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EXTRACELLULAR MATRIX, CELL INTERACTIONS
1Division of Cardiology, Department of Medicine, Medical University of South Carolina, 2Ralph H. Johnson Department of Veterans Affairs Medical Center, and 3Division of Cardiothoracic Surgery, Department of Surgery, Medical University of South Carolina, Charleston, South Carolina
Submitted 24 October 2006 ; accepted in final form 28 July 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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heart failure; extracellular matrix; proteases
The MMPs constitute a diverse set of degradative enzymes that have been stratified into subclasses based upon commonalities in generalized structure and proteolytic portfolio. These subclasses include the collagenases, the gelatinases, stromelysins/matrilysins, and the membrane-type MMPs (MT-MMPs). Of more recent interest is the subclass of MT-MMPs, of which MT1-MMP is the most commonly studied (37, 25, 46, 12). As the name implies, MT1-MMP is a transmembrane protein with a diversity of biological functions that include 1) degradation of a spectrum of matrix structural proteins, 2) proteolytic processing of biologically active molecules such as growth factors and cytokines, and 3) activation of other MMPs (25, 30, 13, 18, 2). A significant increase in the myocardial levels of MT1-MMP has been identified in DCM patients, and the relative magnitude of this increase was greater than that of any other MMP subclass (41). These increased myocardial levels of MT1-MMP likely contribute to changes in matrix structure and function with DCM. However, the molecular basis for the upregulation of MT1-MMP in the context of DCM remains unknown. MT1-MMP is proteolytically active once inserted into the cell membrane, and, therefore, transcription and translation are important molecular steps in the formation of constitutively active MT1-MMP (50, 28, 43, 22, 35, 29). The central hypothesis of this study was that the increased MT1-MMP protein levels observed within the myocardium of DCM patients would be recapitulated in myocardial fibroblast cultures and that contributory molecular mechanisms for the persistent increase in MT1-MMP would be alterations in indices of transcription and translation. Accordingly, the objectives of this study were fourfold. First, we sought to establish primary cultures of myocardial fibroblasts from LV biopsies taken from DCM patients and patients with normal LV function. Second, we sought to compare MT1-MMP steady-state mRNA, protein levels, and subcellular localization in reference normal and DCM myocardial fibroblast cultures. Third, we sought to measure an index of MT1-MMP translational efficiency in both DCM and reference normal myocardial fibroblast cultures. Fourth, we sought to examine the effects of a biological stimulus that may cause alterations in transcriptional activity, such as that of MT1-MMP (1, 22, 33, 40). Specifically, reference normal and DCM fibroblasts were exposed to the cytokine tumor necrosis factor-
(TNF) and determinants of MT1-MMP transcription and translation examined.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Myocardial fibroblast cultures. Primary cultures of myocardial fibroblasts were established using a previously described outgrowth technique (5, 8, 42). Briefly, the LV myocardial samples were minced under a sterile laminar flow hood, transferred to cell culture flasks (75 cm2, Falcon), and allowed to adhere. Sterile growth medium was added to the flasks, and the cells were incubated under standard cell culture conditions (37°C; 21% O2, 5% CO2). Culture media consisted of fibroblast growth medium (FGM) from Promocell (no. C23010 [GenBank] ; Heidelberg, Germany), 20% fetal bovine serum (FBS), and Promocell Supplement Mixture (no. C39315 [GenBank] ) containing amphotericin B, basic fibroblast growth factor, gentamicin, and insulin. After a 2-wk incubation period, myocardial fibroblasts migrated from the initial myocardial plugs and grew to confluency. At this point, the cells were scraped and transferred to 0.2% gelatin-coated (Sigma no. G-2500) tissue culture flasks (150 cm2, Falcon) containing the supplemented FGM and grown to confluency. These initial confluent cultures were then split and passages maintained under identical culture conditions until the studies described below were initiated.
