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Université de Montréal (X.C., S.-L.Z., L.P, J.S.D.C.), Centre hospitalier de l’Université de Montréal (CHUM)-Hôtel-Dieu, Centre de recherche, 3850 Saint-Urbain Street, Montréal, Québec, Canada H2W 1T8; Université de Montréal (J.G.F.), Hôpital Maisonneuve-Rosemont, Centre de recherche, Montréal, Québec, Canada H1T 2M4; and Harvard Medical School (S.-S.T., J.R.I.), Massachusetts General Hospital, Pediatric Nephrology Unit, Boston, Massachusetts 02114-3117
Address all correspondence and requests for reprints to: John S. D. Chan, Université de Montréal, Centre hospitalier de l’Université de Montréal (CHUM)-Hôtel-Dieu, Centre de recherche, Pavillon Masson, 3850 Saint-Urbain Street, Montréal, Québec, Canada H2W 1T8. E-mail: john.chan{at}umontreal.ca
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Abstract |
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Introduction |
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In addition to the well-characterized systemic RAS, the presence of a local intrarenal RAS has now been generally accepted. The messenger RNA (mRNA) components of the RAS, including angiotensinogen (ANG), renin, angiotensin-converting enzyme (ACE) and angiotensin II (Ang II) receptors (AT-1 and AT-2 receptors), are expressed in murine and rat immortalized renal proximal tubular cells (IRPTCs) (7, 8, 9, 10, 11, 12). We have reported that ANG protein is synthesized and secreted from rat IRPTCs (13, 14), providing evidence that intrarenal Ang II is probably derived from ANG that is synthesized within renal proximal tubular cells (RPTCs) in vivo. The local formation of Ang II may play an important role in the development of nephropathy in diabetes.
We have recently reported that ANG expression in IRPTCs is stimulated by high glucose levels (i.e. 25 mM) (15). Inhibitors of aldose reductase (i.e. tolrestat) and protein kinase C (i.e. staurosporine or H-7) block the stimulatory effect of glucose (15). These studies suggest that high glucose levels may activate the local renal RAS via the stimulation of ANG gene expression in vivo. Most recently, we have reported that insulin inhibited the stimulatory effect of high glucose levels and phorbol 12-myristate 13-acetate (PMA, an activator of protein kinase C) on ANG secretion and ANG messenger RNA expression in IRPTCs (16). This inhibitory action of insulin is blocked by PD98059 (an inhibitor of mitogen- activated protein kinase, MAPK) but not by wortmannin (an inhibitor of phosphatidylinositol-3-kinase, PI3K), suggesting that the insulin action is mediated, at least in part, via the MAPK signal transduction pathway, subsequently inhibiting activation of the local renal RAS.
In the present studies, we investigated whether insulin regulates ANG gene expression at the transcriptional level via interaction of the putative insulin-responsive element (IRE) with its binding protein(s) in the 5'-flanking region of the rat ANG gene. We have identified a DNA fragment containing nucleotides N-878 to N-864 (5' CCT TCC CGC CCT TCA 3') upstream of the start site of transcription of the rat ANG gene (16) as a putative rat ANG-IRE. This ANG-IRE was bound to a major nuclear protein with an apparent molecular mass of 48 kDa from IRPTCs incubated in normal glucose (i.e. 5 mM) medium. High glucose levels (i.e. 25 mM) enhanced the expression of 48-kDa nuclear protein and induced an additional 70 kDa nuclear protein expression in IRPTCs. Insulin inhibited both 48- and 70-kDa nuclear protein expression stimulated by high glucose. This inhibitory action of insulin was blocked by PD98059 but not by wortmannin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-[32P-ATP] (3000 Ci/mol) and Na
125I were obtained from Amersham Pharmacia Biotech (Oakville, Ontario, Canada). The Quick Change
Site-Directed Mutagenesis kit was bought from Stratagene
Inc. (La Jolla, CA). Restriction and modified enzymes were acquired
from either Life Technologies, Inc. (Burlington, Ontario,
Canada), Roche Molecular Biochemicals (Dorval,
Québec, Canada) or Amersham Pharmacia Biotech. The plasmid, pRSV-Neo, containing the coding sequence for Neomycin (Neo) with the Rous Sarcoma Virus (RSV) enhancer/promoter sequence fused in the 5'-end of the Neomycin gene was a gift from Dr. Teresa Wang (Department of Pathology, Stanford University, Stanford, CA). The plasmid, pOGH, containing the human GH (hGH) gene as reporter gene without promoter sequence was purchased from the Nichols Institute of Diagnostics (La Jolla, CA).
