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Section of Renovascular Pharmacology and Physiology (INSERM-ULP), University Louis Pasteur School of Medicine, Strasbourg, France
1Correspondence: Pharmacologie et Physiologie Rénovasculaires (Equipe INSERM 0015-Equipe MENRT 2307), 11, rue Humann, Bâtiment 4, 1er étage, F67085 Strasbourg Cédex, France. E-mail: Jean-Jacques.Helwig{at}pharmaco-ulp.u-strasbg.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: mitogenesis • intracrine • nucleolus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, it became apparent that parathyroid hormone-related
protein (PTHrP) is actually produced by virtually all normal cells
during embryonic, fetal, and adult life (1)
. Lessons from
gene knockout experiments emphasize the critical role of PTHrP in
normal life (2
, 3)
. PTHrP is processed by members of the
prohormone convertase family to at least three fragments: the
amino-terminal PTHrP (1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36)
structurally related to PTH,
a mid-region PTHrP(38–94), and a carboxy-terminal PTHrP(107–139),
each having its own biological activities (1
, 4)
. So far,
however, only the receptor binding both PTHrP(1–36) and PTH(1–34),
also called PTH1R, has been characterized (5
, 6)
. The
repertoire of the possible physiological roles of PTHrP is continuously
expanding. First, PTHrP is a regulator of transepithelial calcium
transport in renal tubules, placenta, and the mammary gland. Second,
PTHrP is a potent relaxant of vascular and extravascular smooth muscle.
Finally, PTHrP controls the rate of cell proliferation, apoptosis, and
differentiation, thus affecting local tissue (re)organization under
various physiological and pathophysiological situations. (1
, 7
8
9)
. Most activities of the different PTHrP species were
believed to act exclusively through the auto/paracrine pathways
(1
, 4
, 10
11
12)
. Recently however, PTHrP appeared to have
intracrine effects by translocation of the nascent peptide into the
nucleus. Indeed, PTHrP has been proved to contain a classical basic
bipartite nuclear/nucleolar location signal (NLS) in its 88–107 region
similar to the NLSs in viral and mammalian transcription factors
(13
14
15)
. Nuclear localization of PTHrP has been shown to
delay apoptosis and to regulate differentiation in chondrocytes
(13)
.
In vessels, PTHrP and the PTH1R are expressed in both vascular smooth
muscle cells (VSMC) and endothelial cells (7)
. In VSMC,
PTHrP is rapidly and transiently up-regulated in response to growth
factors, vasoconstrictors, and mechanical forces (16
, 17)
.
PTHrP is also up-regulated in the aorta of genetically hypertensive rat
models (18)
, in atherosclerotic coronary arteries
(19)
, and in restenotic coronaries during neointimal
formation after angioplasty (20)
. In functional terms,
PTHrP decreases blood pressure, dilates blood vessels, and has been
proposed to be involved in the regulation of systemic and regional
hemodynamics (7)
. In strong support of this latter
hypothesis, transgenic mice that selectively overexpress PTHrP or the
PTH1R in smooth muscle exhibit a cardiovascular phenotype, including a
decrease in blood pressure (21
, 22)
. In addition to its
tonic effects, PTHrP inhibits the growth of VSMC either directly
(23
, 24)
by inducing growth arrest at
G1 phase (25)
or indirectly by
opposing the growth-promoting effects of vasoconstrictor agents such as
angiotensin II (16)
. In rat aortic A10 VSMC, exogenously
applied amino-terminal PTHrP species inhibit proliferation by
interacting with the PTH1R. In marked contrast, stable transfection of
A10 cells with full-length PTHrP induces a marked increase in cell
proliferation (15)
. This latter effect is achieved through
nuclear translocation of PTHrP in a NLS-dependent manner
(15)
. Moreover, an association between the up-regulation
of PTHrP and down-regulation of vascular PTH1R mRNA in vessels has
repeatedly been reported (7
, 20)
. Collectively, these
findings strongly indicate that PTHrP might serve as a deleterious
promitogenic factor that participates in the deranged VSMC
proliferation that occurs under various pathophysiological conditions.
The spontaneously hypertensive rat (SHR) model of genetic hypertension
provides many similarities to human essential hypertension with respect
to such important aspects as pathophysiological development, clinical
course, and secondary diseases. Thus, the SHR is now the most widely
used animal model of primary hypertension. Moreover, genetically
determined renal mechanisms play a major role in the development of
primary hypertension in both human and SHR. In particular, evidence
from renal transplantation studies between SHR and normotensive Wistar
Kyoto rats (WKY), as well as studies of human renal graft recipient,
strongly supports the view that the kidney plays an important role in
the development of primary hypertension. These aspects have been
extensively reviewed by Rettig et al. over the past decade
(26
27
28)
. In terms of PTHrP, we reported previously that
this peptide is expressed throughout the entire renal arterial tree
(29)
and that PTHrP(1–36) not only modulates renal
hemodynamics both in vitro and in vivo
(30
31
32)
, but also interacts with the renin-angiotensin
system (30
, 33)
. In SHR, the renal vasodilatation induced
by PTHrP is markedly reduced as compared to age-matched WKY rats,
suggesting that endogenous PTHrP may be more important for the
regulation of VSMC proliferation than tone (34)
. In this
organ, deranged VSMC proliferation under pathophysiological conditions
is preferentially localized in the small arteries. The most striking
example is precisely the almost selective, early, and
pressure-independent wall hypertrophy of arcuate and interlobular
arteries in SHR.