Experimental protocol. For all of the studies, confluent cultures from passages 2–5 were used. The confluent cultures were trypsinized and washed with PBS and were transferred to laminin-coated flasks (25-cm2, Sigma no. L-6274) at a cell density of 1 x 105 cells. In the first set of measurements, alternating passages were used to measure steady-state MT1-MMP mRNA or protein levels. Before collection of the fibroblasts for study, the cultures were maintained under serum-free conditions. The cultures were initially incubated for 24 h, in serum-free media consisting of FGM with 0.1% bovine serum albumin (BSA, Gibco no. 11021-029), 1% insulin-transferrin-selenium-A (ITS, Gibco no. 51300-044), and 1% penicillin/streptomycin/Fungizone (PSF, from BioWhittaker no. 17-745E). The cell cultures were then briefly washed with PBS and incubated for an additional 2 or 24 h with serum-free media (FGM, 0.1% BSA, 1% ITS, and 1% PSF). Fibroblasts were then harvested for steady-state measurements MT1-MMP mRNA and protein.
In the next set of experiments, fibroblast cultures were treated in identical fashion as above, but cultures from identical passages were incubated with or without TNF (Sigma T-6674 human recombinant TNF, 100 ng/ml) for the second serum-free incubation period. This concentration of TNF was chosen based upon previous fibroblast studies (40). For the final set of studies, fibroblasts were prepared in identical fashion (steady-state conditions and TNF stimulation) and then subjected to subcellular fractionation of ribosomes and polysomes, as well as total MT1-MMP mRNA measurements.
To determine whether cell number and/or viability were different between primary cell cultures, between passages, or with TNF treatment, cell number was computed, and a mitochondrial viability assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.1 mg/ml, Sigma)] was performed (5). Fibroblast MTT activity was unchanged over the serum-free incubation periods and in the presence or absence of TNF. Therefore, fibroblast number and viability remained constant during the serum-free incubation conditions, between passages, and with TNF treatment. Cell number and/or viability were also not different when age was used as a covariate in the analysis using the approaches described in the subsequent data analysis section. The time to fibroblast confluence from the initial biopsy placement was 23 ± 3 days, and this culture growth time was similar between reference normal and DCM myocardial specimens (24 ± 7 vs. 22 ± 3 days, respectively). The time to confluence for subsequent passages 2–5 was 14 ± 2 days and was identical between reference normal and DCM cultures.
Cell cultures were confirmed to be pure fibroblasts by immunochemistry using antisera against a panel of antibodies and were positive for the discoidin domain receptor 2 (DDR2) and proly-4-hydroxylase but were negative for Factor VIII (5, 42, 8). In addition, immunoblotting for this panel of antisera was performed routinely on cell passages to ensure phenotypic stability with respect to these fibroblast markers. The immunochemistry and immunoblotting approaches are described in detail in a subsequent section.
Steady-state mRNA levels. Steady-state mRNA levels were determined by one of two methods. In the first method, RNA was extracted from the cell pellets by using the TriZol (Invitrogen, Carlsbad, CA) method (14). Equal volumes of each sample were then aliquoted for one-step RT-PCR analysis. The Qiagen SYBR Green Quantitect RT-PCR kit (Valencia, CA) and gene-specific primers were used to complete real-time RT-PCR on a Bio-Rad iCycler (170-8720XTU; Bio-Rad, Hercules, CA). Following reverse transcription, 35 cycles of PCR were performed with product amount being optically captured during each cycle. A melt curve was produced to verify single product formation, and all primer sets were optimized before use. The primer sequences for human MT1-MMP and GAPDH were as follows: MT1-MMP, 439F 5'-CAATTGGCAGCCTCTCACTAC/2590R 5'-TGACTGAGCAACGAAGACC-3'; GAPDH, 354F 5'-AGGTCATCCCAGAGCTGAAC-3'/491R 5'-CCTGCTTCACCACCTTCTTG-3'.
