CCN3 (NOV) Is a Negative Regulator of CCN2 (CTGF) and a Novel Endogenous Inhibitor of the Fibrotic Pathway in an in Vitro Model of Renal Disease
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.080241
ISSN1525-2191
AutoresBruce L. Riser, Feridoon Najmabadi, Bernard Perbal, Darryl R. Peterson, Jo Ann Rambow, M Riser, Ernest Sukowski, Herman Yeger, Sarah C. Riser,
Tópico(s)Biomarkers in Disease Mechanisms
ResumoFibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-β, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-β treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-β treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-β to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments. Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-β, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-β treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-β treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-β to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments. Renal fibrosis is a common endpoint of chronic injury in the kidney, is central in diabetic renal disease, and thus is critical to the current increase in end-stage renal disease. Although angiotensin II inhibitor therapy is being used to successfully slow progression in many patients, there remains a need for more effective treatments capable of blocking or reversing progression. Recognition of multiple initiating factors in renal fibrosis has led to a search for novel, and common, downstream targets. A molecule recently identified as playing a critical role in fibrosis development is CCN2, originally termed connective tissue growth factor (CTGF). It is a member of a now recognized CCN family of six cysteine-rich proteins demonstrating similarities in their multimodular structure, but differences in function.1Brigstock DR Goldschmeding R Katsube KI Lam SC Lau LF Lyons K Naus C Perbal B Riser B Takigawa M Yeger H Proposal for a unified CCN nomenclature.Mol Pathol. 2003; 56: 127-128Crossref PubMed Scopus (205) Google Scholar, 2Perbal B CCN proteins: multifunctional signalling regulators.Lancet. 2004; 363: 62-64Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar Three of the four constitutive modules present in all members show partial identity with insulin-like growth factor binding proteins, von Willebrand factor, and thrombospondin 1. The C-terminal module contains a cysteine knot motif that is important in the heterodimerization of several growth factors and matrix proteins.3Perbal B The CCN family of genes: a brief history.Mol Pathol. 2001; 54: 103-104Crossref PubMed Scopus (60) Google Scholar, 4Rachfal AW Brigstock DR Structural and functional properties of CCN proteins.Vitam Horm. 2005; 70: 69-103Crossref PubMed Scopus (151) Google Scholar CCN2 is produced by kidney mesangial cells (MCs), a cell type critical in the formation of glomerulosclerosis.5Riser BL Denichilo M Cortes P Baker C Grondin JM Yee J Narins RG Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar We and others have shown that up-regulation of CCN2 in MCs occurs in response to factors known to be responsible for driving fibrosis in renal disease, including a high glucose environment, hypertensive force, and elevated transforming growth factor (TGF)-β,5Riser BL Denichilo M Cortes P Baker C Grondin JM Yee J Narins RG Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar the latter being a well-established profibrotic cytokine in fibrosis.6Bollineni JS Reddi AS Transforming growth factor-beta 1 enhances glomerular collagen synthesis in diabetic rats.Diabetes. 1993; 42: 1673-1677Crossref PubMed Google Scholar, 7Ziyadeh FN Mediators of diabetic renal disease: the case for TGF-β as the major mediator.J Am Soc Nephrol. 2004; 15: S55-S57Crossref PubMed Google Scholar A number of factors, including mechanical strain, appear to stimulate CCN2 expression without the up-regulation of TGF-β, supporting the idea that CCN2 may provide a more downstream and essential target for regulation of matrix metabolism in fibrosis.5Riser BL Denichilo M Cortes P Baker C Grondin JM Yee J Narins RG Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar, 8Hahn A Heusinger-Ribeiro J Lanz T Zenkel S Goppelt-Struebe M Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton.J Biol Chem. 2000; 275: 37429-37435Crossref PubMed Scopus (99) Google Scholar This idea is supported by recent data in several animal models of renal fibrosis, including that induced by diabetes, whereby CCN2 inhibition by antisense oligonucleotides (AS-ODN) is able to block progression.9Yokoi H Mukoyama M Nagae T Mori K Suganami T Sawai K Yoshioka T Koshikawa M Nishida T Takigawa M Sugawara A Nakao K Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis.J Am Soc Nephrol. 