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Regulation of Trabecular Matrix Metalloproteinases and TIMPs
From the Casey Eye Institute, Oregon Health Sciences University, Portland.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is a strong modulator of trabecular meshwork (TM)
matrix metalloproteinase (MMP) and tissue inhibitor (TIMP) expression.
Studies were conducted to identify signal-transduction pathways
involved.
METHODS. Porcine TM cells were treated with TNF, and MMP and TIMP levels were
evaluated by zymography and Western immunoblot. Inhibitors and
activators of several signal-transduction pathways were used to select
pathways that could be involved. Trabecular protein kinase C (PKC)
isoforms were identified and localized by using Western immunoblots and
confocal immunohistochemistry. Changes in subcellular distribution of
PKC isoforms were evaluated. PKC isoform downregulation and additional
inhibition profiles were used to refine the involvement pattern of
different isoforms.
RESULTS. TNF treatment increased MMP-1, -3, and -9 and TIMP-1
expression, whereas MMP-2 expression was not affected and TIMP-2
expression decreased. Agents that modulate protein kinase A (PKA) or
inhibit phosphatidylinositol 3-kinase (PI3K) had minimal effects on
trabecular MMP or TIMP induction by TNF, whereas several agents that
modulate PKC activity were effective. Trabecular cells expressed
several PKC isoforms, which exhibited distinctive subcellular
localization. TNF treatment triggered some PKC isoform
translocations. Exposure of trabecular cells to TNF for 72 hours
differentially downregulated several PKC isoforms. Treatment with a
phorbol mitogen that stimulates most PKC isoforms produced strong
increases in these MMPs. TNF’s effects on MMP and TIMP expression
were completely blocked by only one PKC inhibitor.
CONCLUSIONS. The PKA and PI3K pathways appear not to be involved directly in
transducing this TNF signal, but at least one isoform of PKC seems
to be required. Based on the inhibitor profiles and the downregulation
and translocation studies, PKCµ appears to be critical in transducing
this signal. Unraveling the remaining steps in this and in additional
related TM signal-transduction pathways may provide targets for
developing improved glaucoma treatments.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MMPs and TIMPs are integrally involved in ECM turnover throughout the body. MMP activity is modulated by extracellular zymogen activation, by TIMP inhibition, and probably by changes in MMP protein interactions and turnover.9 10 11 Intricate and complex transcriptional regulation of MMP and TIMP expression provides an additional level of ECM turnover regulation. The 5'-promoter regions of the various MMP and TIMP genes contain a variety of simple and complex enhancer elements, and their expression is modulated by numerous growth factors, cytokines, steroids, integrin ligation, and other extracellular information and conditions.12 13 14 15 16 17 18 This regulation is mediated by signal-transduction pathways that have been identified in several specific cases and partially unraveled in a few others. Protein kinase C (PKC) involvement has been demonstrated in transcriptional regulation of the MMPs and TIMPs in several tissues,19 20 21 22 23 24 25 although PKC’s involvement may not be a universal requirement, and different isoforms have been implicated in different tissues for different regulatory processes.