In the second method, RNA was extracted from cell pellets using the Qiagen RNeasy Kit. RNA samples were reverse-transcribed to generate cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA was amplified with gene-specific TaqMan primer/probe (MT1-MMP, no. Hs00237119_m1; GAPDH, no. Hs99999905_m1; MMP2, no. Hs0023422_m1; c-Jun, no. Hs00277190_s1; Applied Biosystems, Foster City, CA) sets using the TaqMan Universal PCR Master Mix (no. 4364321, Applied Biosystems) on a Bio-Rad iCycler (170-8720XTU, Bio-Rad). Forty cycles of PCR were performed with product amount being optically captured during each cycle.
Irrespective of method, after the RT-PCR was complete, the threshold cycle was chosen based on the log of the baseline-subtracted values. All of the samples were within the linear range of product formation. The relative mRNA amount of a specific gene was normalized to GAPDH mRNA.
MT1-MMP protein abundance. The relative abundance of MT1-MMP was examined in harvested cell pellets. Cell pellets were first lysed using ice-cold MMP extraction buffer (10 mM cacodylic acid, 150 mM NaCl, 0.01 mM ZnCl2, 20 mM CaCl2, 2 mM NaN3, and 0.1% Triton X-100, pH 5.0). Cell pellet lysates (5.0 µg total protein; no. 23227; Pierce, Rockford, IL) were then electrophoresed, transferred, and immunoblotted as previously described (7, 51). Briefly, the membrane was incubated with an MT1-MMP antibody (0.2 µg/ml; no. AB815; Chemicon, Temecula, CA), washed and then incubated with a secondary antibody (1:5,000; Vector Laboratories, Burlingame, CA) conjugated with horseradish peroxidase. Signals were detected by chemiluminescence (Western Lightning; Perkin Elmer, Boston, MA), digitized, and analyzed (Gel Pro Analyzer; Media Cybernetics, Silver Spring, MD). Positive controls were included on each immunoblot using a purified MT1-MMP recombinant protein (Chemicon, CC1043). While these immunoblotting procedures were performed using identical protein concentrations, additional controls for loading conditions were performed by reprobing these membranes for GAPDH (1:5,000 dilution; MAB374, Chemicon).
MT1-MMP ribosomal distribution. The fibroblasts were dounce-homogenized in an ice-cold buffer containing 10 mM Tris, pH 7.5, 250 mM KCl, 10 mM MgCl2, 0.5% Triton X-100, 2 mM DTT, 100 µg/ml cycloheximide, and 2 µl RNAsin (Promega, Madison, WI). Tween-deoxycholate (20%) was added to the cell homogenate, incubated for 15 min, and then centrifuged (12,000 g) to produce a postmitochondrial supernatant, which was then layered onto a 15–50% sucrose gradient containing 20 mM Tris, pH 7.5, 250 mM KCl, 10 mM MgCl2, and 80 units of RNAsin. The samples were then subjected to ultracentrifugation at (100,000 g/100 min), and gradients were fractionated on an ISCO density gradient fractionator (ISCO, Lincoln, NB) yielding eight fractions of equal volumes (1.2 ml). The first four fractions of the gradient separation contained monosomes that include free 40S and 60S ribosome subunits, plus mRNAs assembled into messenger ribonucleoprotein (mRNP) (35). The next four gradient fractions contained polysomes, which consist of mRNAs that are active in translation. The RNA was extracted from each fraction and MT1-MMP and GAPDH mRNA levels determined by RT-PCR as described in the preceding section. For these studies, two different approaches were taken. In the first approach, the absolute amounts of MT1-MMP and GAPDH mRNA undergoing active translation (i.e., the polysomal fractions) were examined. For this approach, the first half of the polysomal gradient (fractions 5 and 6) was considered the light fraction and the second half (fractions 7 and 8) considered the heavy fraction. This categorical approach accounted for small shifts in the resolution of adjacent polysome fractions, following sucrose gradient centrifugation within each experiment. Using this approach, the absolute amounts of MT1-MMP mRNA distributed on the polysomal fractions could be compared between the reference normal and DCM cultures. For the second approach, the entire distribution of MT1-MMP mRNA across both monosomal and polysomal fractions was examined. For both approaches, total translational capacity and ribosomal abundance were taken into account by normalizing the individual mRNAs of interest to the 18S ribosomal RNA (rRNA) subunit using the following primer sequence: 110F 5'-TATGGTTCCTTTGGTCGCTC/240R 5'-GTTGGTTTTGATCTGATCTGATAAAT-3'.
