Static Pressure Regulates Connective Tissue Growth Factor Expression in Human Mesangial Cells*,

Keiichi HishikawaDagger §, Barry S. Oemar, and Toshio NakakiDagger

From the Dagger  Department of Pharmacology, Teikyo University School of Medicine, Tokyo 173-8605, Japan and  Centocor, Inc., Malvern, Pennsylvania 19355

Received for publication, November 28, 2000, and in revised form, January 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders such as renal fibrosis and atherosclerosis. Fibrosis is a common final pathway of renal diseases of diverse etiology, including inflammation, hemodynamics, and metabolic injury. Mechanical strains such as stretch, shear stress, and static pressure are possible regulatory elements in CTGF expression. In this study, we examined the ability of static pressure to modulate CTGF gene expression in cultured human mesangial cells. Low static pressure (40-80 mm Hg) stimulated cell proliferation via a protein kinase C-dependent pathway. In contrast, high static pressure (100-180 mm Hg) induced apoptosis in human mesangial cells. This effect was reversed by treatment with CTGF antisense oligonucleotide but not with transforming growth factor beta 1-neutralizing antibody or protein kinase C inhibitor. High static pressure not only up-regulated the expression of CTGF, but also the expression of extracellular matrix proteins (collagen I and IV, laminin). This up-regulation of extracellular matrix proteins was also reversed by treatment with CTGF antisense oligonucleotide. As judged by mRNA expression of a total of 1100 genes, including apoptosis-associated genes using DNA microarray techniques, recombinant CTGF protein induced apoptosis by down-regulation of a number of anti-apoptotic genes. Overexpression of CTGF in mesangial cells by transient transfection had similar effects. Taken together, these results suggest that high blood pressure up-regulates CTGF expression in mesangial cells. High levels of CTGF in turn enhance extracellular matrix production and induce apoptosis in mesangial cells, and may contribute to remodeling of mesangium and ultimately glomerulosclerosis.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Connective tissue growth factor (CTGF)1 (1, 2) represents the latest addition to the list of growth factors implicated in the pathogenesis of renal fibrosis (3, 4). CTGF mRNA has been shown to be overexpressed in the extracapillary and severe mesangial proliferative lesions of crescentic glomerulonephritis, IgA nephropathy, focal, and segmental glomerulosclerosis and diabetic nephropathy (5). In anti-Thy1 nephritis, an animal model of acute glomerulonephritis, CTGF mRNA was shown to be up-regulated in both mesangial cells and podocytes (6). CTGF mRNA up-regulation in sclerotic glomeruli and fibrotic interstitium was also found in a chronic hypertension model in rats (uninephrectomized spontaneously hypertensive rats) (6). Hence, substantial evidence both in human disease as well as in experimental models of kidney disease suggests an important role of CTGF in renal diseases. Nonetheless, we have just begun to understand the regulation of CTGF expression in renal diseases. In particular, the role of high blood pressure in regulating CTGF expression has not yet been elucidated.

TGF-beta is thought to be the major pathogenic factor in the development of renal fibrotic disorders. However, the development and progression of renal sclerosis is determined by complex interactions of many factors, including growth factors and direct hemodynamic action. In human skin fibroblasts, CTGF mRNA is specifically induced by TGF-beta but not by platelet-derived growth factor, epidermal growth factor, or basic fibroblast growth factor (6). Recent reports also suggest a possible role for hepatic growth factor, interleukin-1beta , interleukin-4, tumor necrosis factor-alpha , and vascular endothelial growth factor (VEGF) in regulation of CTGF (6, 7). Thus far, TGF-beta has been shown to be the strongest inducer of CTGF expression in a variety of cells derived from different organs (8). In fact, Grotendorst et al. (8) found a unique TGF-beta -responsive element within the promoter sequence of the CTGF gene, where point mutation of this responsive element abolished the regulatory effect of TGF-beta on CTGF gene expression. More importantly, in anti-Thy1 nephritis, TGF-beta and CTGF are expressed in coordinate fashion, suggesting the important role of TGF-beta in regulating CTGF expression in vivo (3, 6).

