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Am J Physiol Renal Physiol 293: F157-F165, 2007. First published March 20, 2007; doi:10.1152/ajprenal.00508.2006
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TNF-{alpha}, but not IFN-{gamma}, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis

Laurinda A. Cooker,1 Darryl Peterson,1 Joann Rambow,1 Melisa L. Riser,1 Rebecca E. Riser,1 Feridoon Najmabadi,1 David Brigstock,2 and Bruce L. Riser1,3

1Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois; 2Center for Cell and Vascular Biology, Children's Research Institute, Columbus, Ohio; and 3Baxter Healthcare, Renal Division, McGaw Park, Illinois

Submitted 19 December 2006 ; accepted in final form 13 March 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Connective tissue growth factor (CCN2) is a profibrotic factor acting downstream and independently of TGF-beta to mediate renal fibrosis. Although inflammation is often involved in the initiation and/or progression of fibrosis, the role of inflammatory cytokines in regulation of glomerular CCN2 expression, cellular proliferation, and extracellular matrix accumulation is unknown. We studied two such cytokines, TNF-{alpha} and IFN-{gamma}, for their effects on cultured mesangial cells in the presence or absence of TGF-beta, as a model for progressive renal fibrosis. Short-term treatment with TNF-{alpha}, like TGF-beta, significantly increased secreted CCN2 per cell, but unlike TGF-beta inhibited cellular replication. TNF-{alpha} combined with TGF-beta further increased CCN2 secretion and mRNA levels and reduced proliferation. Surprisingly, however, TNF-{alpha} treatment decreased baseline collagen type I protein and mRNA levels and largely blocked their stimulation by TGF-beta. Long-term treatment with TGF-beta or TNF-{alpha} alone no longer increased CCN2 protein levels. However, the combination synergistically increased CCN2. IFN-{gamma} had no effect on either CCN2 or collagen activity and produced a mild inhibition of TGF-beta-induced collagen only at a high concentration (500 U/ml). In summary, we report a strong positive regulatory role for TNF-{alpha}, but not IFN-{gamma}, in CCN2 production and secretion, including that driven by TGF-beta. The stimulation of CCN2 release by TNF-{alpha}, unlike TGF-beta, is independent of cellular proliferation and not linked to increased collagen type I accumulation. This suggests that the paradigm of TGF-beta-driven CCN2 with subsequent collagen production may be overridden by an as yet undefined inhibitory mechanism acting either directly or indirectly on matrix metabolism.

inflammatory cytokines; chronic kidney disease; pathophysiology of renal disease

GLOMERULOSCLEROSIS IS THE hallmark of progressive renal failure, regardless of the underlying disease, and is found in ~90% of end-stage renal disease (ESRD). Sustained imbalances of cytokines contribute to the development of the glomerulosclerosis and the tubulointerstitial fibrosis that typify the failed kidney. Transforming growth factor-beta (TGF-beta) is a key cytokine in progressive fibrosis/sclerosis in the kidney and other organs (30, 49, 50). Although important in the normal process of wound healing, most likely including acute renal injury, a sustained or chronic elevated activity of TGF-beta in the diseased kidney acts to produce an imbalance in extracellular matrix (ECM) protein metabolism, leading to the abnormal accumulation of these proteins with progressive sclerosis and eventual kidney failure. Altered mesangial cell proliferation is also thought to be a factor influencing progression in at least some forms of acute and chronic renal disease, including some forms of glomerular nephritis and diabetic glomerulosclerosis.

Connective tissue growth factor (CCN2) is a member of the CCN family of proteins. This family, named for the first three members reported (cyr61, CTGF, and nov), consists of six members in humans that share a multimodular domain structure. Collectively, CCN proteins are involved in a wide range of biological processes, including, in some cells, proliferation, adhesion, apoptosis, chemotaxis, angiogenesis, and ECM formation (6, 39). CCN2 was first identified in 1991 (5) and, in addition to roles in embryonic development, is a key component in fibrosis, and likely wound healing, in a variety of cell types (33). It is strongly responsive to TGF-beta in fibroblasts (21), hepatic stellate cells (18), and kidney mesangial cells (MC) (46), and it appears to be a downstream mediator of many known TGF-beta functions (15, 21). However, under certain conditions, CCN2 activity is also stimulated by mechanisms other than TGF-beta and can thus mediate fibrotic effects in vitro in the absence of TGF-beta (17). In fact, evidence suggests that CCN2 is a critical mediator of fibrotic processes in both in vitro and in vivo models of kidney disease (46), as well as in clinical renal disease (26). In the unilateral ureteral obstruction model of renal fibrosis in the rat, blocking the activity of CCN2 by antisense oligodeoxynucleotides reduced the induction of CCN2 and, in turn, ECM genes and their corresponding proteins in these obstructed kidneys (52). CCN2 antisense oligodeoxynucleotides also significantly blocked CCN2 expression and renal interstitial fibrogenesis in the remnant kidney of subtotally nephrectomized mice transgenic for TGF-beta, despite sustained expression of TGF-beta (36).

