Investigative Ophthalmology & Visual Science Cover Image for Volume 47, Issue 4
April 2006
Volume 47, Issue 4
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Glaucoma  |   April 2006
IL-1 and TNF Induction of Matrix Metalloproteinase-3 by c-Jun N-Terminal Kinase in Trabecular Meshwork
Author Affiliations
  • Mojgan Hosseini
    From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; and
  • Anastasia Y. Rose
    From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; and
  • Kaili Song
    From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; and
  • Cynthia Bohan
    From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; and
  • J. Preston Alexander
    Triple Point Biologics, Forest Grove, Oregon.
  • Mary J. Kelley
    From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; and
  • Ted S. Acott
    From the Casey Eye Institute, Oregon Health & Science University, Portland, Oregon; and
Investigative Ophthalmology & Visual Science April 2006, Vol.47, 1469-1476. doi:https://doi.org/10.1167/iovs.05-0451
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      Mojgan Hosseini, Anastasia Y. Rose, Kaili Song, Cynthia Bohan, J. Preston Alexander, Mary J. Kelley, Ted S. Acott; IL-1 and TNF Induction of Matrix Metalloproteinase-3 by c-Jun N-Terminal Kinase in Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2006;47(4):1469-1476. https://doi.org/10.1167/iovs.05-0451.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The cytokines TNF and IL-1 mediate the MMP-3 increase that occurs in response to trabecular meshwork (TM) treatment by laser trabeculoplasty. This MMP-3 increase appears to play a key role in the efficacy of this treatment for open-angle glaucoma. Protein kinase Cμ and the Erk mitogen-activated protein (MAP) kinases are essential signaling components in transducing MMP-3 increases produced by treatment of TM cells with these cytokines. Here, the involvement of the JNK-MAP kinase pathway in this process was evaluated.

methods. Porcine TM cells were treated with TNFα, IL-1α, or IL-1β. Changes in MMP-3 and MMP-9 protein levels in the media were then determined by Western immunoblot. The effect of JNK inhibitor 2 was evaluated. Changes in the level of phosphorylation of JNK, c-Jun, ATF-2, MKK4, and MKK7 were also determined at various times after TNFα or IL-1α treatment. A 2.3-kb MMP-3 promoter fragment was cloned into a secreted alkaline phosphatase reporter vector. This reporter construct was cotransfected into TM cells with a mammalian expression vector containing a dominant-negative mutant of JNK. The involvement of JNK activity in the TNFα and IL-1α induction of MMP-3 expression was then evaluated.

results. TNFα, IL-1α, and IL-1β increase media MMP-3 and MMP-9 protein levels, and JNK inhibitor 2 blocks these increases. JNK1/2, MKK4, c-Jun, and ATF-2 phosphorylation levels increase in response to TNFα and IL-1α treatment. JNK inhibitor 2 pretreatment blocks these c-Jun and ATF-2 phosphorylation increases. Dominant-negative JNK dramatically reduces the MMP-3 promoter–driven reporter activity induced by these cytokines.

conclusions. JNK activity is necessary for the induction of MMP-3 and MMP-9 by TNFα, IL-1α, or IL-1β in TM cells. Phosphorylation of components of the JNK signaling pathway and of the transcription factors c-Jun and ATF-2 support a role for this pathway in the induction of MMP-3 and MMP-9 in the TM in response to these cytokines. Thus, at least three separate signal transduction pathways are necessary in this signaling event in TM cells.

