 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
*
Molecular Pathology Section, Division of Biomedical Sciences, Imperial
College School of Medicine, London, United Kingdom
Service de Néphrologie A, Hopital Tenon,
Paris, France.
Correspondence to Roger M. Mason, Molecular Pathology Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, UK. Telephone: +44 20 7594 3019; Facsimile: +44 20 7594 3022; E-mail: roger.mason{at}ic.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Decorin is a small proteoglycan whose core protein contains leucine-rich repeats of about 24 amino acids in length. Decorin is modified posttranslationally by polymerization of a single glycosaminoglycan chain onto a site near the amino terminus of the core protein and by N-glycosylation at three potential sites (4). It is a component of the extracellular matrix of many tissues and, due to the amphipathic leucine-rich repeats, is thought to interact with several other proteins and lipid molecules. It may be involved in the regulation of fundamental biologic functions, such as matrix assembly (5), modulating fibronectin- and thrombospondin-mediated cell adhesions (6,7), regulating transforming growth factor-ß (TGF-ß) activity, which is a key factor in tissue fibrosis (8,9,10), and in promoting cell proliferation (11). There is also evidence that decorin binds to and causes rapid phosphorylation of the EGF receptor with a concurrent activation of the mitogenic-activated protein (MAP) kinase signal pathway (12).
The decorin gene spans about 38 kbp and has eight exons that encode a protein of 359 amino acids. The human gene has been mapped to chromosome 12q23 (13). It has two promoters, P1(1a) and P2 (1b), each of which lies upstream of a leader exon, exon 1a and 1b, respectively. Both exons are correctly spliced to exon II and encode a portion of the 5' untranslated region of the mRNA (13). Thus, mRNA transcribed from either the P1 or the P2 promoter would be translated into only one protein. However, Iozzo and his collaborators found that only the P2 (1b) promoter was functional in HeLa and MG-63 osteosarcoma cells (14).
The regulation of decorin expression is not well understood. Upregulation of decorin mRNA in colon cancer has been shown to be associated with hypomethylation of the decorin gene (15). Conflicting results were reported when the influence of TGF-ß on decorin expression was investigated. TGF-ß upregulates the expression of decorin in some cell types such as primary mesangial cells (16), lung fibroblasts, and lung epithelial cells (17). TGF-ß also mediates increased decorin protein expression in the anti-Thy-1 model of glomerulonephritis (18) and in experimental hydronephrosis (19). However, marginal effects or even a downregulation of decorin expression by TGF-ß were observed in other types of cells such as human skin fibroblasts, a human osteosarcoma cell line (20), and in dermal and gingival fibroblasts (21,22). This clearly indicates cell type-specific control of decorin expression.
Interestingly, a TGF-ß inhibitor element (TIE) is present in the P2 (1b) promoter region of the decorin gene and has been demonstrated to negatively regulate its expression by TGF-ß (14). A TIE has also been shown to be the negative element in the promoter regions of transin/stromelysin (23), elastase (24), collagenase, gelatinase B, and matrilysin (25), and mediates their downregulation by TGF-ß.
HMC exposed to chronic high glucose conditions express markedly elevated levels of both TGF-ß and decorin at both the transcriptional and protein level (2). This raises the question of how the decorin gene escapes the negative control of TGF-ß under these conditions. The present study aimed to elucidate the molecular basis of upregulation of decorin gene expression in high glucose. Identification of the enhancer element(s) and the transcription factors that mediate overexpression of decorin and other extracellular matrix molecules in high glucose mesangial cells may also point to the signal transduction cascades that are initiated when the cells are exposed to prolonged high glucose concentrations.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RNA Extraction
Total RNA was extracted from 6 x 106 mesangial cells using
the RNAzol B method (AMS Biotechnology, Oxfordshire, United Kingdom). Samples
were treated with DNase I using the Message Clean Kit of BioGene Ltd.
