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Endocrinology Vol. 143, No. 4 1260-1269
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Connective Tissue Growth Factor/IGF-Binding Protein-Related Protein-2 Is a Mediator in the Induction of Fibronectin by Advanced Glycosylation End-Products in Human Dermal Fibroblasts

Stephen M. Twigg, Alison H. Joly, Michelle M. Chen, Junko Tsubaki, Ho-Seong Kim, Vivian Hwa, Youngman Oh and Ron G. Rosenfeld

Department of Pediatrics (S.M.T., J.T., H.-S.K., V.H., Y.O., R.G.R.), Oregon Health Sciences University, Portland, Oregon 97201; and Cardiorenal Cell Biology (A.H.J., M.M.C.), Scios, Inc., Sunnyvale, California 94086

Address all correspondence and requests for reprints to: Stephen M. Twigg, Kolling Institute of Medical Research, Royal North Shore Hospital, Pacific Highway, St. Leonards, New South Wales 2065, Australia. E-mail: . stwigg{at}med.usyd.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expansion of extracellular matrix with fibrosis occurs in many tissues, including skin, as part of the end-organ complications in diabetes. Advanced glycosylation end-products (AGEs) have been implicated as a pathogenic factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as IGF-binding protein-related protein-2, induces extracellular matrix. We have recently shown that CTGF mRNA and protein are up-regulated by AGE treatment of cultured human dermal fibroblasts. The aim of this study was to determine whether CTGF is an autocrine mediator in the induction of fibronectin (FN) by AGE. Primary cultures of nonfetal human dermal fibroblasts in confluent monolayer were treated with synthesized soluble AGE BSA, 0–200 µg/ml. Analysis of mRNA, by quantitative real-time RT-PCR and conditioned media from treated cultures, showed that FN mRNA was increased by approximately 4-fold at 48 h, and FN protein levels by Western immunoblot and FN ELISA were doubled, compared with control. In the same system, added recombinant human CTGF (0–500 ng/ml) induced FN mRNA and protein levels dose dependently and in a rapid time course. To test whether AGE BSA acts through cell-derived CTGF to induce FN, a CTGF neutralizing antibody was shown to significantly attenuate, but not fully inhibit, the AGE induction of FN mRNA. A pan-specific PKC inhibitor, GF109203X, at 0.2 µM, inhibited the induction of FN mRNA by AGE BSA. Although the same inhibitor did not significantly affect the induction of CTGF mRNA by AGE, it blocked the induction of FN mRNA by recombinant human CTGF. In summary, the induction of FN by AGE is partly mediated by the AGE-induced up-regulation of cell-derived CTGF and is dependent on PKC activity. These results have potential implications for the expansion of extracellular matrix in diabetes mellitus by advanced glycosylation end products.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A MECHANISM PROPOSED whereby chronic hyperglycemia contributes to diabetic complications is in the formation of advanced glycosylation end-products (AGEs) (1). AGEs constitute irreversibly formed biochemical end-products of nonenzymatic glycosylation (2) and are elevated in many tissues, including skin (3), in subjects with diabetes (4). One method by which AGEs appear to contribute to pathological end-organ changes that occur in tissues in subjects with diabetes is through the induction of specific cytokines and growth factors, which may act as mediators in causing tissue pathology (5, 6, 7).

One hallmark in most tissues in which diabetic complications occur is expansion of extracellular matrix [ECM (8)]. AGEs (7) and increased cellular PKC activity (9) have each been identified as contributors to ECM expansion in diabetes through increased ECM production. An integral component of ECM is the glycoprotein, fibronectin (FN), which acts as a scaffold for collagens, and contributes to an ECM network involved in cell proliferation and migration (10). FN is increased in vivo in the ECM expansion that occurs in diabetes (11, 12).

Recently, we have reported that the cytokine, connective tissue growth factor (CTGF), is up-regulated at the mRNA and protein level by AGEs in confluent monolayers of cultured human dermal fibroblasts (13). Also known as IGF-binding protein-related protein-2 [IGFBP-rP2 (14)], CTGF is a potent inducer of ECM, including FN (15), in fibroblasts (16) as well as an angiogenic factor (17, 18). A potential role for CTGF in fibrotic disease states is increasingly being described (19, 20), suggesting that CTGF is a mediator in the ECM expansion and fibrosis occurring in diabetes. To date, the cellular mechanism of action of CTGF in enhancing ECM has been inadequately studied (21).

