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Molecular Pathology Section, Division of Biomedical Sciences, Imperial College School of Medicine, London, United Kingdom.
Correspondence to Professor Roger M. Mason, Molecular Pathology Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London, SW7 2AZ, UK. Phone: +44 20 7594 3019; Fax: +44 20 7594 3022; E-mail: roger.mason{at}ic.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are two types of latent TGF-ß1 complex. A small latent complex consists of TGF-ß1 associated noncovalently with a disulfide-linked dimer of the N-terminal part of pro-TGF-ß1, referred to as the latency-associated peptide (LAP) (4,5). A large complex also contains latent TGF-ß1-binding protein (LTBP) disulfide-linked to LAP. LTBP does not itself play any role in latency but is involved in the secretion of TGF-ß1, storage in the extracellular matrix, and eventual activation of the factor (6).
In vitro studies show that the latent TGF-ß1 complex can be activated by conformational changes induced by pH or high temperature (7), limited proteolysis or deglycosylation of LAP (8,9,10), the effect of reactive oxygen species on redox-sensitive elements in the complex (11), or binding to several membrane and extracellular matrix components. Each of these mechanisms could occur in vivo, depending on the conditions in the tissue. However, one activation mechanism, initiated by the binding of the latent complex of TGF-ß1 to the extracellular matrix glycoprotein thrombospondin-1 (TSP-1), may be particularly important in vivo (12). Moreover, we hypothesize that this mechanism of TGF-ß1 activation is likely to be very important in diabetic nephropathy. On the one hand, elevated glucose upregulates the expression and secretion of TSP-1 in mesangial cells (13,14,15); on the other hand, it reduces the likelihood of protease-dependent activation. High-glucose conditions result in decreased activity of plasmin (16,17,18) and cathepsin D (19), the main proteolytic enzymes involved in TGF-ß1 activation.
TSP-1 is a trimer of disulfide-linked 180-kD subunits. It is found in platelet ß-granules and is produced by a number of other cell types, including mesangial cells. Each subunit consists of several domains that bind to matrix and cell surface proteins (20). The TSP-1 site responsible for activating latent TGF-ß1 is localized in a domain that consists of three type 1 repeats (21). These have homology to properdin and are also referred to as properdin repeats. Two amino acid sequences are implicated in TGF-ß1 activation: GGWSHW (amino acids 418 to 423) in the first type 1 repeat (or potentially DGWSPW in the second and GGWGPW in the third type 1 repeat) and KRFK (amino acids 412 to 415) between the first and the second type 1 repeats (22). It has been proposed (22) that latent TGF-ß1 is activated as a result of binding of the KRFK sequence of TSP-1 to the LSKL sequence of the amino-terminal region of LAP (23). However, this is only possible if the TSP-1 molecule is first oriented correctly by initial interaction between the GGWSHW sequence and the mature portion of TGF-ß1. This two-step mechanism of TGF-ß1 activation is based on the results of cell-free experiments with synthetic TSP-1—derived KRFK and GGWSHW peptides (22). It was shown that the minimal sequence able to activate TGF-ß1 is KRFK. GGWSHW peptide, while unable to activate TGF-ß1, is able to bind to the latent complex. Moreover, it blocks activation by TSP-1 and increases activation by KRFK peptide (22).
