From the 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
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ABSTRACT |
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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 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- 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- Cyclic stretching increases production and release of various
vasoactive substances and growth factors in cultured cells, including
TGF- 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- 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.
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- 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- 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- 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 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.
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 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- 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.
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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-
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-1
,
interleukin-4, tumor necrosis factor-
, and vascular endothelial
growth factor (VEGF) in regulation of CTGF (6, 7). Thus far, TGF-
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-
-responsive
element within the promoter sequence of the CTGF gene, where point
mutation of this responsive element abolished the regulatory effect of
TGF-
on CTGF gene expression. More importantly, in anti-Thy1
nephritis, TGF-
and CTGF are expressed in coordinate fashion,
suggesting the important role of TGF-
in regulating CTGF expression
in vivo (3, 6).
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).
, 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
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-neutralizing antibody
was obtained from (R&D Systems). Treatment with TGF-
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).
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ABSTRACT
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RESULTS
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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-
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- -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-
-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).
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- . 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-
-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-
-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-
-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).
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- -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.
-actin. The signal intensity of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase or
-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-
, TGF-
1, TGF-
2, and
TGF-
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- , TGF-
1, TGF-
2, and
TGF-
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.
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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).
-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.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. 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.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are:
CTGF, connective
tissue growth factor;
CMV, cytomegalovirus;
TGF-, transforming
growth factor
;
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.
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