Collectively, these data prompted us to examine the hypothesis that 1) like in the clonal cell line A10 derived from embryonic thoracic aorta, PTHrP exerts paradoxical effects on VSMC derived from small intrarenal arteries, and 2) such effects may play a role in the deranged proliferation of these cells in the SHR model of genetic hypertension. Our study therefore attempted to explore the role of exogenously added and endogenous PTHrP on the proliferation of renovascular smooth muscle cells (RvSMC) derived from small arteries isolated from SHR as compared to age-matched normotensive WKY rats. We have demonstrated that exogenously added PTHrP, a surrogate for autocrine-secreted PTHrP, is antimitogenic, whereas endogenously produced PTHrP is mitogenic on RvSMC. These paradoxical effects of PTHrP on renovascular SMC proliferation appear to be reversed in the SHR model of genetic hypertension.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of intrarenal arteries and culture of RvSMC
All animal studies were performed in compliance with the French
animal use rules. Twelve-week-old male SHR or WKY rats weighing
280–340 g with free access to standard food and water were
anesthetized with ether and decapitated. Small preglomerular arterial
trees consisting of arcuate and interlobular arteries (80–400 µm
diameter) were isolated from excised kidneys exactly as we have
described previously (35)
. Briefly, kidneys were
decapsulated, longitudinally bisected, and the medulla removed. Kidneys
halves were pressed sequentially against stainless steel sieves of 40
and 50 mesh size and nylon sieve of 150 mesh size. The renal vascular
trees, devoid of preglomerular arterioles and glomeruli, were retained
on the grids and further processed for culture of RvSMC. All subsequent
steps were performed in sterile conditions. Four vascular trees were
prepared independently from 4 WKY rats, and 5 vascular trees were
prepared independently from 5 SHR. Vascular trees were incubated for 20
min at 37°C in phosphate-buffered saline (PBS) containing 0.6 mg
ml-1 collagenase (type IA), rinsed with the
buffer successively on 60 and 150 mesh grids, and then transferred in
6-well plates precoated with rat tail collagen (type I) in 0.5 ml DMEM
supplemented with 30% FBS, penicillin (100 U/ml), and streptomycin
(0.1 mg/ml). Explants were cultured at 37°C in humidified air
containing 10% CO2. When a sufficient amount of
cells had grown out from the explants (typically after 10 to 14 days),
cells were passaged by trypsinization. A homogeneous population of
spindle-shaped cells were obtained, which grew in a hill-and-valley
pattern and stained positively for smooth muscle 
-actin, consistent
with the VSMC phenotype (35)
. We have shown previously
(35)
that RvSMC can be successfully passaged more than
20-fold without noticeable changes in morphology, growth
characteristics, and smooth muscle -actin expression. Unless
otherwise specified, RvSMC were used at passages 6–20 and cultured at
37°C in DMEM medium containing 10% FBS and antibiotics in humidified
air containing 10% CO2.
Plasmids and stable transfection of RvSMC
To overexpress PTHrP(1–139), RvSMC were transfected with the
retroviral vector pLJ, which has been used for A10 cell transfection
(15)
. Human PTHrP instead of rat PTHrP was used to
distinguish its expression from that of endogenous PTHrP. To inhibit
endogenous rat (r)PTHrP expression, RvSMC were transfected with the
pcDNA3 vector in which the human PTHrP(1–139) cDNA was subcloned in an
antisense orientation. RvSMC derived from one representative WKY or SHR
explant were stably transfected with these constructs using
lipofectamine according to the manufacturer’s protocol. In this
protocol, cells were challenged with constructs over a period of 3 h in serum-free medium to optimize the efficiency of cell survival and
therefore of cell transfection. Two days after transfection, 50 µg
ml-1 G418 was added to the medium for selection
of transfected cells. The visual evaluation of the number of floating
cells after treatment with G418 indicated that most of the cells were
transfected. Cells transfected with the empty vectors (pLJ and pcDNA3)
served as controls.