(Bolnhurst, Bedfordshire, United Kingdom). RNA was dissolved in
diethylpyrocarbonate—distilled H2O, quantified, and stored at
-70°C until used.
Reverse Transcription-PCR Analysis
Equal amounts of total RNA (2 µg) from each sample were
reverse-transcribed into cDNA, using SuperScript II RNase H+
reverse transcriptase (Life Technologies BRL, Paisley, United Kingdom) and
random primers. Equal amounts (1 µl) of the reverse transcription (RT)
reaction (20 µl) were subjected to PCR amplification in a 100-µl volume
containing 10 µl of 10 x PCR buffer, 16 µl of dNTP (1.25 mM each),
2 mM MgCl2, 20 µM of each specific primer, and 1.25 U of
Amplitaq DNA polymerase. Amplification was started with 5 min of denaturation
at 94°C followed by 30 PCR cycles. Each cycle consisted of 60 s at
94°C, 60 s at 50°C, and 60 s at 72°C. The final extension was 10
min at 72°C in all instances. To quantify PCR products comparatively and
to confirm the use of equal amounts of the RNA, we coamplified the
housekeeping gene, ß-actin or GAPDH. The amount of reverse transcription
reaction used for the amplification (1 µl) was selected as being
nonsaturating for the PCR product of both ß-actin and the gene under
investigation after 30 cycles of amplification.
The sequences of primers were designed from the published sequences of the human genes and are listed in Table 1. After amplification, 10 µl of each PCR reaction mix was electrophoresed through a 2% (wt/vol) agarose gel containing ethidium bromide (0.5 µg/ml).
|
Cloning and Sequence Analysis
The genomic clones DEC-10 and DEC-1 were kindly provided by Dr. Renato V.
Iozzo (Thomas Jefferson University, Philadelphia, PA). The DEC-10 plasmid
contained the decorin gene promoter P2 (1b) and exon 1b, while the DEC-1
plasmid contained DNA sequences for the P1 (1a) promoter and exon 1a
(20). Nested deletions were
generated along the 5' prime end of each promoter by PCR amplification
of the desired segment, using the DEC-1 and DEC-10 plasmids as templates and
the designed primers with KpnI or XhoI sites for
unidirectional cloning. The promoterless secreted human placental alkaline
phosphatase plasmid vector pSEAP2-Basic was used for all promoter studies
(Clontech Laboratories, Palo Alto, CA). An SV40 promoter-ß-galactosidase
reporter vector pßgal-control (Clontech Laboratories) was used as an
internal control for evaluating the efficiency of transfections in each
experiment. An SV40 promoter-secreted placental alkaline phosphatase reporter
vector (pSEAP2-Control) was used to evaluate the transcription strength of the
decorin promoter. PCR products were KpnI/XhoI digested and
subcloned into compatible sites of the SEAP reporter plasmid, upstream of the
SEAP cDNA. The constructs were sequenced to verify the accuracy of ligation
and sequences. For transfection, plasmid DNA was purified using Qiagen
Maxi-prep ion-exchange cartridges (Qiagen, Crawley, United Kingdom).
Transient Transfection
Decorin promoter-reporter constructs (20 µg) or the pSEAP2-Control (10
µg) were transfected with 5 µg of the pßgal-control vector into 25
x 106 C2TM2 cells in 0.5 ml of electroporation buffer (137 mM
NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM D-glucose, 21 mM
Hepes, pH 7.1) per transfection. Briefly, the cells and plasmid DNA were
incubated at room temperature for 10 min and electroporated at 250 V, 500
microfarad, using the Bio-Rad Gene Pulser (Bio-Rad, Hemel Hempstead, United
Kingdom). After an additional 10 min at room temperature, the cells were
seeded into 5 ml of growth medium containing either 4 or 30 mM D-glucose, 4 mM
D-glucose + 1.25 ng/ml TGF-ß1, or 30 mM D-glucose + 1.25 ng/ml
TGF-ß1.
