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Department of Endocrinology, Royal Prince Alfred Hospital (S.V.M., V.M., D.K.Y., S.M.T.), and Discipline of Medicine (S.V.M., X.Y.W., D.K.Y., S.M.T.), University of Sydney, Sydney, New South Wales 2006, Australia
Address all correspondence and requests for reprints to: Dr. S. McLennan, Department of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia. E-mail: sue{at}med.usyd.edu.au.
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Abstract |
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Introduction |
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The enzymes largely responsible for ECM degradation are the matrix metalloproteinases (MMPs). They comprise a large family of some 25 structurally related metal ion-dependent enzymes that are secreted (collagenases, gelatinases, and stromelysins) or membrane-bound, or membrane-type (MT)-MMPs. They are classified according to their substrate specificity: the collagenases (e.g. MMP-1), which cleave native helical collagens type I, II, and III; the stromelysins (e.g. MMP-3), which have broad substrate specificity and degrade fibronectin; and laminin and the gelatinases (e.g. MMP-2 and MMP-9), which degrade collagen IV (7). Another type are the cell membrane-bound forms of MMPs of which MT1-MMP is the best characterized. MT1-MMP has been shown to be responsible for activation of MMP-2 on the cell membrane (8). Because of the importance of these enzymes in the maintenance of ECM balance, their expression and activities are tightly regulated both at the level of transcription and posttranslationally via the actions of specific inhibitors, the tissue inhibitors of MMPs (TIMPs). Four TIMPs have been identified (TIMPs 1–4). Each of these inhibitors can inhibit the active forms of the MMPs by binding to the active site of the enzyme (9). TIMP-2 has an additional role in the activation of MMP-2 (10), whereas TIMP-3 has the unique property of being bound to the ECM (11).
In vivo and in vitro studies have shown that high glucose concentration can alter the expression of some of the MMPs and increase the expression of TIMPs, in particular TIMP-1 (4, 5, 12). Moreover, using a biosynthetically labeled matrix substrate we previously showed that high glucose concentration decreases the matrix-degrading ability of mesangial cells (6). The cellular mechanisms by which high glucose inhibits the activities of the MMPs in these cells are not well understood.
The early response gene of the CCN family termed connective tissue growth factor (CTGF), induces extracellular matrix protein production, including types of collagens, fibronectin, and laminin (13, 14, 15, 16). Up-regulation of CTGF in vivo commonly occurs in pathological states characterized by ECM deposition and fibrosis (17, 18, 19, 20, 21, 22), including human diabetic nephropathy (23) and experimental models of renal diabetes in rodents (24, 25). In vitro, CTGF is up-regulated in mesangial cells in response to high glucose concentration (24, 25). Moreover, CTGF is positively regulated by TGF-ß, and it induces fibronectin and collagen type IV in mesangial cells (15, 24, 25), supporting a role for this growth factor in increased ECM synthesis in the glomerulus, including in diabetes. In contrast to these well documented effects of CTGF on ECM formation, CTGF regulation of matrix degradation in normal glucose conditions, and under the high glucose conditions that occur in diabetes has not been reported, nor has any protease pathway involved in possible CTGF effects on matrix degradation been defined.
We hypothesized that CTGF inhibits matrix degradation, that CTGF is a mediator in the effect of high glucose to inhibit matrix degradation, and that this mechanism is through regulation of MMP activities by CTGF. Studies were designed to investigate the role of CTGF on ECM degradation under normal and high glucose conditions in human mesangial cells. The MMPs and TIMPs involved in the observed effects of CTGF on matrix degradation were determined. We focused on MMP-2, its cell membrane-bound activator MT1-MMP, and TIMP-1, -2, and -3 in this study, because MMP-2 degrades type IV collagen, which is known to accumulate in the mesangium in diabetic nephropathy, TIMP-1 and -2, -3 play important roles in the regulation of MMP-2. In the case of TIMP-2, this is by the inhibition of activation of MT1-MMP, and MMP-2 and TIMP-1 and -3 can inhibit the active form of MMP-2. Because CTGF was shown to regulate TIMP-1 and thus matrix degradation in vitro, the relationship between glomerular CTGF, TIMP-1, and matrix degradation were investigated in vivo in a rodent model of diabetes.