Glaucoma is a leading cause of irreversible blindness. 1 2 A primary risk factor for glaucoma is elevated intraocular pressure (IOP), which can contribute to significant optic nerve damage and vision loss. Laser trabeculoplasty (LTP), a common alternative treatment used to reduce IOP in patients with glaucoma, may owe much of its efficacy to the increased levels of MMP-3 (also called stromelysin-1) within the juxtacanalicular region of the TM. 3 4 Addition or induction of MMP-3 in perfused human anterior segment organ culture increases aqueous humor outflow facility. 5 Blocking the endogenous activity of the MMPs of the TM reduces outflow facility. Thus, ongoing extracellular matrix (ECM) turnover, initiated by one or more of these MMPs, appears to be essential to maintaining IOP homeostasis. 5 MMP-2 and MMP-14 appear to be the important initiators in the process of IOP homeostasis. 6 MMP-3, with possible contributions from MMP-9, appears to be the critical initiator of ECM turnover in the therapeutic effects of LTP in ameliorating glaucomatous IOP elevations. 4 Thus, it seems possible that an agent that increases one or more MMPs within the TM might have beneficial effects on IOP in patients with glaucoma. 
The trabecular MMP-3 increase produced by LTP is mediated by secreted factors identified as the cytokines IL-1β and TNFα. 7 Both are secreted in response to LTP, and blocking the action of either dramatically reduces the MMP-3 increase. 7 IL-1α also increases dramatically with LTP but is not secreted. 7 IL-1α treatment increases outflow facility, and this increase is antagonized by treatment with a tissue inhibitor of metalloproteinase (TIMP). 5  
Although the signal transduction pathways involved in IL-1, TNF, and growth factor induction of the MMPs have been studied extensively, they remain only partially understood, and significant variations are seen between different tissues. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 We have previously shown that protein kinase Cμ 26 and mitogen-activated protein kinase (MAPK) Erk1 or Erk2 (or both) 27 are necessary, but not sufficient, to transduce the increases in MMP-3 produced by TNFα treatment of TM cells. Inhibitors of these same kinases were also shown to block the increases in trabecular MMP-3 production in response to IL-1α treatment. 28 29 c-Jun has been shown in several other tissues to be an important component in the transcriptional activation of MMP-3. 8 9 10 25 Several MMPs, including MMP-3 and MMP-9, have AP-1 transcription enhancer elements in their promoter regions. 8 10 15 16 17 18 19 30 31 32 Because JNK phosphorylation of c-Jun on S63 and S73 has been shown to be one step in activating transcription through AP-1 sites, 33 we evaluated the details and contributions of the JNK pathway to increasing MMP-3 expression by TNFα, IL-1α, and IL-1β. 
Materials and Methods
Porcine eyes were obtained from Carlton Packing Company (Carlton, OR) 2 to 5 hours postmortem. Human TNFα and human and porcine IL-1α and IL-1β were from R&D Systems (Minneapolis, MN). Phosphospecific MKK4 (S257/T261), MKK7 (S271/T275), JNK (T183/Y185), c-Jun (S73 or S63), and ATF-2 (T71 or T71/T69) antibodies, MKK4, MKK7, and JNK1/2 protein antibodies, and conjugated horseradish peroxidase secondary antibodies were from Cell Signaling Technologies (Beverly, MA). Phosphospecific ATF-2 (T71) antibody was also obtained from Santa Cruz Biotechnology (Santa Cruz, CA). MMP-3 and MMP-9 antibodies were from TriplePoint Biologics (Forest Grove, OR), and c-Jun antibody was from Biosource (Camarillo, CA). High- and low-glucose Dulbecco modified Eagle medium (DMEM), antibiotics, and antimycotic 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 were from Rockland (IRDye 800-conjugated; Gilbertsville, PA); and JNK inhibitor 2 was from CalBiochem (SP600125; San Diego, CA). Statistical significance when comparing groups subjected to different treatments used the Student’s t test or the Mann-Whitney U test. 
Cell Culture, Treatment, and Protein Extraction
Porcine TM cells were cultured as previously detailed 26 27 34 35 36 37 in medium glucose (1:1 mix of high and low glucose) DMEM supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic mix and were used by passage 5. Confluent cells were serum starved for 48 hours before and during treatment with recombinant human TNFα (10 ng/mL), recombinant human IL-1α (25 ng/mL), recombinant porcine IL-1α (10 ng/mL), recombinant human IL-1β (10 or 25 ng/mL), or recombinant porcine IL-1β (10, 25, or 50 ng/mL) for 5, 10, 15, 20, or 30 minutes or 1, 4, 24, 48, or 72 hours, as indicated. For inhibitor studies, cells were pretreated with 20 μM JNK inhibitor 2 for 1 hour before and during TNFα, IL1α, or IL-1β treatment. Parallel controls with and without equivalent levels of the JNK inhibitor 2 vehicle (dimethyl sulfoxide [DMSO]) were included in all inhibitor studies. Cellular proteins were extracted with a modified radioimmunoprecipitation assay (RIPA) buffer (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 nM phenylmethyl sulfonyl fluoride (PMSF), 20 μg/mL leupeptin, 20 μg/mL aprotinin, 20 μg/mL pepstatin, and 50 mM Tris, pH 7.5) on ice, flash frozen in liquid nitrogen, and kept at –80°C until use. Aliquots of culture media for MMP analysis were frozen and kept at –20°C. Thawed media aliquots were concentrated 4× using concentration columns (Centricon YM-10; Millipore, Bedford, MA). 
Western Immunoblots
Cellular signal transduction proteins were extracted with modified RIPA buffer and subjected to standard SDS-PAGE on 8% or 12% separating gels. 38 Culture media proteins were subjected to similar SDS-PAGE separation. Proteins were then transferred from gels to polyvinylidene difluoride (PVDF) or nitrocellulose membranes and were blocked with 5% nonfat dry milk before probing with the primary antibody. In some cases, detection was performed with the appropriate secondary antibodies with conjugated horseradish peroxidase using chemiluminescent substrate (SuperSignal West Pico; Pierce). To verify uniform total protein loading and transfer, blots were stained with Ponceau stain (Sigma-Aldrich, St. Louis, MO) after transfer and before the addition of the blocking agent. X-ray films exposed to chemiluminescent blots were scanned (ScanJet II CX/T; Hewlett-Packard, Palo Alto, CA), and relative band densities were determined using commercial software (Labworks; UVP, Upland, CA). 39 In other cases, blocking buffer (Odyssey; Li-Cor Biosciences, Lincoln, NE) was used to block membranes before incubation with primary antibodies. Detection was performed using the appropriate conjugated secondary antibody (Alexa Fluor 680 [Molecular Probes] or IRDye 800 [Rockland]); blots were then scanned, and relative band density was determined on an imaging system (Odyssey Infrared; Li-Cor Biosciences). 
Plasmid Constructs
A 2.3-kb DNA fragment containing the human MMP-3 promoter (hMMP3p) was amplified from human genomic DNA by PCR and subcloned into MluI/BglII restriction sites upstream of the secreted alkaline phosphatase (SEAP) gene in the reporter vector (SEAP-Basic; Clontech, Palo Alto, CA). Correct insertion and sequence of the MMP-3 promoter in the hMMP3p-SEAP construct were confirmed by sequencing. The dominant-negative (T183A and Y185F) JNK construct pCDNA3-Flag-JNK1 (APF) was a gift of Roger Davis (University of Massachusetts Medical Center; Worcester, MA). 33 40 The control pCDNA3.1 plasmid was from Invitrogen (Carlsbad, CA). Vectors and constructs were amplified in Escherichia coli cells (OneShot; Invitrogen) and were extracted (EndoFree Plasmid Maxi Kit; Qiagen Valencia, CA) before transfection. 
Cotransfection of TM Cells and Chemiluminescence SEAP Assay
TM cells were seeded at a density of 80,000 cells per well in 12-well plates and were maintained in DMEM supplemented with 10% FBS overnight. Cells were prewashed twice with serum-free DMEM before cotransfection. TM cells in each well were cotransfected with 0.2 μg hMMP3p-SEAP or with 0.2 μg control SEAP-Basic (Clontech) construct and with either 0.4 μg dominant-negative JNK construct or 0.4 μg control pcDNA3 plasmid using reagent (Transfectam; Promega, Madison, WI) according to the manufacturer’s instructions. After a 2-hour cotransfection period, cells were overlaid with 2 mL DMEM supplemented with 10% serum and allowed to recover overnight. Cells were then serum starved for 48 hours before and during treatment with recombinant human TNFα (20 ng/mL), recombinant porcine IL-1α (10 ng/mL), or vehicle alone. Conditioned medium was collected at 72, 96, and 120 hours after treatment, and promoter activity was determined using detection kits (Great EscAPe SEAP Chemiluminescence; BD Biosciences, San Jose, CA) according to the manufacturer’s directions. DNA analysis with assay kits (Picogreen) was sometimes used after the analysis was completed to verify that the various treatments did not change TM cell numbers. Transfection efficiency optimization and transfection uniformity among the various vectors and constructs were determined by cotransfection with a green fluorescent protein (GFP)-pcDNA3 construct. 
Results
Effects of TNFα, IL-1α, and JNK Inhibitor 2 on MMP-3 Levels
Incubation of porcine TM cells with TNFα or IL-1α for 24, 48, or 72 hours produced significant increases in MMP-3 protein levels in the medium. Western immunoblots of gels showing a band at 63 kDa (the pro–MMP-3 isoform) from typical samples are shown in Figure 1B and 1Cat the indicated times after treatment, and the resultant data from scans of several experiments are shown in Figure 1A . The increases produced by recombinant human or porcine IL-1α were larger than those produced by recombinant human TNFα. The amplitude of the MMP-3 response to human IL-1α, used at 25 ng/mL, was comparable to the response to porcine IL-1α at 10 ng/mL (data not shown). Pretreatment of TM cells with JNK inhibitor 2 dramatically reduced, but did not totally block, the MMP-3 production induced by TNFα or by IL-1α (Fig. 1D 1E 1F)