In addition to TGF-beta and CCN2, other cytokines play a role in glomerulosclerosis. Inflammation is an early event in the sclerotic process in kidney tissues and results in the recruitment of macrophages. These infiltrating cells produce a variety of cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}) andinterferon-{gamma} (INF-{gamma}) (8), which may in turn affect the expression of profibrotic cytokines and/or ECM components. Serum levels of TNF-{alpha} are elevated in patients with chronic kidney failure, even before initiation of dialysis, and range from 20 to 50 pg/ml, generally increasing with declining renal function (14), and are also elevated in patients with diabetes (34). TNF-{alpha} is a mediator of the acute phase reaction of the early inflammatory response. In the kidney, it contributes to the chronic inflammation that often precedes interstitial matrix deposition and is also implicated in obstruction-induced renal injury (35). ANG II production has been reported to upregulate the expression of TNF-{alpha} (29), and increased serum TNF-{alpha} in aged mice has been associated with increased glomerular inflammation (54). TNF-{alpha} activity was also increased in the serum of patients suffering from systemic sclerosis (scleroderma) (48) and was found to suppress the induction of CCN2 by TGF-beta in fibroblasts derived from scleroderma lesions (2). In addition, CCN2 expression was reduced in bovine aortic epithelial cells, fibroblasts, and vascular smooth muscle cells exposed to TNF-{alpha} (31). Thus it is possible that TNF-{alpha}, like TGF-beta, modulates the expression of CCN2 in the kidney, as has been reported in other cell types, although whether as a promoter or an antagonist of CCN2 is not known.

IFN-{gamma} is another cytokine that may be important in the fibrotic processes that lead to kidney failure, perhaps as an antagonist to the actions of TGF-beta and CCN2. IFN-{gamma} indeed acts antagonistically to TGF-beta in human skin fibroblasts and lung fibroblasts (16, 19). Treatment with IFN-{gamma} appeared to inhibit renal fibrosis induced by subtotal nephrectomy in rats (37), whereas a similar treatment was reported to reduce levels of tgf-beta and ccn2 mRNA in lung tissue of nine patients with idiopathic pulmonary fibrosis (55). However, in a larger study IFN-{gamma} treatment was not associated with clinically significant improvements in patient outcomes (42). The specific effect of INF-{gamma} on TGF-beta-induced fibrosis in MC does not appear to have been studied.

Prevention of fibrosis in progressive kidney disease may be a matter of artificially readjusting the balance of cytokines and growth factors that contribute to the fibrotic process. Blocking TGF-beta may have potential for effectively diminishing fibrosis (3, 25) but could also lead to systemic problems due to a reduction in 1) normal wound healing capabilities, 2) tumor surveillance, and 3) ability to downregulate the T-cell overresponse. CCN2, on the other hand, may provide a more attractive target for therapeutic intervention. Blocking CCN2 can reduce the severity of fibrosis in experimental models (36, 52, 56), but it is important to understand the effects of the many cytokines present in the milieu of the fibrotic kidney on the production and control of CCN2 and ECM. Toward that end, we investigated the effects of the proinflammatory cytokines TNF-{alpha} and IFN-{gamma}, alone or in combination with TGF-beta, on CCN2 and type I collagen expression in cultured MC.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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Reagents. Tissue culture reagents (media and components, Nuserum, and FCS) were from Collaborative Research (Bedford, MA). TGF-beta, TNF-{alpha}, and IFN-{gamma} were from R&D Systems (Minneapolis, MN). Purified rat collagen type I was from Upstate Biotechnology (Lake Placid, NY), and polyclonal anti-rat collagen type I from Chemicon International (Temecula, CA). The production of full-length recombinant human CCN2 protein in a baculovirus expression system has been described previously (46).