The transfected cells were incubated at 37°C in 5% CO2 for 48 h, after which media were collected for measuring SEAP activity. The cells were scraped and assayed for ß-galactosidase activity.
Secreted Alkaline Phosphatase and ß-Galactosidase Assays
Enzyme activities were determined as described by the supplier of the
reporter vectors, using chemiluminescent detection kits. The chemiluminescent
signals were detected using a 96-well plate luminometer (Luminoskan Ascent
100-240V; Labsystems, Helsinki, Finland). Measurements were performed after
various incubation times (10 to 60 min), and light signals were recorded as 5-
to 20-s integrals.
Preparation of Nuclear Extracts
Nuclear extracts were prepared as described by Abmayr and Workman
(27). Confluent mesangial
cells that had been maintained under the conditions described above were
scraped from the tissue culture dishes in PBS buffer using a cell scraper and
were collected by centrifugation at 1500 x g for 5 min. The
cell pellets were resuspended in hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], and
0.5 mM dithiothreitol [DTT]) and homogenized with 10 strokes of a glass Dounce
homogenizer. Nuclei were isolated by centrifugation at 3300 x g
for 15 min. Nuclear proteins were extracted by suspending the nuclei in an
equal volume of extraction buffer (20 mM Hepes, pH 7.9, 25% glycerol, 1.5 mM
MgCl2, 0.8 M KCl, 0.2 mM ethylenediaminetetra-acetic acid, 0.2 mM
PMSF, and 0.5 mM DTT). The extract was dialyzed against 20 mM Hepes (pH 7.9),
20% glycerol, 100 mM KCl, 0.2 mM ethylenediaminetetra-acetic acid, 0.2 mM
PMSF, and 0.5 mM DTT. The protein concentration was determined using Coomassie
Plusprotein assay reagent (Pierce and Warriner, Chester, United Kingdom).
Gel Mobility Shift Assay
Oligonucleotides encoding the CRE-like element were synthesized by Perkin
Elmer (Warrington, Cheshire, United Kingdom). Double-stranded oligonucleotide
probes were prepared by mixing equimolar amounts of two complementary
single-stranded DNA, heating to 80°C for 5 min, and allowing the
oligonucleotides to cool slowly to room temperature. The annealed
oligonucleotides were end-labeled with 
-32pATP and T4 DNA
polynucleotide kinase. Both labeling and binding reactions were carried out
using the Promega gel shift assay system according to the manufacturer's
instructions (Promega, Southampton, United Kingdom). Nuclear extract and
oligonucleotide probe mixtures were incubated at room temperature for 20 min,
after which 2 µl of 5x gel loading buffer (20% glycerol, 0.1%
bromphenol blue, 0.1% xylene cyanol in 5x TBE running buffer) was added
before loading onto a 6% nondenaturing polyacrylamide gel. Electrophoresis was
carried out at 100 V in 1x TBE buffer until the bromphenol blue dye had
migrated three-quarters of the way down the gel. The gel was then transferred
onto 3MM filter paper (Whatman International, Ann Arbor, MI), dried, and
autoradiographed. Supershift assays were performed by adding polyclonal
antibodies against either cAMP response element binding protein (CREB), CREB2,
cJun, ATF1, or ATF2 (Autogen Bioclear, Calne, United Kingdom) to the nuclear
extract for 30 min at 4°C before the addition of the probe.
Immunoblotting
Immunoblots of gel shift assays were performed by electroblotting the 6%
polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes
(Immobilon-P; Millipore, Bedford, United Kingdom), using a Bio-Rad transfer
apparatus. Membranes were dried and autoradiographed to locate DNA/protein
complexes. Blots were then hydrated and probed for CREB, CREB2, c-jun, ATF1,
and ATF2.