Hypertension has been shown to independently accelerate the progression of chronic renal failure in humans irrespective of the pathogenesis of the renal disease (9, 10). The potential role of local hemodynamic changes on the progression of glomerulosclerosis and renal fibrosis has also been extensively studied in animal models. In patients with hypertension and diabetic nephropathy, the autoregulation of glomerular pressure is impaired, resulting in exposure of the capillary bed to systemic blood pressure fluctuation reaching unphysiologically high levels of local pressure. In normal tissue, this peak level of pressure is absorbed by the elasticity of the mesangium in the kidney glomeruli. The increased capillary pressure causes the mesangial area, including the mesangial cells to spread and expand. At the same time, the increased pressure also compresses the cells in a tangential direction. Chronic exposure to high blood pressure eventually leads to chronic expansion and remodeling of the mesangial area with accumulation of extracellular matrix and finally glomerulosclerosis. Numerous locally produced factors, including TGF-beta and PDGF have been implicated in this mechanically induced mesangial expansion and remodeling. However, it is impossible to separate the effect of stretch and tangential compressive strain by pressure, in vivo. Therefore, to study these differential effects of mechanical strain on mesangial cells, we have utilized in this study an in vitro model in which only static pressure was applied to mesangial cells in culture without stretch (11-14).

Cyclic stretching increases production and release of various vasoactive substances and growth factors in cultured cells, including TGF-beta , which in turn up-regulates CTGF mRNA expression (15-20). So far, the role of static pressure in regulating CTGF expression in mesangial cells has not been elucidated. We now provide direct evidence that high static pressure up-regulates CTGF levels in CTGF-mediated apoptosis.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- Human renal mesangial cells were obtained from Clonetics (21). Cells were cultured in MsGM (Clonetics), and cells from passages 2 to 4 from three different isolates were used for experiments. All experiments were performed after 24 h of incubation in 1% fetal bovine serum. TGF-beta 1-neutralizing antibody was obtained from (R&D Systems). Treatment with TGF-beta 1 (Roche Molecular Biochemicals) for 48 h reduced cell viability of human mesangial cells in a concentration-dependent manner (1-10 ng/ml), and pretreatment with the neutralizing antibody (10 µg/ml) completely prevented it. On the other hand, treatment with PDGF-BB (Roche Molecular Biochemicals) (5 ng/ml) stimulated cell proliferation in human mesangial cells, but pretreatment with the neutralizing antibody never prevented it. The specificity and efficacy of the neutralizing antibody is available on the Web from R&D Systems. Chelerythrine was purchased from LC laboratories. Recombinant CTGF protein and polyclonal anti-CTGF antibody were generous gift of Japan Tobacco (Osaka Japan).

Exposure of Mesangial Cells to Constant Static Pressure-- The pressure-loading apparatus was set up as previously described (11-14). The culture plates and dishes were placed in the pressure chamber containing 5% CO2, and the chamber was completely sealed using clamps. The internal pressure was raised using compressed helium gas, and the chamber was kept at 37 °C in a thermal incubator. Whereas the helium gas was being pumped into the sealed chamber, no air in the sealed chamber was released, so that the partial pressures of the gases contained in the chamber such as oxygen, nitrogen oxide, and carbon dioxide were kept constant in accordance with Boyle-Charles's law. In fact, there were no significant changes in pH and partial pressure of oxygen in culture medium throughout the experiments (up to 48 h). Mesangial cells were grown under confluent conditions to minimize the possibility of stretch and spreading under high static pressure.

Preparation of Antisense and Scrambled CTGF Oligonucleotide-- 16-mer CTGF antisense phosphorothioate oligonucleotide (5'-TACTGGCGGCGGTCAT-3') containing the initial ATG translation start site was synthesized and purchased from Amersham Pharmacia Biotech as described previously (22). An oligonucleotide containing a scrambled nucleotide sequence (5'-GGTCTAGCTTGCGGAC-3') was used as control. The synthetic oligonucleotides were added directly to the cell culture medium (final concentration, 20 µg/ml).