Cell culture. MC were from a cloned line (16KC2) derived from Fischer rat glomeruli (44). MC were grown long term in RPMI 1640 containing penicillin and streptomycin and 5 mM glucose, plus 20% Nuserum. For experiments, cells were seeded in normal growth medium at 12,500 cells/ml, 0.6 ml/well in 24-well tissue culture plates, and then were grown for 3 days. On day 4, cells were washed with serum-free RPMI and then incubated in RPMI + 0.5% FCS. Twenty-four hours later, the cells were again washed and exposed to RPMI (0.5% FCS) with added cytokines as indicated in the figures. Cells incubated in RPMI (0.5% FCS) served as the unstimulated control. After 24 h, all cells were washed in serum-free RPMI and then incubated in RPMI (0.5% FCS) for 2 additional days (3-day total group) and harvested on day 3. For longer exposures, cells were stimulated, washed, and incubated as above, and on day 3 were washed again, and the cytokine exposure repeated again for 72 h, under the same conditions as the initial stimulation. In this case, on day 6 following the first stimulation (3 days after the second treatment), the cells were again washed and harvested (6-day total group). In all cases, heparin (0.3 µg/well) was added to all wells 18 h before harvest to induce the release of cell- or matrix-bound CCN2 into the media as previously demonstrated (46).

At the end of the incubation periods, cells and media were harvested. Media were collected on ice and centrifuged at 2,000 rpm for 10 min at 4°C and then transferred to storage tubes. Aprotinin (10 µg/ml) and PMSF (1 mM) were added, and samples were stored at –70°C. Cells were washed once with serum-free RPMI and then dispersed by trypsin and harvested using standard techniques. Finally, an aliquot of the harvested cells was removed and counted in a Coulter counter.

For Northern blot analysis, MC were seeded at 40,000 cells/ml in RPMI plus 20% Nuserum, 3.0 ml/well, in six-well tissue culture plates. Cells were grown for 4 days, with one change of media 1 day after seeding. On day 5, cells were washed with serum-free RPMI and then incubated in RPMI (0.5% FCS). Twenty-four hours later, the cells were again washed and stimulated in RPMI (0.5% FCS) containing TGF-beta (5 ng/ml), TNF-{alpha} (1 or 10 nM), or TGF-beta (5 ng/ml) with TNF-{alpha} (0.1 to 10 mM). Cells incubated in RPMI (0.5% FCS) served as the unstimulated control. Cells were incubated under these conditions for 48 h and then harvested for isolation of RNA.

Measurement of CCN2 and collagen protein levels. An indirect ELISA method was used to quantify CCN2 and collagen type I levels in the collected media, similar to that as previously reported (46). In brief, for CCN2, the wells were first coated with a polyclonal, affinity-purified goat anti-CCN2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Following a blockade of unbound sites and extensive washing, the sample or human recombinant standard (in the same medium as the sample) was added and incubated at room temperature to allow binding to the immobilized antibody. After further being washed, a polyclonal anti-CCN2 rabbit antibody produced in our laboratory and directed against the 20-kDa COOH-terminal portion of CCN2 was added (4). After further washing, horseradish peroxidase-conjugated antibody (Jackson Laboratories, West Grove, PA, Cat. Number 111-035-003) was added, followed by substrate (TMB-ELISA, Life Technologies). The reaction was stopped and the color intensity was read at 450 nm using a microplate reader (Thermo Max, by Molecular Devices, Sunnydale, CA). The ELISA method for measurement of secreted collagen type I by ELISA has been described previously in detail (45).

Probes. The ccn2 cDNA probe for Northern analysis was from a sequence of the human ccn2 that is conserved in rat and mouse (46). The collagen probe was a 1.5-kb cDNA of human pro{alpha}1(I) (23). Probes were labeled with 32P by random hexamer priming using the Sigma Prime-1 kit (Sigma, St. Louis, MO).

RNA isolation and Northern blot. Cells were pulverized in a liquid nitrogen-cooled stainless steel mortar and homogenized in 1.0 ml RNA Stat-60 reagent (Tel-Test, Friendswood, TX). RNA samples (20 µg) were denatured in glyoxal/dimethylsulfoxide at 55°C for 1 h. Electrophoresis in 10 mM sodium phosphate/1% agarose gels was followed by staining with ethidium bromide. Stained gels were photographed and blotted onto Genescreen membranes (New England Nuclear Research, DuPont, Boston, MA) using standard methods (47). Membranes were probed for ccn2 or collagen. Autoradiograms of the probed membranes were scanned on a Scanmaster 3+ Densitometer (Howtek, Hudson, NH) and the images were analyzed with NIH Image, version 1.59 (Twilight Clone BBS, Silver Spring, MD).