Effects of IL-1β and JNK Inhibitor 2 on MMP-3 and MMP-9 Levels
In response to IL-1β, MMP-3 increased significantly, but to a smaller extent and more slowly than it did in response to IL-1α or TNFα (Fig. 2A) . Increasing the IL1β dose to 50 ng/mL also enhanced the response. This MMP-3 increase was effectively inhibited by treatment with JNK inhibitor 2 (Fig. 2A)
Treatment with TNFα, IL-1α, or IL-1β also produced significant increases in MMP-9 (Fig. 2B) . The relative potency of these cytokines in inducing MMP-9 was different from that observed for MMP-3. Pretreatment of TM cells with JNK inhibitor 2 dramatically reduced MMP-9 production induced by all three cytokines (Fig. 2B)
Phosphorylation of TM Cell JNK 1 and 2 and MKK4/MKK7
The dual phosphorylation on T183 and Y185, the common kinase activation site for JNK 1 and 2, is relatively rapid after treatment with TNFα or IL-1α (Fig. 3) . The 46-kDa JNK 1 and the 54-kDa JNK 2 were phosphorylated at similar rates; significant increases were achieved by 5 minutes, and maximum was reached by 15 or 30 minutes for TNFα or IL-1α, respectively. 
This dual phosphorylation of JNK 1 and 2 is often achieved by activated MKK4 or MKK7, or both. 41 Evaluation of the activation state of MKK4 as reflected by phosphorylation on S257/T261, at various times after treatment of TM cells with TNFα or IL-1α, is shown in Figure 4A or 4B , respectively. A band migrating at approximately 44 kDa was phosphorylated significantly by 5 minutes, reaching a maximum at 10 or 15 minutes with TNFα and at 30 minutes with IL-1α. MKK4 phosphorylation then declined, becoming insignificantly different from that in controls by 4 hours but rebounding to achieve significant elevation at 24 hours after either treatment. Although significant levels of MKK7 protein were detectable at the expected size of approximately 48 kDa, only modest phosphorylation on S271/T275 was observed, and this did not change significantly with TNFα or IL-1α treatment (data not shown). 
Phosphorylation of Transcriptional Activators c-Jun and ATF-2
Downstream phosphorylations often attributable to JNK included the transcription factors c-Jun and ATF-2. Phosphorylation of c-Jun on S73 increased dramatically, achieving statistical significance by 10 minutes and maximum by 60 minutes after treatment with TNFα (Fig. 5A)or IL-1α (Fig. 5B) . Approximately the same profile was obtained using phosphospecific antibodies, which recognized S63 (data not shown). When TM cells were pretreated with JNK inhibitor 2, phosphorylation of c-Jun on S73 in response to treatment TNFα (Fig. 4C)or IL-1α (Fig. 4D)was blocked. The phosphorylation of c-Jun on S63 was similarly affected by this inhibitor (data not shown). 
Phosphorylation of ATF-2 on T71 after TNFα (Fig. 4E)or IL-1α (Fig. 4F)increased by 5 to 10 minutes and peaked at 15 or 30 minutes, respectively. Pretreatment with JNK inhibitor 2 blocked phosphorylation on T71 after treatment with TNFα (Fig. 4G)or IL-1α (Fig. 4H) . The phosphorylation pattern for ATF-2 on T69 (data not shown) was similar to that shown for T71. 
Effects of Dominant-Negative JNK on TM Cell MMP-3 Promoter Activity after TNFα and IL-1α Treatment
TM cells, which were transfected with the SEAP reporter driven by a 2.3-kb MMP-3 promoter (hMMP3p-SEAP), responded to TNFα or IL-1α treatment by secreting high levels of SEAP (solid bars in Fig. 6A 6B ) when compared with similarly transfected cells not treated with either cytokine (vertical hatched bars) or with cells transfected with control plasmids and treated with these cytokines (clear bars). Cotransfection of hMMP3p-SEAP with the dominant-negative JNK construct dnJNK dramatically blocked TNFα or IL-1α induction of SEAP (horizontal hatched bars in Fig. 6A B ). This effect was highly significant at all three time points evaluated for both cytokines. 
Discussion
Several lines of evidence developed herein support the hypothesis that the JNK MAPK pathway is a necessary component of the TNFα, IL-1α, or IL-1β induction of MMP-3 and MMP-9 in TM cells. Temporal phosphorylation patterns of MKK4, JNK 1 and 2, and c-Jun after TNFα or IL-1α treatment suggest that the JNK pathway may be important in transducing these signals. Inhibition of the TNFα, IL-1α, and IL-1β induction of MMP-3 and MMP-9 by JNK inhibitor 2 provides strong support for the required involvement of the JNK pathway. The effects of dominant-negative (kinase dead) JNK on MMP-3 promoter activity further established a necessary role for this pathway in this signal transduction. Although this conclusion is not surprising, based on signaling in other cell types, it had not been clearly established in the TM. 
We have previously shown that LTP induces relatively sustained MMP-3 expression, specifically within the TM juxtacanalicular region. 3 4 This induction occurs through media-borne factor(s), identified as TNFα and IL-1β. 7 Anterior segment perfusion with the MMPs increases outflow facility, and inhibition of the endogenous MMPs within the TM dramatically decreases outflow facility. 5 It seemed likely that this explains the efficacy of LTP as a treatment for the elevated IOP seen in many patients with glaucoma. To understand the signal transduction involved in this process, we evaluated the roles of several possible protein kinase pathways in signaling. We showed earlier that protein kinase Cμ and the Erk MAPK pathways are required for TNFα induction of MMP-3 in the TM. 26 27 Others have shown that a PKCμ inhibitor or an inhibitor of Erk phosphorylation blocks the induction of MMP-3 by IL-1α. 29 Thus, strong evidence has now been presented supporting a requirement for JNK, Erk, and PKCμ in transducing the trabecular MMP-3 increase in response to treatment with TNFα or IL-1α/β. In other studies, chronic elevations in IL-1 levels have been associated with several forms of glaucoma. 42 Possible relationships between this chronic cytokine elevation and our current studies of relatively short-term elevation remain unclear, but some of the same signaling pathways appear to be involved. 
The actual MMP-3 promoter elements and the specific transcriptional activator proteins that act through them—c-Jun, c-Fos, ATF-2, Ets-1/2, Elk-1—during this signaling in the TM remain incompletely defined. In some other tissues or with the use of other primary signals, an AP-1 site, a pair of head-to-head polyomavirus enhancer A-binding protein-3 (PEA-3) sites, and a novel stromelysin PDGF-responsive element (SPRE) site have been identified in MMP-3 transcriptional activation. 8 10 12 13 22 25 30 43 44 45 46 47 Other enhancer or repressor sites have been identified and may be involved as well. We have identified 2 additional sites, a repressor and an enhancer, in addition to the AP-1 and Ets sites, that are critical to MMP-3 induction by these cytokines (Song K, et al. IOVS 2005;46:ARVO E-Abstract 1356). The SPRE element 24 does not appear to be involved in this signaling process in the TM (Song K, et al. IOVS 2005;46:ARVO E-Abstract 1356). Thus, the specifics of MMP-3 induction in the TM remain only partially understood. Our studies and earlier studies by another group 28 29 provide evidence for a role for c-Jun in trabecular MMP-3 and MMP-9 induction by these cytokines. An involvement of ATF-2, which is activated in a JNK-dependent manner, in mediating some effects of these cytokines in the TM is clear. However, it has not yet been demonstrated that ATF-2 is acting on MMP-3 or MMP-9 transcription. 
The observation that at least 3 parallel protein kinase pathways are necessary, but not sufficient, to induce MMP-3 in response to TNF or IL-1 treatments suggests one of several possibilities. One possibility is that each of these kinases phosphorylates a different set of transcriptional activator proteins, which then bind to different or interacting enhancer sites in the MMP-3 promoter. Such an interaction between the Ets and AP-1 sites has been reported. 25 48 The Ets site has been shown to enhance the effects of the AP-1 site under other conditions in other cell types. 49 50 It may also be that several different phosphorylation sites on the same transcriptional activator protein (eg, c-Jun has at least 7 phosphorylation sites) must be phosphorylated to achieve full activation. 51 Effects beyond transcriptional activation—mRNA half-life or translational regulation—have also been demonstrated for the MMPs 52 53 and could be involved here. 
The exact mechanism of phosphorylation of JNK on T183 and Y185 is also controversial but was thought to require either of the dual-specificity kinases, MKK4 or MKK7. Each of these kinases was thought to be able to phosphorylate T183 and Y185 in the JNK activation loop. However, a recent study 41 suggests that what occurs may be a concerted event requiring both kinases. The increased phosphorylation of MKK4 implicates it in the phosphorylation and activation of JNK. MKK7 exhibits modest constitutive levels of phosphorylation without treatment, and this is not significantly affected by these treatments. It may be that MKK7 is not involved in activating JNK in the TM in response to these treatments. It may also be that this low level of MKK7 activity is able to maintain T183 phosphorylation, which could maintain JNK in a constitutive “prepared but not active” state awaiting complete activation by MKK4 phosphorylation of Y185. Our results are consistent with either possibility and do not allow us to differentiate between these two hypothesized mechanisms. 
Although LTP has been a relatively noninvasive alternative treatment for elevated IOP in glaucoma, developing a drug that could mimic its action would be of considerable therapeutic interest. Defining the signaling pathways mediating the therapeutic effect of a treatment such as LTP, which has the outflow pathway as a target, remains an attractive goal. 
 