Cell Viability-- Cell viability was evaluated using an MTT assay kit (Roche Molecular Biochemicals) according to the manufacturer's instruction (23). After exposure to increased static pressure (40-160 mm Hg), cells were incubated for an additional 4 h in the presence of MTT reagent under atmospheric pressure. Cells were then lysed with lysis buffer provided in the kit, incubated overnight at 37 °C, and absorbance was measured at A550 nm to A690 nm.

Nuclear Morphology-- Both floating and trypsinized adherent mesangial cells were collected, washed with phosphate-buffered saline, fixed with fresh 10% paraformaldehyde for 30 min, and incubated in Hoechst 33258 (Sigma) at room temperature for 30 min (final concentration, 30 µg/ml) (23). Nuclear morphology was examined using fluorescence microscopy with standard excitation filters. To calculate the percentage of apoptotic cells, all cells from four randomly selected microscopic fields were counted at 400× magnification.

DNA Fragmentation-- Tdt-mediated dUTP biotin nick end-labeling (TUNEL) was performed with the In Situ Cell Death Detection Kit from Roche Molecular Biochemicals according to the manufacturer's instructions (23). To calculate the percentage of TUNEL-positive cells, all cells from four randomly selected microscopic fields were counted at 100× magnification. DNA fragmentation was measured using the Cell Death Detection ELISAPLUS kit (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Western Blot Analysis-- Cell lysates (20 µg) were subjected to 12.5% percentage gel SDS-polyacrylamide gel electrophoresis (Ready Gel, Bio-Rad), transferred to polyvinylidene difluoride membranes (Bio-Rad), and incubated with anti-CTGF antibody at 1:250 dilution for 1 h as previously described (24). Equal amounts of protein loading were confirmed by Coomassie Brilliant Blue staining before blotting. The membranes were visualized using an ECL kit (Amersham Pharmacia Biotech). Semi-quantitative analyses of the blots were performed using the public domain IMAGE 1.60 program for Apple Macintosh from the National Institutes of Health.

Plasmid Constructs-- To overexpress the CTGF protein in human mesangial cells, a mammalian expression vector (pCMV-CTGF) containing the complete open reading frame of the CTGF gene, driven by the CMV promotor, was constructed (23, 24). Mesangial cells transfected with pCMV vector alone were used as controls. Transient transfection was performed using Superfect reagent (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. Transfection efficiency was evaluated by fluorescence microscopy in cells co-transfected with plasmid containing the green fluorescence protein gene (pEGFP-C1)(CLONTECH). The average transfection efficiency using 1 µg of pEGFP-C1 and 1 µg of pCMV-CTGF in 1 × 105 human mesangial cells was calculated to be about 30%.

DNA Microarray-- The DNA microarray hybridization experiments were performed using Intelligene DNA chips (codes X101, X102, X103) (Takara, Tokyo, Japan) according to the manufacturer's protocol. The protocol and the complete listing of genes on Intelligene DNA chips are available on the Web. The DNA arrays were scanned using ScanArray (GS Lumonics), and the data were analyzed using the Quant Array (BM BIO, Tokyo, Japan). Results of important genes are shown in Figs. 4 and 7, and all results are provided in the supplemental material.

Statistics-- Statistical analysis of the data was performed using analysis of variance followed by Fisher's test. p < 0.05 was considered statistically significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Low Static Pressure Stimulates Cell Proliferation, but High Static Pressure Reduced Cell Viability-- Exposure of mesangial cells to low static pressure (80 mm Hg) for 48 h significantly increased the number of viable cells as compared with cells exposed to atmospheric pressure (Fig. 1A). This increase was inhibited by chelerythrine (CHE, 0.6 µM), a selective protein kinase C inhibitor, but not by CTGF antisense oligonucleotide (AS, 20 µg/ml), scrambled oligonucleotide (SC, 20 µg/ml), or TGF-beta 1-neutralizing antibody (nAb, 10 µg/ml) (Fig. 1B). In contrast, exposure of human mesangial cells to high static pressure (140 and 180 mm Hg) significantly reduced cell viability as compared with cells exposed to atmospheric pressure (Fig. 1A), and this effect was reversed by treatment with CTGF antisense oligonucleotide but not by scrambled oligonucleotide, protein kinase C inhibitor, or TGF-beta 1-neutralizing antibody at the same concentrations (Fig. 1C).