Total protein synthesis rates. To examine whether and to what degree global changes in translational capacity occurred within DCM fibroblasts, total protein synthesis rates were computed in reference normal (n = 3) and DCM cultures (n = 3) using a radioactive pulse-label approach described in detail previously (14). The cultures were prepared as described in the preceding section and stabilized in serum-free media for 24 h. Following which, medium containing 10 µCi/ml of [3H]phenylalanine (TRK648; Amersham Biosciences) at a final concentration of 0.4 mM phenylalanine was substituted, and the cells were incubated under routine culture conditions for 4 h. The cells were then scraped in 1 ml of buffer (10 mM Tris, pH 7.5, 250 nM KCl, 0.5% Triton X-100) and dounce homogenized. Total protein was precipitated by adding HClO4 (0.5 nM) and then centrifuged, and the resultant cell pellet washed twice with HClO4 and finally resuspended in a 0.5 N NaOH solution. Radioactivity (dpm, LS6000; Beckman Coulter, Fullerton, CA) and total protein concentration were measured and the specific radioactivity (dpm/mg) determined. Specific radioactivity of [3H]phenylalanine of the conditioned medium was also determined (dpm/nmol). Total protein synthesis rates (nmol phenylalanine·mg protein–1·h–1) were calculated by dividing the specific radioactivity of the cell sample by the specific radioactivity of the culture media. The [3H]phenylalanine-labeling experiments were performed in duplicate for each independent cell culture, and the total protein synthesis rates were computed in triplicate from each labeling experiment. These values were averaged to achieve a final protein synthesis rate for each independent normal reference and DCM fibroblast culture.
MT1-MMP proteolytic processing. An important proteolytic substrate for MT1-MMP is the proform of MMP-2 (28, 37, 46). Specifically, pro-MMP-2 complexes with MT1-MMP at the cell surface and, through a cooperative process, results in cleavage of pro-MMP-2 (72 kDa) to the active form of MMP-2 (64 kDa) (28, 37, 46). Since pro-MMP-2 is synthesized and released into the extracellular space, the relative abundance of the proform and active form of MMP-2 was examined in conditioned media from normal reference control and DCM fibroblasts, using substrate-specific gelatin zymography and immunoblotting (41, 47). For these studies, conditioned medium was concentrated by using Centriplus centrifugal filtering devices at 4°C (Millipore, Bedford, MA). Protein concentration was determined by using a standardized colorimetric assay based on the Bradford method (Bio-Rad). Electrophoresis and zymography were performed as described previously (8, 16, 21). An equivalent amount of protein was loaded for these experiments in which the average total protein loaded for each sample was 2.2 ± 0.2 µg with no difference in the total protein loaded between reference normal and DCM media samples. Proteolytic activity appeared as clear lytic bands against a dark blue background. Since this approach utilizes detergents and electrophoretic separation, denaturing of the proform of MMP-2 occurs and exposes the catalytic domain. Accordingly, proteolytic zones corresponding to the proform (72 kDa) and the active form (64 kDa) were visualized and digitized (8, 16, 21). To further confirm and validate the identification of the high and low molecular weights of MMP-2, immunoblotting of the conditioned medium was performed. Conditioned media samples of equivalent protein concentrations were subjected to electrophoresis and membrane transfer as described previously. The membrane was incubated with MMP-2 antisera (IM33, Calbiochem, CA; 0.4 µg/ml), washed, and visualized as described in the previous paragraph.
