K. Song, None; B. Lystrup, None; J.W. Samples, None; T.S. Acott, Alcon Labs (F)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Mary J. Kelley, Casey Eye Institute, Oregon Health & Science University, 3375 SW Terwilliger, Portland, OR 97239-4197; kelleyma{at}ohsu.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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