Quantitative competitive reverse transcriptase-polymerase chain
reaction (RT-PCR) assay for endogenous rat PTHrP
To measure the expression of the rat PTHrP transcript in
untransfected WKY and SHR RvSMC and to assess the efficiency of the
transfection with the PTHrP antisense construct, endogenous rat PTHrP
mRNA was quantified using a sensitive competitive RT-PCR according to
the protocol recently described by Pirola et al. (17)
with
slight modifications. Briefly, to construct the competitive template
(competitor), a 166 bp EcoN1 fragment was cleaved from the rat
PTHrP(1–141) cDNA clone rPLPm10 (17)
. Rat PTHrP sense and
antisense primers (17)
were designed bracketing the EcoN1
cleavage sites and were predicted to give a 160 bp competitor fragment
compared to 320 bp of target PTHrP upon amplification of total cDNA.
RvSMC were grown to 70 to 80% confluence. Total RNA was extracted using the TRIzol method according to the manufacturer’s protocol. Reverse transcription was performed with 10 µg denatured total RNA using nonspecific P(dT)15 primer (2 µM) at 37°C for 1 h. PCR was performed using 0.75 µg of reverse transcribed RNA and serial dilutions of the competitor ranging from 0.755 to 0.001 attamole were added to the PCR reactions. All reactions were done with 1.5 mM MgCl2 and 0.15 µM of each primer. The PCR began with denaturation at 94°C for 4 min. PCR cycles were programmed as follows: 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. PCR was run for 40 cycles and the last cycle was followed by an additional incubation at 72°C for 7 min. Amplified products were separated by electrophoresis on a 2% agarose gel containing 0.5 µg ml-1 ethidium bromide in the presence of Tris acetate EDTA buffer. PCR products were identified by their expected size of 320 bp (endogenous PTHrP) and 160 bp (competitor). Control reactions were done by omitting reverse transcriptase. Agarose gels were recorded with a video system. Band intensities were quantified by means of a gel analysis software (Sigma Gel®, Jandel Scientific, Erkrath, Germany) and ratios of competitor/rPTHrP were calculated.
Semi-quantitative RT-PCR assay for the rat PTH1R and human PTHrP
Total RNA was extracted from subconfluent RvSMC as described
above. Expression of the rat PTH1R transcript in untransfected WKY and
SHR RvSMC and human PTHrP transcript in RvSMC transfected with human
PTHrP(1–139) were analyzed by RT-PCR using glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) expression as a reference for semi-quantitative
analysis. In each case, RT was performed with 15 µg denatured total
RNA using nonspecific P(dT)15 primer (2 µM) at
37°C for 1 h. The concentrations of reverse-transcribed RNA in
PCR reactions were adjusted in preliminary experiments to obtain
similar product amplifications in the presence of 0.4 µM of the
corresponding primers. Thus, the rat PTH1R and human PTHrP were
detected with 4.4 µg of reverse transcribed RNA, and GAPDH was
detected with 1.5 µg of reverse transcribed RNA. PCR reactions were
performed with the primers specific to rat PTH1R (36)
,
human PTHrP (sense: ATG CGA CGG AGA CTG GTT CAG; antisense: TCA ATG CCT
CCG TGA ATC GAG CTC CAG CGA CGT), or GAPDH (37)
. The cycle
times for PCR were as described above for competitive RT-PCR. PCR was
performed for 36 cycles, followed by an additional 7 min extension at
72°C. Amplified products were separated by electrophoresis on a 2%
agarose gel containing 0.5 µg ml-1 ethidium
bromide in the presence of Tris acetate EDTA buffer. PCR products were
identified by their expected size of 535 bp (hPTHrP), 817 bp (rPTH1R),
and 415 bp (GAPDH). Control reactions were done by omitting reverse
transcriptase. Agarose gels were recorded with a video system and band
intensities were measured. Ratios of rat PTH1R/GAPDH or human
PTHrP/GAPDH were calculated.
Radioimmunoassay of amino-terminal PTHrP species in conditioned
medium
RvSMC were grown to 70 to 80% confluence. Conditioned medium
was harvested and centrifuged at 1000 g for 2 min at 4°C
to pellet cell debris. Immunoreactive PTHrP (iPTHrP) was directly
measured on the supernatant with a commercially available
radioimmunoassay (RIA) kit, using an affinity-purified antibody
directed against chicken PTHrP(1–36). We checked that this antibody
cross-reacted with neither PTH(1–34) nor PTHrP(7–36). The results are
expressed as pmol. l-1 of medium.
Western blot analysis
The expression of PTH1R protein in RvSMC was evaluated by
Western blot analysis as compared to COS7 cells taken as a negative
control. RvSMC and COS-7 cells were grown to 70 to 80% confluence.
Cells were washed twice with ice-cold PBS, scraped, and lysed in
ice-cold lysis buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, 0.02%
sodium azide, 100 µg ml-1 PMSF, 1 µg
ml-1 aprotinin, and 1% Nonidet P-40, pH 7.0.