2004; 15: 1430-1440Crossref PubMed Scopus (224) Google Scholar, 10Okada H Kikuta T Kobayashi T Inoue T Kanno Y Takigawa M Sugaya T Kopp JB Suzuki H Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis.J Am Soc Nephrol. 2005; 16: 133-143Crossref PubMed Scopus (174) Google Scholar, 11Guha M Xu ZG Tung D Lanting L Natarajan R Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes.FASEB J. 2007; 21: 3355-3368Crossref PubMed Scopus (169) Google Scholar The biological process of fibrosis initiation is not unlike normal wound healing. However in fibrosis, perhaps because of the chronic insult, there is an inability to terminate the cytokine up-regulation that drives the remodeling of the extracellular matrix (ECM). We hypothesized that an endogenous regulatory molecule(s) exists that may be active in shutting down this process in normal wound healing, ie, after new tissue and matrix are generated. If identified, such a factor might be used therapeutically to prevent or reverse the progression of fibrosis. Toward this end, we suspected that one possibility for negative regulation of CCN2 might be CCN3 [formerly known as nephroblastoma overexpressed gene (NOV)]. CCN3 has not been previously studied as an element of the fibrotic pathway, or as a CCN2 regulatory molecule. Whereas CCN2 has often been associated with proliferative disease, CCN3 has anti-proliferative effects in a number of cell systems, and likely in disease.12McCallum L Price S Planque N Perbal B Pierce A Whetton AD Irvine AE A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation.Blood. 2006; 108: 1716-1723Crossref PubMed Scopus (59) Google Scholar, 13Li CL Martinez V He B Lombet A Perbal B A role for CCN3 (NOV) in calcium signalling.Mol Pathol. 2002; 55: 250-261Crossref PubMed Scopus (54) Google Scholar We also noted that cell types expressing high levels of CCN2 generally expressed low levels of CCN3 (Li CL, Perbal B, and Riser B, unpublished observations). This study was therefore conducted to test the hypothesis. RPMI from Invitrogen (Grand Island, NY) and fetal bovine serum (FBS) from Gemini (Woodland, CA) were used for growth medium. TGF-β1 was from R&D Systems (Minneapolis, MN). Purified rat collagen type I (COLI) was from Upstate Biotechnology (Lake Placid, NY) and polyclonal anti-rat COLI from Chemicon International (Temecula, CA). The production of full-length recombinant human CCN2 protein in a baculovirus expression system has been described previously.5Riser BL Denichilo M Cortes P Baker C Grondin JM Yee J Narins RG Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar Recombinant human and mouse CCN3 (rhCCN3 and rmCCN3) were from R&D Systems and were generated from a DNA sequence encoding a mature CCN3 protein expressed in a mouse myeloma cell line. Anti-CCN3 antibodies included a rabbit polyclonal produced by us14Kyurkchiev S Yeger H Bleau AM Perbal B Potential cellular conformations of the CCN3(NOV) protein.Cell Commun Signal. 2004; 2: 9Crossref PubMed Scopus (37) Google Scholar and monoclonals from R&D Systems. The MCs used were from a cloned line (16KC2) derived from Fischer rat glomeruli as previously described.15Riser BL Cortes P Yee J Sharba AK Asano K Rodriguez-Barbero A Narins RG Mechanical strain- and high glucose-induced alterations in mesangial cell collagen metabolism: role of TGF-beta.J Am Soc Nephrol. 