The RIA kit for hGH was a gift from NIDDK (National Institutes of Health, Bethesda, MD). The RIA procedures have been described previously (17). NIAMDD-hGH-I-1 (afp-4793B) was used for both iodination and hormone standard. The limit of sensitivity of the assay was 0.1 ng/ml. The interassay and intraassay coefficients of variation were 12% (n = 10) and 10% (n = 10), respectively.
Oligonucleotides for rat ANG N-882 to N-855 (5' CCT CCC TTC CCG CCC TTC ACT TTC TAG T 3') (17), mutants of ANG N-882 to N-885 (M1, 5' CCT CCC TTC CAT TAC TTC ACT TTC TAG T 3', M2, 5' CCT CCC TTA AAT AAG ACC ACT TTC TAG T 3', M3, 5' CCT CCC TTC CCT TCC TTC ACT TTC TAG T 3', M4, 5'CCT CCC TTC CCT CCC TTC ACT TTC TAG T 3', and M5, 5' CCT CCC TTC CCG TCC TTC ACT TTC TAG T 3'), rANG-IRE motif N-878 to N-864 (5' CCT TCC CGC CCT TCA 3'), rANG-5' IRE N-901 to N-880 (5' CAG GGA CTG CTC TGC CAA TACC 3'), rANG-3' IRE N-862 to N-841 (5'TTT CTA GTG CCA CTT TAG GGT 3'), IRE of the human glyceraldehyde phosphate dehydrogenase gene (hGAPDH-IRE, N-473 to N-477, 5' CCA ACT TTC CCG CCT CTC AGC CTT TGA A 3') (18) and IRE of the rat glucagon gene (N-267 to N-242, 5' AGT TTT CAC GCC TGA CTG AGA TTG A 3') (19) were synthesized by Life Technologies, Inc.
Construction of fusion genes
The method of construction of the rANG-GH fusion genes, pOGH
(rANG N-1498/+18), pOGH (rANG N-1120/+18), pOGH (rANG N-820/+18), pOGH
(rANG N-688/+18) and pOGH (rANG N-53/+18) has been described previously
(17, 20). To construct the fusion genes, pOGH (rANG
N-882/+18) and pOGH (rANG N-854/+18), PCR were used to synthesize the
DNA fragments N-882 to N+10 and N-854 to N+10 by employing the fusion
gene pOGH (rANG N-1498/+18) as template and forward oligonucleotides
corresponding to the nucleotides N-882 to N-855 and N-854 to N-829 with
HindIII enzyme restriction site on the 5'-end and a reversed
oligonucleotide corresponding to N-13 to N+10 of the rat ANG gene
(17, 20). The PCR-DNA fragments N-882 to N+10 and N-854 to
N+10 were then digested with the restriction enzymes HindIII
and XhoI. The digested DNA fragments N-882 to N-35 and N-854
to N-35 were isolated by a QIAGEN spin column
(QIAGEN, Mississauga, Ontario, Canada) and then inserted
into the fusion gene pOGH (rANG N-1498/+18) which had been digested
previously with HindIII and XhoI.
To construct the fusion genes pOGH (rANG-IRE/-53/+18) and its mutant (M2), the double-strand DNA fragments ANG N-882 to N-855 and its mutant with the HindIII and XbaI enzyme restriction site on the 5' and 3' ends, respectively, were inserted upstream of the promoter (N-53/+18) of the rat ANG gene in pOGH (rANG N-53/+18) which had been digested previously with the restriction enzymes HindIII and XbaI.