Figure 1.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on MMP-3 production by TM cells. (A) Media levels of MMP-3 were measured by Western immunoblot at the indicated times after treatment with TNFα (10 ng/mL; horizontally hatched bars) or IL-1α (25 ng/mL; solid bars). Mean ± SEM is shown with t test significance as indicated (n = 6). (B, C) Examples of individual Western immunoblots of the 63-kDa pro-MMP-3 isoform. (D) MMP-3 levels determined after 48 hours of treatment with or without JNK inhibitor 2 (JNK I II). Mean ± SEM (n = 6) with significance as indicated above comparison pairs. (E, F) Typical MMP-3 immunoblots, with treatments as indicated.
Figure 1.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on MMP-3 production by TM cells. (A) Media levels of MMP-3 were measured by Western immunoblot at the indicated times after treatment with TNFα (10 ng/mL; horizontally hatched bars) or IL-1α (25 ng/mL; solid bars). Mean ± SEM is shown with t test significance as indicated (n = 6). (B, C) Examples of individual Western immunoblots of the 63-kDa pro-MMP-3 isoform. (D) MMP-3 levels determined after 48 hours of treatment with or without JNK inhibitor 2 (JNK I II). Mean ± SEM (n = 6) with significance as indicated above comparison pairs. (E, F) Typical MMP-3 immunoblots, with treatments as indicated.
Figure 2.
 