For Western blot analysis, nuclear proteins were run on a 4 to 12% sodium dodecyl sulfate-polyacrylamide gradient gel and electroblotted onto PVDF membranes as above. Membranes were blocked in 5% nonfat dry milk for 2 h, then incubated overnight at 4°C in the recommended concentrations of primary antibodies (1/1000 of each anti-P-CREB and anti-CREB) (New England Biolabs, Hitchin, Hertfordshire United Kingdom). Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunodetection was performed essentially as described by Towbin et al. (28). Bound antibodies were visualized with Phototope-horseradish peroxidase Western detection kit (New England BioLabs).
Immunocytochemistry
Cells were grown on glass coverslips for 2 d in medium containing 0.5% FCS
and 4 mM D-glucose. Cells were then exposed to medium containing 0.5% FCS and
either 30 mM D-glucose, 4 mM D-glucose + 26 mM mannitol, or 4 mM D-glucose for
24 h. Cells were rinsed briefly in PBS and fixed in paraformaldehyde (3.7% in
PBS) for 20 min at 4°C. Cells were washed once with PBS, then
permeabilized with Triton X-100 (0.1% in PBS) for 10 min at room temperature,
after which they were washed again 3 times for 5 min each with PBS. Cells were
then blocked overnight at 4°C with goat serum (5% in PBS), aspirated, and
incubated with anti-phospho-p38 MAP kinase (New England Biolabs) or
Anti-ACTIVE mitogen-activated protein kinase (MAPK) pAb (Promega) primary
antibodies (at the recommended dilution in PBS containing 5% BSA) overnight at
4°C, or for 1 h at 37°C, and then washed 3 times for 5 min each with
PBS. Cells were then incubated in the dark for 1 h with fluorescein-conjugated
goat anti-rabbit secondary antibody (Sigma-Aldrich, Dorset, United Kingdom) at
a dilution of 1:200 in PBS containing 5% BSA, before being washed 3 times for
5 min each with PBS. Coverslips were mounted on glass slides with anti-fade
mounting media (Vector Laboratories, Peterborough, United Kingdom) and
examined using a fluorescence microscope (Olympus AX70).
Statistical Analyses
Results are given as mean ± SEM. Unpaired t test was used
to assess differences between means. A value of P < 0.05 was
accepted as indicating a significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
Because endogenous TGF-ß expression is elevated in HMC exposed to high glucose (2), we investigated whether the stimulatory effect of 30 mM glucose on decorin expression is mediated by the endogenous growth factor. Cultures were maintained in medium containing either 4 mM glucose, 30 mM glucose, or 30 mM glucose and anti-TGF-ß neutralizing antibody for 1 wk, after which decorin 1a and 1b transcripts were estimated by RT-PCR. The concentration of antibody (2.6 µg/ml) was calculated to be at least fourfold greater than that needed to neutralize the endogenous TGF-ß. The expected amount of TGF-ß was calculated from previous experiments. Media were changed every 2 d. The level of decorin 1a transcripts in 30 mM D-glucose cultures treated with anti-TGF-ß antibody was still elevated (1.7x) compared with 4 mM D-glucose, but was less than that in 30 mM cultures without antibody (2.6x) (Figure 4). This suggests that the stimulatory effect of glucose on decorin transcription driven by the P1 promoter is mediated partly by endogenous TGF-ß and partly by TGF-ß-independent signaling pathways. The inhibitory effect of 30 mM D-glucose on the production of decorin 1b transcripts via promoter 2 was, if anything, slightly alleviated by the anti-TGF-ß antibody (Figure 4).
|
The molecular basis of this marked regulation of transcription of the decorin gene in HMC was investigated by analyzing the two promoter sequences for transcription factor binding sites using the Transcription Element Search database (http:// pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html). Putative response elements are indicated in Figure 5.
|
Functional Activity of the Decorin Promoters
We subcloned the two promoters of decorin into the Great Escape Reporter
System. In this system, any cloned promoter will drive the transcription of
the SEAP reporter gene, which encodes a form of human placental alkaline
phosphatase. This form is secreted, heat stable, and resistant to alkaline
phosphatase inhibitors. Transient expression of SEAP driven by each promoter
was determined after transfecting C2TM2 mesangial cells with the appropriate
construct. SEAP activity was assayed 48 h after transfection and was
normalized to ß-galactosidase activity to correct for any difference in
transfection efficiency.