Lysates were centrifuged at 12,000 g for 5 min at 4°C;
supernatants were used for Western blot analysis. Protein
concentrations were determined according to the method of Lowry et al.
(38)
with bovine serum albumin (BSA) as standard. Samples
were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). After addition of Laemmli’s SDS-PAGE
sample buffer (final concentrations: 32 mM Tris-HCl, 1% SDS, 5%
glycerol, 0.5
bromphenol blue, 3.25% 2-mercaptoethanol, pH 6.8),
the cell lysates were heated at 100°C. Lysate aliquots containing 10
µg of protein were separated on a 10% polyacrylamide gel. Proteins
were then transferred onto ECL nitrocellulose membrane. The membrane
was stabilized by incubation overnight in TSBT containing 10 mM Tris,
100 mM NaCl, 5% non-fat dry milk, and 0.1% Tween-20, pH 7.5. Using
the same buffer, the membranes were then washed three times, incubated
for 60 min at 4°C with a polyclonal rabbit anti-rat PTH1R antibody at
1/200 dilution, and again washed three times. Thereafter, blots were
incubated for 1 h with peroxidase-conjugated donkey anti-rabbit
antibody at 1/10,000 dilution, followed by 3 washes in TBST.
Immunoreactivity was visualized with the ECL Western blotting detection
kit. Blots were exposed to Hyperfilm-ECL for 5 to 20 min. Films were
digitally scanned and band intensities were quantified using a gel
analysis software (Sigma Gel®, Jandel
Scientific, Erkrath, Germany).
Cell proliferation
The growth of RvSMC was determined by measuring the
incorporation of bromo-deoxyuridine (BrdU) according to the
manufacturer’s protocol. Briefly, RvSMC were grown in 96-well plates
in serum-containing medium until 70 to 80% confluence and rendered
quiescent by culture in serum-free medium containing 0.1% BSA for
48 h. Quiescent RvSMC were exposed to 1–100 pM hPTHrP(1–36)
without or with 0.5 µg ml-1 cholera toxin or
pertussis toxin for 24 h in the presence of BrdU, in serum-free
medium containing 0.1% BSA. In each experiment, 3 wells were used for
each concentration except for the effect of cholera or pertussis toxins
alone, where 8 wells were used. BrdU incorporation was then determined
colorimetrically using a microplate reader.
Growth of untransfected and transfected RvSMC was determined not only by incorporation of BrdU as described above, but also by cell counting. For cell counting, 104 RvSMC per well were grown in 24-well plates in serum-containing medium for 12 to 14 days. Cells were harvested every 2 days by trypsinization and counted using an hemocytometer under light microscopy. Two wells were used for each time point in each experiment. In all cases, cell viability was above 90% as assessed by trypan blue exclusion. The cell population doubling time (PDT), representing the cell cycle duration during the exponential growth (typically between day 2 and day 6), was used as an index of cell growth.
PTHrP immunohistochemistry
RvSMC were plated in 4-well Tissue-Tek chamber slides and
cultured until 50% confluence in serum-containing medium. Cells were
fixed and permeabilized for 20 min in ice-cold methanol-acetone (1:1),
air-dried, rehydrated for 15 min in PBS, and blocked for 20 min with
0.1% BSA in PBS containing 0.2% Triton X-100 (PBST). Cells were then
washed in PBST and incubated for 1 h at room temperature with an
affinity-purified polyclonal rabbit anti-PTHrP(34–53) antibody at 5
µg ml-1 in PBST. Avidin-biotin
immunoperoxidase complex/labeled streptavidin biotin was used for
detection. For in vivo PTHrP localization, 12-wk-old WKY
rats and SHR kidneys were fixed in situ with 3%
paraformaldehyde for 15 min, minced into 3 mm pieces, and further fixed
for 1 h at room temperature and embedded in paraffin. Paraffin
blocks were sectioned (10 µm), paraffin was removed, sections were
incubated for 1 h at room temperature with the PTHrP(34–53)
antibody diluted at 10 µg ml-1 in PBST, and
detection was performed as described above. Sections were lightly (5 s
immersion) counterstained with hematoxylin. As a competition control,
the primary antibody was preincubated overnight at 4°C with 1 µM
PTHrP(34–53) peptide. As an additional control, some sections or cells
were processed with nonimmune serum (rabbit IgG) in the place of
primary antibody.
Statistical analysis
All values are expressed as mean ± SE. In
RT-PCR and Western-blot studies, direct band intensity values or band
intensity ratios were statistically evaluated using a paired Student’s
t test. Absolute and relative growth values of RvSMC were
compared using multifactorial analysis of variance, followed by the
Student-Newman-Keul’s test for multiple comparisons. P
values less than 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and PDT (Fig. 1B
), were studied on 4 to 5
independent explants prepared from separate WKY rats and SHR.