Statistical analysis. In each experiment, the averages of four replicates per treatment group were calculated and compared by single-factor ANOVA, using Microsoft Excel 97 SR-1 for Windows. If ANOVA returned a P value <0.05, a post hoc Tukey test determined significant differences, with 95% confidence, among the different treatment groups.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of TNF-{alpha} on CCN2 protein and mRNA levels. We previously observed that the stimulatory effect of TGF-beta on collagen secretion by MC required 72 h for maximal effect. However, the removal of TGF-beta after the first 24 h did not alter the final stimulation of collagen production (unpublished observations, Riser BL), indicating that the signaling event(s), as measured by the collagen response (72 h later), is (are) accomplished within the first 24 h. In the present experiments, we began with this same protocol to examine the effect of TNF-{alpha} on CCN2 expression. As expected, a single 24-h exposure to TGF-beta (5 ng/ml) stimulated the production of CCN2 protein per cell with levels twofold greater than those in untreated controls, measured at 72 h after the initiation of treatment (Fig. 1A), and the replacement of TGF-beta with TNF-{alpha} alone (1 nM) produced a similar twofold increase in CCN2 levels. When cells were then exposed to increasing concentrations (0.1 to 10 nM) of TNF-{alpha} in combination with TGF-beta (fixed at 5 ng/ml), the level of secreted CCN2 protein per cell rose about eightfold over that of the unstimulated control cultures. To determine whether these effects were linked to cell proliferation, we also determined the cell number at the termination of the experiment, i.e., after 3 days of treatment. TGF-beta treatment had no significant effect on proliferation, whereas TNF-{alpha} produced quite a strong inhibitory effect (Fig. 1B). This inhibitory effect of TNF-{alpha} was not significantly altered when TGF-beta was also present. Thus TNF-{alpha} greatly inhibited proliferation while stimulating the CCN production/release per cell, whereas TGF-beta did not significantly change proliferation but did stimulate CCN2 secretion. Combined, the inhibitory effect of TNF-{alpha} on cell growth predominated, and there was a synergistic effect on CCN2 release. A detailed examination of viability by trypan blue exclusion method demonstrated that the observed reduction in cell numbers induced by cytokine exposure was due to an inhibition of proliferation and not an induction of cell death (data not shown).


Figure 1
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  		Fig. 1. Effect of TNF-{alpha} on TGF-beta-induced connective tissue growth factor (CCN2) protein secretion and cellular proliferation after short-term exposure to cytokines. The y-axes: CCN2 protein levels (ng/106 cells; A) and number of cells (x106; B) for each treatment group; all results are means ± SE. The TGF-beta concentration was 5 ng/ml in all cases. Results reflect a 48-h collection following the initial 24-h stimulation period. *Significant difference compared with untreated cells. **Significant difference compared with TGF-beta-treated cells. ***Significant difference compared with TNF-{alpha}-treated cells.

 
To explore the effects of long-term exposure, the cytokine treatment was repeated at day 3, and the response was observed an additional 3 days later. Under these conditions, TGF-beta mildly (but significantly) stimulated MC proliferation (Fig. 2B) but surprisingly no longer increased CCN2 release per cell (Fig. 2A). TNF-{alpha} alone continued to inhibit proliferation and, like TGF-beta, no longer stimulated CCN2 secretion per cell (Fig. 2A). When added in combination, TNF-{alpha} continued to produce a significant blockade of the TGF-beta-induced proliferation, to levels below that of control cultures with increasing doses of TNF-{alpha}. This long-term exposure to the combination of cytokines, however, strongly and synergistically induced (greater than 4-fold over that of either individual cytokine) CCN2 release per cell (Fig. 2A).


Figure 2
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  		Fig. 2. Effect of TNF-{alpha} on TGF-beta-induced CCN2 protein secretion and cellular proliferation after long-term exposure to cytokines. The y-axes: CCN2 protein levels (ng/106 cells; A) and number of cells (x106; B) for each treatment group; all results are means ± SE. The TGF-beta concentration was 5 ng/ml in all cases. Results reflect a 72-h collection with concurrent stimulation, following the initial short-term exposure period. Significant differences indicated as in Fig. 1.