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Fig. 1.   Effects of static pressure on mesangial cell viability as measured by MTT assay. A, exposure of mesangial cells to low static pressure (up to 80 mm Hg) for 48 h increased the number of viable cells. However, high static pressure reduced the number of viable cells in a pressure-dependent manner with the largest reduction at 180 mm Hg. B, the increase in the number of viable mesangial cells exposed to low static pressure (80 mm Hg) was significantly inhibited by treatment with protein kinase C inhibitor (CHE: 0.6 µM) but not with CTGF antisense oligonucleotide (AS), scrambled oligonucleotide (SC), or anti-TGF-beta -neutralizing antibody (nAB). C, the reduction of the number of viable mesangial cells exposed to high static pressure (140 mm Hg) could be reversed by treatment with CTGF antisense oligonucleotide only but not with protein kinase C inhibitor (CHE), scrambled oligonucleotide (SC), or anti-TGF-beta -neutralizing antibody (nAB). Results are presented as the mean ± S.E. of four independent experiments. *, p < 0.05 compared with 0 (atmospheric pressure) (n = 8). **, p < 0.05 compared with 80 (80 mm Hg) (n = 8). ***, p < 0.05 compared with 140 (140 mm Hg) (n = 8).

High Static Pressure Induces Apoptosis via CTGF-- To investigate the mechanisms of high static pressure-induced reduction in cell viability, we performed nuclear staining of mesangial cell culture using Hoechst nuclear stain. As shown in Fig. 2A, exposure to high static pressure (140 mm Hg) significantly increased the number of apoptotic cells. DNA fragmentation analysis (Fig. 2B) and TUNEL staining (Fig. 2C) confirmed that the reduced cell number is due to the increase in the number of apoptotic cells. This effect was inhibited by treatment with CTGF antisense oligonucleotide (20 µg/ml). Control experiments using scrambled oligonucleotide (20 µg/ml) or TGF-beta 1-neutralizing antibody (10 µg/ml) had no effect (Fig. 2, A-C), suggesting that CTGF directly mediates the high static pressure-induced apoptosis in these cells.


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Fig. 2.   High static pressure induces apoptosis via CTGF independent of TGF-beta . A, exposure to high static pressure increased the number of apoptotic mesangial cells as judged by nuclear morphology. Treatment with CTGF antisense oligonucleotide (AS), but not with scrambled oligonucleotide (SC) or anti-TGF-beta -neutralizing antibody (nAB) reduced the number of apoptotic cells. B, similar effect on histone-associated DNA fragmentation was found in mesangial cells exposed to high static pressure. Again, treatment with CTGF antisense oligonucleotide (AS), but not with scrambled oligonucleotide (SC) or anti TGF-beta -neutralizing antibody (nAB), reduced the histone-associated DNA fragmentation. C, the result of both nuclear staining and DNA fragmentation was confirmed by TUNEL staining. The number of TUNEL-positive mesangial cells was reduced only in cells treated with CTGF antisense oligonucleotide (AS), but not with scrambled oligonucleotide (SC) or anti TGF-beta -neutralizing antibody (nAB). Results are presented as the mean ± S.E. of four independent experiments. *, p < 0.05 compared with 0 (atmospheric pressure) (n = 8); **, p < 0.05 compared with 140 (140 mm Hg) (n = 8).