Treatment of cultured mesangial cells with TSP-1 leads to an increase in active TGF-ß1, elevating the production of fibronectin (14). TGF-ß1 expression is increased in stretched mesangial cells, and extracellular matrix synthesis is enhanced. The stretch-induced matrix expression is inhibited by TSP-1—blocking peptide (24). TSP-1 is also a major activator of TGF-ß1 in vivo as demonstrated with TSP-1 null mice (12). In addition, TSP-1—blocking peptides are active in vivo, inhibiting mesangial cell proliferation in the anti-Thy1 model of glomerulonephritis in rats, a process driven by TGF-ß1 (25). Moreover, TSP-1 expression is related closely to the development of fibrosis in proliferative glomerulonephritis (26) and in tubulointerstitial fibrosis (27). Collectively, this evidence suggests a major role for TSP-1 in pathologic fibrosis by its activation of TGF-ß1. However, direct evidence for TSP-1—activating TGF-ß1 in mesangial cells in high-glucose conditions is lacking. Thus, we investigated TGF-ß1 activation by endogenous TSP-1 in human mesangial cell cultures maintained in media containing normal (4 mM) and elevated (30 mM) glucose levels. We used the GGWSHW peptide to inhibit activation by TSP-1 and TSP-1 antisense oligonucleotide to inhibit TSP-1 synthesis in the mesangial cells. Secreted and cell layer—associated fibronectin and plasminogen activator inhibitor-1 (PAI-1) were measured as markers of TGF-ß1—dependent gene activation in the mesangial cell cultures. The results indicate that the elevated TGF-ß1 activity in mesangial cells in high-glucose conditions is dependent on TSP-1 activation of the growth factor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental Design
Early confluent cultures of HMC were maintained in either normal glucose (4
mM) or high glucose (30 mM) containing media supplemented with 10% FCS for up
to 3 wk (28). The
TSP-1—blocking peptide GGWSHW or a negative control peptide, GGYSHW, was
added to media throughout the experiment. Media were changed every 48 h. At
the end of each week, the medium was changed to serum-free and ITS-free medium
containing the appropriate concentration of glucose and peptide. After 24 h,
secreted TSP-1, TGF-ß1 (total and active), FN, and PAI-1 were measured in
the medium by enzyme-linked immunosorbent assay (ELISA). The cell layers were
used to estimate mRNA (RT-PCR) and protein (Western blot) levels of cell
layer—associated TSP-1, FN, and PAI-1. The biologic activity of
TGF-ß1 was determined using an Mv1Lu mink lung epithelial cell growth
inhibition assay (29).
A TSP-1 mRNA antisense oligonucleotide (2 µM, as recommended by the manufacturer) or a CG-matched randomized sequence oligonucleotide (negative control) was added directly to normal- and high-glucose cultures for 1 wk. The medium containing oligonucleotide was renewed every 48 h. All subsequent analytical procedures were as for the peptide addition experiments.
Mesangial Cell Culture
Primary HMC were maintained at 37°C with 5% CO2/95% air in
RPMI 1640 medium, containing 4 mM glucose, 10% FCS, 2 mM glutamine, 100
µg/ml streptomycin, 100 U/ml penicillin, 1 µg/ml Amphotericin B, and ITS
(5 µg/ml, 5 µg/ml, 5 ng/ml, respectively). Cells were routinely passaged
with a 1:4 split and were used for experiments at the seventh to ninth
passages.
At the end of each experimental period, HMC-conditioned medium was collected under sterile conditions, centrifuged (3000 x g, 10 min), and aliquoted. Protease inhibitors were added to give final concentrations of 10 mM aminohexanoic acid, 1 mM ethylenediaminetetraacetate, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide. Inhibitors were not added to samples assigned for measurement of TGF-ß1 bioactivity. Samples were stored at -70°C before ELISA assay or TGF-ß1 bioactivity assay.
Cell layers (5 to 6 x 105 cells) were washed extensively with phosphate-buffered saline at 4°C and were solubilized either in a reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer containing protease inhibitors for subsequent analysis of protein expression by Western blotting or in RNAzolB (AMS Biotechnology, Oxfordshire, UK) for investigating gene expression by RT-PCR. In the first case, samples were frozen and stored at -20°C before analysis. In the second case, total RNA was extracted, dissolved in diethyl pyrocarbonate—treated water, and stored at -70°C before assay.
Assay for TGF-ß1 Bioactivity
TGF-ß1 bioactivity in the conditioned media was assayed by its ability
to inhibit the growth of mink lung epithelial (Mv1Lu) cells
(29). Mv1Lu cells were
maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, and antibiotics
(as above). Cells were passaged with a 1:10 split.
For bioassays, Mv1Lu cells (100 µl, 7 to 10 x 104 cells/ml) were seeded in 96-well plates in DMEM with 10% FCS. After 2 to 3 h, the plates were washed and DMEM with 10% FCS (100 µl), and HMC-conditioned medium (100 µl) were added to the wells. After 3 d, the number of cells in each well was determined by sulforhodamine B assay (30). A standard curve was constructed for 1 x 10-4 - 30 x 10-4 cells. A further standard curve was set up for each assay with 0.01 to 1 ng/ml of human platelet TGF-ß1.