1998; 9: 827-836PubMed Google Scholar MCs were long term cultured in RPMI 1640 medium containing antibiotics and 5 mmol/L glucose, plus 10% FBS. For many experiments, cells were seeded in normal growth medium in 24-well tissue culture plates, and grown for 3 days. On the day 4, cells were washed with RPMI containing 1% FBS (RPMI-1), then incubated in fresh RPMI-1. Twenty-four hours later, the cells were washed and exposed to RPMI-1 with added cytokines, as indicated in the figures. After 48 hours, all cells were again washed and incubated in RPMI-1 plus cytokines for 1 or 2 additional days before harvest. In all cases, heparin (50 μg/ml) was added to all wells before harvest to induce the release of cell- or matrix-bound CCN2, as previously described.5Riser BL Denichilo M Cortes P Baker C Grondin JM Yee J Narins RG Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar For determination of cellular proliferation and standardization of enzyme-linked immunosorbent assay (ELISA) data, results were expressed per cell, based on the amount of DNA determined using the CyQuant cell proliferation assay kit (catalog no. C-7026; Molecular Probes, Eugene, OR). By running samples against a standard curve generated using increasing numbers of MCs, the cell number in each test well could be determined. To generate a stable cell line expressing hCCN3, MCs were seeded into culture plates and grown for 24 hours. The transfecting mixture containing 20 μg of CCN3 expression vector PCB6+16Chevalier G Yeger H Martinerie C Laurent M Alami J Schofield PN Perbal B novH: differential expression in developing kidney and Wilm's tumors.Am J Pathol. 1998; 152: 1563-1575PubMed Google Scholar was incubated with 15 μl of transfection reagent, Lipofectamine 2000 (Invitrogen, Carlsbad, CA), in 1 ml of serum-free RPMI for 30 minutes at room temperature. This preparation was then added to cells with fresh medium and incubated. The medium was replaced the next day and, after two additional days, cells were replated with 1 mg/ml of G418 antibiotic, to select for stable antibiotic-resistant colonies. These G418-resistant cell lines were then analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using human-specific CCN3 primers to assess expression of hCCN3. Cells were seeded on 24-well plates and transfected 18 hours later with COL1a2 constructs kindly provided by Dr. William Schnaper, Northwestern University, Evanston, IL.17Poncelet AC de Caestecker MP Schnaper HW The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.Kidney Int. 1999; 56: 1354-1365Crossref PubMed Scopus (139) Google Scholar Renilla-luciferase pRL-SV40 was used as a control to normalize for transfection efficiency. Transfection was performed with the Invitrogen reagent Lipofectamine 2000. Briefly, 0.8 μg of collagen promoter constructs and 0.01 μg of pRL-SV40 control constructs were mixed with 1 μl of Lipofectamine 2000 in 100 μl of serum-free medium. The mixtures were incubated for 30 minutes at room temperature and added to the cells with 1 ml of fresh medium. After 18 hours the medium was replaced with one containing 2% FBS, and cells were incubated for an additional 18 hours. Either TGF-β (2 ng/ml) or control vehicle was added to the cells. In some experiments, the transfected cells were pretreated for 4 hours with 0 to 300 ng/ml CCN3 before adding TGF-β. Then, 24 hours later, the cells were washed with PBS and extracts were prepared using 150 μl of reporter lysis buffer (Promega Inc., Madison, WI). Luciferase activity of the promoter construct and the internal control were measured by adding 15 μl of extract with 50 μl of luciferase substrate and 50 μl of stop-and-go reagent. The luciferase activity determined in the assay was normalized using the transfection efficiency. The experiments were performed with triplicate samples. RNA extraction was performed using Trizol reagent under methods provided by Invitrogen.18Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar Synthesis of cDNA was performed using random hexamers and Moloney murine leukemia virus reverse transcriptase at 42°C for 1 hour starting with 5 μg of total RNA. Two μl of cDNA was then used for PCR. The sequences of primers used in PCR amplification are shown in Table 1. PCR analyses were done using a thermocycler (Applied Biosystems, Foster, CA). Electrophoresis of the amplification products was in 1 to 2% agarose gels. Bands were visualized by ethidium bromide staining, and intensities determined by densitometer scanning with subsequent analysis using the NIH Image program (National Institutes of Health, Bethesda, MD).Table 1Sequences of Primers Used in PCR AmplificationGeneStrandPCR primer