Effects of IL-1β and JNK inhibitor 2 on MMP-3 levels and of TNFα, IL-1, and JNK inhibitor 2 on MMP-9 levels. (A) Media levels of MMP-3 after treatment of TM cells with IL-1β (50 ng/mL) with or without JNK inhibitor 2 were determined by Western immunoblot. Mean ± SEM is shown with t test significance as indicated (n = 4–7). (B) Media levels of MMP-9 were determined by Western immunoblot after 48 hours of treatment with TNFα (horizontally hatched bars), IL-1α (solid bars), IL-1β (vertically hatched bars), and JNK inhibitor 2 (JNK I II) as indicated. Mean ± SEM (n = 4–7) and t test significance for pairs above the connecting lines as indicated. Typical Western immunoblot below the graph shows the 92-kDa pro–MMP-9 band and a very light 88-kDa activated form.
Figure 2.
 
Effects of IL-1β and JNK inhibitor 2 on MMP-3 levels and of TNFα, IL-1, and JNK inhibitor 2 on MMP-9 levels. (A) Media levels of MMP-3 after treatment of TM cells with IL-1β (50 ng/mL) with or without JNK inhibitor 2 were determined by Western immunoblot. Mean ± SEM is shown with t test significance as indicated (n = 4–7). (B) Media levels of MMP-9 were determined by Western immunoblot after 48 hours of treatment with TNFα (horizontally hatched bars), IL-1α (solid bars), IL-1β (vertically hatched bars), and JNK inhibitor 2 (JNK I II) as indicated. Mean ± SEM (n = 4–7) and t test significance for pairs above the connecting lines as indicated. Typical Western immunoblot below the graph shows the 92-kDa pro–MMP-9 band and a very light 88-kDa activated form.
Figure 3.
 