Because AGEs and CTGF can both up-regulate fibronectin, and AGEs also up-regulate CTGF in human fibroblasts, the aim of this study was to determine whether the induction of FN by AGEs is mediated through up-regulation of endogenous CTGF and to explore the cellular secondary messenger systems involved in mediating this effect on FN expression. Our work shows that CTGF contributes significantly to AGE up-regulation of FN in human dermal fibroblasts, through a PKC-dependent mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The antifibronectin mouse monoclonal antibody against human fibronectin was purchased from Neomarkers Inc. (Union City, CA). Polyclonal anti-IGFBP-rP2 (CTGF) antibody (8800), was generated in New Zealand White rabbits, using full-length human CTGF (IGFBP-rP2) as the immunogen, as previously described (22). The anti-AGE polyclonal antiserum generated in New Zealand White rabbits, which neutralizes the activity of AGE BSA (23), was a generous gift from Dr. Miyata (Kissei Pharmaceutical Co. Ltd., Nagano, Japan). The antisera to human CTGF, and to AGE, as well as nonimmune normal rabbit serum (Sigma, St. Louis, MO) were each affinity purified by protein A affinity chromatography, using protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden). In each case, the eluted IgG protein was dialyzed against PBS, using a low-molecular-mass-cut-off membrane (Spectrapor 1, 6–8 kDa, Spectrum Industries, Los Angeles, CA), and confirmed to be immunoglobulin by migration characteristics on nonreducing SDS-PAGE, followed by Coomassie staining. The total amount of IgG protein was quantitated using the DC protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA) and was further confirmed by spectrophotometer absorbance readings at 280 nm. D-Glucose, BSA (fraction V, fatty acid and endotoxin free), human plasma fibronectin, and aminoguanidine were purchased from Sigma. TGF-ß1 was purchased from Austral Biologicals (San Ramon, CA). The pan-specific PKC inhibitor, GF109203X, was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).

AGE synthesis
Advanced glycosylation end-products were synthesized in vitro, following methods previously described (13, 23, 24, 25). BSA (Sigma, RIA grade, fraction V) at 10 mM, was coincubated in sterile PBS with 0.5 M D-glucose for 10 wk, with 1.5 mM phenylmethylsulfonyl fluoride, under aerobic conditions at 37 C. To generate control BSA for comparison with AGE treatments, tubes were prepared with simultaneous incubations under the same conditions without the addition of the D-glucose. Additionally, in parallel preparations, aminoguanidine at 100 mM, as an inhibitor of formation of products of nonenzymatic glycosylation (7), was added to the BSA and glucose. All preparations were extensively dialyzed in PBS, using a low-molecular-mass-cut-off membrane (Spectrapor 1, 6–8 kDa, Spectrum Industries; Ref. 7).

The AGE content in the preparations was assessed by means of fluorescence, SDS-PAGE analysis, and ELISA. The fluorescence content, measured with a fluorescence spectrometer at 390 nm emission after a 450-nm excitation, in relative fluorescence units per milligram of BSA, was 11.2 ± 2.5 for control BSA, 52.3 ± 6.3 for AGE BSA, and 9.7 ± 1.2 for aminoguanidine added to BSA and glucose (termed aminoguanidine BSA) (25). By SDS-PAGE analysis under reducing conditions, followed by Coomassie staining, the AGE BSA produced was shown to have high-molecular-mass species, consistent with the intermolecular cross-linking ability of AGE, as described (23). In contrast, the control BSA and aminoguanidine BSA preparations did not have these high-molecular-mass forms (data not shown). By competitive ELISA [as described in (26)] performed by Dr. P. Foiles (Alteon Inc., Ramsey, NJ), using a synthetic N-{epsilon} carboxymethyl lysine (CML) analog as the standard, the CML content of the preparations (picomole CML per microgram of BSA ± 95% confidence interval) was: 13 ± 1.4 for AGE BSA from glucose and was undetectable (<1) for control BSA and also undetectable when aminoguanidine at 100 mM was coincubated with BSA and glucose.

Synthesis and purification of recombinant human CTGF
The recombinant human CTGF [rhCTGF (IGFBP-rP2)] protein was produced using a baculovirus expression system (Invitrogen Corp., Carlsbad, CA). 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 XhoI sites in the baculovirus recombination vector, pFastBac1 (Life Technologies, Inc., Rockville, MD), and insert presence and orientation were verified by DNA sequencing. Recombinant baculovirus stocks were isolated and produced in increasing titer, as recommended by the supplier of the expression system. The rhCTGF protein was then produced by infecting HIGH Five insect cells with recombinant virus under serum-free conditions and collecting the conditioned media. To purify rhCTGF protein from filtered media, heparin-Sepharose affinity chromatography, using HiTrap columns (Pharmacia Biotech) with a step-up salt gradient in the elution, was employed as previously described (15, 27). Peak fractions containing rhCTGF were determined by Western immunoblotting and CTGF protein was quantitated using Coomassie Blue-stained gels with BSA as standard, as previously described (13, 28).

Cell culture
Primary cell cultures of nonfetal human dermal fibroblasts, CRL-2097, and CRL-1474 were purchased from ATCC (Manassas, VA). Cells were maintained in MEM supplemented with 10% FBS and were used in these studies between passages 4 and 12. The human primary cultures of dermal fibroblasts, designated A35 (derived from the forearm of a 70-yr-old male) and A305 (newborn foreskin fibroblasts) were generous gifts from Dr. S. Goldstein, Memorial Veteran’s Hospital (Little Rock, AR). These cells were maintained in DMEM containing 450 mg/dl glucose and 15% FBS.

Cell treatment
After trypsinization, cells were grown in 12-well plates for 5 d in their respective media with FBS until they were confluent. For experiments requiring the use of blocking antibodies to AGE or CTGF, or using control normal rabbit serum IgG, cells were grown in 24-well plates under the same conditions. Cells were then incubated in their respective serum-free media for 16 h and were then treated on d 0 under serum-free conditions using fresh media. Unless otherwise indicated, the conditioned media were not changed after adding the treatments. Cell lysates and conditioned media were harvested up to 3 d after treatments. For experiments involving the use of blocking antibodies or PKC inhibitors, cells were preincubated with the antibody or reagent for 2 h under serum-free conditions before the addition of AGE or control BSA.