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Materials and Methods |
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Animal studies
Diabetes was induced in 12 Wistar Firth rats by the iv injection of streptozocin (60 mg/kg). Another six animals were used as nondiabetic controls. One week after induction of diabetes all diabetic rats received 2 U/d long-acting insulin (Ultratard, Novo-Nordisk, Malmo, Sweden), known as maintenance insulin regimen, to maintain body weight and prevent ketoacidosis. To determine the effect of more intensive insulin treatment, six of the diabetic animals were randomly selected and allocated to receive twice-daily insulin injections (2 U actrapid and 4 U monotard) in addition to the Ultratard.
After 8 wk, rats were anesthetized with pentobarbital and the kidneys removed. The cortex was dissected from one kidney from each animal and stored at –80 C until use. Blood obtained at death was used for determination of HbA1c using the Bio-Rad DC protein assay (Bio-Rad, New South Wales, Australia). All aspects of the experiment were approved by the Animal Ethics Committee of the University of Sydney, Australia.
Synthesis and purification of rhCTGF
Recombinant human (rh)CTGF protein was produced using a recombinant human adenoviral expression system, AdEasy as provided by Prof. Vogelstein (26). The CTGF 1047-bp cDNA open reading frame was cloned from an Hs578T human breast cancer cDNA library and sequenced. The resulting fragment coding for full-length nontagged human CTGF was subcloned into BamHI and Kpn1 sites of the AdEasy vector, and insert presence and orientation were verified by DNA sequencing. Recombinant adenovirus stocks were isolated and produced in increasing titer, as recommended. The rhCTGF protein was then produced by infecting human 911 cells with recombinant virus under serum-free conditions. To purify rhCTGF protein from filtered media, heparin-Sepharose affinity chromatography, using HiTrap columns (Pharmacia, Uppsala, Sweden) with a step-up salt gradient in elution, was used (15, 24). Bands observed in eluted fractions by Coomassie Brilliant Blue-stained gels were confirmed to be rhCTGF by Western blotting using anti-CTGF antibody, and peak fractions containing rhCTGF were as previously described (27, 28).
Polyclonal anti-CTGF antiserum (8800) was generated in New Zealand White rabbits, using full-length recombinant human CTGF as the immunogen, as previously described (29). This antibody has been shown to fully inhibit the induction of fibronectin by rhCTGF added to human fibroblasts (29).
Effect of glucose on CTGF expression
Confluent mesangial cells were cultured in RPMI medium containing 0.1% BSA, in the presence of either 5 or 25 mM glucose for 72 h. Cells cultured in a mixture of 5 mM glucose and 20 mM mannitol were used as an osmotic control. After 72 h, the media were collected and the cells washed with PBS before extraction of RNA with Tri Reagent (Sigma). The expression of CTGF was measured by quantitative real-time RT-PCR as previously described (30).
Effect of CTGF cell degradative capacity
Mesangial cells were cultured in serum-free RPMI in the presence of rhCTGF (0–1000 ng/ml). After 72 h, the media were collected for study of MMP activities, and the cells were washed with cold PBS before extraction of RNA for measurement of MMP and TIMP gene expression, as described in detail below.
The total degradative capacity of mesangial cell-conditioned medium was determined using a biosynthetically prepared mesangium matrix substrate as previously described (6). This method enables the analysis of medium MMP activities in the presence of the TIMPs. Briefly, conditioned medium obtained from mesangial cells cultured in the presence of rhCTGF was incubated with the radiolabeled matrix substrate for 24 h. The degradative capacity was determined by counting the radioactivity released to the culture media, and results were expressed as a percentage of the total count incorporated into the matrix. Using specific inhibitors, we have previously shown that the majority of degradative capacity measured by this assay is because of the action of MMPs (5).