sequencehCCN3Sense5′-ATGCAGAGTGTGCAGAGCAC-3′Anti-sense5′-TTACATTTTCCCTCTGGTAGTCTTCA-3′rCCN3Sense5′-TCTGTGGGATCTGCAGTGAC-3′Anti-sense5′-ATTGTTCTGAGGGCAGTTGG-3′rCCN2Sense5′-AAGGGTCTCTTCTGCGACTT-3′Anti-sense5′-ATTTGCAACTGCTTTGGAAG-3′rCOL1Sense5′-TGCTGCCTTTTCTGTTCCTT-3′Anti-sense5′-AAGGTGCTGGGTAGGGAAGT-3′r18s rRNASense5′-GACCATAAACGATGCCGACT-3′Anti-sense5′-AGACAAATCGCTCCACCAAC-3′ Open table in a new tab An ELISA was used to quantify levels of cytokines and collagen. An indirect ELISA was used for CCN2 protein measurements for the conditioned media, as we have previously described.5Riser BL Denichilo M Cortes P Baker C Grondin JM Yee J Narins RG Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar For CCN3, a direct ELISA was used for tissue-culture samples from rat cells, and an indirect ELISA was used for rat cells transfected to express hCCN3. In brief, for the direct ELISA, the samples and recombinant standards (diluted in the same medium as the samples) were incubated at room temperature to allow binding to the 96-well plate. The unbound sites were then blocked with 1% bovine serum albumin (BSA) + 0.05% Tween 20. After washing, a primary antibody was added (MAB1640, R&D Systems) or K19-immunized rabbit serum.14Kyurkchiev S Yeger H Bleau AM Perbal B Potential cellular conformations of the CCN3(NOV) protein.Cell Commun Signal. 2004; 2: 9Crossref PubMed Scopus (37) Google Scholar For the indirect ELISA, the plate was first coated with MAB1640, then blocked, then incubated with sample and standard before giving K19-immunized rabbit serum. After further washing, horseradish peroxidase-conjugated secondary antibody (anti-rabbit 111-035-003 or anti-mouse 115-035-003; Jackson ImmunoResearch, West Grove, PA) was added, followed by more washing and horseradish peroxidase substrate (Enhanced K-βlue TMB substrate, 308175; Neogen Corp., Lexington, KY). The color intensity was allowed to develop before being read at 650 nm using a microplate reader (Thermo Max; Molecular Devices Corp., Sunnydale, CA). The measurement of COL1 was done by direct ELISA, as previously described19Cooker LA Peterson D Rambow J Riser ML Riser RE Najmabadi F Brigstock D Riser BL 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.Am J Physiol. 2007; 293: F157-F165Google Scholar and similar to the method described here for CCN3, except that incubations were performed at 4°C to minimize aggregation of the molecule, and the samples and standards were incubated in the plate overnight. The blocking solution and secondary antibody dilution buffer contained 5% nonfat dry milk + 0.05% Tween 20. The BSA blocking solution above was used to dilute the primary antibody (AB755P, Chemicon). The substrate color was developed at room temperature, and read as above. Biological samples were prepared by mixing 1 vol of a sample with 1 vol of the loading buffer with 10% 2-mercaptoethanol and 2% sodium dodecyl sulfate. Twenty μl of each prepared sample was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad, Hercules, CA) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was blocked with 5% nonfat dried milk in Tris-buffered saline (TBS) + 0.1% Tween 20 for 1 hour at room temperature and then incubated with monoclonal anti-CCN3 antibody (1:500 dilution, R&D Systems). For phospho-Smad3 activity a phospho-Smad3-specific rabbit monoclonal antibody (Cell Signaling, Danvers, MA) was used. A horseradish peroxidase-conjugated secondary antibody (1:15,000 dilution; Amersham, Piscataway, NJ) and horseradish substrates (Pierce, Rockford, IL) were used to label the bands, which were enhanced with the chemiluminescence system (Pierce) and were developed using Amersham X-ray film. A single cell suspension was prepared from the cell cultures and fixed in ice-cold 70% ethanol for 2 hours. Cells were then washed with PBS and incubated in PBS containing 0.1% Triton X-100, 0.2 mg/ml RNase A, and 5 μg/ml propidium iodide for 30 minutes at the room temperature. Fluorescence-activated cell sorting analysis was conducted using a Becton Dickinson (Mountain View, CA) LSRII system using PC-based FACSDiva acquisition and analysis software with ModFit program for determining cell cycle. MCs grown on chamber slides were fixed in methanol. Immunoperoxidase labeling was performed at ambient temperature in a humidified chamber. Endogenous peroxidase was first blocked in 0.3% hydrogen peroxide/methanol. Cells