Site-directed mutagenesis was used to construct the mutant of pOGH (rANG N-1498/+18) with the nucleotides N-874 to N-867 (5' CCC GCC CTT 3') mutated to 5' AAA TAA GAC 3' by employing the Quick Change Site-Directed Mutagenesis kit (Stratagene Inc.) according to the manufacturer’s instruction manual. Briefly, the mutated oligonucleotide N-892 to N-850, 5' CTC TGC CAA TCC TCC CTT AAA TAA GAC CAC TTT CTA GTC CCA C 3', and its complementary strand were used in the PCR by employing pOGH (rANG N-1498/+18) as template. The following PCR conditions were followed: denaturation at 95 C for 30 sec, annealing at 55 C for 60 sec, and then extension at 68 C for 12 min. Twelve cycles of PCR reaction were conducted. Then, the PCR mixture was digested with the restriction enzyme Dpn1 at 37 C for 1 h and subsequently transformed the bacteria XL-1 Blue. The plasmids were then isolated and identified by restriction enzyme digestion mapping and DNA sequencing. The sequence and orientation for the fusion genes were also confirmed by dideoxy sequencing with the SP-6 primer (Promega-Fisher Inc., Saint-Laurent, Québec, Canada) and restriction enzyme digestion mapping.
Cell culture
IRPTCs at passages 11–18 were used in the present studies. The
characteristics of IRPTCs have been described previously
(21). These cells express the mRNA and protein of ANG,
renin, ACE, and Ang II receptors (21).
IRPTCs were grown in 100 x 20 mm plastic Petri dishes (Life Technologies, Inc.) in normal glucose (i.e. 5 mM) DMEM (pH 7.45), supplemented with 10% FBS, 100 U/ml of penicillin and 100 µg/ml of streptomycin. The cells were propagated in a humidified atmosphere in 95% air, 5% CO2 at 37 C. For subculturing, the cells were trypsinized (0.05% trypsin and EDTA) and plated at 2.5 x 104 cells/cm2 in 100 x 20 mm Petri dishes.
DNA transfection
Plasmids or ANG-GH fusion genes were transfected into IRPTCs
using Lipofectamine reagent according to the instruction manual
provided by the supplier (Life Technologies, Inc.). We
have optimized the DNA concentration for gene transfection at 2 µg
per 0.5 to 1 x 106 cells. Thus, in the
present studies, a total of 2 µg of supercoiled DNA (i.e.
1 µg of ANG-GH fusion gene and 1 µg of pRSV/CAT) was used routinely
in cell transfection. The plasmid pRSV/CAT served as an internal
control to monitor the efficiency of transfection of various ANG-GH
fusion genes in the absence of insulin. The level of transfection
efficiency for pRSV/CAT in IRPTCs ranged from 60–90%, i.e.
the percentage of conversion of 14-C
chloramphenicol to mono- and di-acetyl chloramphenicol. The method for
chloramphenicol acetyltransferase (CAT) assay has been described
previously (22, 23, 24).
Effect of insulin on fusion genes expression in IRPTCs
After DNA transfection, the cells were synchronized with
serum-free 5 mM glucose DMEM for 24 h. Then, the cells
were incubated for 24 h in 5 mM glucose or 25
mM glucose medium containing 1% depleted FBS (dFBS) in the
absence or presence of
10-7 M
insulin. At the end of the incubation period, the media were collected
and stored at -20 C until assayed for immunoreactive human GH
(IR-hGH). The cells were harvested for CAT assay. To maintain constant
isotonicity or osmolality, the 5 mM glucose media were
supplemented with D-mannitol (20 mM) final
concentration. The dFBS (i.e. depletion of endogenous
steroid and thyroid hormones) was prepared by incubation with 1%
activated charcoal and 1% AG 1 x 8 ion-exchange resin
(Bio-Rad Laboratories, Inc., Richmond, CA) for 16 to
24 h at room temperature, as described by Samuels et
al. (25).