Enzyme-Linked Immunosorbent Assay
The concentration of TSP-1, FN, and PAI-1 in the conditioned media was
measured by ELISA
(28,31,32)
with some modifications to the original method. Briefly, microtiter plates
were coated overnight at 4°C with serial dilutions of standard proteins in
coating buffer (0.15 M NaCl, 1.5 mM KH2PO4, 10.8 mM
Na2HPO4, 2.7 mM KCl [pH 7.3] for TSP-1 and 15 mM
Na2CO3, 35 mM NaHCO3 [pH 9.6] for FN and
PAI-1) and with HMC-conditioned media, diluted with the appropriate coating
buffer. Nonspecific proteins were blocked with 2% bovine serum albumin at
37°C for 1 h. The optimal dilutions of the primary antibodies were found
to be 1:1000, 1:500, and 1:2000 for mouse monoclonal anti—TSP-1, goat
anti—PAI-1, and rabbit anti-human FN, respectively. The dilutions of
secondary antibodies were 1:2000 (rabbit anti-mouse) and 1:1000 (rabbit
anti-goat and swine anti-rabbit). The bound antibodies were detected with
2,2'-azinobis-3-ethylbenzthazoline-6-sulfonic acid at a wavelength of
405 nm.
TGF-ß1 was determined by ELISA using TGF-ß1—soluble receptor type II (33). To measure active TGF-ß1 in the media, the samples were assayed directly, whereas to determine total TGF-ß1, samples were acidified before the measurement.
Western Blotting
Samples were boiled for 5 min and diluted with reducing loading buffer to
standardize for cell number. Samples were resolved by SDS-PAGE (15% wt/vol
gels for PAI-1 and 7.5% for FN)
(28). Prestained
molecular-mass standards were used to monitor protein migration. Proteins were
transferred onto poly(vinylidene difluoride) membrane (Immobilon-P; Millipore,
Bedford, UK) with a Bio-Rad transfer apparatus (Hercules, CA). Immunodetection
was performed as described
(34), and bound antibodies
were revealed using the enhanced chemiluminescence reagent ECL+Plus (Amersham
Pharmacia Biotech, Little Chalfont, UK). The results were normalized against
the intensity of the ß-actin band for each sample.
Reverse Transcription-PCR
Equal amounts (2 µg) of DNA-free total RNA from each sample were
converted to cDNA by SuperScriptTM II RNase H- reverse
transcriptase (Life Technologies) with random primers in a 20-µl reaction
volume. The reverse transcription reaction (0.5 µl) was subjected to PCR
amplification using 2.5 U of Taq DNA polymerase in a 100-µl
reaction volume with 0.5 µM of each dNTP, 0.5 µM of each specific
primer, and 1.5 mM MgCl2. The amount of amplified
reverse-transcribed cDNA (0.5 ml) was determined to be nonsaturating. The
"house-keeping" gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was amplified simultaneously with TSP-1, PAI-1, and FN genes.
The sequences of primers were designed from the human genes: TSP-1 (496 bp), sense: 5'-AAA GCG TCT TCA CCA GAG ACC T-3', antisense: 5'-GCA GAT GGT AAC TGA GTT CTG ACA-3'; PAI-1 (396 bp), sense: 5'-GTA TCT CAG GAA GTC CAG CC-3', antisense: 5'-TCT AAG GTA GTT GAA TCC GAG C-3'; FN (639 bp), sense: 5'-CGA AAT CAC AGC CAG TAG-3', antisense: 5'-ATC ACA TCC ACA CGG TAG-3', GAPDH (195 bp), sense: 5'- CCA TGG AGA AGG CTG GGG-3', antisense: 5'- CAA AGT TGT CAT GGA TGA CC-3'. The amplification program was 94°C for 3 min, 35 cycles consisting of 94°C for 60 s, 55°C for 60 s, 72°C for 90 s, and 72°C for 10 min.