A number of PKC isoforms have been identified that exhibit distinct
regulation, subcellular distribution, and translocation patterns. They
are differentially involved in diverse regulatory phenomena in various
cell types.26
27
These isoforms are grouped as
conventional (, ßI/ßII, and 
), novel (
, 
, 
, and
), and atypical (
and 
/
). The µ isoform is somewhat
unique in that it is membrane associated and does not fit completely
into any of the previous categories.28
All isoforms
require phosphatidylserine; the conventional, novel, and PKCµ
isoforms are activated by diacylglycerols or their analogue,
12-tetradecanoylphorbol-13-acetate (TPA), and the conventional isoforms
are activated by calcium.26
TPA activation of PKC is
controversial and its direct activation of PKC/ is thought not to
occur. Although there is some isoform variability, PKC activation
generally involves phosphorylation, ligand binding, proteolytic removal
of the autoinhibitory pseudosubstrate, and subcellular translocation,
often directed to the membrane by diacylglycerol and
phosphatidylserine.26
RACKs are proteins thought to target
PKC isoforms to specific subcellular structures or
substrates.29
30
31
Trabecular cells respond to treatment with a variety of growth factors
and cytokines by changing MMP and TIMP expression.32
33
TNF, IL-1, and TPA are among the most effective agents we identified in
producing these changes, after comparing a number of common
extracellular signaling molecules. The effects of laser trabeculoplasty
on trabecular MMP levels were recently shown to require mediation by
IL-1 and/or TNF.34
Thus, a study was undertaken to
identify the signal-transduction pathways involved in the TNF
modulation of trabecular MMPs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 and
-1ß were from R&D (Minneapolis, MN); TPA, 3-isobutyl-1-methylxanthine
(IBMX), dibutyryl cAMP, H-89, KT-5720, forskolin, wortmannin,
leupeptin, aprotinin, pepstatin, and fluorescein isothiocyanate
(FITC)– and horseradish peroxidase–conjugated secondary antibodies
were from Sigma (St. Louis, MO); GF 109203X (bisindolylmaleimide I or
Gö 6850), Ro 31-8220, Gö 6976, and Gö 6983 were from
CalBiochem (San Diego, CA); double-stranded DNA quantitation
reagent (PicoGreen) was from Molecular Probes (Eugene, OR);
protein kinase A, MMP, and TIMP antibodies were from Triple Point
Biologics (Portland, OR); protein kinase C isoform and RACK-1
antibodies were from Transduction Laboratories (San Diego, CA);
Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and
antimycotics were from Gibco BRL (Grand Island, NY); fetal bovine serum
was from HyClone (Logan, UT); chemiluminescence detection kits were
from NEN Life Sciences (Boston, MA); and NIH-3T3 fibroblasts were from
the American Type Culture Collection (Rockville, MD).
Cell and Organ Culture, Treatments, and Extractions
Porcine and human TM cells and NIH-3T3 fibroblasts were cultured
as previously described.35
36
For one group of studies,
stationary human anterior segment organ culture was used as previously
described.37
The cultured trabecular cells were used as
confluent monolayers at passage 3 and were maintained serum free for 48
hours before and during treatments. Except as specifically indicated,
all the data shown are from porcine TM cells. The observations in
Figures 1 4
and 5
were replicated in humans, showing no significant
species differences. Five human cell lines and more than 20 different
porcine cell lines each pooled from 20 to 40 eyes were studied.
Double-stranded DNA analysis to estimate cell density in parallel
flasks was conducted for some studies, as directed by the manufacturer.
Because the differences between flasks were always less than ±10%,
this procedure was not used in all studies. The lane-to-lane
consistency of the protein-banding patterns on Western blot analysis
(see description later), which were stained for 15 minutes (Ponceau S
stain; Sigma), destained in 5% acetic acid, rinsed, and air-dried
before probing, further verified uniform gel loading. MMP and TIMP
analysis was conducted on culture medium collected 24, 48, or 72 hours
after treatments and stored in aliquots frozen at -20°C until use.