Cellular nuclear extract preparation
IRPTC nuclear extracts were prepared from 20 plates (150 x
20 mm) each of confluent IRPTCs that had been incubated in DMEM with 5
mM glucose and 20 mM D-mannitol, 25
mM glucose, or 25 mM glucose plus insulin
(10-7 M) with
or without PD98059 (10-5
M) or wortmannin
(10-5 M)
according to the method of Henninghausen and Lubon (26)
with slight modification, as we have described elsewhere
(27).
Gel mobility shift assay
The assay was performed according to the methods presented
elsewhere (27, 28), employing the labeled ANG-IRE motif
(N-878 to N-864) as probe. Briefly, the DNA fragment was 5'-end labeled
with [-32P]-ATP by T4
polynucleotide kinase. IRPTC nuclear proteins (10 µg) or BSA (10
µg) in the presence of 0.3 U of poly(dI/dC) in 20 mM
HEPES (pH 7.6), 1 mM EDTA, 50 mM KCl, 2
mM spermidine, 1 mM DTT, 0.5 mM
PMSF, and 10% glycerol (vol/vol) were incubated for 30 min at room
temperature. Then, the 5'-labeled probe (
0.1 pmol) was added and
further incubated for 30 min at room temperature. After being chilled
on ice, the mixture was run on 8% (wt/vol) nondenaturing
polyacrylamide gel and exposed for autoradiography.
In competition assays, 100- to 300-fold molar excess of unlabeled DNA fragments was added to the reaction mixture and incubated for 30 min at room temperature before incubation with the labeled probe.
Southwestern blotting
Southwestern blotting analysis was performed according to the
procedures of Kwast-Weldfeld et al. (29) with
slight modifications (27, 28). Briefly, IRPTC nuclear
proteins (50–200 µg) were resolved on a 4 to 20% gradient of
SDS-PAGE (30) and were then electrotransferred to a Hybond
C-extra membrane (Amersham Pharmacia Biotech). The
membrane was incubated with 10% (wt/vol) nonfat milk proteins in a
binding buffer containing 10 mM HEPES, pH 7.0, 10
mM MgCl2, 50
mM NaCl, 0.25 mM EDTA and
2.5% glycerol (vol/vol) for 24 h at 4 C. The membrane was then
washed at least twice with binding buffer containing 0.25% nonfat milk
proteins. Subsequently, the membrane was hybridized overnight with
32P-labeled double-stranded oligonucleotides
(1.0 to 2.0 pmol; 106 cpm/ml) in
binding buffer containing 0.25% nonfat milk proteins and 300
µg/ml nondenatured herring sperm DNA at 4 C. The membrane was finally
washed, air-dried and exposed for autoradiography.
Statistical analysis
The DNA transfection experiments were performed at least three
times in triplicate. The data were analyzed by Student’s t
test or ANOVA. A probability level of P 
0.05 was
regarded as significant.
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Results |
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depicts the IRE consensus
sequence in hGAPDH gene (18), rat glucagon gene
(19), rat apolipoprotein AI (apo AI) gene
(31), and rat ANG gene (17). It is apparent
that the putative rANG-IRE motif 5' CCT TCC CGC CCT TCA 3' is
approximately 80% homologous with hGAPDH-IRE (18), 60%
homologous with rat Apo AI-IRE (31), and 53% homologous
with rat glucagon-IRE (19). The mutation of the putative
IRE of rat ANG gene is shown in Fig. 2.
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schematically depicts the
structure of various ANG-GH fusion genes. These fusion genes were
transiently cotransfected with the internal control plasmid pRVS/CAT to
monitor the efficiency of transfection.