High Static Pressure Induces CTGF-- To clarify whether high pressure induced CTGF expression at protein level as well, we performed Western blot analysis of CTGF protein expression in human mesangial cells exposed to 80 or 140 mm Hg static pressure for 48 h. As shown in Fig. 3, exposure to high static pressure (140 mm Hg), but not to low static pressure (80 mm Hg), significantly increased CTGF protein expression. This effect was significantly reduced by treatment with CTGF antisense oligonucleotide (20 µg/ml) but not by scramble oligonucleotide (20 µg/ml) or TGF-beta 1-neutralizing antibody (10 µg/ml) (Fig. 3B). Treatment with protein kinase C inhibitor Chelerythrine up to 0.6 µM also had no effect (data not shown).


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Fig. 3.   High static pressure induced CTGF protein expression in human mesangial cells as judged by Western blot analysis. A, exposure of mesangial cells to low static pressure (80 mm Hg) showed no effect on CTGF protein expression. B, exposure of mesangial cells to high static pressure (140 mm Hg) markedly increased CTGF protein level. This increase in CTGF protein could be reversed by treatment with CTGF antisense oligonucleotide (AS), but not with scrambled oligonucleotide (SC) or anti TGF-beta -neutralizing antibody (nAb). Upper panel, representative Western blot is shown. Lower panel, semiquantitative analysis of three independent experiments. Results are presented as mean ± S.E. *, p < 0.05 compared with atmospheric pressure (0) (n = 4). **, p < 0.05 compared with 140 (140 mm Hg) (n = 4). 0, 0 mm Hg; 140, 140 mm Hg.

High Static Pressure Induces Mesangial Cell Matrix Production-- To investigate the effects of pressure-induced CTGF overexpression on matrix production, human mesangial cells were incubated in the presence and absence of CTGF antisense oligonucleotide for 24 h. Again, scrambled oligonucleotide was used as control. The expression of extracellular matrix mRNA was analyzed using Intelligene DNA chips. The signal intensity of the DNA arrays between treated and non-treated samples was adjusted to that of glyceraldehyde-3-phosphate dehydrogenase or beta -actin. The signal intensity of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase or beta -actin was unchanged in treated versus non-treated samples and was arbitrarily set to 1 for use as a control. As compared with cells exposed to atmospheric pressure, exposure to 140 mm Hg static pressure up-regulated not only CTGF mRNA but also collagen types I and IV and laminin mRNA expression in human mesangial cells (Fig. 4 A). On the other hand, mRNA expression of TGF-alpha , TGF-beta 1, TGF-beta 2, and TGF-beta 3 was never changed by high static pressure (Fig. 4A). Under this condition (140 mm Hg), addition of CTGF antisense oligonucleotide (20 µg/ml) to the culture medium down-regulated CTGF, collagen types I and IV and laminin mRNA but never modulated that of fibroblast growth factor, hepatocyte growth factor, vascular endothelial growth factor, and platelet-derived growth factor (Fig. 4B). Additional experiments using human mesangial cells treated with recombinant CTGF protein (10 µg/ml) showed similar effects (see Fig. 7A), suggesting an important and direct effect of CTGF in high static pressure-induced extracellular matrix production in these cells.


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Fig. 4.   CTGF mediates overexpression of extracellular matrix mRNA expression induced by high static pressure. A, exposure to high static pressure (140 mm Hg) did not change the mRNA expression of TGF-alpha , TGF-beta 1, TGF-beta 2, and TGF-beta 3, but increased CTGF mRNA expression up to 6-fold in human mesangial cells as compared with cells grown in atmospheric pressure. At the same time, type I and type IV collagen as well as laminin mRNA expression was induced up to 6-fold. B, the increase in CTGF, type I and type IV collagen, and laminin mRNA expression in human mesangial cells exposed to high static pressure was reversed by treatment with CTGF antisense oligonucleotide. In contrast, treatment with CTGF antisense oligonucleotide did not change mRNA expression of FGF, hepatocyte growth factor, VEGF, and PDGF. Results are presented as mean ± S.E. (n = 3-5). The -fold increase in mRNA expression is indicated in arbitrary units representing the ratio of mRNA expression in test conditions versus control conditions. Fluorescence intensity was normalized to that of housekeeping genes.