Equal volumes of the amplification products were analyzed by agarose gel (1.2%) electrophoresis with ethidium bromide (0.5 µg/ml) staining. A 1-kb DNA ladder was used as a size marker. Gels were photographed and analyzed with Image software (NIH Image). The results were normalized to the intensity of GAPDH bands.
Statistical Analysis
Results were compared using unpaired t test. A P value of
0.05 or less was regarded as denoting a significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TSP-1 Expression in Mesangial Cells Exposed to Low- and High-Glucose
Conditions
TSP-1 mRNA levels, measured by semiquantitative RT-PCR, were increased in
HMC exposed to 30 mM D-glucose for 1, 2, and 3 wk over levels detected in
cultures maintained in 4 mM D-glucose conditions
(Figure 1, lanes 1 and 2).
Densitometry of the cDNA bands from three independent experiments showed a
mean increase of 1.8-fold after 1 wk, 2-fold at 2 wk, and 2.5-fold at 3 wk
(P < 0.01 to 0.05; data not shown). Treatment of the 30 mM
D-glucose cultures with either the TGF-ß1 activation-blocking peptide
(peptide W, 1 µM) or the control peptide (peptide Y, 1 µM) had no effect
on the mRNA expression level of TSP-1 at any time point
(Figure 1, lanes 5 and 6).
However, treatment of the high-glucose cultures with TSP-1 antisense
oligonucleotide for 1 wk reduced TSP-1 expression to approximately or below
the level found in 4 mM D-glucose cultures
(Figure 1, lane 3). A control
oligonucleotide had no effect on TSP-1 mRNA expression level in high glucose
(Figure 1, lane 4).
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The increased levels of TSP-1 mRNA in high-glucose cultures were reflected in increased levels of TSP-1 protein in the culture media (Figure 2). The greatest increase occurred after 1 wk (2.3-fold, P < 0.001) and was 2.2-fold at 2 wk (P < 0.001) and 1.6-fold at 3 wk (P < 0.02), despite the gradually increasing levels of TSP-1 mRNA in high-glucose cultures at these time points (see above). This may be because of increased retention of TSP-1 in the cell layer matrix of cultures with increasing time. Mesangial cell cultures treated with 4 mM D-glucose + 26 mM mannitol had the same levels of TSP-1 in the medium after 1, 2, and 3 wk as cultures maintained in 4 mM D-glucose alone (data not shown). Therefore, the increased levels of TSP-1 in cultures maintained in 30 mM D-glucose are not due to the hyperosmotic effect of high-glucose concentration.
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Effect of Blocking Peptide W on TSP-1 and TGF-ß1
The effect of peptide W on TSP-1 synthesis was investigated. Concentrations
of the peptide up to 10 µM had no effect on TSP-1 levels in the media of
either normal- or high-glucose cultures after 1 wk of treatment (data not
shown). Moreover, neither 1 µM peptide W nor the control peptide Y had any
significant effect on the elevated TSP-1 mRNA levels in high-glucose cultures
(Figure 1, lanes 5 and 6) or on
TSP-1 protein levels in normal- or high-glucose media
(Figure 2) when cultures were
treated with either peptide over 3 wk.
ELISA assay of culture media for total TGF-ß1 showed that high-glucose—treated HMC synthesized 2.5-, 2.3-, and 1.8-fold, more growth factor after 1, 2, and 3 wk, respectively, than 4 mM D-glucose—treated cells (P < 0.01), and this was not affected by 1 µM peptide W (Figure 3) or concentrations of peptide up to 10 µM (data not shown). The amount of naturally "active" TGF-ß1, detected by ELISA, accounted for less than 5% of total TGF-ß1 in 4 or 30 mM D-glucose—treated cultures. The levels were not affected by the presence of the control peptide Y in either low- or high-glucose conditions. In contrast, concentrations of peptide W between 0.1 and 10 µM markedly reduced the level of "active" TGF-ß1 in HMC cultures maintained in either 4 or 30 mM D-glucose. Maximum reduction was achieved with 1 µM peptide W (data not shown).