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shows the expression of
pOGH (rANG N-1498/+18), pOGH (rANG N-1120/+18), pOGH (rANG N-882/+18),
pOGH (rANG N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-688/+18) and
pOGH (rANG N-53/+18) in IRPTCs incubated in 5 mM glucose
plus 20 mM D-mannitol or 25 mM
glucose in the absence or presence of
10-7 M
insulin. A high level of glucose (25 mM) enhanced the
expression of pOGH (rANG N-1498/+18), pOGH (rANG N-1120/+18) and pOGH
(rANG N-882/+18) by greater than 2.0-fold compared with normal glucose
(i.e. 5 mM) (P
0.005). The addition of insulin to the culture medium abolished the
high glucose (25 mM)-stimulated expression of
pOGH (rANG N-1498/+18), pOGH (rANG N-1120/+18) and pOGH (rANG
N-882/+18. In contrast, neither 25 mM glucose nor
insulin (10-7
M) had any effect on the expression of pOGH (rANG
N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-688/+18) and pOGH (rANG
N-53/+18) in IRPTCs. These studies demonstrate that the inhibitory
effect of insulin on the expression of the ANG gene may be mediated, at
least in part, via the DNA sequence located between nucleotides N-882
and N-855 upstream of the start site of transcription in the
5'-flanking region of rat ANG gene.
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). As shown in Fig. 5, the expression of the mutant of pOGH
(rANG N-1498/+18) was increased (i.e. 2.0-fold,
P 0.005) in 25 mM glucose
medium compared with 5 mM glucose medium. Insulin
(10-7
M), however, did not inhibit the stimulatory
effect of the 25 mM glucose medium on mutant pOGH
(rANG N-1498/+18) expression. These studies provide evidence that the
DNA sequence, N-878 to N-864 is a putative rat ANG-IRE motif.
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). Figure 5 shows that the
expression of pOGH (rANG-IRE/-53/+18) and the mutant of pOGH
(rANG-IRE/-53/+18) did not change significantly in 25 mM
glucose compared with 5 mM glucose medium. The addition of
insulin (10-7
M), however, decreased the expression of pOGH
(rANG-IRE/-53/+18), but had no effect on the expression of mutant of
pOGH (rANG-IRE/-53/+18). These data further supported the postulate
that the DNA sequence N-874 to N-867 is a functional rat ANG-IRE
motif.
Gel mobility shift assay
Interaction of the putative rat ANG-IRE (N-878 to N-864)
with IRPTC nuclear proteins was examined by gel mobility shift assay,
as shown in Figs. 6 and 7. When the labeled rANG-IRE motif N-878
to N-864 was incubated with nuclear proteins of IRPTCs in 5
mM glucose medium, 1 major band and 1 minor band appeared
with retarded mobility (Fig. 6A). No slowly migrating band was observed
when the labeled DNA was incubated with BSA. The addition of an
unlabeled DNA fragment, rANG-IRE (WT, N-882 to N-855), rANG-IRE motif
(N-878 to N-864) or hGAPDH-IRE, was effective in competing with the
binding of labeled ANG N-878 to N-864 to the nuclear proteins(s)
(i.e. at 100- and 300-fold molar excess of unlabeled DNA
fragment) but not the unlabeled DNA fragment of rat glucagon-IRE or
the 5'- and 3'-ends of rANG-IRE (Fig. 6B). Unlabeled mutants (M3, M4,
and M5) of rat ANG-IRE (N-878 to N-864) were effective in competing
with the binding of labeled ANG N-878 to N-864 but not the unlabeled
mutants (M1 and M2) of rANG N-882 to N-855 (Fig. 7). These studies
showed that the mutation of 4 nucleotides in N-872 to N-869 was
sufficient to completely abolish binding with the nuclear protein(s) of
IRPTC but not a single mutation of nucleotides in N-70 or N-71 or a
mutation of both nucleotides in N-70 to N-71. Our experiments indicate
that the rANG N-874 to N-867 sequence localized within nucleotides
N-878 to N-864 (i.e. 5' CCT TCC CGC CCT TCA 3') is important
for binding to nuclear proteins of IRPTCs incubated in 5
mM glucose medium.