Analysis of PKC isoforms was conducted on extracts of cells at the
times indicated. For these extractions, media were replaced with 0.5 ml
of 4°C modified RIPA buffer38
39
(2 mM EDTA, 2 mM EGTA,
1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
[SDS], 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride [PMSF], 20
µg/ml leupeptin, 20 µg/ml aprotinin, 20 µg/ml pepstatin, and 50
mM Tris, [pH 7.5]) per T-75 flask (BD Biosciences, Oxnard, CA);
flasks were immediately placed on ice. Cells were scraped from the
flasks, and the extract was sonicated, centrifuged, and frozen. For
subcellular fractionation studies, cells were extracted at the
indicated times by rinsing in 4°C phosphate-buffered saline, scraping
the cells from the flasks in translocation buffer (2 mM EDTA, 2 mM
EGTA, 50 mM NaF, 2 mM dithiothreitol [DTT], 1 mM sodium
orthovanadate, 10 mM
NaP4O7, 1 mM PMSF, 20
µg/ml leupeptin, 20 µg/ml aprotinin, 20 µg/ml pepstatin, and 30
mM Tris, [pH 7.4]) and sonicating on ice. The cytosolic fraction was
the supernatant, after centrifugation at 100,000g for 30
minutes. The pellet was then resuspended in translocation buffer
containing 0.1% Triton X-100, incubated at 4°C for 30 minutes, and
centrifuged at 100,000g to separate the membrane and the
insoluble particulate fractions.
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All experiments presented were repeated at least three times and typical gels or micrographs were selected for presentation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment treatments produced dose-dependent increases in
trabecular cell MMP-9 and -3 and TIMP-1 expression without affecting
MMP-2 levels (Fig. 1)
. TNF but not TPA, decreased TIMP-2 expression. At higher
concentrations of culture medium, modest TPA and significant TNF
induction of MMP-1 (interstitial collagenase) could be detected. These
changes were also time dependent, becoming significant by 24 hours and
reaching maximum changes by 72 hours at moderate doses (not shown).
Involvement of PKC in TPA and TNF Responses
Treatment of trabecular cells with agents that modulate PKA signal
transduction, such as dibutyryl cAMP, forskolin, KT-5720, IBMX, or H-89
and attempts to block TPA’s or TNF’s effects with these agents
produced no significant effects, although trabecular cells express the
typical PKA isoforms and subunits as detectable on Western immunoblots
(not shown). A wide range of doses, based on common literature usage
for each agent,
was added for 24, 48, or 72 hours in these studies (Table 1)
.
Treatment with wortmannin was also ineffective in changing MMP or TIMP
expression, in either the presence or absence of TPA or TNF (data
not shown). However, several relatively nonspecific PKC inhibitors,
such as staurosporine, affected trabecular MMP and TIMP expression
patterns as modulated by TNF or TPA (data not shown). These
observations, plus the strong responsiveness to TPA, prompted a more
detailed analysis of trabecular PKC.
|
, -, -/, -, -,
and -µ at relatively high levels. PKC was detectable, although at
very low levels. Only human cells in culture produced detectable
amounts of either ßI or II. Low levels of the ß isoform could be
detected in highly concentrated extracts from human or porcine cells
(data not shown). These cells also expressed high levels of the
putative PKC-anchoring protein, RACK-1 (Fig. 2A)
. All these PKC
isoforms and RACK-1 were observed at the predicted
Mr, based on observations in other tissues
reported in the literature. Some small species, and possibly
phosphorylation-dependent Mr differences, were
apparent (data not shown).
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, -, -/, and -
immunostaining was associated with filamentous cytoplasmic strands,
which was apparently the trabecular cytoskeleton. However, very
distinctive patterns were seen with each isoform. Note that the
different isoforms are shown in Figure 2
at different magnifications to
accentuate the most distinctive aspect of their distribution, with the
scale bar signifying 10 µm. PKC, -/, and - showed a
punctate scattering throughout or at the surface of the cell. PKC
staining was most intense over and around the nucleus, whereas staining
for PKC and -/ were negative in the nucleus. From optical
sections through the nucleus, it was apparent that PKC was not
actually predominantly within the nucleus (data not shown).
Immunostaining for PKC/, and to a lesser extent for PKC and
-, was apparent at the cell periphery. PKC was clearly associated
with filamentous strands, which are concentrated in some cells in a
wide zone around the nucleus. Some of this immunostaining may also be
associated with the Golgi–endoplasmic reticulum. Strong PKCµ
immunostaining was associated with what appeared to be the Golgi
apparatus in the confocal images. Punctate, probably
cell-surface–associated, PKCµ immunostaining was apparent across the
cells. Very distinctive PKCµ immunostaining also appeared within the
nucleus, apparently surrounding and within the nucleoli.