Total RNA isolation and analysis by quantitative real-time RT-PCR
Total RNA was isolated from duplicate wells, (RNeasy minikit, QIAGEN, Valencia, CA) and analyzed by quantitative real-time PCR using an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA) as previously described (13, 29). This system is based on the ability of the 5' nuclease activity of Taq polymerase to cleave a nonextendable dual-labeled fluorogenic hybridization probe during the extension phase of PCR. The following sequence specific primers and probes for human CTGF, FN, and 18S rRNA were designed, using Primer Express software 1.0 (PE Applied Biosystems): for FN, forward 5'-TCCTTGCTGGTATCATGGCAG-3', reverse 5'-AGACCCAGGCTTCTCATACTTGA-3', and probe 5'-6FAM-CCACGTGCCAGGATTACCGGCTACAT-TAMRA-3'; for CTGF, forward 5'-GAGGAAAACATTAAGAAGGGCAAA-3', reverse 5'-CGGCACAGGTCTTGATGA-3', and probe 5'6FAM-TTTGAGCTTTCTGGCTGCACCAGTGT-TAMRA3'; for 18S, forward 5'-CGGCTACCACATCCAAGGAA-3', reverse 5'-GCTGGAATTACCGCGGCT-3', and probe 5'-6FAM-TGCTGGCACCAGACTTGCCCTC-TAMRA3'. Primers were used at a concentration of 200 nM and probes at 100 nM in each reaction. Multiscribe reverse transcriptase and Amplitaq gold polymerase (PE Applied Biosystems) were used in all RT-PCR. Each RNA sample was analyzed in triplicate. Relative quantitation of 18S rRNA and human FN and CTGF mRNAs were calculated, using the comparative threshold cycle number for each sample fitted to a five-point standard curve (ABI prism 7700 user bulletin no. 2, PE Applied Biosystems). The standard curve was constructed using a serial dilution of total RNA extracted from human cardiac fibroblasts that had been treated with TGF-ß1 at 1 ng/ml for 24 h. Expression levels were normalized to 18S rRNA and related to relevant controls as indicated in the text.

Preparation of conditioned media and cell lysates
Conditioned media were collected after cell treatment and centrifuged at 10,000 x g for 10 min at 4 C, and then supernatants from duplicate wells within each experiment were pooled and stored at -20 C until analysis. Cell lysate samples were harvested after treatment by washing cells with PBS and then adding 100 µl cold RIPA lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% NaDOC, 0.1% SDS) plus a protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) directly to each well. Plates were rocked for 15 min at 4 C, and lysates were collected and centrifuged at 10,000 x g for 10 min at 4 C. The supernatants from duplicate wells within each experiment were pooled and stored at -20 C until analysis. Total protein concentration was determined for each sample by use of the DC protein assay reagent (Bio-Rad Laboratories, Inc.). Twenty micrograms of total protein were then loaded per lane for SDS-PAGE analysis.

Western immunoblot analysis
Conditioned media samples were separated on 7.5% nonreducing SDS-PAGE. Proteins were electrotransferred onto nitrocellulose, and membranes were blocked with 5% nonfat dry milk/TBS with 0.1% (vol/vol) Tween 20 for 1 h at 22 C, then incubated overnight, 4 C, in FN antiserum (1:800 dilution; Neomarkers, Inc.). After incubation of membranes with a horseradish peroxidase-labeled secondary antibody for 1 h at 22 C, immunoreactive proteins were detected by use of enhanced chemiluminescence (NEN Life Science Products, Boston, MA).

ELISA for fibronectin
A competitive ELISA was developed based on previously described methods (30). Pure human plasma FN (100 ng/well) in 10 mM sodium carbonate, pH 9.6, was adsorbed to 96-well immunoplates (Nalge Nunc International, Rochester, NY) by a 20-h incubation at 4 C. The wells were then blocked by incubation with PBS, 0.1% (vol/vol) Triton X-100 (buffer A) containing 10 g/liter BSA for 2 h at 37 C, and washed four times with buffer A. Purified human FN in buffer A was used to generate standard curves. Standard and samples (125 µl/tube) were incubated with a limiting amount (1:2500 titer, 0.005 µl/tube) of antifibronectin antibody (Neomarkers, Inc.) at 22 C for 1 h. The plate was then incubated with the FN standards (0–500 ng) and samples (each at 100 µl/well) for 30 min at 22 C. After washing, the plate was incubated with streptavidin horseradish peroxidase at 1:3000 (Sigma) for 30 min at 22 C and, after four more washes in buffer A, with substrate [0.1 g/liter 3,3',5,5'-tetramethyl benzidine in 0.2 M sodium acetate, pH 6, containing 0.06% (wt/wt) H2O2] for 10 min at 22 C. The reaction was stopped by the addition of 2 M H2SO4, and the absorbance was measured at 450 nm using a microplate reader. Linearity in the assay was achieved over the range of 15–250 ng/well of FN.

Densitometric analysis
To quantify the relative induction of FN following SDS-PAGE and Western immunoblotting, densitometric measurement was performed using GS-700 imaging densitometer with MultiAnalyst Software (Bio-Rad Laboratories, Inc.).