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. The labeled rat ANG-IRE motif
interacted with 1 major molecular species with an apparent molecular
mass of 48 kDa from IRPTCs incubated in 5 mM glucose medium
(Fig. 8A). High glucose levels (25 mM) enhanced 48-kDa
species expression and induced an additional molecular species
expression with an apparent molecular mass of 70 kDa that interacted
with the labeled rat ANG-IRE in IRPTCs (Fig. 8B). Insulin inhibited the
stimulatory effect of high glucose on both 48- and 70-kDa nuclear
proteins expression in IRPTCs. Unlike wortmannin, the addition of
PD98059 reversed the inhibitory action insulin on both 48- and 70-kDa
nuclear proteins expression in IRPTCs incubated in high glucose medium
(Fig. 8B).
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Discussion |
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pOGH (rANG-1498/+18) expression in IRPTCs was increased 2.0-fold in the
presence of high-glucose (25 mM) medium compared with
normal glucose (5 mM) medium (Figs. 3 and 4). This level of
stimulation is similar to that in our previous studies, which showed
that high glucose levels stimulate the expression of the pOGH
(rANG-1498/+18) and ANG mRNA 1.5- to 2.0-fold in opossum kidney (OK)
proximal tubular cells (32) and in IRPTCs
(15), respectively. Studies by Chang and Perlman
(33) and Aubert et al. (34) have
demonstrated that insulin attenuates ANG mRNA expression in rat
hepatoma cells and cultured adipose tissue in vitro,
respectively. We have also reported that insulin inhibits ANG and ANG
mRNA expression in IRPTCs (16) and inhibits the expression
of human ANG gene promoter activity in OK cells in high glucose medium
(35). Consistent with these findings, we have observed
that insulin inhibits the stimulatory effect of glucose on rat ANG-GH
fusion genes expression in IRPTCs (Figs. 2 and 3). These results, taken
together with those of Chang and Perlman (33) and Aubert
et al. (34), suggest that insulin
down-regulates ANG gene expression at the transcriptional level. We did
not observe, however, any significant inhibition of ANG-GH fusion genes
expression in IRPTCs treated with
10-7
M insulin-like growth factor (IGF)-I or IGF-II
(data not shown), indicating that the inhibitory action of insulin on
ANG-GH fusion genes expression in IRPTCs is specific for insulin and
the insulin receptor. The addition of insulin
(10-7
M) also had no significant effect on ANG-GH
fusion gene in IRPTCs when cells were grown in 5
mM glucose medium (data not shown). Moreover, we
did not observe any significant effect of hGH (i.e. 1 to 100
ng/ml) on the expression of fusion gene pOCAT(rANG N-1498/+18)
(24) containing the full-length 5'-flanking region of rat
ANG gene inserted upstream of the chloramphenicol acetyl transferase
gene in IRPTCs (unpublished results). These studies demonstrated that
hGH had no effect on ANG gene expression in IRPTCs.
The present studies demonstrate that insulin
(10-7 M)
inhibited the expression of pOGH (rANG-1498/+18), pOGH (rANG
N-1120/+18) and pOGH (rANG N-885/+18), but not of pOGH (rANG
N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-688/+18) and pOGH (rANG
N-53/+18) in IRPTCs in high (25 mM) glucose medium (Fig. 4), suggesting that a putative IRE is probably located between
nucleotides N-884 to N-855 in the 5'-flanking region of the rat ANG
gene. Sequence analysis of nucleotides in the 5'-flanking region of the
rat ANG gene (17) revealed that nucleotides N-878 to N-864
(5' CCT TCC CGC CCT TCA 3') are 80% homologous with nucleotides N-469
to N-455 (5' CTT TCC CGC CTC TCA 3') of hGAPDH-IRE (Fig. 1), indicating
that N-878 to N-864 might be a putative IRE. Indeed, site-directed
mutagenesis of the nucleotides N-874 to N-867 in the 5'-flanking
region of the rat ANG gene abolished the response to insulin in IRPTCs
(Fig. 5). Furthermore, insulin inhibited the expression of pOGH
(rANG-IRE/-53/+18) but not that of a mutant (M2) of pOGH
(rANG-IRE/-53/+18) in IRPTCs (Fig. 5). These studies further support
the notion that ANG N-878 to N-864 contains the functional IRE of the
rat ANG gene.