were then rinsed in TBS and nonspecific binding was blocked in 1% BSA/TBS. Primary antibodies were anti-CCN3 (K19, 1:250 dilution in 1% BSA/TBS), anti-CCN2 (polyclonal, affinity-purified rabbit antibody recognizing the COOH-terminal portion of CCN2), and anti-rat polyclonal COLI (all described above) were applied, then cells washed 5× in TBS/0.025% Brij35. Secondary antibody conjugate (1:10 in BSA/TBS; horseradish peroxidase polymer conjugate, broad spectrum; Zymed Laboratories, San Francisco, CA) was applied, and then washed in TBS/Brij and 1 in TBS only. Stable diaminobenzidine (Invitrogen, Burlington, Canada) was applied while monitoring color development. Cells were counterstained in hematoxylin and dehydrated before mounting. The procedure MIXED (SAS software; SAS Institute, Cary, NC) was used to perform specific analyses. Differences of least squares means estimates were performed for the pairwise comparisons of groups. P values less than 0.05 were considered statistically significant. For analyses in which there were only two groups, a Student's t-test was performed and P values less than 0.05 were considered statistically significant. Analysis of variance was used with a Tukey test when appropriate. To first examine the potential effect of CCN3 on CCN2 and the resulting fibrosis, we used a previously reported in vitro model.20Riser BL Cortes P Yee J Modelling the effects of vascular stress in mesangial cells.Curr Opin Nephrol Hypertens. 2000; 9: 43-47Crossref PubMed Scopus (42) Google Scholar In this model cultured MCs are grown to near confluence, then the medium is replaced for 24 hours with one containing a low concentration (2%) of FBS to place the cells in a near growth-arrested state. Cells are then exposed for 24 to 96 hours to medium containing TGF-β1 (2 ng/ml), or control medium only. This TGF-β treatment results in an increase in CCN2 mRNA and protein, and is followed some hours later by a concomitant increase in the baseline level of type I collagen (COL1) mRNA and in turn increased secreted COL1.20Riser BL Cortes P Yee J Modelling the effects of vascular stress in mesangial cells.Curr Opin Nephrol Hypertens. 2000; 9: 43-47Crossref PubMed Scopus (42) Google Scholar, 21Riser BL Cortes P Connective tissue growth factor and its regulation: a new element in diabetic glomerulosclerosis.Ren Fail. 2001; 23: 459-470Crossref PubMed Scopus (59) Google Scholar TGF-β stimulation of collagen synthesis can be blocked by CCN2-specific antisense oligonucleotides (ODNs), demonstrating a critical role for CCN2 in this pathway in vitro22Riser B Foroni A Karoor S Morgensen C Cortes P CCN2 (CTGF) in the Pathogenesis of Diabetic Renal Disease: A Target for Therapeutic Intervention. Kluwer Academic Publishers, Boston2006Google Scholar, 23Abdel-Wahab N Weston BS Roberts T Mason RM Connective tissue growth factor and regulation of the mesangial cell cycle: role in cellular hypertrophy.J Am Soc Nephrol. 2002; 13: 2437-2445Crossref PubMed Scopus (105) Google Scholar similar to that shown in vivo in models of renal fibrosis.9Yokoi H Mukoyama M Nagae T Mori K Suganami T Sawai K Yoshioka T Koshikawa M Nishida T Takigawa M Sugawara A Nakao K Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis.J Am Soc Nephrol. 2004; 15: 1430-1440Crossref PubMed Scopus (224) Google Scholar, 10Okada H Kikuta T Kobayashi T Inoue T Kanno Y Takigawa M Sugaya T Kopp JB Suzuki H Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis.J Am Soc Nephrol. 2005; 16: 133-143Crossref PubMed Scopus (174) Google Scholar, 11Guha M Xu ZG Tung D Lanting L Natarajan R Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes.FASEB J. 2007; 21: 3355-3368Crossref PubMed Scopus (169) Google Scholar To test the effects of CCN3 in the above in vitro model, we used two different preparations. The first was a conditioned medium from the NCI-H295R human cell line. This cell expresses biologically active CCN3 at high levels, as we have described.24Thomopoulos GN Kyurkchiev S Perbal B Immunocytochemical localization of NOVH protein and ultrastructural characteristics of NCI-H295R cells.J Submicrosc Cytol Pathol. 