Treatment of trabecular cells with TPA or TNF did not produce simple
interpretable changes in these immunostaining patterns. Some
differences were apparent, but they were modest and quantitative rather
than qualitative or absolute (not shown). The TPA-triggered increase in
membrane-associated PKC, -, and - observed in the
translocation studies (described in the next section) was modestly
apparent in the immunostaining localization also (data not shown).
Differences in PKC Isoform Translocation Induced by TPA and TNF
Because one step in the activation and action of some PKC isoforms
involves translocation between subcellular compartments, we evaluated
the distribution of several PKC isoforms among the membrane, cytosolic,
or particulate fractions at various times after TPA or TNF
treatment. The membrane fraction at various times after treatment is
shown in Figure 3
. Trabecular cells show an unusually high proportion of all the PKC
isoforms in the particulate fraction and a very low proportion in the
cytosolic fraction. More than 90% of trabecular PKC, -, -,
-/, and - and more than 75% of trabecular PKCµ were found
in the particulate fraction, and continuous or transient cytoplasmic
levels were very low (data not shown). To eliminate the possibility
that this was a methodologic rather that a cell-type phenomenon, we
conducted parallel studies to compare trabecular cells to NIH-3T3
fibroblasts, with and without TPA and TNF treatments (data not
shown). In the fibroblasts, we found distributions similar to those
normally reported in the literature. Fibroblast PKC is still
predominantly particulate, but dramatically more is seen transiently in
the cytosolic fraction after treatments (data not shown).
|
, -, and - over time, reaching a maxima at 30
minutes and declining modestly at 60 minutes (Fig. 3)
. PKC
associated with the membrane increased by 5 minutes, reached a maximum
at 30 minutes and declined slightly by 60 minutes after TPA treatment.
TNF, by contrast, caused a modest reduction in membrane-associated
PKC, -, -, and - isoforms compared with control at early
times with a return to baseline by 30 to 60 minutes. PKC/ levels
in the membrane fraction were relatively high and appeared unchanged in
response to both TPA and TNF. The membrane fraction of PKCµ was
high in controls and remained high after either TPA or TNF
treatment. After either treatment, although more pronounced after TPA,
PKCµ became a doublet, with the appearance of a slightly
slower-migrating band reaching a maximum at 30 minutes The cytosolic
RACK-1 levels (data not shown) were several times as high as in the
membrane fraction, and neither TPA nor TNF changed this distribution
dramatically.
Downregulation of PKC Isoforms by Extended Treatments.
PKC downregulation often provides an indication of PKC involvement in a
regulatory process. Although shorter treatment times had less dramatic
effects on PKC isoform levels, by 72 hours of treatment several
isoforms were strongly downregulated (Fig. 4) . PKC, -, and - levels were almost undetectable, whereas
PKC/, -µ, or - levels are only modestly affected by TPA
treatment. TNF had modest effects on PKC, -, and -/
levels; moderate effects on PKC levels; and strong effects on PKCµ
and - levels. IL-1, another important modulator of trabecular MMP
and TIMP expression, had similar, but not identical, downregulating
effects on these isoforms.
Differential Effects of Synthetic PKC Inhibitors on TPA- and
TNF-Induced MMP and TIMP Expression.