To investigate whether ANG N-878 to N-864 interacts with nuclear
proteins in IRPTCs, we performed gel mobility shift assays. These
assays revealed that labeled putative rANG-IRE interacted with IRPTC
cellular nuclear protein(s) (Fig. 6). The addition of unlabeled rANG
N-882 to N-855, ANG N-878 to N-864 and hGAPDH-IRE effectively displaced
labeled ANG-IRE at or greater than a 100-fold molar excess of
unlabelled DNA, whereas unlabeled rat glucagon-IRE and 5'- or 3' of
rANG-IRE at 100-fold molar excess were slightly effective in competing
with labeled DNA. Similarly, the unlabeled mutants M1 and M2 of
rANG-IRE were not effective in displacing the labeled putative rANG-IRE
at 100- or 300-fold molar excess (Fig. 7). In contrast, M3, M4, and M5
were effective in competing with labeled ANG-IRE (Fig. 7). These
studies further demonstrate that the nucleotides N-878 to N-864
represent the rANG-IRE motif, which is essential for binding to IRPTC
nuclear proteins.
Most interestingly, our Southwestern blotting experiments revealed that
labeled ANG N-878 to N-864 bound to 1 major IRPTC nuclear protein(s)
with an apparent molecular mass of 48 kDa (Fig. 8A). High glucose
levels (25 mM) enhanced the expression of the 48 kDa
molecular species and induced an additional molecular species of 70 kDa
(Fig. 8B). Insulin suppressed the expression of both 48- and 70-kDa
molecular species induced by high glucose. PD98059 but not wortmannin
reversed the insulin action. The addition of PD 98059
(10-5 M) or
wortmannin (10-5
M) had no effect on cell viability after a 24 h-incubation
period (i.e. >95% viability as determined by Trypan blue
exclusion method) (data not shown). These studies indicate that 48- and
70-kDa IRE-binding protein(s) may mediate the stimulatory and
inhibitory effect of high glucose and insulin respectively on ANG gene
expression in IRPTCs. Furthermore, we have found that the 48 kDa IRE-BP
is present in nuclear extracts of rat brain, lung, testis, spleen,
kidney, and liver. The larger molecular species of IRE-BP, however, is
found only in the nuclear extracts of rat brain, kidney and liver (our
unpublished results). These observations raise the possibility
that the distribution of these two IRE-BPs is distinct in different
tissues and might be differentially regulated. More studies are
warranted to study the regulation of these IRE-BPs in different
tissues.
The molecular structure of the 48- and 70-kDa IRE-binding protein is
unknown. It is not clear whether these two nuclear proteins are
structurally related. It is possible that the 48-kDa species might be
the mature product of 70-kDa species. Molecular cloning of these
proteins are required to elucidate their relationship. The apparent
molecular masses of these nuclear proteins are not similar to either
Sp-1-related proteins (i.e. 106 kDa) (36, 37)
or the binding proteins to the hGAPDH-IRE reported by the group of
Alexander-Bridge (38, 39). These investigators identified
the binding protein to hGAPDH-IRE as a member of the HMG class of
transcriptional factors. It has an apparent molecular mass of
approximately 10 kDa and is homologous with SRY and TCF-1 
.