CTGF Induces Apoptosis in Human Mesangial Cells-- To investigate the direct effects of CTGF on cell survival, human mesangial cells were treated with recombinant human CTGF (0.05-10 µg/ml) for 48 h. As shown in Fig. 5 (A-C), treatment of mesangial cells with recombinant CTGF protein significantly reduced cell viability with increased DNA fragmentation and TUNEL-positive cells. In addition, transient transfection of human mesangial cells with an expression vector containing the complete open reading frame of the human CTGF gene driven by the CMV promoter (pCMV-CTGF) resulted in CTGF overexpression and significant reduction of cell viability, as well as increases in DNA fragmentation and TUNEL-positive cells (Fig. 6, A-C). Transfection of human mesangial cells with vector alone (pCMV) had no effect.


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Fig. 5.   Recombinant CTGF protein dose dependently induced apoptosis in human mesangial cells. Recombinant CTGF protein dose dependently decreased the number of viable human mesangial cells, as judged by MTT assay (A). Treatment of human mesangial cells with recombinant CTGF protein also dose dependently increased the number of apoptotic cells as judged by histone-associated DNA fragmentation (B) and increased TUNEL staining (C). Results are presented as the mean ± S.E. of four independent experiments. *, p < 0.05 compared with control (cont = vehicle) (n = 8).


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Fig. 6.   Overexpression of CTGF mRNA induced apoptosis in human mesangial cells. Transient overexpression of CTGF in human mesangial cells significantly decreased the number of viable cells as judged by MTT assay (A). Overexpression of CTGF also increased the number of apoptotic mesangial cells as judged by histone-associated DNA fragmentation (B) and TUNEL staining (C). Results are presented as the mean ± S.E. of four independent experiments. *, p < 0.05 compared with pCMV (control vector)-transfected cells (n = 8).

CTGF Down-regulates Anti-apoptotic Genes-- To elucidate the mechanism by which CTGF induces apoptosis in human mesangial cells, we performed DNA microarray experiments using Intelligene DNA chips. Cells grown in atmospheric pressure were treated with recombinant CTGF (10 µg/ml) for 24 h, and mRNA expression of 1100 genes was analyzed. As before, the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase or beta -actin were used as control. As shown in Fig. 7A, recombinant CTGF up-regulated mRNA expression of several extracellular matrix protein. Moreover, several anti-apoptotic genes, including BCL2-like2, BCL2A1, DAD1, apoptosis inhibitor 1, apoptosis inhibitor 2, and survivin were considerably down-regulated (up to 5-fold), and several caspase genes (caspases 1, 3, 7, 8, and 9) were up-regulated (up to 1.7-fold) in human mesangial cells treated with recombinant CTGF protein.


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Fig. 7.   Recombinant CTGF protein down-regulated the expression of anti-apoptosis genes as judged by DNA microarray analysis. In human mesangial cells treated with recombinant CTGF (10 µg/ml) for 24 h, several extracellular matrix genes were markedly up-regulated (up to 6-fold). The expression of several anti-apoptotic genes was markedly down-regulated (up to 5-fold) and that of several caspases was up-regulated (up to 2-fold). The -fold increase and decrease in mRNA expression was measured as arbitrary units and represents the ratio of mRNA expression exposed to recombinant CTGF versus vehicle control. Results are presented as mean ± S.E. (n = 3-5). Fluorescence intensity was normalized to that of housekeeping genes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we provide for the first time evidence that high static pressure induces up-regulation of CTGF expression and that this up-regulated CTGF expression is involved in overproduction of extracellular matrix and apoptosis in human mesangial cells. This is based on the following observations: (i) exposure to high static pressure increased CTGF mRNA and protein expression in human mesangial cells; (ii) exposure to high static pressure induced apoptosis in human mesangial cells, and this effect was reversed by treatment with CTGF antisense oligonucleotide; (iii) exposure to high static pressure as well as treatment with recombinant CTGF protein increased mRNA expression of extracellular matrix protein in human mesangial cells, and this effect was inhibited by treatment with CTGF antisense oligonucleotide; (iv) treatment with recombinant CTGF protein or transient overexpression of the CTGF gene induced apoptosis in human mesangial cells; and (v) treatment with recombinant CTGF protein down-regulated anti-apoptotic genes.