SHR-derived RvSMC grew faster than WKY-derived RvSMC (PDT of 43±1.8 h
in WKY vs. 32±3.2 h in SHR), a finding consistent with the known
hyperplasia/hypertrophy of the small renal arteries in SHR. Subsequent
experiments were performed on RvSMC-derived from one representative WKY
or SHR explant, indicated as ‘selected’ in Fig. 1B
,
exhibiting a PDT value comparable to the overall mean value, resulting
in a similar relative increase in proliferation of SHR-derived RvSMC as
compared to WKY-derived RvSMC.
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Expression of PTHrP and the PTH1R in WKY- and SHR-derived RvSMC
Previous studies have shown that renal vessels express
immunoreactive (i)PTHrP (26
, 31)
and display pharmacologic
evidence for PTH(1–34) and PTHrP(1–36) binding (39)
. We
therefore first asked whether RvSMC derived from small intrarenal
arteries express PTHrP and PTH1R and transcripts. Typical examples of
gels of competitive RT-PCR products for PTHrP in RvSMC derived from
both strains of rats are shown in Fig. 2A
, B
. According to quantitative analysis, the PTHrP mRNA
level in SHR RvSMC was clearly up-regulated by 
threefold as compared
to WKY (Fig. 2C
). In conditioned media, iPTHrP(1–36) was
between 0 and 6 pmol l-1 but failed to exhibit
any significant strain difference (Fig. 2D
). Despite the
clear difference in the transcript level, the absence of strain
difference is probably related to iPTHrP(1–36) values close to the
detection limit of the RIA (2–3 pmol l-1). In
cell extracts, iPTHrP(1–36) was below the detection limit (results not
shown).
|
The PTH1R mRNA levels were comparable in both strain of rats
(Fig. 3A
). The presence of PTH1R was further documented by Western
blotting. Western blot analysis of membrane proteins of RvSMC derived
from both strains of rats showed a band with an apparent molecular mass
of 90 kDa (Fig. 3B
), which corresponds to the expected
molecular weight of a functional glycosylated PTH1R (6
, 40)
. Consistent with RT-PCR studies, there was no strain
difference in the expression levels of PTH1R protein. That the Western
blot band was really the PTH1R was further supported by the virtual
absence of its expression in COS-7 cells taken as negative controls, as
well as by the absence of signal when the primary antibody was replaced
by non-immune rabbit IgGs (not shown).
|
Expression of PTHrP in transfected WKY- and SHR-derived RvSMC
Figure 4A
shows that the expression of human PTHrP transgene was
readily observable in RvSMC transfected with the pLJ-hPTHrP(1–139)
construct. In RvSMC that had been transfected with the empty pLJ
vector, human PTHrP transcript was undetectable.
|
The efficiency of the antisense technology on the expression of
endogenous rat PTHrP was quantified by competitive RT-PCR (Fig. 4B
, C
, D
, E
) Transfection with the pcDNA3-PTHrP(1–139) antisense
construct virtually abolished the expression of endogenous rat PTHrP in
both WKY- and SHR-derived RvSMC lines (Fig. 4F
). It
should be stressed that, like in untransfected cells, the cells
transfected with empty vectors displayed a similar threefold
up-regulation of endogenous PTHrP in SHR-derived cells as compared to
WKY-derived cells.
Proliferation of pLJ-hPTHrP- and pcDNA3-PTHrP antisense-transfected
RvSMC
Figure 5A
shows the PDT values of pLJ-hPTHrP- and pcDNA3-PTHrP
antisense-transfected RvSMC. As expected, transfection with the empty
vectors pLJ or pcDNA3 did not influence the PDT in either WKY- or
SHR-derived RvSMC. Despite the clear presence of the transgene,
transfections with the PTHrP sense construct (pLJ-hPTHrP) had no effect
either. On the other hand, transfections with the PTHrP antisense
construct (pcDNA3-AS:PTHrP) markedly affected the growth of RvSMC. In
WKY-derived RvSMC, PDT increased from 89 ± 5% in
vector-transfected cells to 129 ± 4% in AS:PTHrP-transfected
cells. In striking contrast, in SHR-derived RvSMC, PDT decreased from
100 ± 4% in vector-transfected cells to 81 ± 1% in
AS:PTHrP-transfected cells.
|
BrdU incorporation studies in serum-deprived quiescent cells confirmed
these observations (Fig. 5B
). Again, transfection with empty
vectors or transfection with the PTHrP sense construct (pLJ-hPTHrP) did
not influence BrdU incorporation in WKY- or SHR-derived RvSMC.
Consistently, transfection with AS:PTHrP, decreased BrdU incorporation
from 122 ± 16% in vector-transfected cells to 73 ± 8% in
transfected WKY cells and markedly increased the incorporation of BrdU
from 112 ± 4% in vector-transfected cells to 157 ± 9% in
transfected SHR cells. These results demonstrate that endogenously
produced PTHrP increases growth of WKY-derived RvSMC and decreases
growth of SHR-derived RvSMC.