Moreover, the unidentified 48- and 70-kDa nuclear proteins are smaller
than the 75-kDa FKHR nuclear protein (40) that binds to
the IRE motif (i.e. TG/ATT) of IGF binding protein-1
(41, 42, 43). Finally, both 48- and 70-kDa nuclear proteins
are also smaller than the 78-kDa glucose-regulated proteins
(44, 45, 46). Thus, our studies suggest that 48- and 70-kDa
IRE-binding proteins may represent unidentified IRE-binding proteins.
The exact physiological role(s) of 48- and 70-kDa nuclear proteins is
unknown at present. Experiments such as cloning and expression of these
proteins are definitely warranted to demonstrate their biological
activity.
In summary, these studies shown that insulin inhibited the expression of the rat ANG gene at the transcriptional level. Insulin effect is mediated, at least in part, via interaction of IRE (ANG N-878 to N-864) in the 5'-flanking region of the rat ANG gene with a major 48-kDa nuclear protein in IRPTCs incubated in normal (i.e. 5 mM) glucose medium. High glucose and insulin enhanced and suppressed 48- and 70-kDa nuclear protein expression in IRPTCs, respectively. PD98059 but not wortmannin abolished the insulin effect. These observations indicate that the unidentified 48- and 70-kDa nuclear proteins may play an important role in mediating the stimulatory and inhibitory effect of high glucose and insulin respectively on the expression of the ANG gene in the kidney. Finally, our studies raise the possibility that hyperglycemia stimulates renal ANG gene expression in vivo, mediated via the expression of unidentified 48- and 70-kDa IRE-binding proteins. Consequently, the increased local formation of renal Ang II may contribute to renal remodeling (i.e. renal hypertrophy observed in early diabetes). Moreover, insulin therapy may attenuate this event by inhibiting ANG gene expression via the down-regulation of 48- and 70-kDa IRE-binding proteins and subsequently suppressing activation of the local renal RAS.
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Acknowledgments |
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Footnotes |
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Received December 18, 2000.
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 C.-C. Wei, S.-L. Zhang, Y.-W. Chen, D.-F. Guo, J. R. Ingelfinger, K. Bomsztyk, and J. S. D. Chan Heterogeneous Nuclear Ribonucleoprotein K Modulates Angiotensinogen Gene Expression in Kidney Cells J. Biol. Chem., September 1, 2006; 281(35): 25344 - 25355. [Abstract] [Full Text] [PDF]
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C.-C. Wei, D.-F. Guo, S.-L. Zhang, J. R. Ingelfinger, and J. S.D. Chan Heterogenous Nuclear Ribonucleoprotein F Modulates Angiotensinogen Gene Expression in Rat Kidney Proximal Tubular Cells J. Am. Soc. Nephrol., March 1, 2005; 16(3): 616 - 628. [Abstract] [Full Text] [PDF]
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 T.-J. Hsieh, P. Fustier, C.-C. Wei, S.-L. Zhang, J. G Filep, S.-S. Tang, J. R Ingelfinger, I G. Fantus, P. Hamet, and J. S D Chan Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells J. Endocrinol., December 1, 2004; 183(3): 535 - 550. [Abstract] [Full Text] [PDF]
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 T.-J. Hsieh, P. Fustier, S.-L. Zhang, J. G. Filep, S.-S. Tang, J. R. Ingelfinger, I. G. Fantus, P. Hamet, and J. S. D. Chan High Glucose Stimulates Angiotensinogen Gene Expression and Cell Hypertrophy via Activation of the Hexosamine Biosynthesis Pathway in Rat Kidney Proximal Tubular Cells Endocrinology, October 1, 2003; 144(10): 4338 - 4349. [Abstract] [Full Text] [PDF]
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S.-L. Zhang, X. Chen, C.-C. Wei, J. G. Filep, S.-S. Tang, J. R. Ingelfinger, and J. S. D. Chan Insulin Inhibits Dexamethasone Effect on Angiotensinogen Gene Expression and Induction of Hypertrophy in Rat Kidney Proximal Tubular Cells in High Glucose Endocrinology, December 1, 2002; 143(12): 4627 - 4635. [Abstract] [Full Text] [PDF]
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