Immunofluorescence and confocal microscopy. To examine the relative content and distribution of MT1-MMP in the myocardial fibroblast cultures, immunohistochemistry and confocal microscopy were performed (8, 51). An aliquot of fibroblasts from each passage were placed on plated on glass slides (Lab-Tek II; Nalge Nunc Int, Naperville, IL) and incubated for 24 h in serum-free FGM. The cells were then fixed for 30 min with 3.7% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100 for 10 min. All steps were performed at room temperature. The fibroblasts were first incubated for 60 min with blocking serum containing 3% BSA, washed, and then incubated with the primary MT1-MMP antisera (1:100, 2 h). After a washing step was completed, the cells were incubated for 30 min with a conjugated detection antibody (1:500, Molecular Probes, Alexa Fluor 488 no. A-11008). The cells were washed again and then incubated with a DNA probe for 10 min (1:1,000, Molecular Probes, To-Pro-3 no. T3605). In additional fibroblast preparations, costaining of the actin cytoskeleton was performed using phalloidin (Alexa Fluor 546 phalloidin, Molecular Probes no. A12380). Briefly, following the steps above, the fibroblasts were incubated for 20 min with a 1:40 dilution of phalloidin and then washed. The stained cultures were then coverslipped and images obtained on a Zeiss LSM 510 Meta Confocal microscope using Plan-Neofluar x40, 1.3 oil DIC objective. Negative controls for the immunostaining procedure included substitution of the primary antisera with nonimmune serum.
Data analysis. For these studies, initial evaluations were performed in which the main treatment effect was the presence and absence of DCM, and the second treatment effect was cell passage, by 2-way analysis of variance (ANOVA). However, results from both control and LV failure myocardial fibroblast cultures revealed that all biological response variables (viability, growth rates, mRNA levels, MT1-MMP protein levels, and ribosomal fractionation) were similar between passages, i.e., there was no significant passage effect. Thus the statistical modeling was compressed to a single main treatment effect. The results from each passage and each isolation were pooled to obtain one value for each response variable. Thus, although multiple observations were performed on each independent culture, and on different passages, these multiple observations were not used for the purposes of sample sizes and statistical power. The sample sizes for this study were based upon patient number, i.e., five DCM preparations and six reference normal control preparations. For the immunoblotting results, the DCM fibroblast results were computed as a percentage of control values. Since the all-response variables were summarized as 2 group comparisons, an unpaired t-test was then performed. For comparisons between steady-state values and TNF-treated values, a one-way ANOVA was performed. For the ribosomal MT1-MMP fractionation studies, an ANOVA using a split-plot design was performed. Post-ANOVA pair-wise comparisons were performed using Bonferroni adjusted t-test. All of these approaches were performed using a statistical package (STATA; Statacorp, College Station, TX). Results are presented as means ± SE. Values of P < 0.05 were considered significant.