Since the effect of endogenously produced PTHrP in A10 VSMC has been
associated with translocation of PTHrP into the nucleus
(15)
, we asked whether PTHrP could actually be detected
into the nucleus of RvSMC. In immunohistochemical studies, specific
staining for PTHrP was seen not only in the cytoplasm of virtually all
RvSMC, but also in the nucleolus of 2% of the wild RvSMC, whatever
the strain of rats (Fig. 6A
, C
, C
, D
). The nucleolar presence of PTHrP was also detected
in situ in paraffin-embedded kidney sections in the media of
small arteries of 12-wk-old WKY or SHR (Fig. 6E
, F
).
PTHrP staining was specific in that preincubation with the primary
antibody with PTHrP(34–53) virtually abolished staining and
replacement of the primary antiserum with non-immune antisera led to
the absence of staining (not shown).
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Effect of added hPTHrP(1–36) on WKY- and SHR-derived RvSMC
proliferation
To ask whether the above-described effects of endogenously
produced PTHrP on RvSMC proliferation occur through an autocrine
pathway, we next examined the growth effect of exogenously added
PTHrP(1–36) in WKY- and SHR-derived RvSMC (Fig. 7
). The addition of 1–100 pM hPTHrP(1–36), blunted BrdU incorporation
by 20% in WKY-derived RvSMC. This effect was reversed in
SHR-derived RvSMC; 1–100 pM hPTHrP(1–36) stimulated BrdU
incorporation by 22%.
|
Since PTH1R has been proved to be coupled to both Gs and Gi-proteins
(41)
and because Gi-protein has repeatedly been proved to
be up-regulated in SHR as compared to Gs (42)
, we asked
whether cholera toxin, a Gs-protein activator, and pertussis toxin, a
Gi-protein inhibitor, were able to influence the growth effects of
exogenous PTHrP on WKY- and SHR-derived RvSMC (Fig. 8
). Neither cholera nor pertussis toxin affected the basal proliferation
rates of WKY- and SHR-derived RvSMC. In WKY RvSMC, however, both toxins
potentiated the inhibitory effect produced by 1 pM hPTHrP(1–36) upon
cell proliferation. In SHR-derived RvSMC, both toxins abolished the
proliferative effect of 1 pM hPTHrP(1–36).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We then used
this technique to study the role of PTHrP in the abnormal proliferation
of VSMC in the renal vasculature of SHR. Studies have shown that VSMC
derived from a wide array of vascular beds proliferate faster in SHR
than in WKY (43–46). In the present study we report that SHR-derived
RvSMC proliferate faster than WKY-derived RvSMC, an observation that is
in accordance with a number of studies showing the hypertrophy and
hyperplasia of the small intrarenal arteries in the SHR model of
genetic hypertension (47)
.
Expression of PTHrP is believed to be up-regulated in the aorta in
response to increased blood pressure in experimental (48)
as well as the SHR model of genetic hypertension (18)
. In
the present studies, PTHrP mRNA was strongly increased in RvSMC
cultured from SHR as compared to WKY. We previously demonstrated that
the vasodilation caused by PTHrP(1–36) is markedly reduced in the
in vitro perfused kidney of mature hypertensive SHR
(34)
, suggesting down-regulation of PTH1R in these
animals. In the present studies, the expression of the PTH1R transcript
and protein in smooth muscle cells cultured from intrarenal small
arteries failed to exhibit any strain difference. Whether PTH1R
expression and/or coupling are altered in situ awaits
further experiments. The iPTHrP(1–36) concentrations in cell extracts
and conditioned culture media were close to the detection limit of the
RIA kit, which may explain the inability to reach significant
difference in the content and secretion levels between WKY- and
SHR-derived RvSMC. Further studies are required to elucidate these
particular points. In any event, the present studies demonstrate that
RvSMC cultured from small intrarenal arteries isolated from WKY and SHR
express PTHrP and the PTH1R. Furthermore, PTHrP transcript is
overexpressed in SHR-derived RvSMC whereas PTH1R exhibited similar
expression in both cell lines.
The first objective of the present studies was to determine whether the
paradoxical effects of PTHrP evidenced earlier in A10 aortic VSMC
(15)
also applies to VSMC cultured from small intrarenal
arteries. In A10 VSMC, exogenously added PTHrP has been proved to
inhibit cell growth, whereas transfection with the full-length PTHrP
cDNA stimulated cell growth (15)
. The present studies
demonstrate that PTHrP indeed exerts paradoxical effects on RvSMC
proliferation similar to those reported in aortic VSMC (Table 1
). Therefore, the dual opposite autocrine and intracrine proliferative
effects of PTHrP in VSMC can be observed not only in cells derived from
large vessels like the aorta, but also in cells derived from small
resistance arteries.