Statistical analysis
Results are expressed as mean ± SD or mean ± SEM as indicated. Differences between groups were assessed using a two-tailed paired t test in Microsoft Corp. (Redmond, WA) Excel 98, where shown. A level of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether FN mRNA steady-state levels are up-regulated by AGE in primary cultures of human dermal fibroblasts, confluent monolayers of CRL-2097 fibroblasts were treated with soluble AGE BSA under serum-free conditions. In response to 100 µg/ml of AGE BSA, an increase in FN mRNA was observed at 24 h of AGE treatment, and a progressive increase occurred over the 3-d time course of the study (Fig. 1AGo). In contrast, no change in FN mRNA over time was seen after treatment with the same concentration of control BSA (Fig. 1AGo). A dose-response study with AGE BSA from 0 to 200 µg/ml in 2097 cells, with continuous AGE treatment and RNA collection at 48 h after initial AGE addition, showed that increases in FN mRNA were detectable using 10 µg/ml or greater AGE BSA, but increasing concentrations of control BSA did not show any change in FN mRNA in comparison with no addition (Fig. 1BGo).



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  		Figure 1. Time course and dose-response induction of FN mRNA by AGE BSA. Soluble AGE BSA or control BSA was added in fresh media to duplicate wells of confluent primary cultures of human fibroblasts (CRL-2097) under serum-free conditions. Total RNA was collected, and FN mRNA was determined by quantitative RT-PCR in triplicate for each sample, as described in Materials and Methods. The FN mRNA level is expressed in arbitrary units normalized to 18S. A, Time course from 1 to 3 d, with AGE BSA or control BSA treatment, each at 100 µg/ml. B, Dose response with AGE BSA or control BSA from 0 to 200 µg/ml added to wells followed by RNA collection at 48 h. The mean of three independent experiments ± 1 SD is shown in A and B. *, P < 0.05; **, P < 0.01 vs. the respective control BSA.

 
Formation of products of nonenzymatic glycosylation are inhibited by the dihydrazine compound aminoguanidine (7). When cells were treated with BSA that had been coincubated for 10 wk with both glucose and aminoguanidine, as described in Materials and Methods, no increase in FN mRNA was observed, compared with control BSA treatment alone or with serum-free media without any addition (Fig. 2AGo). This result confirms that the active component in the AGE reagent used is a product of nonenzymatic glycosylation. As a positive control reagent in this system for the induction of FN, TGF-ß1 (1 ng/ml) was also seen to induce FN mRNA (Fig. 2AGo).



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  		Figure 2. AGE effect on FN mRNA occurs through nonenzymatic glycosylation of BSA and is blocked by an anti-AGE-neutralizing antibody. A, Soluble AGE BSA or control BSA at 100 µg/ml, or no treatment, was added to duplicate wells of confluent primary cultures of human fibroblasts under serum-free conditions. In other wells BSA that had previously been coincubated with glucose and aminoguanidine before dialysis, termed aminoguanidine BSA, as described in Materials and Methods, was added. Treatment with TGF-ß1 (1 ng/ml) as a positive control is shown for comparison. Total RNA was collected at 48 h, and FN mRNA was determined by quantitative RT-PCR in triplicate for each sample. The FN mRNA level is expressed in arbitrary units. The mean ± SD of three independent experiments is shown. **, P < 0.01 vs. no addition, control BSA, and aminoguanidine BSA. In B, wells were preincubated with anti-AGE polyclonal neutralizing IgG, at 100 µg/ml IgG or 100 µg/ml NRS IgG for 2 h. Soluble AGE BSA or control BSA at 100 µg/ml was then added to the wells. Total RNA was collected at 48 h and FN mRNA determined by quantitative RT-PCR in triplicate for each sample. The means ± 1 SD of four independent experiments are shown. *, P < 0.05 vs. AGE BSA added alone.

 
To determine whether early or advanced glycosylation end-products mediate the effect of the AGE reagent on FN in this cell system, cells were preincubated with anti-AGE IgG before addition of the AGE reagent. Using the anti-AGE antibody, the AGE induction of FN mRNA was inhibited on average by 81% (Fig. 2BGo). In contrast, normal rabbit serum had no effect (Fig. 2BGo). Because the anti-AGE antibody is specific for AGE and does not bind to Amadori products (23), which are early products of nonenzymatic glycosylation, these results showed that AGE is the active component in the synthesized reagent responsible for increasing FN in these studies.

To test whether the changes seen in FN mRNA in human foreskin fibroblast CRL-2097 cells after AGE treatment can also be observed in human dermal fibroblasts, primary human skin fibroblasts from other donors were studied. When these other cells were treated with 100 µg/ml of AGE BSA, increases in FN mRNA were observed 48 h after treatment, compared with control BSA, in all of the fibroblast cell lines studied, whether they were derived from neonatal foreskin (A305), a child’s abdomen (CRL-1474), or the forearm of a mature adult (A35) (Fig. 3Go).



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  		Figure 3. AGE BSA effects on FN mRNA are observed in human skin fibroblasts. Soluble AGE BSA or control BSA at 100 µg/ml was added to duplicate wells of confluent primary cultures of human skin fibroblasts from multiple donors under serum-free conditions, and total RNA was collected at the time points shown. FN mRNA was then determined by quantitative RT-PCR in triplicate for each sample. The FN mRNA level is expressed in arbitrary units, normalized to 18S. The donor age and skin site of the fibroblasts studied are: 7-yr-old male abdomen (CRL-1474); 70-yr-old male forearm (A35); and newborn foreskin (A305). The means ± 1 SD of three independent experiments are shown.