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RESULTS |
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LV fibroblast MT1-MMP translational efficiency. MT1-MMP translational efficiency was examined following a 24-h incubation period under steady-state conditions or following a 24-h exposure to TNF. The absolute values for MT1-MMP and GAPDH mRNA distribution on ribosomal fractions undergoing active translation (polysomes) were first examined. Since the polysomal fractions were collected from a gradient, the first half of the polysomal fraction would represent the lower mass fraction (Light), and the second half would represent the heavier mass fraction (Heavy). The absolute values for MT1-MMP and GAPDH contained on these Light and Heavy polysomal fractions are shown in Fig. 3. In normal reference fibroblast cultures, the steady-state distribution of MT1-MMP mRNA was primarily located on the Heavy polysomal fraction. In the DCM fibroblast cultures, the absolute amounts of MT1-MMP mRNA located on the Heavy polysomal fraction were significantly reduced. TNF exposure for 24 h did not significantly alter the absolute content or distribution of MT1-MMP mRNA from steady-state values in either normal reference or DCM fibroblast cultures. In contrast to MT1-MMP, GAPDH mRNA was equally distributed between the Light and Heavy polysomal fractions (Fig. 3). The absolute amounts and distribution of GAPDH mRNA were equivalent between reference normal and DCM fibroblast cultures. Exposure to TNF for 24 h did not significantly alter the content or distribution of GAPDH mRNA in these fibroblast culture systems. The distribution of MT1-MMP in ribosomal fractions, as a function of monosomes to polysomes, for both normal reference control and DCM fibroblast cultures is shown in Fig. 4. The distribution of MT1-MMP mRNA in reference normal fibroblasts was predominantly in polysomal fractions. Exposure to TNF for 24 h did not result in a substantial shift in the relative distribution of MT1-MMP mRNA in ribosomal fractions in reference normal fibroblast cultures. Overall, the relative content of MT1-MMP mRNA in ribosomal fractions was significantly reduced when considered as a composite value (Fig. 4, P < 0.05). The distribution of MT1-MMP mRNA in monosome fractions decreased by over twofold in DCM fibroblasts, compared with reference normal values (P < 0.05), and remained lower in polysomal fractions. For example, in polysomal fraction 6, the relative distribution of MT1-MMP mRNA was 15.7 ± 5.2% in reference normal fibroblasts and 1.4 ± 0.6% in DCM fibroblasts (P < 0.05). With TNF exposure, a noticeable shift in relative MT1-MMP mRNA in polysomal fraction 8 was observed in DCM fibroblasts, and the overall mean value fell outside the 95% confidence interval. However, when this TNF value was subjected to pair-wise comparisons with steady-state polysomal fraction 8 in DCM fibroblasts, this did not reach statistical significance (P = 0.25).
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DISCUSSION |
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MT1-MMP proteolytic activity. The MT-MMPs, notably MT1-MMP, proteolytically process a wide portfolio of matrix proteins, enzymes, and biologically active molecules (2, 13, 18, 30). For example, MT1-MMP has been demonstrated to be a fundamental mechanism for activation of soluble proforms of MMPs, such as the gelatinase MMP-2 (25, 28, 46). It has also been demonstrated that MT1-MMP can proteolytically process membrane-bound cytokines to a soluble, active form such as TNF (30). Thus, MT1-MMP not only degrades collagens, basement membrane components, and proteoglycans, but also can induce a localized proteolytic and biological signaling cascade that would alter myocardial structure and function. To examine whether and to what degree the increased MT1-MMP protein observed in DCM fibroblasts was proteolytically competent, the concentration of the proform and active form of MMP-2 was determined. A robust amount of total MMP-2 was released into the cell culture media of both reference normal and DCM fibroblasts, which is consistent with past cultured myocardial fibroblast studies from this laboratory and others (5, 8, 40). However, while the total amount of MMP-2 released into the culture media over a 24-h period appeared equivalent between the normal and DCM fibroblast cultures, a significantly greater amount of the lower molecular weight form of MMP-2, consistent with the activated form of MMP-2, was observed in the DCM fibroblast cultures. Since MT1-MMP is a critical pathway for proteolytic processing of MMP-2, these results would then suggest that the increased MT1-MMP protein in the DCM fibroblast cultures was proteolytically competent and translated to greater overall proteolytic activity at the fibroblast cell surface.
MT1-MMP transcriptional processes.