Effects of TNFα and IL-1α on JNK 1 and 2 phosphorylation. Western immunoblots of TM cell extracts at the indicated times after treatment with TNFα (A, C, E) or IL-1α (B, D, F) were probed with a phosphospecific antibody that recognized activated JNK 1 (C, D) and JNK 2 (A, B) phosphorylated on T183 and Y185. Mean, SD, and t test significance are shown (n = 9 or n = 6). Untreated controls, which had not been treated but which were incubated for each of the times indicated, were not different from the 0-minute control and thus are not shown. (E, F) Representative Western immunoblots are shown with the phosphorylated 54-kDa JNK 2 band and the 46-kDa JNK 1 bands as labeled between the blots.
Figure 3.
 
Effects of TNFα and IL-1α on JNK 1 and 2 phosphorylation. Western immunoblots of TM cell extracts at the indicated times after treatment with TNFα (A, C, E) or IL-1α (B, D, F) were probed with a phosphospecific antibody that recognized activated JNK 1 (C, D) and JNK 2 (A, B) phosphorylated on T183 and Y185. Mean, SD, and t test significance are shown (n = 9 or n = 6). Untreated controls, which had not been treated but which were incubated for each of the times indicated, were not different from the 0-minute control and thus are not shown. (E, F) Representative Western immunoblots are shown with the phosphorylated 54-kDa JNK 2 band and the 46-kDa JNK 1 bands as labeled between the blots.
Figure 4.
 
Phosphorylation of MKK4 after TNFα and IL-1α treatment. At the indicated times after treatment with TNFα (A) or IL-1α (B), TM cell extracts were subjected to Western immunoblots and probed with phosphospecific antibodies for S257/T261 of MKK4. Mean, SEM, n, and t test significances are shown at the indicated times.
Figure 4.
 
Phosphorylation of MKK4 after TNFα and IL-1α treatment. At the indicated times after treatment with TNFα (A) or IL-1α (B), TM cell extracts were subjected to Western immunoblots and probed with phosphospecific antibodies for S257/T261 of MKK4. Mean, SEM, n, and t test significances are shown at the indicated times.
Figure 5.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on phosphorylation of the transcriptional activator proteins c-Jun and ATF-2. Phosphorylation of c-Jun on S73 was assessed on Western immunoblots of TM cell extracts after treatment with TNFα (A) or IL-1α (B) for the indicated times. Mean ± SEM of relative band densities are shown for a band(s) migrating at approximately 48 kDa; t test significance and n are as indicated. The phosphorylation pattern of S63 (not shown) was similar to that of S73. The effect of pretreatment with JNK inhibitor 2 (JNK I II) on c-Jun phosphorylation measured 1 hour after treatment with TNFα (C) or IL-1α (D) is shown and t test significance (P < 0.001) is indicated above the lines linking the respective treatment pairs compared. Phosphorylation of ATF-2 on T71 is shown for the indicated treatment times with TNFα (E) or IL-1α (F). ATF-2 migrates at approximately 70 kDa. Effects of pretreatment with JNK inhibitor 2 are shown for TNFα (G) and IL-1α (H). Significance from t test comparisons is shown above the lines indicating which treatment pairs were compared.
Figure 5.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on phosphorylation of the transcriptional activator proteins c-Jun and ATF-2. Phosphorylation of c-Jun on S73 was assessed on Western immunoblots of TM cell extracts after treatment with TNFα (A) or IL-1α (B) for the indicated times. Mean ± SEM of relative band densities are shown for a band(s) migrating at approximately 48 kDa; t test significance and n are as indicated. The phosphorylation pattern of S63 (not shown) was similar to that of S73. The effect of pretreatment with JNK inhibitor 2 (JNK I II) on c-Jun phosphorylation measured 1 hour after treatment with TNFα (C) or IL-1α (D) is shown and t test significance (P < 0.001) is indicated above the lines linking the respective treatment pairs compared. Phosphorylation of ATF-2 on T71 is shown for the indicated treatment times with TNFα (E) or IL-1α (F). ATF-2 migrates at approximately 70 kDa. Effects of pretreatment with JNK inhibitor 2 are shown for TNFα (G) and IL-1α (H). Significance from t test comparisons is shown above the lines indicating which treatment pairs were compared.
Figure 6.
 