 
The 2097 fibroblasts were used in all subsequent experiments. The addition of pure rhCTGF protein (500 ng/ml) caused an induction of FN mRNA. Up-regulation was observed at 24 h and 48 h after reagent addition (Fig. 4AGo). In a parallel fashion, CTGF mRNA was autoinduced by rhCTGF treatment (Fig. 4AGo). Such autoinduction of CTGF mRNA, observed with the addition of recombinant CTGF protein, is consistent with a similar effect of added CTGF protein previously described in cultured rodent renal mesangial cells (27). A dose-response study from 0 to 500 ng/ml rhCTGF showed that statistically significant increases in FN mRNA at 24 h after treatment occurred when 100 ng/ml or more rhCTGF protein was added to the conditioned media (Fig. 4BGo). In parallel with the induction observed for FN, a dose-response induction of CTGF mRNA by added rhCTGF was also seen with a statistically significant increase occurring from 250 ng/ml added protein (Fig. 4BGo).



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  		Figure 4. Induction of FN mRNA and CTGF mRNA autoinduction by soluble rhCTGF. RhCTGF was added to human foreskin fibroblasts (2097 cells) under serum-free conditions. Total RNA was collected and FN mRNA and CTGF mRNA were both analyzed in A and B by quantitative RT-PCR in triplicate for each sample, with correction in each sample for 18S. A, Time course over 2 d, at 500 ng/ml rhCTGF. B, Dose-response induction of FN mRNA and CTGF mRNA using 0–500 ng/ml rhCTGF with isolation of total RNA at 48 h. The means ± 1 SD of four independent experiments are shown in A and B. *, P < 0.05 vs. no addition on the same curve.

 
To determine whether AGE and rhCTGF treatment up-regulated FN protein levels, conditioned media were collected and analyzed by Western immunoblots and by FN ELISA. Immunoblot analysis indicated that in the presence of no addition, with serum free media alone, basal levels of FN protein accumulated in the conditioned media (Fig. 5AGo), a finding that has been observed by others in human fibroblasts (28). After treatment with rhCTGF, increased FN protein, compared with the no addition control, was detected in a time- and dose-dependent manner (Fig. 5Go, A and B). The addition of control BSA resulted in an accumulation of soluble FN protein (Fig. 5CGo). Over the 3 d of the study, after treatment of cells with AGE BSA, FN protein levels were further increased above that of the control BSA (Fig. 5CGo). In whole-cell lysates, an increase in FN protein was also observed after rhCTGF and AGE treatment, compared with either no addition or control BSA (data not shown). Quantitation by FN ELISA confirmed the media Western immunoblot results (Fig. 5Go, D through E). FN protein in conditioned media after 48 h treatment with 500 ng/ml rhCTGF was approximately 1.6-fold higher than in cells in which serum-free media were added alone (Fig. 5DGo). Increases in FN media protein by ELISA after AGE treatment peaked at 1.9-fold at 48 h and remained significant at 72 h at 1.4-fold (Fig. 5EGo). The progressive accumulation of soluble FN protein in the media seen after control BSA addition did not differ statistically from that of the no reagent addition, serum-free control (Fig. 5Go, D and E). When measured by ELISA in media derived from three independent experiments in which both controls were studied, the FN protein level averaged 2.49 µg/ml at 24 h by no addition and 1.95 µg/ml after control BSA addition and 5.04 µg/ml at 48 h by no addition and 4.11 µg/ml at 48 h after control BSA addition. Thus, after both AGE and CTGF treatment, the increases in FN-soluble protein were increased above the controls, and these increases were somewhat less marked than the observed increases in FN mRNA.



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  		Figure 5. Increases in FN protein in conditioned media by AGE and rhCTGF. Duplicate wells of confluent primary cultures of human fibroblasts (CRL-2097) under serum-free conditions were incubated with rhCTGF (500 ng/ml) for 24–48 h (A) and increasing amounts of rhCTGF (0–500 ng/ml) for 24 h (B). Conditioned media were analyzed for FN immunoreactivity by Western immunoblot after SDS-PAGE under reducing conditions. C, Cells cultured as in A were treated with AGE BSA or control BSA at 100 µg/ml for 24–72 h, and then FN immunoreactivity was detected in the conditioned media by Western immunoblot. For A–C, molecular mass markers are shown to the left of the figure, and representative immunoblots are shown from three experiments, each showing equivalent results. D, Media levels of FN measured by FN ELISA, as described in Materials and Methods, after treatment of cells with rhCTGF for 24 and 48 h, compared with no addition. *, P < 0.05 vs. no addition on the respective day. E, Media levels of FN measured by FN ELISA, after treatment of cells with AGE BSA or control BSA each for 24–72 h. *, P < 0.05 for AGE BSA vs. control BSA on the respective day. For D and E, data shown are means ± 1 SD from three independent experiments.