 
Stimulated changes at the mRNA level were expected to precede changes in the production of the corresponding protein. Therefore, for Northern blot analysis of ccn2 mRNA from cells exposed to TNF-{alpha}, TGF-beta, or a combination, a single 48-h exposure to the cytokines was used. As was the case for CCN2 protein after short-term exposure, ccn2 transcript levels increased markedly when cells were stimulated with TGF-beta alone (Fig. 3). Treatment with TNF-{alpha} alone failed to increase ccn2 mRNA levels. However, as was observed for CCN2 protein secretion (short- and long-term exposure), a combination of TGF-beta and TNF-{alpha} resulted in a synergistic increase (2-fold) in the ccn2 transcript levels over those stimulated by TGF-beta alone and approximately eightfold over those of untreated cells.


Figure 3
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  		Fig. 3. Northern blot analysis of ccn2 mRNA in mesangial cells following 48-h exposure to TGF-beta and/or TNF-{alpha}. Shown are an autoradiograph of the ccn2 mRNA bands (top) and results obtained from densitometer scanning of the ccn2 mRNA bands, adjusted for the 18S RNA in the same lane to standardize for loading differences (bottom). The bars depict means ± range from 2 blots.

 
Effects of TNF-{alpha} on collagen protein and mRNA levels. To investigate the relationship between cytokine stimulation (or repression) of CCN2 activity and ECM changes, collagen type I secretion was determined following the treatments as described above. As previously reported, collagen secretion was strongly increased in MC following short-term stimulation with TGF-beta, reaching levels five- to sixfold greater than in control untreated cells (Fig. 4A). In cultures treated with TNF-{alpha} alone, there was a mild but inconsistent reduction in baseline collagen levels (Fig. 4A). In additional experiments, not shown, concentrations up to 10 nM did not produce a consistent, significant effect. Nevertheless, TNF-{alpha} was a consistent and potent inhibitor (up to ~80%) of the collagen augmentation stimulated by TGF-beta (Fig. 4A). This inhibitory result appeared to reach a maximum at 1.0 nM concentration. A similar but somewhat reduced TNF-{alpha} inhibitory effect (~60%) of collagen type I accumulation was observed following a long-term exposure to the cytokines (Fig. 4B).


Figure 4
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  		Fig. 4. Effect of TNF-{alpha} on TGF-beta-induced collagen type I protein after short-term (A) or long-term (B) exposure to cytokines. The y-axis records average collagen protein levels (µg/106 cells) for each treatment group, plus SE. The TGF-beta concentration was 5 ng/ml in all cases. Results reflect a 48-h collection following the initial 24-h stimulation period. Significant differences indicated as in Fig. 1.

 
Northern blot analysis was performed to examine the effects on collagen type I at the gene transcription level. The results (Fig. 5) from mRNA analysis in all cases mirrored those observed for collagen protein levels, albeit at a lower magnitude. That is, treatment with TGF-beta alone markedly increased collagen mRNA levels (over 2-fold). There was little or no change in baseline collagen mRNA expression in response to TNF-{alpha} alone, but TNF-{alpha} used in combination with TGF-beta again substantially reduced (~50%) the stimulatory effect of TGF-beta on the collagen gene.


Figure 5
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  		Fig. 5. Northern blot analysis of collagen type I mRNA in mesangial cells (MC) following 48-h exposure to TGF-beta and/or TNF-{alpha}. Shown are an autoradiograph of the collagen type I mRNA bands (top) and results obtained from densitometer scanning of the collagen I mRNA bands, adjusted for the 18S RNA in the same lane to standardize for loading differences (bottom).

 
Effects of IFN-{gamma} on CCN2 protein levels. To determine the comparative effect of the cytokine INF-{gamma} on the parameters described above, similar experiments were conducted. The short-term exposure (24 h followed by a 48-h follow-up period) to INF-{gamma} alone (50 U/ml) had no effect on constitutive CCN2 levels (Fig. 6A). Furthermore, IFN-{gamma} did not alter the CCN2 protein levels stimulated by TGF-beta. Likewise, there was no significant effect of INF-{gamma} on either constitutive or TGF-beta-stimulated cell growth (Fig. 6B). Similarly, INF-{gamma} failed to have a significant effect on CCN2 protein levels following long-term treatment (total 6 days). Whereas there was a tendency for TGF-beta to reduce CCN2, although not significantly, CCN2 protein levels did not appear altered by the addition of IFN-{gamma} (Fig. 7A). There was no effect on MC growth of long-term exposure to INF-{gamma} alone or in combination with TGF-beta (Fig. 7B).