Effects of dominant-negative JNK on MMP-3 promoter activity stimulated by TNFα or IL-1α. TM cells were cotransfected with the reporter vector without any promoter (SEAP Basic) or with the MMP-3 promoter (hMMP3p-SEAP) and with the control plasmid (pCDNA) or the dominant-negative JNK construct (dnJNK). SEAP reporter activity as measured in the media is shown in relative chemiluminescence units at the indicated times after the indicated treatments with ΤNFα (A) or IL-1α (B). Mean ± SEM for triplicate determinations from 2 separate experiments are shown (n = 6). Significance, evaluated by t test, was determined as indicated by the lines between the compared groups. *P < 0.001.
Figure 6.
 
Effects of dominant-negative JNK on MMP-3 promoter activity stimulated by TNFα or IL-1α. TM cells were cotransfected with the reporter vector without any promoter (SEAP Basic) or with the MMP-3 promoter (hMMP3p-SEAP) and with the control plasmid (pCDNA) or the dominant-negative JNK construct (dnJNK). SEAP reporter activity as measured in the media is shown in relative chemiluminescence units at the indicated times after the indicated treatments with ΤNFα (A) or IL-1α (B). Mean ± SEM for triplicate determinations from 2 separate experiments are shown (n = 6). Significance, evaluated by t test, was determined as indicated by the lines between the compared groups. *P < 0.001.
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Figure 1.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on MMP-3 production by TM cells. (A) Media levels of MMP-3 were measured by Western immunoblot at the indicated times after treatment with TNFα (10 ng/mL; horizontally hatched bars) or IL-1α (25 ng/mL; solid bars). Mean ± SEM is shown with t test significance as indicated (n = 6). (B, C) Examples of individual Western immunoblots of the 63-kDa pro-MMP-3 isoform. (D) MMP-3 levels determined after 48 hours of treatment with or without JNK inhibitor 2 (JNK I II). Mean ± SEM (n = 6) with significance as indicated above comparison pairs. (E, F) Typical MMP-3 immunoblots, with treatments as indicated.
Figure 1.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on MMP-3 production by TM cells. (A) Media levels of MMP-3 were measured by Western immunoblot at the indicated times after treatment with TNFα (10 ng/mL; horizontally hatched bars) or IL-1α (25 ng/mL; solid bars). Mean ± SEM is shown with t test significance as indicated (n = 6). (B, C) Examples of individual Western immunoblots of the 63-kDa pro-MMP-3 isoform. (D) MMP-3 levels determined after 48 hours of treatment with or without JNK inhibitor 2 (JNK I II). Mean ± SEM (n = 6) with significance as indicated above comparison pairs. (E, F) Typical MMP-3 immunoblots, with treatments as indicated.
Figure 2.
 
Effects of IL-1β and JNK inhibitor 2 on MMP-3 levels and of TNFα, IL-1, and JNK inhibitor 2 on MMP-9 levels. (A) Media levels of MMP-3 after treatment of TM cells with IL-1β (50 ng/mL) with or without JNK inhibitor 2 were determined by Western immunoblot. Mean ± SEM is shown with t test significance as indicated (n = 4–7). (B) Media levels of MMP-9 were determined by Western immunoblot after 48 hours of treatment with TNFα (horizontally hatched bars), IL-1α (solid bars), IL-1β (vertically hatched bars), and JNK inhibitor 2 (JNK I II) as indicated. Mean ± SEM (n = 4–7) and t test significance for pairs above the connecting lines as indicated. Typical Western immunoblot below the graph shows the 92-kDa pro–MMP-9 band and a very light 88-kDa activated form.
Figure 2.
 
Effects of IL-1β and JNK inhibitor 2 on MMP-3 levels and of TNFα, IL-1, and JNK inhibitor 2 on MMP-9 levels. (A) Media levels of MMP-3 after treatment of TM cells with IL-1β (50 ng/mL) with or without JNK inhibitor 2 were determined by Western immunoblot. Mean ± SEM is shown with t test significance as indicated (n = 4–7). (B) Media levels of MMP-9 were determined by Western immunoblot after 48 hours of treatment with TNFα (horizontally hatched bars), IL-1α (solid bars), IL-1β (vertically hatched bars), and JNK inhibitor 2 (JNK I II) as indicated. Mean ± SEM (n = 4–7) and t test significance for pairs above the connecting lines as indicated. Typical Western immunoblot below the graph shows the 92-kDa pro–MMP-9 band and a very light 88-kDa activated form.
Figure 3.
 
Effects of TNFα and IL-1α on JNK 1 and 2 phosphorylation. Western immunoblots of TM cell extracts at the indicated times after treatment with TNFα (A, C, E) or IL-1α (B, D, F) were probed with a phosphospecific antibody that recognized activated JNK 1 (C, D) and JNK 2 (A, B) phosphorylated on T183 and Y185. Mean, SD, and t test significance are shown (n = 9 or n = 6). Untreated controls, which had not been treated but which were incubated for each of the times indicated, were not different from the 0-minute control and thus are not shown. (E, F) Representative Western immunoblots are shown with the phosphorylated 54-kDa JNK 2 band and the 46-kDa JNK 1 bands as labeled between the blots.
Figure 3.
 