 
We have recently reported that in the same dermal fibroblast system and under the same cell culture conditions, AGE BSA induces up-regulation of CTGF mRNA and protein (13). Increases in CTGF mRNA were detectable from 8 h onward after AGE addition to the media and became most marked after 72 h of AGE treatment and were seen using 10 µg/ml or more of the AGE BSA (13). Increases in CTGF protein in the conditioned media were detectable within 24 h of AGE treatment and continued to increase for the study duration of 72 h (13).

Based on our present observation that both AGE and rhCTGF up-regulate FN and that AGE increases endogenous CTGF in fibroblasts, we tested whether cell-derived CTGF, in an autocrine manner, is a contributor to the observed increase in FN mRNA following AGE treatment. Using an IgG affinity purified fraction of polyclonal antibody against rhCTGF, the induction of FN at 48 h by exogenously added rhCTGF (250 ng/ml) was fully inhibited (Fig. 6AGo). In contrast, no inhibition of FN mRNA up-regulation by rhCTGF occurred when the same amount of normal rabbit serum IgG was added exogenously to cells (Fig. 6AGo). In other experiments, the anti-CTGF antibody inhibited rhCTGF-induced increases in FN mRNA after 24 h of rhCTGF treatment (data not shown). In addition, the autoinduction of CTGF mRNA by rhCTGF, described in Fig. 4Go, was fully blocked by the CTGF antibody (data not shown). These results confirm that the anti-CTGF antibody specifically neutralizes the bioactivity of CTGF in this system.



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  		Figure 6. AGE induction of FN mRNA is partly mediated by endogenous CTGF. A, Duplicate wells of confluent primary cultures of human fibroblasts (CRL-2097) under serum-free conditions were preincubated with anti-CTGF IgG (40 µg/ml), NRS-derived IgG (40 µg/ml), or no IgG. Then AGE BSA or control BSA each at 100 µg/ml or rhCTGF (250 ng/ml) was added. Total RNA was collected 48 h later and FN mRNA analyzed by real-time quantitative RT-PCR. The mRNA levels are expressed in arbitrary units. The means ± 1 SD of four independent experiments are shown. **, P < 0.01 for rhCTGF with no IgG addition *, P < 0.05 vs. AGE BSA with no IgG addition. B, The same protocol was followed as in A, except that a further addition of anti-CTGF IgG or NRS IgG, each at 40 µg/ml, was made to the media bathing the cells, followed by RNA collection at 96 h and analysis for FN mRNA. Results are the means ± 1 SD from two independent experiments. *, P < 0.05; **, P < 0.01 for AGE BSA with anti-CTGF IgG preincubation vs. AGE BSA with preincubation of NRS IgG, on the respective day.

 
The ability of the CTGF-neutralizing antibody to block AGE induction of FN mRNA was then assessed. When the CTGF antibody was added before AGE treatment of cells, the induction of FN mRNA at 48 h by AGE, was attenuated by about 42%, compared with no IgG addition (Fig. 6AGo). In contrast to the effect of the anti-CTGF IgG, the same amount of normal rabbit serum IgG had no obvious effects (Fig. 6AGo). A longer-term protocol to 96 h was then performed, with the anti-CTGF IgG or normal rabbit serum IgG also added at 48 h (Fig. 6BGo). These studies showed that anti-CTGF IgG abrogated the induction of FN mRNA by AGE by about 82% at 96 h, compared with the effect of normal rabbit serum IgG (Fig. 6BGo). Higher concentrations of CTGF antibody and more frequent treatments did not produce any greater inhibitory effect on AGE induction of FN (data not shown). These data show that endogenous CTGF contributes to the induction of FN by AGE in this cell system.

Recent studies have implicated PKC in the induction of FN in a diabetic environment (12, 31). Because AGE can regulate PKC isoforms in some systems (32), we tested whether PKC activity might be involved in the induction of FN by AGE and/or by rhCTGF. Cells were preincubated with a PKC inhibitor for 2 h, followed by addition of reagent (rhCTGF or AGE), and total RNA was collected at 48 h. Preincubation of cell monolayers with the pan-specific PKC inhibitor GF109203X at 0.2 µM caused an inhibition of AGE induction of FN mRNA by about 72% (Fig. 7AGo). In contrast, there was no statistically significant inhibition of CTGF mRNA induction by AGE (Fig. 7BGo). In a similar manner, preincubation of cells with GF109203X reduced rhCTGF induction of FN mRNA to basal levels (Fig. 7CGo). Autoinduction of CTGF mRNA by rhCTGF, however, was not significantly inhibited by preincubation with GF109203X (Fig. 7DGo). The effects of GF109203X were observed at relatively low concentrations (0.2 µM), consistent with specificity of this inhibitor for PKC isoforms (33). In addition, no significant effects on basal (unstimulated) FN mRNA were observed, suggesting that the PKC blocker was specifically inhibiting the activity of these reagents on FN mRNA induction (Fig. 7Go, A and C). No further specific inhibitory effects on FN mRNA or CTGF mRNA were seen with higher concentrations (up to 5 µM) of GF109203X (data not shown). These results indicate that in this cell system, cellular PKC activity is required for optimal induction of FN mRNA by both AGE and rhCTGF but not for the induction of CTGF mRNA by either AGE or rhCTGF.