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In A10 cells, transfection with PTHrP led to a marked increase in cell
proliferation (15)
, whereas transfection with PTHrP
antisense led to marked decrease in A10 proliferation (Massfelder T.,
and Stewart A. F., unpublished observations). However, in contrast
to these studies, the proliferative effect of endogenously produced
PTHrP in WKY-derived RvSMC could only be deduced from studies using an
antisense technology. In fact, transfection with the PTHrP construct
was unable to affect the growth of WKY-derived RvSMC. Moreover, the
strong inhibitory effect of transfection with antisense PTHrP cDNA on
PTHrP expression is evidenced by quantitative competitive RT-PCR
analysis. Taken together, these observations strongly indicate that
endogenous PTHrP responsible for the increase of cell proliferation in
wild RvSMC is at maximal effective level as compared to aortic VSMC. In
support of this, in A10 cells a full mitogenic activity was obtained
not only with high level of PTHrP overexpression (15)
, but
also with a low level of PTHrP overexpression (Massfelder T., and
Stewart A. F., unpublished observations), suggesting that PTHrP is
close to but not at the maximal effective level in wild A10 cells. The
proliferative effect of endogenous PTHrP deduced from antisense
approaches could not occur through an autocrine pathway, as exogenously
applied PTHrP exerted an opposite antiproliferative effect. These
observations also imply that the mitogenic properties of endogenously
produced PTHrP is preponderant compared to the antiproliferative
properties of secreted PTHrP.
The major new finding of the present studies is the demonstration that
the dual opposite effects of PTHrP on RvSMC are reversed in RvSMC
derived from the SHR model of genetic hypertension. Exogenously added
PTHrP stimulated, whereas endogenously produced PTHrP inhibited, the
proliferation of SHR-derived RvSMC. As for WKY-derived RvSMC, the
antiproliferative effect of endogenous PTHrP in SHR-derived RvSMC could
only be deduced from studies using the potent antisense technology. The
antiproliferative effect of endogenous PTHrP deduced from antisense
approaches could not occur through an autocrine pathway, since
exogenously applied PTHrP exerted an opposite proliferative effect.
Moreover, the effects of endogenous PTHrP are serum independent as they
occur not only in cycling cells grown in serum-containing medium (PDT
measurements), but also in serum-deprived quiescent cells (BrdU
incorporation). In SHR-derived cells, these observations again imply
that the antimitogenic properties of endogenously produced PTHrP is
preponderant compared to the proliferative properties of exogenously
added PTHrP. Endogenous and secreted PTHrP species in VSMC have not yet
been characterized. Therefore, the possibility exists that species
different from PTHrP(1–36) might be involved in the paradoxical
effects of endogenous PTHrP in both cell lines. However, such a
hypothesis is not supported by our preliminary unpublished experiments.
In these experiments, neither PTHrP(38–94), PTHrP(107–139), the known
secreted species of PTHrP in non-SMC (4)
, nor
PTHrP(74–113) added to the culture medium were able to display the
effect of endogenous PTHrP in WKY- and SHR-derived RvSMC (results
not shown). In our previous study, PTHrP(38–94), PTHrP(67–86),
PTHrP(107–139), PTHrP(109–138), and PTHrP(141–173) were unable to
affect the proliferation of A10 cells (15)
.
Several arguments prompted us to explore the involvement of Gi-protein
in the unexpected proliferative effect of PTHrP in SHR-derived RvSMC.
First, the cyclic AMP system is acknowledged as the cellular pathway
accounting for the antiproliferative effect of PTHrP in VSMC (15
, 23
, 49)
. Second, the PTH1R is coupled to Gs- and Gi-proteins in
a variety of cell types (41)
. Finally, the RPTH1-dependent
stimulation of Gi-protein by PTH has been proved to decrease the
production of cyclic AMP (50)
. We therefore tested the
hypothesis that the PTH1R is differently coupled to Gs- and Gi-protein
in WKY- and SHR-derived RvSMC. Results obtained with cholera and
pertussis toxins strongly indicate that in WKY-derived RvSMC, PTH1R is
preferentially coupled to Gs-protein whereas in SHR-derived RvSMC,
PTH1R is mostly coupled to Gi-protein, resulting in the opposite
effects of PTHrP on the proliferation of WKY- and SHR-derived RvSMC.