The relative activity of the decorin promoters in driving transcription in the presence of 4 mM D-glucose, 30 mM D-glucose, 4 mM D-glucose + 1.25 ng/ml TGF-ß, and 30 mM D-glucose + 1.25 ng/ml TGF-ß was tested. Figure 6 summarizes the results of six independent experiments. Under the 4 mM D-glucose conditions, P1 promoter activity was almost twice that of the P2 promoter activity (P = 0.002). Significantly, both high glucose (P = 0.0001) and TGF-ß (P = 0.006) enhanced the activity of the P1 promoter, with maximal response when both are present together (threefold, P = 0.0001). In contrast, high glucose and TGF-ß had no effect on the activity of the P2 promoter. The latter result fails to confirm the suppression effect of high glucose and TGF-ß on the P2 promoter, demonstrated by RT-PCR experiments using primary mesangial cells (Figures 2 and 3). This may be due to the low functional activity of the P2 promoter in the transient cell transfection experiments, using the immortalized human mesangial cell line.
|
Minimal Sequence Required for Promoter P1 Response to High
Glucose
A series of nested 5' deletions of the promoter was tested for the
ability to drive the enhanced expression of the SEAP reporter gene in the
transiently transfected mesangial cell line exposed to high glucose conditions
or to TGF-ß1. The results of detailed studies using six independent
experiments, each run in duplicate, are given in
Figure 7. Deleting the promoter
region from -2102 to -387 bp resulted in a statistically significant decrease
in basal activity in the presence of 4 mM D-glucose. This suggests that the
core promoter elements responsible for basal P1 promoter activity in HMC are
located within the proximal 387 bp. Both high glucose and TGF-ß enhanced
the relative activity of the promoter constructs within the -2102 to -583-bp
region, with maximal response when added together. Deletion from position
-1275 to -1021 led to a 15% reduction in the stimulatory effect of high
glucose, TGF-ß, or both together. This region contains putative HSF2 and
SP1 sites, and an E-box. Deletion from position -683 to -583 bp led to the
loss of the high glucose and TGF-ß stimulation effects. This indicates
that this region contains an important positive regulatory element.
|
Identification of a CRE-Like Response Element and Proteins Binding to
It
Examination of the -683 to -583 region indicated the presence of a putative
cAMP response element (CRE)-like sequence (TGACGTTT) located between -625 to
-632 bp. This CRE variant is two nucleotides different from the CRE consensus
(TGACGTCA). Gel mobility shift assays were performed with a
32P-labeled oligonucleotide containing the CRE-like sequence and
revealed multiple complexes with mesangial cell nuclear proteins. No
differences were observed in the mobility of complexes formed with nuclear
proteins extracted from low glucose and high glucose-treated mesangial cells
(Figure 8). However, binding
activity, as assessed by the intensity of the DNA-protein band, appears to be
stronger with extracts from high glucose cells compared with those from low
glucose cells. Excess unlabeled CRE-like oligonucleotide competed for these
complexes, indicating specific binding. Excess unlabeled consensus CRE
oligonucleotide was able to compete with only the upper band of the complexes
(Figure 8A). Parallel
experiments were performed using labeled consensus CRE oligonucleotide and
gave similar results (Figure
8B). Interestingly, the binding activity of the complexes that
were formed were also stronger with extracts from high glucose cells. Excess
unlabeled CRE-like oligonucleotides competed only partially for these
complexes, indicating differences in the composition of the complexes formed
with CRE and CRE-like oligonucleotides.