Figure 6
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  		Fig. 6. Effect of IFN-{gamma} on TGF-beta-induced CCN2 protein after short-term exposure to cytokines. The y-axes: CCN2 protein levels (ng/106 cells; A) and number of cells (x106; B) for each treatment group; all results are means ± SE. The TGF-beta concentration was 5 ng/ml in all cases. Results reflect a 48-h collection following the initial 24-h stimulation period. *Significant difference compared with untreated cells. **Significant difference compared with TGF-beta-treated cells. ***Significant difference compared with IFN-{gamma}-treated cells.

 

Figure 7
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  		Fig. 7. Effect of IFN-{gamma} on TGF-beta-induced CCN2 protein secretion and cellular proliferation after long-term exposure to cytokines. The y-axes: CCN2 protein levels (ng/106 cells; A) and number of cells (x103; B) for each treatment group; all results are means ± SE. The TGF-beta concentration was 5 ng/ml in all cases. Results reflect a 72-h collection with concurrent stimulation, following the initial short-term exposure period. Significant differences indicated as in Fig. 6.

 
Effects of IFN-{gamma} on collagen type I protein levels. As was the case for the CCN2 response, a single treatment with IFN-{gamma} had no effect on collagen type I protein secretion, whereas TGF-beta strongly induced secretion of the ECM protein (Fig. 8). Only at the highest dose (500 U/ml) tested was IFN-{gamma} able to produce a statistically significant (albeit mild) reduction in the collagen type I secretion stimulated by TGF-beta, an effect not observed on CCN2 secretion.


Figure 8
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  		Fig. 8. Effect of IFN-{gamma} on TGF-beta-induced collagen type I protein after short-term exposure to cytokines. The y-axis records average collagen protein levels (µg/106 cells) for each treatment group, plus SE. The TGF-beta concentration was 5 ng/ml in all cases. Results reflect a 48-h collection following the initial 24-h stimulation period. Significant differences indicated as in Fig. 6.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The first observation from the present study was that TNF-{alpha} alone stimulated the secretion of CCN2 protein at 72 h poststimulation (Fig. 1). This appears to be in contrast to reports using several other cell types, modeling other disease, where TNF-{alpha} in the absence of other added cytokines had no effect on constitutive CCN2 protein levels (2), and/or reduced ccn2 mRNA levels (13, 31, 32). Interestingly, our observed increase in CCN2 protein in response to TNF-{alpha} occurred in the absence of increased ccn2 mRNA levels (Fig. 3). This posttranslational effect on CCN2 gene expression might be explained via the modulation of levels of proteases or protease inhibitors, for example TIMP-1 and/or MMP-3 (22, 38). Also, the observation that, unlike with TGF-beta, TNF-{alpha} increases CCN2 secretion per cell while at the same time reducing proliferation supports the idea that these two cytokines are working through different mechanisms to increase CCN2 production.

One of the primary goals of this study was to determine in MC the effect of TNF-{alpha} on CCN2 activity stimulated by TGF-beta. That is, modeling in vitro the chronic upregulation of TGF-beta, the resulting increase in CCN2 and ECM, and the subsequent worsening of renal fibrosis. We found a synergistic effect of TNF-{alpha} in combination with TGF-beta on CCN2 protein secretion after short-term exposure, and while the long-term exposure to each cytokine individually had little effect on CCN2, the combination greatly stimulated release. There is limited information available on the contribution of TNF-{alpha} to TGF-beta-stimulated CCN2 activity in any cells, and no study appears to have simultaneously investigated the effects on collagen production in MC. Pawluczyk and Harris (38) examined the effects of TNF-{alpha} on MC and whereas they did not examine the CCN2 response, they did report a mild increase of TGF-beta mRNA and protein and an autoinduction by TGF-beta alone. In the same study, TGF-beta in combination with TNF-{alpha} synergistically upregulated TGF-beta mRNA and protein, above the levels of induction by either cytokine by itself. One might then anticipate a subsequent elevation in CCN2 levels, as we observed, in response to the increased TGF-beta activity. Although the effect on collagen was not examined, they did report that the combined treatment increased both TIMP-1 and fibronectin mRNA levels (38). Our finding of an upregulation of CCN2 protein by TNF-{alpha} in the presence of TGF-beta (both short- and long-term exposure) was supported by our analysis of ccn2 mRNA levels. This suggests mechanisms operating at the levels of transcription/translation, but our results could also involve changes in catabolism, involving changes at the levels of both MMPs and TIMPs (22, 38).