To address a potential role for CTGF in the inhibition of matrix degradation by high glucose, mesangial cells were cultured in the presence of either 5 or 25 mM glucose, each with 30 µg/ml of an anti-CTGF antibody (known to neutralize rhCTGF (500 ng/ml) (30), or with 30 µg/ml of control IgG. For each experiment, the anti-CTGF antibody or control IgG was added 30 min before addition of either 25 mM glucose or the rhCTGF. After another 72 h of cell culture, the conditioned medium was collected for degradation studies and the RNA was extracted from the cells for measurement of gene expression of the MMPs and TIMPs.
Effect of CTGF on MMP and TIMP gene expression
Cells were cultured in the presence of rhCTGF (0–1000 ng/ml) as described. After 72 h, the cells were washed with cold PBS and the RNA isolated using TRI Reagent (Sigma). RNA was transcribed to cDNA using oligo dT (10 pmol) (Invitrogen, Carlsbad, CA) and Superscript II RNase H– (Invitrogen). At least three independent experiments were carried out to generate data for each figure. For each experiment, the treatment conditions were tested in triplicate wells of cells, and these samples were analyzed in duplicate by quantitative, real-time RT-PCR. The expression of MMPs, TIMPs, and fibronectin (31) were determined by quantitative real-time RT-PCR using SYBR green fluorophore. Briefly, all amplicons were amplified using Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and 20 pmol of each forward and reverse primer (Table 1
) and the following PCR conditions: for MMP-2, MT1-MMP, TIMP-2, and TIMP-3 (1 µl first-strand cDNA), 50 C for 2 min and 95 C for 5 min, followed by 45 cycles of 95 C for 15 sec, 58 C for 30 sec, and 72 C for 30 sec; and for TIMP-1, fibronectin, and ß-actin (0.5 µl first-strand cDNA), 50 C for 2 min and 95 C for 5 min, followed by 45 cycles of 95 C for 15 sec, 60 C for 30 sec, and 72 C for 30sec. The expression of the housekeeping gene ß-actin was also measured as control for loading and RT efficiency. The primers used generated a single appropriately sized cDNA band on DNA agarose gels. Negative control RT-PCRs were performed by omitting either the reverse transcriptase enzyme or the RNA from the reaction mixture. All negative controls failed to produce PCR amplicons (data not shown). All samples for analysis generated cycle thresholds that were on the linear part of the serial dilution curves, and an equivalent gradient of dilution curves with the housekeeper was confirmed in each case. The interassay coefficient of variation for real-time runs was maximally 8.2 ± 3.1%. Results were expressed using the delta-delta method as a ratio corrected for abundance of the housekeeping gene ß-actin (31).
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TIMP-1 and TIMP-2 protein levels in the cell-conditioned media were determined by ELISA (Chemicon, Boronia, Australia) according to the manufacturer’s instructions. These assays measure the MMP/TIMP complexes as well as free TIMP-1 or TIMP-2, respectively. TIMPs in the conditioned media were also determined by reverse zymography, using standard methods (34).
Effect of anti-TIMP-1 antibody on degradative capacity
To evaluate the role of TIMP-1 in matrix degradation caused by high glucose and rhCTGF, cells were cultured in serum-free RPMI media in the presence of either 25 mM glucose or rhCTGF (0–1000 ng/ml) and an anti-TIMP-1 antibody (20 µg/ml total IgG, clone 147-6D11, ICN Biomedicals) or control IgG (20 µg/ml). After 72 h, the medium was removed and its degradative capacity measured.
Role of CTGF in TGF-ß-mediated effects
Mesangial cells were cultured in serum-free RPMI containing 5 mM glucose and TGF-ß (10 ng/ml) alone or in combination with 30 µg/ml of the anti-CTGF antibody or with 30 µg/ml of control IgG. After 72 h, the medium was removed for measurement of degradative capacity and the RNA extracted from the cells for determination of TIMP-1 and TIMP-3 mRNA.