Circumstantial evidence suggests an important role of CTGF in the development of glomerulosclerosis in patients with diabetic nephropathy (3, 5). Although the mechanism by which CTGF affects renal function in vivo is not yet clear, Murphy et al. (22) recently demonstrated that high glucose stimulates mesangial CTGF expression and matrix production by inducing TGF-beta . Hypertension and hyperglycemia are well known independent pathogenic factors in the development of glomerulosclerosis in diabetes patients. Indeed, the present study provides the evidence that CTGF expression in cultured mesangial cells is regulated by static pressure. Increased CTGF activity in turn increased extracellular matrix production and induced apoptosis in these cells, suggesting the important role of CTGF overexpression induced by high static pressure in mesangial expansion, mesangial matrix accumulation, remodeling, and, ultimately, glomerulosclerosis. Therefore, it is conceivable that high blood pressure may also affect the renal function via CTGF pathway similar to hyperglycemia.

The increase in proliferation of human mesangial cells induced by exposure to low static pressure (80 mm Hg) was abolished by treatment with the specific protein kinase C inhibitor chelerythrine (14). These results were in line with recent studies showing the inhibitory effect of protein kinase C inhibitors in proliferation of rat mesangial cells and rat vascular smooth muscle cells (13, 25). In contrast, our results showed that increased CTGF expression and apoptosis in human mesangial cells exposed to high static pressure is protein kinase C-independent, contrary to the effect of high glucose-induced CTGF expression in rat mesangial cells, which is protein kinase C-dependent (26). Interestingly, Fan et al. (27) reported that activation of protein kinase C inhibited CTGF induction. A recent report by Suzuma et al. (7) indicated that vascular endothelial growth factor (VEGF) induced up-regulation of CTGF expression in bovine retinal capillary cells. This effect was mediated primarily by phosphatidylinositol 3-kinase activation, whereas involvement of protein kinase C and ERK pathways were only minimal. These results suggest heterogeneity in the regulation of CTGF by a variety of signaling pathway in different cell types.

CTGF has been shown to be mitogenic to NRK cells and chemotactic to NIH3T3 cells (1) and to induce connective tissue cell proliferation and extracellular matrix synthesis in skin fibroblasts (reviewed in Ref. 2). On the other hand, CTGF is also expressed at very high levels in non-proliferating proliferating cell nuclear antigen-negative smooth muscle cells in atherosclerotic lesions in human (28). In human chondrosarcoma, proliferating cell nuclear antigen expression is negatively correlated with CTGF expression (29). Taken together, these findings support a pro-apoptotic function of CTGF in a variety of human cells. Indeed, we showed that transient overexpression of CTGF in human aortic smooth muscle cells (24) and human breast cancer cells (MCF-7) (23) as well as treatment of these cells with recombinant human CTGF protein induced apoptosis (23, 30). The recombinant CTGF protein used in the present study was able to stimulate cell proliferation of normal rat kidney cells in a dose-dependent manner.2 However, this same CTGF preparation has no mitogenic effect on rat or human mesangial cells, or rat or human vascular smooth muscle cells.2 Thus far, the reasons for these opposite effects of CTGF on different cell type are not clear. In our hands, transient overexpression of CTGF by transfection with the CTGF gene construct appears to be more effective than using purified recombinant CTGF protein in inducing apoptosis. Recently, Kubota et al. (31) found that overexpressed CTGF protein was predominantly localized intracellularly in COS-7 cells transfected with similar construct and that intracellular CTGF acts as an antimitogenic factor in these cells by modulating the cell cycle. Taken together, these observations corroborate our findings and suggest multiple functions for CTGF depending on the location and the cell types where CTGF is found.