Many reports in the literature confirm that treatment with TGF-beta increases production of collagen in MC and other cell types (30, 40, 49), but whether it acts directly or through the upregulation of an intermediate factor is not known. We believe that in MC, TGF-beta-mediated collagen upregulation proceeds primarily through the intermediary CCN2, while direct upregulation of collagen by TGF-beta signaling is either absent or a minor mechanism (Fig. 9A). Our hypothesis is supported by reports of markedly reduced fibrosis in animals with experimental kidney disease treated with CCN2-blocking agents (36, 52), as well as by reports of dramatically decreased collagen synthesis in mesangial (43) and nonrenal cultured cells (15) exposed to CCN2-blocking agents.


Figure 9
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  		Fig. 9. Depiction of the possible mechanisms of action for the observed TNF-{alpha} modification of TGF-beta-stimulated CCN2 and collagen activities in vitro. A, top: current paradigm for TGF-beta upregulation of CCN2 gene expression leading to increased CCN2 protein production followed by upregulation of collagen gene expression and ultimately the corresponding protein, as a pathway for the development of fibrosis. We speculate, as depicted in B, bottom diagram in dotted arrows, that TNF-{alpha} may greatly modify this pathway acting to decrease collagen protein production by one or more of the following mechanisms: 1) production of an as yet unidentified intermediate molecule (gray box) that is able to block normal CCN2 signaling; 2) direct inhibition of collagen gene expression or its protein products; 3) enhanced CCN2 protein turnover, i.e, stimulating CCN2 breakdown (to fragments of CCN2), thus more than compensating for the elevated synthesis and leading to decreased collagen gene transcription; or 4) a biphasic effect on collagen activity due to excessive amounts of biologically active CCN2.

 
An unanticipated finding from the current study was the divergent effects of TNF-{alpha} on the activities of CCN2 (including those stimulated by TGF-beta) and collagen type I. The reduction of collagen secretion in cells treated with both TGF-beta and TNF-{alpha} was not surprising in itself, as it is consistent with other reports of decreased collagen production in a variety of cell types treated with both cytokines (10, 12, 20, 27, 30, 51). However, given the hypothesis that CCN2 mediates the TGF-beta-induced upregulation of collagen in MC, the finding of increased CCN2 secretion in the presence of decreased collagen seemed counterintuitive.

What then is the explanation for this apparent contradictory finding? We envision several possible mechanisms. The first possibility (Fig. 9B) is that a third molecule, as yet undefined, produced in response to TNF-{alpha}, is able to alter the context in which CCN2 is received or recognized by the target cell. Our unpublished preliminary observations support this concept.

A second possibility (Fig. 9B) for decreased collagen in the presence of increased CCN2 is that the normal TGF-beta-driven collagen accumulation is overridden by an inhibitory mechanism(s) acting directly on matrix metabolism or matrix gene expression. However, it seems unlikely that this is due simply to a stimulation of protease/collagenase activity, as we found that collagen type I mRNA levels were also reduced in response to TNF-{alpha}. At the matrix gene level, signal transduction molecules of the Smad family appear to be required for TGF-beta-induced ccn2 and collagen type I gene expression in a variety of cell types (10, 12, 20, 27, 30, 51). We do not believe that TNF-{alpha} induces a general reduction in Smad signaling in MC because we did not observe decreased ccn2 expression and CCN2 protein production. It is possible that TNF-{alpha} interferes with expression of the transcription factor TIEG-1 (TGF-beta-inducible early gene 1), a repressor of the gene encoding the inhibitory Smad7 (1), which is upregulated by CCN2 in primary human MC, enhancing the fibrotic response to TGF-beta. Alternatively, TNF-{alpha} may change the binding activity of transcription factor SP-1, reported to be required for TGF-beta-induced collagen upregulation (41), but without affecting CCN2 expression (9), in MC. TNF-{alpha} can influence SP-1 binding activity in several cell types.

A third possible mechanism for collagen inhibition in the presence of increased CCN2 (Fig. 9B) is that the CCN2 produced in response to TNF-{alpha} has reduced biological activity and is therefore unable to effectively stimulate production of ECM. It appears from our data that TNF-{alpha} given alone may act on CCN2 at the translational or posttranslational level, but not at the level of transcription. A TNF-{alpha}-induced increased turnover of the CCN2 protein might result in elevated synthesis of the protein accompanied by a simultaneous increase in breakdown (i.e., increased turnover). These breakdown products, while conceivably detectable by ELISA, may no longer be biologically active. CCN2 fragments, apparently representing one or more modules of CCN2, have been reported in both cell culture media (including that from MC) and biological fluids (7, 46). The biological function(s) of these partial CCN2 molecules, which may represent degradation products, is (are) presently unknown.