|
To characterize the protein complex binding to the CRE-like sequence, nuclear extracts from high glucose-treated mesangial cells were incubated with antibodies against several common CRE transcription factors before detecting DNA-protein interactions by gel mobility shift assay. As shown in Figure 9, antibodies against CREB, c-Jun, ATF1, and CREB2 (lanes 5, 6, 7, and 8) induced supershift of the labeled consensus CRE probe. In contrast, only the antibody against CREB (lane 1) shows a slight interaction with the protein complex bound to the CRE-like oligonucleotide, as shown by the reduction of the DNA/protein complex band intensity but not by the formation of supershift band as with the CRE oligonucleotide. This may be due to the antibody binding site being very near to the CREB/other transcription factor binding site, which in turn could affect binding to the DNA protein complex. Immunoblots of the electrophoretic mobility shift assay gels confirmed these results. Immunoblots probed with antibodies to CREB, c-Jun, CREB2, ATF-1, and ATF-2 revealed the presence of CREB protein in the CRE-like protein complexes. Figure 10 shows a gel shift mobility assay performed in the absence or presence of nuclear extracts from low glucose-treated mesangial cells (Panel A, lanes 1 and 2). This gel was blotted and probed with antibody against CREB. The antibody cross-reacted with a band localized to the vicinity of the CRE-like protein complexes (Panel B, lane 2).
|
|
Phosphorylation of CREB in High Glucose Conditions
Because phosphorylation of CREB at serine 133 is thought to be obligatory
for its activation, we examined nuclear proteins prepared from 4 and 30 mM
glucose-treated mesangial cells for the level of activated CREB.
Figure 11A shows that the
anti-phospho-CREB (Ser 133) antibody detects activated CREB and also the
phosphorylated form of the CREB-related protein ATF-1. Nuclear extract
prepared from 30 mM glucose-treated mesangial cells contains high levels of
activated CREB compared with nuclear extract prepared from either 4 mM
glucose-treated mesangial cells or HeLa cells. To ensure that the increased
reactivity is not due to increased amounts of protein loaded onto the gel, we
adjusted the amount of loaded samples to give equal signals with CREB
antibody, as shown in Figure
11B. Serine 133 of CREB has been shown to be phosphorylated
through a number of signal transducing pathways. Thus, we examined the ability
of high glucose to activate two of these pathways in HMC.
Figure 12 shows that high
glucose causes the activation and the nuclear translocation of both the
p38MAPK and MAPK (extracellular signal-regulated kinase) pathways.
Interestingly, 4 mM D-glucose supplemented with 26 mM mannitol as an osmotic
control also resulted in some activation and nuclear translocation of both
kinases, although this was much less than that observed with 30 mM
D-glucose.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RT-PCR experiments indicated that both high glucose and exogenous TGF-ß1 upregulate decorin expression in HMC through the P1 promoter and downregulate expression through the P2 promoter. Thus, we investigated whether increased transcription from P1 resulted from increased levels of endogenous TGF-ß produced by mesangial cells exposed to 30 mM D-glucose. A large excess of anti-TGF-ß neutralizing antibody only partially reduced the increased level of decorin in transcripts from P1 in high glucose, indicating that upregulated transcription depends on stimulation by both TGF-ß-dependent and TGF-ß-independent pathways. Transient transfection experiments with an SEAP-reporter gene driven by the P1 promoter confirmed that high glucose concentration and TGF-ß1 stimulate gene expression via this promoter in HMC. However, the P2 promoter was very poor in driving the transcription of SEAP reporter gene. Thus, we were unable to confirm downregulation of transcription via the P2 promoter by high glucose and TGF-ß1, as observed in the RT-PCR experiments.