Effects of TNFα and IL-1α on JNK 1 and 2 phosphorylation. Western immunoblots of TM cell extracts at the indicated times after treatment with TNFα (A, C, E) or IL-1α (B, D, F) were probed with a phosphospecific antibody that recognized activated JNK 1 (C, D) and JNK 2 (A, B) phosphorylated on T183 and Y185. Mean, SD, and t test significance are shown (n = 9 or n = 6). Untreated controls, which had not been treated but which were incubated for each of the times indicated, were not different from the 0-minute control and thus are not shown. (E, F) Representative Western immunoblots are shown with the phosphorylated 54-kDa JNK 2 band and the 46-kDa JNK 1 bands as labeled between the blots.
Figure 4.
 
Phosphorylation of MKK4 after TNFα and IL-1α treatment. At the indicated times after treatment with TNFα (A) or IL-1α (B), TM cell extracts were subjected to Western immunoblots and probed with phosphospecific antibodies for S257/T261 of MKK4. Mean, SEM, n, and t test significances are shown at the indicated times.
Figure 4.
 
Phosphorylation of MKK4 after TNFα and IL-1α treatment. At the indicated times after treatment with TNFα (A) or IL-1α (B), TM cell extracts were subjected to Western immunoblots and probed with phosphospecific antibodies for S257/T261 of MKK4. Mean, SEM, n, and t test significances are shown at the indicated times.
Figure 5.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on phosphorylation of the transcriptional activator proteins c-Jun and ATF-2. Phosphorylation of c-Jun on S73 was assessed on Western immunoblots of TM cell extracts after treatment with TNFα (A) or IL-1α (B) for the indicated times. Mean ± SEM of relative band densities are shown for a band(s) migrating at approximately 48 kDa; t test significance and n are as indicated. The phosphorylation pattern of S63 (not shown) was similar to that of S73. The effect of pretreatment with JNK inhibitor 2 (JNK I II) on c-Jun phosphorylation measured 1 hour after treatment with TNFα (C) or IL-1α (D) is shown and t test significance (P < 0.001) is indicated above the lines linking the respective treatment pairs compared. Phosphorylation of ATF-2 on T71 is shown for the indicated treatment times with TNFα (E) or IL-1α (F). ATF-2 migrates at approximately 70 kDa. Effects of pretreatment with JNK inhibitor 2 are shown for TNFα (G) and IL-1α (H). Significance from t test comparisons is shown above the lines indicating which treatment pairs were compared.
Figure 5.
 
Effects of TNFα, IL-1α, and JNK inhibitor 2 on phosphorylation of the transcriptional activator proteins c-Jun and ATF-2. Phosphorylation of c-Jun on S73 was assessed on Western immunoblots of TM cell extracts after treatment with TNFα (A) or IL-1α (B) for the indicated times. Mean ± SEM of relative band densities are shown for a band(s) migrating at approximately 48 kDa; t test significance and n are as indicated. The phosphorylation pattern of S63 (not shown) was similar to that of S73. The effect of pretreatment with JNK inhibitor 2 (JNK I II) on c-Jun phosphorylation measured 1 hour after treatment with TNFα (C) or IL-1α (D) is shown and t test significance (P < 0.001) is indicated above the lines linking the respective treatment pairs compared. Phosphorylation of ATF-2 on T71 is shown for the indicated treatment times with TNFα (E) or IL-1α (F). ATF-2 migrates at approximately 70 kDa. Effects of pretreatment with JNK inhibitor 2 are shown for TNFα (G) and IL-1α (H). Significance from t test comparisons is shown above the lines indicating which treatment pairs were compared.
Figure 6.
 
Effects of dominant-negative JNK on MMP-3 promoter activity stimulated by TNFα or IL-1α. TM cells were cotransfected with the reporter vector without any promoter (SEAP Basic) or with the MMP-3 promoter (hMMP3p-SEAP) and with the control plasmid (pCDNA) or the dominant-negative JNK construct (dnJNK). SEAP reporter activity as measured in the media is shown in relative chemiluminescence units at the indicated times after the indicated treatments with ΤNFα (A) or IL-1α (B). Mean ± SEM for triplicate determinations from 2 separate experiments are shown (n = 6). Significance, evaluated by t test, was determined as indicated by the lines between the compared groups. *P < 0.001.
Figure 6.
 
Effects of dominant-negative JNK on MMP-3 promoter activity stimulated by TNFα or IL-1α. TM cells were cotransfected with the reporter vector without any promoter (SEAP Basic) or with the MMP-3 promoter (hMMP3p-SEAP) and with the control plasmid (pCDNA) or the dominant-negative JNK construct (dnJNK). SEAP reporter activity as measured in the media is shown in relative chemiluminescence units at the indicated times after the indicated treatments with ΤNFα (A) or IL-1α (B). Mean ± SEM for triplicate determinations from 2 separate experiments are shown (n = 6). Significance, evaluated by t test, was determined as indicated by the lines between the compared groups. *P < 0.001.
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