Finally (Fig. 9B), a biphasic effect of CCN2 might explain the divergent results we observed. Such an effect, but on proliferation, has been reported in many cell types with TGF-beta and over a relatively narrow range of cytokine concentration, with lower levels stimulating and higher levels inhibiting (11). While a similar biphasic effect of CCN2, resulting in reduced collagen secretion at high doses, has not yet been demonstrated, a dose-response effect over a wide range of concentrations does not appear to have been clearly investigated. Our results from experiments using repeated administration of TGF-beta (i.e., the long-term groups) indicate that such a reverse effect on continued dosing is possible. Further studies should determine which, if any, of these inhibitory mechanisms are responsible for the observed decline in collagen observed in this fibrosis model in the presence of increased CCN2. In a number of animal models of inflammation-driven glomerular disease, i.e., glomerulonephritis, there is an abundance of data pointing to the long-term presence of TNF-{alpha} as a causal factor in pathology produced, including glomerulosclerosis. It will be interesting to determine in these models whether CCN2 is increased commensurate with the increase in collagen that often characterizes the late stage glomerular lesion.

In the second part of the present study, we investigated the effect of IFN-{gamma} on TGF-beta-induced CCN2 and collagen levels in MC, an effect that has not apparently been previously studied. In our experiments, this cytokine did not have an observable effect on CCN2 production after either short- or long-term TGF-beta stimulation, or on unstimulated MC. There was a statistically significant inhibitory effect of TGF-beta- induced collagen secretion only at the highest dose of IFN-{gamma} used (500 U/ml). An antagonistic effect of IFN-{gamma} on TGF-beta is consistent with reports of results in other cells types (12, 16, 19, 24, 27, 28), and in the lungs of patients with idiopathic pulmonary fibrosis (27), as described previously.

In previous studies examining the effect of TGF-beta exposure on CCN2 in cultured MC, reported by our laboratory and others, the periods of stimulation were typically limited to 1–3 days. In the present study when the TGF-beta treatment time was extended to 6 days, there was a failed continuation of stimulation, i.e., of CCN2 protein levels (Fig. 2A). Other investigators (9, 53) reported levels of ccn2 mRNA that fell substantially following, respectively, 24 or 48 h of exposure to TGF-beta in a rat MC line and normal rat kidney fibroblasts. We might predict from these previous reports that the reduced CCN2 protein we observed at 6 days may be a result of falling mRNA levels, although we did not perform a time course study. Alternatively, decreased CCN2 protein in our long-term exposure experiments might be due to a posttranslational mechanism not previously identified.

In conclusion, we demonstrated a divergent effect of TNF-{alpha} on CCN2 production, cell replication, and collagen type I activities in cultured rat MC exposed to TGF-beta. Under these conditions, TNF-{alpha} stimulates CCN2 release while simultaneously inhibiting cell growth as well as collagen type I gene expression and protein production. The mechanisms responsible are unknown, but the present study and previous work support the hypothesis that TNF-{alpha} upregulates CCN2 at a posttranscriptional level and downregulates collagen at the level of gene transcription. This could conceivably occur by one or several mechanisms that include 1) TNF-{alpha} stimulation of an endogenous collagen inhibitory factor, 2) alteration of signal transduction pathways, 3) increased breakdown or fragmentation of CCN2, and 4) a CCN2-biphasic effect on collagen induction resulting from very high concentrations of the former. Thus TNF-{alpha} may act as a natural regulator of the fibrotic response either (or both) by its effect on CCN2 itself, or the downstream events normally mediated by CCN2. Unlike TNF-{alpha}, IFN-{gamma} only slightly reduced the TGF-beta-mediated induction of collagen type I protein in rat MC, and only at the highest dose tested, while CCN2 protein levels remained unchanged. These experiments demonstrate the complexity involved in understanding the interplay of multiple cytokines and support the idea of a dual role for molecules such as TNF-{alpha} in inflammation and repair vs. inflammation and chronic disease, including that resulting in fibrosis.


   GRANTS
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 ABSTRACT
 METHODS
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This work was supported by grants from the Juvenile Diabetes Research Foundation (B. L. Riser) and the American Diabetes Association (B. L. Riser). Portions of this work were presented at the 2006 American Society of Nephrology Annual Meeting.


   ACKNOWLEDGMENTS
 
The authors thank Drs. E. Sykowski and F. Tam for critical readings of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. L. Riser, Baxter Healthcare, Renal Division, McGaw Park, IL 60085 (e-mail: bruce_riser{at}baxter.com)

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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