Functional analysis of the human decorin P1 promoter showed that both high glucose and TGF-ß exert their effects through the same two sets of positive regulatory sequence. One set is located at - 1275 to - 1021 bp from the transcription start site and accounts for approximately 15% of the enhancement activity. This region contains putative HSF2 and SP1 sites and an E-box. The other set is located between position -683 and -583 bp and accounts for most of the high glucose/TGF-ß stimulation effect. This region contains a CRE-like sequence that differs in its last two bases from the consensus CRE sequence. The latter is an 8-bp palindromic sequence (TGACGTCA) (32). Several genes that are regulated by a variety of endocrinologic stimuli contain similar sequences in their promoter regions, although at different positions. A comparison of CRE sequences identified to date shows that the 5' half of the palindrome, TGACG, is the best conserved, whereas the 3' TCA motif is less constant (33).
Multiple CRE-binding proteins have been detected in cellular extracts, and at least 10 cDNA of CRE-binding proteins have been cloned. All of the CRE-binding proteins contain similar functional domains, called Bzip. These consist of a basic DNA binding region followed by a leucine zipper motif. Dimerization between two of these molecules through the leucine zipper is required for DNA binding (34). All of the CRE-binding proteins form homodimers and certain members form heterodimers (35). Different dimers exhibit different binding affinities for variant CRE (36), and thus transactivate such variants to different degrees.
Gel mobility shift assays revealed the formation of multiple complexes between mesangial cell nuclear proteins and an oligonucleotide containing the decorin CRE-like sequence. Although there were no differences in the mobility of complexes between the oligonucleotide and proteins isolated from 4 and 30 mM D-glucose-treated cells, the binding activity was stronger for the latter. High glucose conditions increased the mRNA levels of several CRE-binding proteins such as CREB, CREB2, CRE-BP1, and c-jun (N. Abdel Wahab, R. M.Mason, unpublished results), but supershift assays and immunoblots identified only CREB as a transcription factor involved in the formation of these complexes.
Interestingly, high glucose conditions and TGF-ß have been reported to stimulate fibronectin gene expression in HMC through a consensus CRE located at - 170 bp of the fibronectin gene (37,38). Nuclear extracts from HMC treated for up to 48 h with either high glucose conditions or with TGF-ß1, or with both, contained proteins that formed complexes with an oligonucleotide corresponding to the fibronectin CRE motif. The mobility and abundance of these complexes in gel shift assays were the same for both treatments. The CREB and ATF1 transcription factors were identified as components of the CRE-protein complexes, and, as for many other CRE-containing genes, induction of fibronectin transcription was due primarily to the phosphorylation of CREB (38). We also found that CREB is phosphorylated in HMC exposed to high glucose for the period of 21 d.
Phosphorylation of CREB at serine 133 appears to be obligatory for its
activation. A number of signaling pathways can induce phosphorylation of this
residue, including activation of the adenyl cyclase pathway
(39). Phosphorylation of CREB
by protein kinase A (PKA) causes a modest increase in its binding to
high-affinity CRE sites and a stronger enhancement in binding to low-affinity
CRE (40). Wang et al.
(41) found that TGF-ß
stimulation of fibronectin expression in murine mesangial cells was blocked in
cells overexpressing an inhibitory peptide of PKA. They proposed that PKA
activation contributes to the TGF-ß stimulation of fibronectin expression
by phosphorylating CRE-binding factors. However, Kreisberg et al.
(37,38)
reported that the phosphorylation of CREB in HMC treated with high glucose or
TGF-ß was independent of PKA activation. Nevertheless, we found that the
mRNA levels for both the PKA regulatory subunit (RIß) and the catalytic
subunit (C
) are increased in HMC under high glucose conditions
(unpublished results).