The present study demonstrated that steady-state mRNA levels of MT1-MMP were increased in DCM myocardial fibroblast cultures. Although this is the first study to report these observations in human myocardial fibroblasts, persistent changes in mRNA and protein expression patterns in fibroblasts obtained from the myocardium of animal models of cardiac disease states are not without precedent (5, 8, 9, 27, 39, 42, 48). For example, differences in the portfolio of genes, as identified by differential display, were identified in cardiac fibroblasts cultured following the induction of volume overload in rabbits (48). The underlying basis for the persistently increased expression of MT1-MMP mRNA and protein in human cardiomyopathic fibroblasts remains unclear, but likely contributory mechanisms include changes in mRNA promoter activity, mRNA stability, and overall MT1-MMP protein stability and turnover. It has been demonstrated previously that growth factors such as transforming growth factor-
can modify MMP mRNA stability (6, 31, 43). In human heart failure, such as cardiomyopathic disease, increased and prolonged neurohormonal activity and the release of biologically active molecules occurs, which in turn may induce prolonged changes in MMP mRNA post-transcriptional modification. Another potential mechanism for the elevated levels of total MT1-MMP mRNA in DCM fibroblast cultures would be change in transcriptional activity. In general, the MMP promoter regions contain a large number of response elements, and, therefore, transcriptional activity would be affected by a large number of signal transduction pathways (50, 22). The MT1-MMP promoter region is unique from other MMP types in that it contains a number of distinct response elements (1, 22). Past studies have demonstrated a selective induction of MMPs in cardiomyopathic disease (20, 21, 41, 47, 49). Furthermore, a specific biological stimulus does not induce an equivalent effect on MMP mRNA levels in vitro (42, 8, 40). Moreover, the relative increase in MT1-MMP mRNA in DCM fibroblasts was not due to a global increase in transcriptional activity, as steady-state levels of GAPDH were equivalent between reference normal and DCM fibroblasts. Thus, although future studies are required, the increased MT1-MMP mRNA levels in DCM fibroblasts may be due to increased promoter activity.
MT1-MMP translational processes. To determine if this increased MT1-MMP abundance in DCM fibroblasts may have been due to alterations in translational efficiency, ribosomal fractionation studies were performed. The results from these sets of studies were rather unexpected. First, the total amount of MT1-MMP mRNA-bound polysomes, indicative of translational efficiency, was unchanged in DCM fibroblast cultures compared with reference normal values. These results would suggest that the increased MT1-MMP protein levels that were observed in the DCM fibroblast cultures in the present study, as well as the increased levels observed in past in vivo studies (41, 51), were not likely due to increased translational activity or changes in efficiency. Rather, these results would imply that changes in MT1-MMP transcriptional and posttranslational processes occur in the context of cardiomyopathic disease. Specifically, the findings from the present study suggest that recruitment of MT1-MMP mRNA into polysomes is compromised in DCM fibroblasts, but MT1-MMP mRNA abundance increased. If a larger pool of mRNA is available for translational initiation, then increased protein synthesis would occur by virtue of a mass effect that would facilitate recruitment into polysomes. The present study demonstrated that overall protein synthesis rates were equivalent between reference normal and DCM fibroblasts. These observations would further support the hypothesis that the increased MT1-MMP protein levels in DCM fibroblasts are not due to an augmentation of translational processes, but rather primarily one of increased MT1-MMP transcriptional and potentially posttranslational processes.
MT1-MMP posttranslational processing.
MT1-MMP is a fully active enzyme once inserted into the cell membrane and contains an extracellular catalytic domain, a transmembrane domain, and an intracellular domain, all of which are critical for full functioning of the protease (10, 19, 25, 29, 32, 34, 36, 44). Thus posttranslational modification is an important event in processing MT1-MMP to a mature enzyme and for trafficking to the membrane. It has been demonstrated that trans-Golgi processing and intracellular activational steps play a critical role in processing the mature, full-length MT1-MMP (10, 29, 44). The present study measured the mature, full-length (
64 kDa) MT1-MMP by immunoblotting of whole cell extracts. Immunofluorescent localization studies demonstrated MT1-MMP in both the cytosol and membrane compartments of both reference normal and DCM fibroblasts. Moreover, the relative abundance of MT1-MMP within the cytosolic compartment appeared increased in DCM fibroblasts. Using an interstitial microdialysis technique, this laboratory has identified that MT1-MMP activity is increased following ischemia and reperfusion, which was associated with increased trafficking to the cell membrane (7). In a study by Remacle and colleagues (35), it was demonstrated that posttranslational modification of MT1-MMP, specifically O-glycosylation, altered MT1-MMP protein stability through a reduction in autocatalysis. In another study by Remacle et al. (34), it was demonstrated that MT1-MMP could be internalized from the cell surface by both clathrin and clathrin-independent pathways, thus providing a pool of recruitable MT1-MMP. Thus a number of posttranslational events can alter the stability and turnover of MT1-MMP, and it is likely that these posttranslational processes contributed to the persistently elevated protein levels of MT1-MMP measured in DCM fibroblast cultures.
