Departments of Medicine and Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267
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ABSTRACT |
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In this study, we investigated the mechanisms responsible for the growth-inhibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascular smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysis of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) demonstrated an enrichment of cells in G1 and a reduction in the S phase. Measurement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibition by 12-16 h, consistent with blockade in mid-G1. cAMP treatment blunted the serum-induced rise in cyclin D1 during cell cycle progression without altering levels of the cyclin-dependent kinase cdk4 or cyclin E and its associated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a reduction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting of extracts from cAMP-treated cells with antibodies to cdk inhibitors revealed a striking increase in p27kip1 abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepared from cAMP-treated cells followed by immunoblotting with antisera to p27kip1 disclosed a threefold increase in p27kip1 associated with cyclin D1 compared with lysates treated with serum alone. We conclude that PTHRP, by increasing intracellular cAMP, induces VSMC cycle arrest in mid-G1. This occurs secondary to a suppression in cyclin D1 and induction of p27kip1 expression, which in turn inhibits Rb phosphorylation.
vascular smooth muscle cell proliferation; cell cycle; retinoblastoma gene product; cyclin D1; p27 kip1
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INTRODUCTION |
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PARATHYROID HORMONE-RELATED PROTEIN (PTHRP) was originally discovered as the principal causative factor of the syndrome of humoral hypercalcemia of malignancy (21). Subsequently, the protein was shown to be produced in many normal nonmalignant tissues and is now recognized as a secreted product that functions in an autocrine/paracrine fashion to modulate the developmental program of several tissues, including skin (37), mammary gland (38), and cartilage (15). PTHRP is produced in vascular smooth muscle cells (VSMC), and its expression is induced in response to mitogens (11), vasoconstrictors (24), and mechanical stretch (25). PTHRP exerts potent vasorelaxant actions by activation of the G protein-linked parathyroid hormone (PTH)/PTHRP type I receptor, which is expressed in VSMC and which is coupled exclusively to adenylyl cyclase and cAMP production.
In addition to its vasorelaxant properties, PTHRP is a potent inhibitor of VSMC growth. PTHRP decreases mitogen-activated DNA synthesis in primary arterial VSMC (11, 14) and in A10 VSMC (18, 19). The antiproliferative effects of PTHRP in VSMC are mimicked by treatment with forskolin and analogs of cAMP, suggesting that PTHRP functions through a cAMP-dependent pathway to oppose mitogenic stimuli in vascular smooth muscle. However, the mechanisms by which cAMP impinges on cell cycle regulatory components in VSMC have not been characterized in any detail.
Like other mesenchymal cells, VSMC require the continuous presence of
growth factors throughout G1 until they pass the restriction point,
after which time they are committed to DNA synthesis. Regulation of
proliferation by extracellular signals occurs during G1 when growth-stimulatory and growth-inhibitory signals influence specific cell cycle regulatory components (31, 32). In
mid-G1, mitogens induce the synthesis of D-type cyclins and promote
their assembly with the cyclin-dependent kinases cdk4 or cdk6. The
active holoenzymatic complexes phosphorylate the retinoblastoma gene
product (Rb) later in G1 (1, 28), which
releases transcription factors such as E2F, that in turn activate genes
whose products are required for S phase progression. Cyclin E is
expressed later in G1 and, upon association with cdk2, induces maximal
Rb kinase activity at the G1/S boundary (29). By contrast,
a family of cdk inhibitors, which includes p21cip1 and
p27kip1, negatively regulate G1 phase progression by
forming complexes with cdks to prevent S phase entry (12,
33). p21cip1 is transcriptionally induced by
p53 and is the primary mediator of G1 growth arrest that occurs after
DNA damage (3, 5, 8), whereas
p27kip1 is thought to be mainly responsible for regulating
cdk activity in response to extracellular anti-proliferative signals
(34, 36). For example, transforming growth
factor- induces G1 arrest in several mesenchymal cell types by
initially inhibiting cdk4, which then frees p27kip1 to bind
cyclin E-cdk2 complexes, thereby blocking the activity of both cdk4 and
cdk2 (7).
The purpose of the present study was to characterize the mechanisms by which PTHRP impacts cell cycle regulatory events to inhibit VSMC proliferation. We demonstrate that PTHRP induces mid-G1 cell cycle arrest through a cAMP-dependent mechanism. The block occurs secondary to an increase in p27kip1, which impairs the activity of the cyclin D1-cdk4 holoenzyme.