To gain insight into the biological role of CTGF in human mesangial cells, we used DNA microarrays containing 1100 genes, including apoptosis-associated genes to analyze CTGF-regulated genes in human mesangial cells. As shown in Fig. 7, recombinant CTGF protein failed to up-regulate the 1100 genes contained in the DNA microarrays by more than 2.0-fold except extracellular matrix protein. In contrast, several anti-apoptotic genes were down-regulated by up to 5-fold, and several caspase genes were up-regulated by up to 1.7-fold in human mesangial cells treated with recombinant CTGF. Recombinant CTGF protein used in this study has also been shown to induce apoptosis in MCF-7 cells and human vascular smooth muscle cells by down-regulating bcl2 expression and increasing caspase 3 activity (24, 30). Antiapoptotic genes such as apoptosis inhibitor protein and bcl2 are known to inhibit caspase 3 activity. A specific inhibitor of caspase 3 such as Z-Asp(Ome)-Glu- (Ome)Val-Asp(Ome)-FMK (32) was also able to prevent CTGF-induced apoptosis in human mesangial cells.2 Hence, down-regulation and diminished inhibitory effect of inhibitor protein and bcl2 in human mesangial cells induced by high static pressure or CTGF overexpression may increase caspase 3 activity and mediate the apoptotic effect of high static pressure via the CTGF-dependent pathway.

Recently, Hahn et al. (33) reported that the regulation of CTGF expression in mesangial cells was mediated by changes in actin cytoskeleton. It is conceivable that static pressure may also modulate cytoskeleton and thus modulate CTGF expression. Mechanical stimuli such as shear stress, cyclic stretch, and static pressure could induce different alterations of the cell surface, and activate different signaling pathways, but the precise mechanism remains to be determined. For example, stretch increased 1,4,5-inositol trisphosphate concentration only transiently (34), but static pressure induced sustained high levels of 1,4,5-inositol trisphosphate production (13). Stretch also rapidly increased intracellular calcium concentration, but shear stress and static pressure did not (13, 35). These differences in response to mechanical stimuli may explain why high static pressure increased sustained high level CTGF mRNA expression and cyclic stretch only induced transient overexpression of CTGF mRNA in rat mesangial cells (20).

In conclusion, high static pressure appears to induce CTGF expression in human mesangial cells. The precise mechanism by which high static pressure regulates CTGF expression and apoptosis remains to be elucidated. Nevertheless, our results clearly show that hypertension regulates extracellular matrix production and apoptosis in human mesangial cells via CTGF and thus may play a more substantial role in accelerating the development of glomerulosclerosis than previously thought. Our results also suggest that CTGF could serve as a potential therapeutic target for the prevention and treatment of hypertension-induced mesangial expansion, remodeling, and sclerotic process in kidney diseases.

    FOOTNOTES

* 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.

The on-line version of this article (available at http://www.jbc.org) contains a table.

§ To whom correspondence should be addressed: Dept. of Pharmacology, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo, 173-8605, Japan. Tel.: 81-3-3964-1211 (ext. 2253); Fax: 81-3-3964-0602; E-mail: hisikawa@med.teikyo-u.ac.jp.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010722200

2 K. Hishikawa, B. S. Oemar, and T. Nakaki, unpublished data.

    ABBREVIATIONS

The abbreviations used are: CTGF, connective tissue growth factor; CMV, cytomegalovirus; TGF-beta , transforming growth factor beta ; TUNEL, TdT-mediated dUTP biotin nick end-labeling; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CHE, chelerythrine; SC, scrambled oligonucleotide; nAb, neutralizing antibody; ERK, extracellular signal-regulated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Bradham, D. M., Igarashi, A., Potter, R. L., and Grotendorst, G. R. (1991) J. Cell Biol. 114, 1285-1294[Abstract]
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