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The levels of biologically active TGF-ß1 produced in mesangial cell cultures maintained in high- and low-glucose conditions over 3 wk was investigated using the mink lung cell growth inhibition bioassay. Bioactive TGF-ß1 levels increased by approximately 10-fold during the first 2 wk of exposure to 30 mM D-glucose and thereafter remained at the same high level (Figure 4). In contrast, bioactive TGF-ß1 concentrations increased only marginally over 3 wk culture in 4.0 mM D-glucose conditions. Treatment of high-glucose cultures with peptide W reduced the level of bioactive TGF-ß1 in these cultures at each time point over 3 wk to the same low levels found in 4 mM D-glucose cultures. In contrast, the control peptide Y had no effect on TGF-ß1 bioactivity in high-glucose cultures (Figure 4). The results indicate that all of the additional TGF-ß1 bioactivity in high-glucose—treated cultures over baseline TGF-ß1 bioactivity in 4 mM D-glucose cultures is dependent on TSP-1 activation of the growth factor.
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Dependence of FN and PAI-1 Expression on Activation of TGF-ß1 by
TSP-1
We investigated next whether the increased expression of FN and PAI-1 in
HMC cultures with prolonged exposure to high glucose is dependent on TSP-1
activation of TGF-ß1. mRNA levels for FN and PAI-1 were increased in 30
mM compared with 4 mM D-glucose cultures at weeks 1, 2, and 3
(Figure 1, lanes 1 and 2 and
Figures 5 and
6), confirming previous results
(17,28).
Treatment with peptide W reduced mRNA levels in 30 mM D-glucose, whereas
control peptide Y had no significant effect
(Figure 1, lanes 5 and 6). The
protein levels of FN and PAI-1 were measured in the media of cultures by ELISA
(Figures 5 and
6). In each case, the protein
levels reflected the mRNA levels for FN and PAI-1, being elevated in
high-glucose conditions and reduced to the basal levels of 4 mM D-glucose
cultures when high-glucose cultures were treated with peptide W. They were
unaffected by the control peptide Y. Thus, we conclude that the elevated
expression of both FN and PAI-1 in mesangial cells exposed to high glucose is
dependent on TGF-ß1 activation by TSP-1.
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Effects of TSP-1 Deletion with an Antisense Oligonucleotide
Treatment of high-glucose cultures for 1 wk with a TSP-1 antisense
oligonucleotide counteracted the overexpression of TSP-1 mRNA in these cells,
whereas a randomized oligonucleotide used as a control had no effect
(Figure 1, lanes 3 and 4). The
level of TSP-1 protein in the medium of TSP-1 antisense
oligonucleotide-treated cells decreased by 85% in high-glucose cultures and
was 70% lower than in untreated 4.0 mM D-glucose cultures
(Figure 7A). The total
TGF-ß1 level remained unchanged
(Figure 7B), but levels of
bioactive TGF-ß1 fell dramatically in the 30 mM D-glucose-treated cells
(Figure 7C). Moreover, treating
HMC with the TSP-1 antisense oligonucleotide greatly reduced FN in mRNA levels
in high-glucose cultures, whereas the control oligonucleotide had no effect
(Figure 1, lanes 3 and 4).
Protein FN levels were correspondingly reduced in the medium (2.5-fold) and
cell layer of high-glucose cultures treated with antisense oligonucleotide but
not with control oligonucleotide (Figure 7,
D and E). Collectively, these results confirm a key role for TSP-1
activation of TGF-ß1 and, downstream of this, for the increased
expression and synthesis of FN in high-glucose conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß3 chain, fibrillin 2
(38), decorin
(39), and TGF-ß1
latency-associated peptide
(23). Its interaction with the
latent TGF-ß1 complex induces activation of the growth factor in
vitro
(21,22,23),
and it is a major activator of TGF-ß1 in vivo
(12). Many studies show that TGF-1ß is a profibrotic cytokine and an important mediator of kidney fibrosis (40,41). There is much evidence to implicate TGF-ß1 as a factor that drives fibrosis in diabetic nephropathy (2,42). Increased levels of glomerular TGF-ß1 mRNA in diabetic nephropathy biopsies correlate with the hyperglycemic status of the patient (43), and glomerular TGF-ß1 protein is elevated in the disease (44). Moreover, treatment of diabetic mice with anti-TGF-ß1 antibodies attenuates renal expression of FN and type IV collagen and prevents glomerular hypertrophy (45). However, TGF-ß1 is secreted in an inactive form (3), raising questions about the mechanism of its activation in the diabetic glomerulus.