2001; 33: 251-260PubMed Google Scholar The second preparation used was a purified recombinant mouse CCN3 (rmCCN3). Although recombinant proteins can lack the biological activity of the native molecule, this CCN3 has been shown to induce cellular adhesion of Balbc/3T3 cells, thus confirming the capability for, at least this biological function.25Ball DK Moussad EE Rageh MA Kemper SA Brigstock DR Establishment of a recombinant expression system for connective tissue growth factor (CTGF) that models CTGF processing in utero.Reproduction. 2003; 125: 271-284Crossref PubMed Scopus (46) Google Scholar After a 96-hour total treatment period, there was little or no measurable constitutive CCN2 present, but a strong stimulation of CCN2 production followed the exposure to TGF-β, as expected (Figure 1A). Under these TGF-β-treated conditions, the addition of conditioned medium-CCN3 at increasing concentrations produced a clear dose-dependent reduction in CCN2 levels that began at the lowest concentration tested (0.5 ng/ml). The effect reached an approximate 50% inhibition at the highest CCN3 concentration (50 ng/ml) used (Figure 1A). A similar level of CCN2 blockade was observed when 5 ng/ml of the purified rmCCN3 was used. As the dose of this purified molecule was increased to 50 ng/ml, total blockade of CCN2 was achieved, and remained unchanged as the dose was further increased (500 ng/ml rmCCN3). A similar, but less potent, CCN3 dose-inhibitory response was seen after a 24-hour exposure to the same preparations (data not shown). Baseline secretion of COL1 protein was also increased in response to the same TGF-β exposure (Figure 1B). As was observed for CCN2, the conditioned medium-CCN3 produced a dose-dependent inhibition of collagen production. In this case, there was a total blockade of collagen at the highest concentration used (50 ng/ml). The purified rmCCN3 produced a similar inhibition, but this time required 500 ng/ml for a complete blockade (Figure 1B). A similar, but less potent, collagen-inhibitory dose response to CCN3 was seen after 24 hours of exposure, reaching an approximate 40% and 80% reduction with the conditioned medium-CCN3 and rmCCN3, respectively, at the highest concentrations tested (data not shown). As a second method for examining the effects of CCN3 on collagen regulation, the human col1 promoter linked to luciferase was expressed in this MC line using a transient transfection method. The cells were then exposed to either 1 or 100 ng/ml of rmCCN3 24 hours after transfection, and the luciferase activity was measured at three subsequent periods. Results showed little collagen gene activation at 24 hours after transfection and little or no effect of CCN3 at the same period (Figure 1C). However, col1 activation increased greatly at 48 hours after transfection, and there was a dose-dependent inhibition of this strong promoter activity in response to the added CCN3, reaching ∼50% inhibition at 100 ng/ml of rmCCN3. By 72 hours after transfection, as the cells became confluent, the level of collagen activation began to fall on its own (Figure 1C, control). Nevertheless, exogenous CCN3 treatment further reduced, in a dose-dependent manner, the col1 promoter activity. Last, immunohistochemical staining was used to examine the localization of CCN2 and COL1 in MCs, and the response to CCN3 treatment. Results showed that both CCN2 and COL1 protein in control unstimulated MCs were present and localized primarily to discrete peripheral areas, ie, near the cell membrane (Figure 2). After 48 hours of TGF-β exposure there was a loss of this discrete localization, and a new and more intense staining was now apparent and diffusely distributed throughout the cytoplasm, with the greatest concentration appearing paranuclear in distribution. Our previous results (Figure 1, A–C) support the idea that this represents new synthesis of both molecules. A 1-hour pretreatment of the cells with CCN3 virtually blocked this TGF-β-induced effect on both CCN2 and COL1 (Figure 2), thus supporting the previous results (Figure 1, A and B). We reasoned that because exogenous CCN3 was able to block the stimulation of CCN2 and collagen by TGF-β, then if these MCs are capable of producing and secreting the molecule, paracrine activity might be important in shutting down the wound healing and/or fibrotic processes. In contrast, to up-regulate ECM metaboli
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