Recent evidence suggests that PTHrP localizes in the nucleus/nucleolus
owing to its intermediate 88–107 region, a functional NLS domain
similar to the NLSs found in transcription factors, viral proteins, and
growth factors (13
14
15)
. The use of alternative
translational initiation start site disrupting the leader sequence
(51)
and endocytosis of secreted PTHrP via the PTH1R
(52)
or via a still undefined receptor (53)
have been described as possible mechanisms for PTHrP to gain access to
the cytoplasmic compartment and, from there, into the nucleus. An
isoform of the PTH1R lacking the signal peptide and present in the
cytosol has recently been described in the kidney (40)
,
leaving open the possibility of an interaction between this isoform and
cytosolic PTHrP. As PTHrP has been shown to be able to interact with
RNA in COS-1 cells via a core motif present in its NLS
(54)
, the possibility exists that PTHrP regulates
transcription, processing, and/or trafficking of RNA. However, the only
biological properties that have been associated with the nuclear
translocation of PTHrP are the inhibition of apoptosis in chondrocytes
and the stimulation of proliferation in VSMC (13
, 15)
.
By extrapolation from the above information, it seemed conceivable that
nuclear translocation of PTHrP would be able to modulate the
proliferation of WKY and SHR-derived RvSMC. We therefore asked whether
PTHrP is able to translocate in the nucleus in these cells. In
immunohistochemical studies, we indeed detected PTHrP not only in the
cytoplasm of all RvSMC, but also in the nucleus of 2% of the cells
derived from both strains of rats. However, unlike A10 cells, where
nuclear PTHrP localized mostly outside the nucleolus, nuclear PTHrP
exclusively localized into the nucleolus in RvSMC. Whether this
discrepancy is related to difference in cell origin (aortic vs. renal)
or to a difference in antibody epitope remains unclear. However,
Henderson et al. (13)
described a similar nucleolar
localization of PTHrP using the same antibody as the one used here. In
the present studies, we also observed the presence of PTHrP in the
nucleolus of SMC from small renal arteries, demonstrating that the
nucleolar translocation of PTHrP is detected not only in
vitro, but also in situ, further supporting a
(patho) physiological significance of the nucleolar presence
of PTHrP. The low percentage of cells with PTHrP in the nucleolus in
randomly cycling cells is reminiscent of a cell cycle-dependent
nucleolar localization of PTHrP. Such dependency has been described in
chondrocytes (13)
and A10 cells (15)
.
Together, these findings strongly suggest, but do not prove, that
translocation in the nucleolus may account for the trophic effects of
PTHrP in WKY and SHR RvSMC. Clearly, further studies are required to
assess this possibility.
The mechanism whereby the growth-stimulatory action of endogenous PTHrP
in WKY-derived RvSMC is converted into an inhibitory effect in
SHR-derived RvSMC remains unclear, but presumably reflects strain
differences in the trophic effects induced by the translocation of the
peptide into the nucleolus. Since PTHrP species, which do not contain
the amino terminus, are unable to influence proliferation of VSMC
(15)
, the possibility that such species influence
proliferation in a way opposed to the effect of amino-terminal PTHrP
fragment through an autocrine pathway appears unlikely. The nucleolus
is the site of rRNA transcription and processing and ribosome assembly
(55
, 56)
. The nucleolus also participates in many other
aspects of gene expression (55
, 56)
. The presence in
nucleoli of trophic factors is an emerging concept, and little is known
about the way by which nucleolus-localized factors regulate cell
proliferation. It is therefore tempting to speculate that PTHrP may be
involved in some of these events affecting different pathway(s) between
both strains of rats. The response to this crucial question will
undoubtedly arise from studies aiming at determining directly the
molecular target(s) of PTHrP within the nucleolus in VSMC.
In conclusion, the present studies demonstrate that as in A10 aortic SMC, PTHrP displays paradoxical effects on the proliferation of intrarenal VSMC. Thus, PTHrP inhibits RvSMC proliferation through the classical autocrine pathway and stimulates the growth of these cells through an intracrine pathway. The major new finding is the demonstration that these dual opposite effects of PTHrP on RvSMC are reversed in the SHR model of genetic hypertension. The mechanism by which the growth inhibitory action of exogenous PTHrP in WKY-derived RvSMC is converted into a stimulatory effect in SHR-derived RvSMC involves a strain difference in the coupling of PTH1R to Gi and Gs. On the other hand, the mechanism by which the growth-stimulatory action of endogenous PTHrP in WKY-derived RvSMC is converted into an inhibitory effect in SHR-derived RvSMC remains unclear. Nevertheless, the possibility exists that a translocation of the peptide into the nucleolus underlie the mechanism of endogenous PTHrP. The present findings also indicate that PTHrP might play, via an intracrine process, a beneficial role as a negative feedback regulator of renal vascular wall hyperplasia that contributes to the progression of the hypertensive state in the SHR model of genetic hypertension. A new concept emerges from these results, according to which a single molecule may have opposite paradoxical effects on VSMC proliferation under physiological and pathophysiological conditions. It predicts a role for PTHrP in regulating vascular wall remodeling that may be related to its localization in the nucleolus in vivo.
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ACKNOWLEDGMENTS |
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Received for publication March 16, 2000.
Revision received July 31, 2000.
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REFERENCES |
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