Previously, we showed that when human glomerular mesangial cells are exposed chronically, in vitro, to high concentrations of glucose, they continuously express increased levels of TGF-ß1 mRNA and protein, of which at least a fraction is active, as detected by a specific ELISA (28). It seems unlikely that plasmin is involved in the activation process (8) because we also found that expression of PAI-1 is induced in these cells by high glucose, eliminating the activity of plasminogen activator in such conditions (17). However, we also found that TSP-1 expression is upregulated in mesangial cells that are exposed to high glucose (13), a finding confirmed subsequently by others (14,15). It is noteworthy that plasma levels of TSP-1 are significantly higher in patients who have diabetes mellitus with secondary complications, e.g., nephropathy, than they are in patients without such complications. The TSP-1 levels in the latter group are indistinguishable from the level in nondiabetic controls (46). Thus, TSP-1 seems to be a good candidate for activating latent TGF-ß1 in glomerular mesangial cells that are exposed to high glucose in vitro or in the diabetic glomerulus in vivo.
Tada and Isogai (14) showed that when HMC cultures were treated with 1 to 5 µg/ml TSP-1, their active TGF-ß1 levels increased by 1.3- to 2.1-fold without any concomitant increase in total TGF-ß1 level. Moreover, the addition of TSP-1 to the cultures was accompanied by an increase in FN production, and this was blocked in the presence of an anti-TGF-ß1 neutralizing antibody. This provided good evidence for activation of latent TGF-ß1 produced by HMC by exogenous TSP-1. However, although these investigators showed that exposure to high-glucose conditions induced increased mesangial cell synthesis of TSP-1 (14), their experiments did not establish whether the endogenous protein was responsible for activating TGF-ß1 produced in these conditions. The importance of our results is that they show that endogenous TSP-1 expression is upregulated in mesangial cells that are cultured in high glucose over 3 wk, simulating chronic hyperglycemia. Moreover, this TSP-1 activates all of the additional TGF-ß1 produced by the cells in high glucose, generating chronically elevated levels of bioactive growth factor.
Although the greatest increase in mRNA and protein for TSP-1 occurred after 1 wk of exposure to high glucose (Figures 1 and 2), the production of bioactive TGF-ß1 increased to reach plateau levels after 2 and 3 wk of exposure. However, as demonstrated by the blocking effect of peptide W, the increased bioactivity was totally dependent on TSP-1 activation of the growth factor at all times. Assuming that a similar situation occurs in vivo, prolonged periods of poor glycemic control in diabetics are likely to induce in mesangial cells levels of TSP-1 synthesis that are sufficient to activate the elevated levels of TGF-ß1 produced during chronic hyperglycemia.
Inhibition of TSP-1 activation of TGF-ß1 in high-glucose cultures by peptide W reduced the synthesis of both FN and PAI-1 to similar levels as were found in 4 mM D-glucose cultures, at all times up to 3 wk (Figures 6 and 7). Semiquantitative RT-PCR showed that this is due to normalization of gene transcription for FN and PAI-1 in high-glucose cultures treated with peptide W. Likewise, inhibiting the increase of TSP-1 transcripts in high-glucose cultures with an antisense oligonucleotide reduced FN synthesis to the same level as found in 4 mM D-glucose cultures. This is consistent with the notion that elevated FN transcription and synthesis in high glucose depends on TSP-1 activation of TGF-ß1, which, after receptor binding, triggers intracellular signaling. The signaling pathway seems to involve activation of C-Jun N-terminal kinase in response to TGF-ß1 and subsequent binding of C-Jun-ATF2 heterodimer to the FN promoter (47). However, this pathway, which is independent of smad 4, has not as yet been confirmed in mesangial cells.