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  		Figure 7. FN mRNA up-regulation by AGE BSA and by rhCTGF is blocked by the pan-specific PKC inhibitor, GF109203X. A, Duplicate wells of confluent primary cultures of human fibroblasts (CRL-2097) under serum free conditions were preincubated for 2 h in the presence or absence of the PKC inhibitor GF109203X (0.2 µM). Then AGE or control BSA (each at 100 µg/ml) in A and B or rhCTGF (250 ng/ml) in C and D was added, and RNA was collected at 48 h. FN mRNA in A and C and CTGF mRNA in B and D, each analyzed by real-time quantitative RT-PCR and corrected for 18S are shown. For A–D, the means ± 1 SD of three independent experiments are shown. *, P < 0.05 vs. same reagent addition but with no addition of GF109203X.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes the stimulation of FN mRNA and protein following treatment of human skin fibroblasts with advanced glycosylation end-products and with rhCTGF and the demonstration that CTGF contributes to AGE up-regulation of FN mRNA. The effect of the synthesized AGE reagent on FN mRNA induction was seen to be caused by products of nonenzymatic glycosylation because coincubation of aminoguanidine, an inhibitor of nonenzymatic glycosylation, with glucose and BSA did not affect FN mRNA, and no effect was seen with control BSA alone. The up-regulation of FN mRNA in this cell model by the synthesized AGE reagent was mediated by AGE rather than by earlier products of nonenzymatic glycosylation, such as Amadori products, because the use of an antibody specific for AGE, which does not bind Amadori products, inhibited the AGE induction of FN gene expression. The increase in soluble FN protein in this system by AGE and rhCTGF were greater than the accumulation of FN protein over time, which was seen in the presence of either no addition or after the addition of control BSA.

The up-regulation of FN by AGE is generalizable to skin fibroblasts from differing sources and passage number. In each of the four fibroblast cell lines studied, AGE up-regulated FN. In the cell line most extensively studied, CRL-2097, FN was regulated by AGE in early passages (passage 4) and also at later passage (passage 12). The concentrations of AGE BSA used in these experiments approximate those used in vitro in other studies exploring biological effects of AGE on cells (7, 34). In addition to AGE BSA synthesized from glucose, we have also synthesized AGE BSA from glycolaldehyde as substrate (13) and have confirmed that AGE synthesized from glycolaldehyde also induces FN mRNA (data not shown). BSA was used as the protein for synthesizing AGE adduct because it is highly purified and delipidated and has commonly been used in AGE experiments by others (23, 24, 25). We have not yet studied other proteins made from AGE, such as AGE synthesized using extracellular matrix proteins. Few AGE components have to date been defined biochemically, and the specific glycosylation end-product(s) that might be mediating the effect on FN were not identified in this work.

Recent studies using AGE in human dermal fibroblasts have focused on AGE effects on type 1 collagen, rather then FN. These reports have shown that in contrast to the observed up-regulation of FN by AGE BSA in the current work, type 1 collagen mRNA and protein synthesis were inhibited by soluble AGE BSA treatment (35). This prior report and the current work are not inconsistent because regulation of FN and type 1 collagen gene transcription differs. Specifically, epidermal growth factor receptor activation, which was shown to mediate AGE inhibition of type 1 collagen gene expression in the previous work (35), is known to have a role in positively regulating FN transcriptional activity in fibroblasts (36, 37). What role the epidermal growth factor receptor may play in AGE up-regulation of FN, possibly in cooperation with CTGF, is an important topic to address in future studies.

CTGF is known to induce FN and to be profibrotic (16). The concentrations of soluble CTGF recombinant protein required in this cell system to induce FN mRNA and protein, at and above 100 ng/ml of rhCTGF, appear higher than others have employed in human fibroblasts (15). It is possible that the purified rhCTGF used in the current study is somewhat less bioactive than endogenous CTGF protein and that used by other groups and/or that quantitation of the purified protein differs between groups. Nonetheless, the current work shows that added CTGF induces FN mRNA and protein in this cell system. That the anti-CTGF IgG specifically and completely blocked FN induction by rhCTGF indicates that this antibody is efficient at blocking CTGF effects on the fibroblast cells. This same antibody was then seen to significantly attenuate AGE induction of FN in a specific manner.

This is the first study demonstrating that CTGF is a mediator in the induction of ECM by AGE, and it provides a potentially important link among AGE, growth factors, and fibrosis. The CTGF neutralizing antibody studies do not implicate CTGF as the only mediator of AGE induction of FN: that AGE induction of FN was only partially inhibited by CTGF-neutralizing IgG also implicates CTGF-independent pathways in AGE-induced increases in FN. We have previously reported that in the same system, a TGF-ß1 neutralizing antibody did not inhibit the induction of AGE by CTGF and that total TGF-ß1 levels were not detectably increased over the time course of the study (13). These results suggest that TGF-ß1 is not involved in the role played by CTGF in contributing to the AGE induction of fibronectin in this cell system. Up-regulation of CTGF in tissues in rodent models of diabetic nephropathy has recently been reported (27, 38). Although AGE has been shown to up-regulate FN in vivo (12), AGE as a reagent has not yet been reported in vivo to induce CTGF mRNA or protein. In contrast to our experimental model, AGE appears to accumulate slowly in vivo, particularly in long-lived proteins (1, 2), and in vivo studies are now required to further substantiate a role for CTGF in mediating diabetic and, specifically, AGE-related ECM expansion.