Effect of rodent diabetes on glomerular CTGF, TIMP-1, and degradative capacity
To investigate the regulation of CTGF, TIMP-1, and matrix degradation in diabetes, the effect of more intensive insulin treatment on the expression of CTGF and TIMP-1 in rat kidney cortex, compared with cortex from maintenance insulin treatment and nondiabetic control animals, was studied. Cortical sections (100 mg) of rat kidney were homogenized in cold PBS containing 20% SDS, and the protein concentration was determined. The capacity of the isolated tissue to degrade matrix was measured using a biosynthetically prepared radiolabeled matrix as substrate, as described. RNA was extracted from an additional 100 mg of cortical tissue and used for analysis of CTGF and TIMP-1 mRNA by semiquantitative RT-PCR. ß-actin was used as a housekeeping gene (33).
Statistics
Cell culture experiments were performed independently at least three times, in triplicate, and the results expressed as mean ± SD. All data were compared using ANOVA followed by post hoc comparisons using Duncan’s multiple range test. Statistical significance was accepted at the P < 0.05 level.
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Results |
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). This increase was not observed when mannitol was used as an osmotic control. That elevated glucose is known to inhibit matrix degradation by mesangial cells (6) was initially verified in this system. High glucose inhibited matrix degradation compared with conditioned media derived from cells treated with normal glucose (Fig. 1C). To then examine the role of endogenous CTGF in this process, anti-CTGF antibody was added to cells grown in the presence of 25 mM glucose. The ability of high glucose treatment to inhibit mesangial cell degradative capacity was partially prevented (73.2 ± 7.8%) by the addition of CTGF-neutralizing antibody (Fig. 1C). Higher concentrations of CTGF antibody showed no further ability to reverse high glucose effects (not shown). In contrast to effects seen with high glucose, endogenous CTGF was not involved in any matrix degradation occurring in the presence of normal glucose (Fig. 1C).
Effect of CTGF on mesangial cell expression of MMPs and TIMPs
Because initial results showed that CTGF plays a role in regulation of matrix degradation, cells were then treated with rhCTGF, and gene and protein expression of mesangial cell MMP-2, MT1-MMP, and TIMP-1, -2, and -3 were examined. Addition of rhCTGF resulted in a dose-dependent increase in the gene expression of TIMP-1 and TIMP-3 but had no effect on TIMP-2 (Fig. 2A). By reverse zymography (Fig. 2B), rhCTGF increased the media concentration of TIMP-1 and had no effect on media TIMP-2. No band was detected at the expected molecular size for TIMP-3, indicating that TIMP-3 was not detectably secreted into the media. Similar to these gene expression and protein profiles, analysis of media TIMP-1 and TIMP-2 levels by ELISA showed that TIMP-1 was increased and no change occurred in TIMP-2 (Fig. 2, C and D, respectively).
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). A similar pattern was also seen when the total MMP-2 protein level (inset) was determined by Western analysis (i) or by zymography (ii). In contrast, rhCTGF had no clear effect on the expression of MT1-MMP, the membrane-bound activator of MMP-2, at either the gene (Fig. 3B) or protein level (inset).
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) but had no effect on TIMP-2 (Fig. 4B). Addition of anti-CTGF antibody prevented the increase in TIMP-1 gene expression induced by high glucose (Fig. 4, A and D) and had no effect on TIMP-2 gene expression (Fig. 4B). Consistent with the mRNA findings, the CTGF-neutralizing antibody blocked the induction of TIMP-1 protein in the media by high glucose (Fig. 4D) but had no effect on TIMP-2 (Fig. 4E). As a positive antibody control, the induction of fibronectin by rhCTGF was prevented by the anti-CTGF antibody (Fig. 4F). Glucose is known to inhibit the gene expression of MT1-MMP (35); however, the addition of anti-CTGF antibody did not prevent the decrease in MT1-MMP gene expression by high glucose (Fig. 4C), a result consistent with our observed lack of effect for any regulation of MT1-MMP by rhCTGF.
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). The ability of high glucose treatment to inhibit degradative capacity was partially prevented by the addition of TIMP-1-neutralizing antibody by 74.3 ± 9.8% compared with control IgG (Fig. 5B).