Overall, our results indicate that endogenous TSP-1 plays a key role in activating TGF-ß1 when HMC are exposed to high glucose in vitro. It is likely that TSP-1 has a similar role in hyperglycemic diabetic patients, promoting the mesangial fibrosis that is characteristic of diabetic nephropathy. Moreover, it is likely that TSP-1 is an intrinsic component of fibrosis in other regions of the kidney (27) and where fibrosis occurs in other organs. For example, liver homogenate from patients with congenital hepatic fibrosis have higher levels of TSP-1 and TGF-ß1 than in normal livers (48).
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Acknowledgments |
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(3)ß(1) integrin recognition sequence in thrombospondin-1.
J Biol Chem 274:24080
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T. Naito, T. Masaki, D. J. Nikolic-Paterson, C. Tanji, N. Yorioka, and N. Kohno Angiotensin II induces thrombospondin-1 production in human mesangial cells via p38 MAPK and JNK: a mechanism for activation of latent TGF-{beta}1 Am J Physiol Renal Physiol, February 1, 2004; 286(2): F278 - F287. [Abstract] [Full Text] [PDF]
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D. Fraser, N. Brunskill, T. Ito, and A. Phillips Long-Term Exposure of Proximal Tubular Epithelial Cells to Glucose Induces Transforming Growth Factor-{beta}1 Synthesis via an Autocrine PDGF Loop Am. J. Pathol., December 1, 2003; 163(6): 2565 - 2574. [Abstract] [Full Text]
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S. Wang, X. Wu, T. M. Lincoln, and J. E. Murphy-Ullrich Expression of Constitutively Active cGMP-Dependent Protein Kinase Prevents Glucose Stimulation of Thrombospondin 1 Expression and TGF-{beta} Activity Diabetes, August 1, 2003; 52(8): 2144 - 2150. [Abstract] [Full Text] [PDF]
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C. Hugo The thrombospondin 1-TGF-{beta} axis in fibrotic renal disease Nephrol. Dial. Transplant., July 1, 2003; 18(7): 1241 - 1245. [Full Text] [PDF]
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R. L. Meek, S. K. Cooney, S. D. Flynn, R. F. Chouinard, M. H. Poczatek, J. E. Murphy-Ullrich, and K. R. Tuttle Amino acids induce indicators of response to injury in glomerular mesangial cells Am J Physiol Renal Physiol, July 1, 2003; 285(1): F79 - F86. [Abstract] [Full Text] [PDF]
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R. M. Mason and N. A. Wahab Extracellular Matrix Metabolism in Diabetic Nephropathy J. Am. Soc. Nephrol., May 1, 2003; 14(5): 1358 - 1373. [Abstract] [Full Text] [PDF]
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), 30 mM
D-glucose (
), 30 mM + TSP-1 blocking peptide W (
), and 30 mM +
control peptide Y (
) for up to 3 wk. At the end of each week, the medium
was changed to serum-free medium containing the same concentration of glucose
and peptide. After an additional 24 h, media were collected and used to
measure the secreted level of TSP-1 by enzyme-linked immunosorbent assay
(ELISA) as described in the Materials and Methods section. The data represent
mean ± SEM for three separate experiments with quadruplicate cultures
for each condition in each experiment. TSP-1 levels are increased in 30 mM
conditions compared with 4 mM conditions. *, P < 0.02;
**, P < 0.001.
) of
1 µM TSP-1 blocking peptide for up to 3 wk. At the end of each week, the
medium was changed to serum-free medium containing the same concentration of
glucose and peptide. After an additional 24 h, media were collected,
acidified, and used to measure the total level of TGF-ß1 by ELISA as
described in the Materials and Methods section. The data represent mean
± SEM for three separate experiments with quadruplicate cultures for
each condition in each experiment. There were no significant differences
between cultures maintained with or without the blocking peptide.
) or 30 mM (
) and
Y (
) peptides for up to 3 wk. At the end of each week, the medium was
changed to serum-free medium containing the same concentration of glucose and
peptide. After an additional 24 h, media were collected and used to measure
TGF-ß1 bioactivity with the mink lung epithelial cell assay as described
in the Materials and Methods section. The data represent mean ± SEM for
three separate experiments with quadruplicate cultures for each condition in
each experiment. Bioactive TGF-ß1 is increased in 30 mM conditions
compared with 4 mM conditions. *, P < 0.01.
0.02.