MT1-MMP and TNF exposure. In the present study, myocardial fibroblasts were exposed to TNF for a 2- or 24-h period. These studies were not intended to be a response study with respect to time of exposure or different TNF levels. Instead, an established concentration of TNF was utilized over standardized exposure periods in serum-free conditions (40). The present study demonstrated that TNF exposure for 2 h caused a significant induction of c-Jun, in both reference normal and DCM fibroblast cultures. The induction of this particular transcription factor is consistent with activation of the TNF receptor pathway and demonstrates that the fibroblast cultures under study were responsive to this cytokine stimulus.
These set of studies demonstrated that the relative mRNA and protein MT1-MMP levels were not increased in the reference normal or DCM myocardial fibroblasts following TNF exposure for 2 h. In contrast, although mRNA levels appeared unchanged, total MT1-MMP protein levels were increased significantly in DCM fibroblasts following a 24-h TNF exposure. In the present study design, the relative prolonged period of TNF exposure and measurement at 24 h may have precluded identifying any early induction of MT1-MMP mRNA in these fibroblast cultures. For example, it has been demonstrated previously that endothelial cell cultures exposed to TNF caused a peak level of MT1-MMP mRNA at 6 h and a return to near basal levels by 24 h (33). Moreover, this past study demonstrated a detectable increase in MT1-MMP protein levels by 24 h of TNF incubation. In the present study, TNF exposure did not appear to significantly shift MT1-MMP translational efficiency in reference normal fibroblasts, but a trend for increased efficiency was observed in DCM fibroblasts. Nevertheless, the most likely explanation for the increased MT1-MMP protein levels in the DCM fibroblasts is that TNF caused an amplification of intracellular events that would increase MT1-MMP protein stability. Whether this robust and differential response in DCM fibroblasts is unique to TNF, or is a generalized response to a number of biological stimuli, warrant further study.
Summary. In 1994, Sato et al. (37) first described a membrane-bound MMP in a tumor cell line that degraded matrix proteins as well as proteolytically processed putative pro-MMPs, such as MMP-2, to an active form. Additional studies have demonstrated that MT1-MMP is an important determinant for local matrix degradation and cell invasion (2, 12, 13, 18, 25, 28, 30, 46). It is now recognized that the portfolio of substrates for MT-MMPs, such as MT1-MMP, are the most diverse of the MMP family (2, 13, 18, 30). Transgenic studies suggest that MT1-MMP serves critical biological functions since MT1-MMP null mice display significant skeletal dysplasia and connective tissue disorders, culminating in early mortality (12). However, basic and clinical reports have demonstrated a significant increase in myocardial MT1-MMP following myocardial infarction and in cardiomyopathic disease (7, 39, 41, 44, 51). The present study moved beyond these in vivo observational studies and directly demonstrated that MT1-MMP protein levels were increased in myocardial fibroblasts obtained from patients with DCM, compared with reference normal myocardial fibroblasts, irrespective of passage number. The outcomes from these studies suggest that myocardial fibroblasts harvested and expanded in culture from patients with end-stage heart failure demonstrate persistent alterations in signaling, transcriptional and posttranslational pathways requisite for MT1-MMP processing. These studies likely provide a human myocardial cellular platform for identifying molecular signals and pathways that cause persistent abnormalities in matrix degradative pathways in cardiomyopathic disease.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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