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). Coincubation of mesangial cells with both CTGF-neutralizing antibody and TGF-ß inhibited this effect of TGF-ß by 69.1 ± 7.3% (Fig. 6A). In parallel, whereas the CTGF antibody alone had no effect on basal TIMP-1 or mRNA levels at 5 mM glucose, it inhibited the induction of TIMP-1 by TGF-ß (Fig. 6B). These data show that CTGF mediates effects of TGF-ß on matrix degradation and expression of TIMP-1.
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. Diabetes was associated with an increase in HbA1c and kidney weight (P < 0.001) and a decrease in body weight (P < 0.05). More intensive insulin treatment attenuated the increase in HbA1c and kidney weight, and reduction in body weight, compared with values in control animals. Similar to our in vitro studies with high glucose, diabetes increased the gene expression of both TIMP-1 and CTGF (Fig. 7 for representative data and Table 2 for group quantitated data),and decreased matrix degradative capacity of the glomerular tissue (Table 2) compared with control animals. More intensive insulin treatment in diabetes resulted in changes in CTGF, TIMP-1, and matrix degradation toward normal (Table 2).
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Discussion |
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CTGF protein induces the gene expression and protein levels of many ECM components in vitro and in vivo (36), and it also regulates vascular neogenesis (39, 40), an essential requirement for the development of fibrosis. We have recently shown that CTGF is up-regulated by advanced glycation end-products (AGEs) in human renal mesangial cells (30) and that CTGF is a mediator in the induction of fibronectin by AGEs in cultured human fibroblasts (16), and others have shown that CTGF contributes to the induction of collagens by high glucose in renal fibroblasts (41). Combined with the evidence that CTGF is increased in the diabetic kidney (24, 25), these data collectively implicate CTGF in contributing to the increase in ECM that occurs in the diabetic kidney.
The current report now shows that CTGF may contribute to matrix accumulation not only through ECM formation but, via the MMP system, also through the inhibition of matrix degradation. Whereas TIMP-1 and TIMP-3 are induced in a dose-dependent and progressive manner by rhCTGF, MMP-2 mRNA and protein are increased in a biphasic pattern. This profile of MMP-2 increase by rhCTGF has also been observed in vascular smooth muscle cells (42). That the effect of CTGF in the current study to inhibit matrix degradation occurred despite increased expression of the protease MMP-2 is consistent with the known role of TIMP-1 and -3 to inhibit MMP-2 activity. In contrast to effects seen on TIMP-1 and -3 and MMP-2 by CTGF, CTGF does not appear to be involved in the high-glucose-induced down-regulation of MT1-MMP expression (35). This lack of effect of CTGF on MT1-MMP indicates that CTGF is a mediator in only some of the effects of high glucose on the MMP system in mesangial cells.
The decrease in MMP activation and increase in TIMPs is central to the effects of high glucose on matrix degradation in renal cells and in diabetes. Similar to the work of others and our previously published studies (4, 5), we have shown in this work that exposure of human mesangial cells to high glucose concentration increases the expression of TIMP-1. The addition of a TIMP-1 neutralizing antibody to the conditioned media demonstrated that CTGF works entirely though TIMP-1 to inhibit matrix degradation in this system. The effect of high glucose on matrix degradation was only partially dependent on both endogenous CTGF and TIMP-1.
In addition to its effect on TIMP-1 we have also shown that CTGF increases the gene expression of TIMP-3. By reverse zymography and despite increased gene expression of TIMP-3, there was no detectable TIMP-3 in the mesangial cell-conditioned media. TIMP-3 is unique among the TIMPs in that it binds avidly to ECM (11). Because TIMP-3 was not detected in the medium it would not be expected to play a role in the high-glucose- or CTGF-induced decrease in matrix degradation in this model system, further supporting the notion that in this system the CTGF effect is mediated by TIMP-1. That we cannot measure effects of regulated TIMP-3 in our matrix degradative studies is a limitation of the method used. Whether the CTGF-induced increase in TIMP-3 can alter ECM degradation at the cell surface or in the pericellular environment remains to be more fully elucidated.