Although a large number of PKC inhibitors have been developed and
characterized, most exhibit only limited differential effects on the
various PKC isoforms and most have limited specificity for PKCs over
other kinase families or limited cell permeability. Our initial studies
with staurosporine were suggestive, but the differential specificity of
this inhibitor for PKCs over myosin light-chain kinase was only 2-fold
and over PKA was only 10-fold, and interpretations are therefore
difficult. In addition, added without TPA or TNF, staurosporine had
effects on MMP and TIMP expression; also, at low doses, it was
synergistic with TPA (data not shown). When light-activated calphostin
C was added, it killed trabecular cells before expression changes could
be analyzed; thus, its usefulness in this study was limited. Several
third-generation PKC inhibitors have been developed with increased PKC
specificity compared with other protein kinases and that show
significant PKC isoform differential effectiveness (Table 1)
.27
42
43
44
45
46
47
48
Thus, we evaluated the effects of these inhibitors on trabecular MMP
and TIMP expression induced by TPA and TNF (Fig. 5)
. None of the inhibitors had appreciable affects on MMP or TIMP levels
in the absence of the stimulatory agents. Bis I (GF109203X), Gö
6976, and Ro 31-8220 showed dose-dependent inhibition of all the
changes in expression induced by TPA. However, differential effects
were seen with these inhibitors’ ability to block TNF’s effects on
trabecular expression of these proteins (Fig. 5)
. Bis I was unable to
block any of TNF’s effects. Gö 6976 was very potent, blocking
the TNF-induced increases in MMP-3 and -1 and TIMP-1 and the
decrease in TIMP-2. It was only partially effective in blocking MMP-9
induction by TNF. At the highest dose, approximately 10 times its
50% inhibitory concentration (IC50), Ro 31-8220
slightly reduced the MMP-9 and TIMP-1 increases and markedly reduced
MMP-3 and -1 immunostaining, without changing MMP-3 activity or the
TIMP-2 level decrease caused by TNF. To further evaluate the
possible involvement of PKC in TNF’s effect, we repeated similar
studies with another PKC inhibitor, Gö 6983, which inhibited
PKC, -ß, -, -, -, and - at 6 to 10 nM and PKCµ at 20
µM. This inhibitor was effective against TPA’s effects, but had no
effect on TNF’s effects (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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’s induction of trabecular MMPs and TIMPs appears to
require PKC and not PKA or phosphatidylinositol 3- or
4-kinases.49
50
51
Based primarily on the inhibitor studies,
TNF appeared to cause trabecular MMP-3, -9, and -1 and TIMP-1
increases with an associated TIMP-2 decrease through a
signal-transduction pathway(s) that included PKCµ as a required step.
Although no selective PKCµ inhibitor is available, the combination of
inhibitor specificities that we used have been studied in considerable
detail. Based on the combination of these specificities, PKCµ appears
to be the only PKC isoform that is required in this signal-transduction
process. One caveat to this assignment is that the inhibition profile
of PKC/ is incomplete (Table 1)
; thus, we cannot be absolutely
certain that it is not involved. Because PKC/ is in the same
family as PKC, it should share this isoform’s inhibition profile.
However, this has not been demonstrated.
The TM expressed a discrete but not particularly unique profile of PKC
isoforms. We found PKC, -, -/, -, -, -µ, and -
to be detectable in human and pig tissue and cell culture, each at the
appropriate Mr, based on data in the literature.
PKCß was barely detectable, and we did not detect other isoforms. For
reasons that are not apparent, we clearly saw several isoforms not
detected in a previous study, in which only PKC and - were
found.52
The apparent PKC anchoring protein, RACK-1, was
present at high levels in the trabecular cytosolic, membrane, and
particulate fractions.