Up-regulation of PKC activity and PKC isoforms in a diabetic environment has been shown in vitro and in vivo in cells and in tissues that are susceptible to diabetic complications (31), and inhibition of PKC activity may attenuate chronic diabetes-related events (12). Although effects on PKC activity have been well described for high extracellular D-glucose (9) and early products of nonenzymatic glycosylation (39, 40), only recently has AGE been implicated in up-regulating PKC activity (32) and potentially using PKC pathways in inducing diabetic complications (41), indicating that the pathological effects of AGE and PKC on tissues may be interrelated at the level of induction of PKC by AGE. In addition, PKC pathways have been shown to regulate FN induction in various cell types, including human fibroblasts (30). The PKC inhibitor concentrations of GF109203X used in the current work (0.2 µM) are consistent with the published amounts required for specifically blocking PKC activity of both conventional and novel PKC isoforms (33). Although the isoform(s) of PKC that is mediating the effect of AGE on FN remains to be described, this study demonstrates a link between AGE and PKC in human dermal fibroblasts, two major proposed mechanisms involved in the pathogenesis of diabetic complications.

The intracellular second messenger systems mediating the up-regulation of ECM by CTGF have been studied to a limited extent only. Inhibition of the induction of type 1 collagen by cAMP in human dermal fibroblasts has been described without evidence of regulation by cellular PKC activity (42). Second messenger systems affecting the previously observed up-regulation of FN mRNA by rhCTGF in human fibroblasts (15) have not been reported before this work. The current studies show that the induction of FN mRNA by rhCTGF is fully blocked by a pan-specific PKC inhibitor in this cell system. These data using the PKC inhibitor do not indicate the number of cellular signaling intermediates involved and the sequence of the effect. Further work is needed to better define the second messenger pathways involved, and the specific PKC isoform(s) mediating the effect is also part of an ongoing study. CTGF cellular signaling in fibroblast cells has been linked to CTGF-induced activation of cell surface receptors, such as the platelet-derived growth factor receptor (43) and, when in solid phase, the integrin receptor, {alpha}6ß1 (44). Which of these receptors, if any, is involved in FN up-regulation by CTGF, through a PKC-dependent mechanism, will require further study.

Recently, PKC activity has been shown to regulate CTGF mRNA in fibroblasts. A previous report showed that blocking conventional and novel PKC isoform activity in combination by using GF109203X and other PKC inhibitors, or PKC depletors, caused an induction of CTGF mRNA under conditions of high FCS in the conditioned media (45). The FCS content likely contributed to prominent basal PKC activity and the detection of the inhibition of CTGF gene expression by basal PKC activity (46). That only a slight and nonsignificant induction of CTGF mRNA was observed by GF109203X in the current study in the basal state (Fig. 7Go) may reflect low cellular PKC activity in these serum-deprived cells. Low PKC activity under serum-free conditions has been observed previously by other groups (47). As well as having no significant effect in the basal state, GF109203X also did not significantly affect the autoinduction of CTGF mRNA by rhCTGF in the current work (Fig. 7DGo).

The finding that CTGF is a mediator in AGE induction of FN in vitro may have relevance in diabetic complications. Based on previous work, there is a rationale to potentially link AGE effects and diabetic complications with the induction of CTGF and ECM in skin and, by association, with pathology in other tissues. A feature commonly present in human diabetes is skin thickening and contracture, termed diabetic sclerosis. This affects mainly the distal extremities and is characterized by expansion of extracellular matrix, fibroblast proliferation, and angiogenesis (48). The presence of overt diabetic sclerosis of skin is correlated with the presence and future development of other end-organ complications (49). AGE products are increased in human diabetic skin (3) as is FN (50, 51), and the levels of AGE in skin also correlate positively with the presence of diabetic microvascular kidney and eye disease (3, 4). That the ability of CTGF to induce fibrosis has been well characterized in skin (15, 42) makes skin fibroblasts a relevant cell model for the current study.

Our work provides potential links in diabetic complications characterized by ECM expansion. Interactions between AGE effects and PKC activity have been described, and AGE induced up-regulation of the profibrotic agent CTGF, which itself contributes to AGE-induced ECM expansion through a PKC-dependent mechanism, has been observed. This work contributes toward further understanding mechanisms involved in the development of chronic diabetes complications, particularly those characterized by ECM expansion and fibrosis.


   Acknowledgments
 
We thank Alteon, Inc. (Ramsey, NJ) for measuring the CML adduct concentration in the AGE reagent by their CML ELISA. The generous gift of the anti-AGE antiserum from Dr. Miyata at Kissei Pharmaceutical Co. Ltd. (Hotaka, Japan) is gratefully acknowledged.


   Footnotes
 
This work was supported by National Health and Medical Research Council of Australia (S.M.T., C. J. Martin Postdoctoral Fellowship); NIH Grants CA-58110 and DK-51513 and U.S. Army Medical Research Grant DAMD 17-00-1-0042 (to R.G.R.); and American Cancer Society Grant RPG-99-103-01-TBE (to Y.O.).

Abbreviations: AGE, Advanced glycosylation end-products; CML, carboxymethyl lysine; CTGF, connective tissue growth factor; ECM, extracellular matrix; FN, fibronectin; IGFBP-rP, insulin-like growth factor binding protein related protein; NRS, normal rabbit serum; rhCTGF, recombinant human connective tissue growth factor.

Received August 27, 2001.

Accepted for publication December 12, 2001.


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