Phosphorylation of CREB-serine 133 also occurs via p42/44 MAP kinase and by p90 (MAPKAP-KI) (42), as well as via p38 MAP kinase and MAPKAP-K2 (42). TGF-ß activates p38 MAP kinase (43), implicating this pathway in the activation of CREB in high glucose conditions. Recently, the new kinase MSK1 (mitogen and stress-activated protein kinase) was shown to be activated by both the p42/44 MAPK and p38 MAPK pathways and thus mediate both the growth factor and stress-induced activation of CREB (44). In this study, we found that the p42/44 and p38 kinases are activated and nuclear-translocated when mesangial cells are exposed to high glucose conditions. Another signal that can induce CREB-serine 133 phosphorylation is increased intracellular Ca2+. This activates CaMKII and CaMKV, which subsequently phosphorylate CREB (45). However, there is little evidence for elevated Ca2+ levels in mesangial cells exposed long term to high glucose conditions (46).
In summary, this study enhances our understanding of the molecular mechanisms involved in the upregulation of decorin gene expression in mesangial cells exposed to high glucose concentration. High glucose induces increased expression of TGF-ß isoforms, which then stimulate decorin transcription via a CRE-like element in the P1 promoter of the gene. CREB was identified as a major transcription factor interacting with this site and may be phosphorylated by the TGF-ß-activated p38 MAPK. Other non-TGF-ß-dependent signaling pathways may also contribute to the phosphorylation of CREB in high glucose mesangial cells, and additional evidence that TGF-ß-independent pathways are involved in stimulating decorin transcription in high glucose was obtained with a TGF-ß neutralizing antibody. A second element in the P1 promoter (- 1275 to - 1021 bp) is also responsive to high glucose and TGF-ß, but plays a relatively minor role in upregulating decorin transcription compared with the CRE-like element (-683 to -583 bp). Clearly, the cell-specific use of the P1 promoter in mesangial cells and the effects of high glucose and TGF-ß on these two elements in that promoter far outweigh any potential for TGF-ß to inhibit overall decorin transcription via the TIE in the P2 promoter.
The biologic effect of increased expression of decorin in high glucose HMC remains to be investigated. Decorin mRNA levels are increased in the cortex of kidneys of streptozotocindiabetic rats (30). Because there is evidence that decorin can neutralize the profibrotic effects of TGF-ß (47), it was proposed that its overexpression in diabetic kidneys may counter-act sclerosis induced by the growth factor (30). However, there is also evidence that in other situations decorin has no effect on TGF-ß bioactivity (48) or may even stimulate it (9); hence, additional experimental work is required to determine its function in the hyperglycemic mesangium.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
rd D:
Specific inhibition of type I and type II collagen fibrillogenesis by the
small proteoglycan of tendon. Biochem J223
: 587-597,1984[Medline]
This article has been cited by other articles:
 |
|
 |
|
  K. J. Williams, G. Qiu, H. K. Usui, S. R. Dunn, P. McCue, E. Bottinger, R. V. Iozzo, and K. Sharma Decorin Deficiency Enhances Progressive Nephropathy in Diabetic Mice Am. J. Pathol., November 1, 2007; 171(5): 1441 - 1450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Fischer, S. A. Steitz, P. Y. Johnson, A. Burke, F. Kolodgie, R. Virmani, C. Giachelli, and T. N. Wight Decorin Promotes Aortic Smooth Muscle Cell Calcification and Colocalizes to Calcified Regions in Human Atherosclerotic Lesions Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2391 - 2396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. G. Banerjee, I. Bhattacharyya, W. M. Lydiatt, and J. K. Vishwanatha Aberrant Expression and Localization of Decorin in Human Oral Dysplasia and Squamous Cell Carcinoma Cancer Res., November 15, 2003; 63(22): 7769 - 7776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 G. La Greca, A. Amore, U. Armato, G. Coles, C. Esposito, R. Deppisch, M. Feriani, R. Mason, D. Noonan, E. Schleicher, et al. The un-physiology of peritoneal dialysis solution and the peritoneal membrane: from basic research to clinical nephrology Nephrol. Dial. Transplant., May 1, 2001; 16(5): 905 - 912. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