The trabecular subcellular distribution pattern of PKC isoforms suggests that each fulfills separate trabecular functions. In general, the trabecular cell localization of PKC is more distinctive by isoform than that reported for NIH 3T3 cells,53 which may reflect the highly differentiated state of trabecular cells. Changes in localization with these treatments did not provide clear indications of which isoforms were involved. The novel PKCµ distribution is intriguing. The distinctive apparent Golgi localization that we observed for PKCµ is compatible with a prior study, which localized this isoform specifically to the Golgi compartment.54 These investigators also suggested that it may be involved in glycosaminoglycan or glycoprotein posttranslational processing or at least in basal protein transport and secretion. The MMPs and TIMPs have been shown to exhibit strong vectorial secretion in the confluent endothelial cell.55 In addition, many of the MMPs and TIMPs have glycosylated and unglycosylated forms. However, this does not seem likely to be the primary site of the critical PKCµ involvement in this specific signal-transduction process, because the ratio of glycosylated and unglycosylated MMP forms is not affected by the PKC inhibitors. Thus, the portion of trabecular PKCµ that is critically involved in transducing this signal could be the apparent Golgi-associated fraction, but seems more likely to be the punctate fraction that is apparently dispersed on the cell surface. The apparent nucleolar PKCµ immunostaining was not observed in previous studies.54 This apparent nucleolar immunostaining was reproducible and specific for this antibody, but it could very well be an artifact. We have not attempted the difficult studies necessary to further clarify this point.
When the subcellular distribution of PKCµ after TNF or TPA
treatment was evaluated, a significant bandshift was observed (Fig. 3)
.
Presumably, this transient upper band reflects a posttranslational
modification, probably a phosphorylation. This could reflect an
activation of this isoform and is probably of significance in the
regulatory process. Multiple phosphorylation sites have been shown to
be important in modulating this isoform’s activity.56
The different translocation patterns of PKC isoforms observed in signal
transduction by TPA and TNF is intriguing. To the extent that
isoform translocation provides information about isoform utilization,
TPA could be using PKC, -, -, - and/or -µ to induce
trabecular MMP and TIMP changes. Although TNF produced a very small
shift of several PKC isoforms away from the membrane fraction at early
times, this may or may not reflect functional effects. If the shift was
to a particulate position, this small change was not detectable on the
very large background level in trabecular cells.
The PKC isoform downregulation is also of interest. The implication,
commonly accepted in the literature, is that a PKC isoform that is
downregulated by extended treatment with an agent is probably actively
involved in some aspect of signal transduction by this agent. These
downregulation studies provide support for a PKCµ step in the
process. However, it can be assumed that multiple PKC isoforms can be
involved in several trabecular processes triggered by TPA or TNF,
whether or not they are required for this MMP-TIMP effect. It is
interesting that the time required to achieve trabecular PKC isoform
downregulation was longer than that observed in many other cells,
suggesting a slower protein turnover rate.
Because carefully regulated increases in trabecular ECM turnover by these MMPs would increase outflow facility,1 studies further unraveling the steps in this and in other pathways involved in this process may allow the development of improved therapies for glaucoma.
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Acknowledgements |
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Footnotes |
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Submitted for publication February 20, 2001; revised July 13, 2001; accepted July 20, 2001.
Commercial relationships policy: N.
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: Ted S. Acott, Casey Eye Institute (CERES), Oregon Health Sciences University, 3375 SW Terwilliger, Portland, OR 97201. acott{at}ohsu.edu
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PKC plays a critical role during stromelysin promoter activation by platelet-derived growth factor through a novel palindromic element J Biol Chem 269,10044-10049 Invest Ophthalmol Vis Sci 41,422-430, novel PKC- and PKC-, but not atypical PKC- are MARCKS kinases in intact NIH 3T3 fibroblasts J Biol Chem 272,4072-4078 as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes J Biol Chem 272,30075-30082
by various inhibitors: differentiation from protein kinase c isoenzymes FEBS Lett 392,77-80[Medline][Order article via Infotrieve]
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J. P. Alexander and T. S. Acott Involvement of the Erk-MAP Kinase Pathway in TNF{alpha} Regulation of Trabecular Matrix Metalloproteinases and TIMPs Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 164 - 169. [Abstract] [Full Text] [PDF]
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T. L. Anthony, J. D. Lindsey, and R. N. Weinreb Latanoprost's Effects on TIMP-1 and TIMP-2 Expression in Human Ciliary Muscle Cells Invest. Ophthalmol. Vis. Sci., December 1, 2002; 43(12): 3705 - 3711. [Abstract] [Full Text] [PDF]
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