Collectively, these findings suggest that CTGF is one mediator of high glucose effects on matrix degradation and that the CTGF mechanism of effect is dependent on TIMP-1. Whether other growth factor and protease systems independent of CTGF and TIMP-1 are also involved in regulation of ECM degradation by high glucose remains to be determined. Although advanced glycation on matrix that occurs in diabetes is known to inhibit matrix degradation (32), the studies in the current work used preformed matrix from mesangial cells cultured under normal glucose conditions to assess matrix degradative capacity, and thus AGEs would not be present to any degree in the matrix studied.
The concentration of rhCTGF required to detect a biological effect in this study is not dissimilar to other published bioactivities of CTGF. RhCTGF has been shown to vary in its activity, from 10 ng/ml to 5 µg/ml (30, 43), depending on the rhCTGF preparation and the cellular end-point studied. The current work shows that effects on matrix degradation were observed at the lowest concentration (62.5 ng/ml) of rhCTGF studied, in a dose-responsive manner. That a neutralizing antibody directed against CTGF was also used provides additional evidence that cell-derived CTGF plays an important role in mediating high glucose effects on matrix degradation.
Other than CTGF, factors that may contribute to the regulation of TIMP-1 by elevated glucose include TGF-ß1. It is recognized that TGF-ß, which is increased in response to high glucose concentration, can up-regulate TIMP-1 expression (9, 44). The known relationship between TGF-ß1 and CTGF is complex; TGF-ß1 is a potent inducer of CTGF (13, 45), and CTGF can mediate or magnify the bioactivity of TGF-ß1 in a variety of cell systems (36, 46, 47). In addition, CTGF has recently been shown to potentiate the bioactivity of TGF-ß1 by facilitating the presentation of TGF-ß1 to the type 2 TGF-ß1 receptor (47). The inhibitory effect of TGF-ß1 on matrix degradative capacity and the induction of TIMP-1 by TGF-ß1 are mediated by endogenous CTGF in this system, suggesting that CTGF contributes to the effect of both high glucose and TGF-ß1.
Other workers have described a role for CTGF in regulation of TIMP-1, -2, and -3 in other cell systems (48, 49). That CTGF regulates TIMP-1 in renal mesangial cells and that this mechanism explains an inhibition of matrix degradation by CTGF have potential applications to other conditions characterized by fibrosis, particularly other forms of chronic renal impairment occurring secondary to glomerular sclerosis or fibrosis (50). CTGF itself may also be regulated by the MMP system (51), and proteolysis of CTGF by MMPs can affect CTGF bioactivity (51), adding another level of complexity to feedback systems involved in ECM degradation. Carboxyl-terminal fragments of CTGF generated by MMP activity, like the intact protein, also have effects on ECM formation (51). We have observed regulation of CTGF at the mRNA level by high glucose, and it will be of interest to determine in future work whether the altered MMP activity present in a high glucose environment regulates CTGF at the protein level and its bioactivity.
The relevance of the in vitro findings in this work to diabetes is supported by our in vivo findings in rodent diabetes. That the increased CTGF and TIMP-1 and reduced matrix degradative capacity seen in renal diabetes are prevented by more intensive insulin treatment causing lesser hyperglycemia is consistent with the concept that CTGF regulates matrix degradation through TIMP-1, not only in vitro by high glucose but also in diabetes. To definitively prove such a causal effect in diabetes will require CTGF-neutralizing studies in diabetes, which have not yet been reported by any research group.
This study shows that CTGF is a mediator in the inhibition of ECM degradation by high glucose and that this mechanism occurs in this system through the regulation of TIMP-1. This work now requires that in future studies, the MMP system is viewed as a target of CTGF and that the role of CTGF in ECM accumulation in diabetes is expanded to include degradation of ECM.
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Acknowledgments |
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Footnotes |
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Abbreviations: AGE, Advanced glycation end-product; CTGF, connective tissue growth factor; ECM, extracellular matrix; MMP, matrix metalloproteinase; MT, membrane-type; rh, recombinant human; TIMP, tissue inhibitor of MMP.
Received April 6, 2004.
Accepted for publication August 25, 2004.
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