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METHODS |
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Materials. Synthetic PTHRP-(1---34)NH2, was purchased from Bachem (Torrance, CA). Dibutyryl-cAMP (DBcAMP) and aphidicholin were from Sigma Chemicals (St. Louis, MO). The glutathione-S-transferase (GST)-pRb and the antibodies against the cdks, cyclins, and cdk inhibitors were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Rb antibody was from Pharmingin (San Diego, CA).
Cell culture. A10-14 VSMC stably expressing the PTH/PTHRP receptor (18) were cultured in DMEM containing 5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a water-jacketed incubator with a humidified atmosphere (5% CO2-air) and were maintained at 37°C. Experiments were performed in cultures grown to 70% confluence in serum-containing medium, and then cells were deprived of serum by replacing the medium with DMEM without serum (serum-free DMEM) for 24-36 h before the addition of test agents for all experiments.
Cell cycle analysis. Cell cycle distribution was analyzed by flow cytometry. Cells (1-2 × 106) incubated in serum-free DMEM or stimulated with 5% FBS with or without PTHRP-(1---34)NH2 or DBcAMP were trypsinized, washed one time with PBS, and fixed with 70% ethanol for at least 1 h at 4°C. Fixed cells were washed with PBS and incubated with a solution containing 0.05 mg/ml propidium iodine, 0.1% sodium citrate, 20 µg/ml RNase A, and 0.3% Nonidet P-40, pH 8.3, for 30 min at 4°C in the dark. The stained cells were analyzed by FACScan (Becton-Dickinson, Mountain View, CA). To monitor cell cycle progression into S phase, cells were plated at 1 × 105 on four-well chambered slides (Nunc, Naperville, IL), starved for 24 h, and then restimulated with platelet-derived growth factor (PDGF, 10 ng/ml) in the presence of 10 µM bromodeoxyuridine (BrDU). DBcAMP or aphidicholin was added at the indicated times after PDGF treatment. Cells were fixed in 70% ethanol in glycine buffer (pH 2.0) and reacted with an anti-BrDU mouse monoclonal antibody followed by treatment with a fluorescein-conjugated anti-mouse antibody (BrDU Labeling and Detection Kit 1; Boehringer Mannheim Biochemicals). A minimum of 500 cells was counted at each time point.
Immunoblot analysis. Cells were rinsed two times with ice-cold PBS and were lysed in 1 ml SDS lysis buffer [125 mM Tris · HCl (pH 6.8), 2% SDS, 1 mM dithiothreitol (DTT) containing Complete protease inhibitor cocktail, and 20 µg/ml (4-amidinophenyl)-methanesulfonyl fluoride (APMSF); Roche, Indianapolis, IN]. Cells were collected by scraping on ice and were sonicated for 2 × 8 s; lysates were cleared by centrifugation at 16,000 g for 5 min at 4°C (total cell lysate). The protein concentration was measured using the Pierce bicinchoninic acid assay reagent (Pierce Chemical, Rockford, IL). For immunoblot analysis, protein samples (50 µg) were boiled for 5 min in Laemmli buffer [62.5 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 0.01% bromphenol blue, and 100 mM DTT] and separated with 7.5 or 12.5% SDS-PAGE. Gels were then transferred to a 0.2-µm nitrocellulose membrane using a semidry transfer method. The transferred membranes were temporarily stained with Ponceau-S solution (Sigma) to check sample loading and the homogeneity of transfer. After being blocked with Tris-buffered saline (TBS; pH 7.4)-0.05% Tween 20 containing 5% low fat milk, the membranes were incubated with primary antibody overnight in blocking buffer, incubated with secondary antibody conjugated with horseradish peroxidase for 2 h, and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). For the cyclin/cdk Western blot (Fig. 3), an evenly loaded immunoblot was sequentially reprobed by antibody stripping using 2% SDS, 100 mM DTT, and TBS for 30 min at 50°C. Quantitation of immunoblots was performed using a computer scanner equipped with a transparency adapter and Scion Image software.
Immunoprecipitations.
Cells were collected by trypsinization, washed two times with cold PBS
containing protease inhibitors, pelleted by centrifugation, frozen with
liquid N2, and stored at 80°C. Cell pellets were solubilized with 50 mM HEPES, pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1 mM
EDTA, O.1% Tween 20, 10% glycerol, 1 mM DTT, 10 mM
-glycerophosphate, 1 mM NaF, 0.1 mM NaVO4, Complete
protease inhibitors, and 20 µg/ml APMSF (IP buffer). Lysate
(0.7-1.0 mg) was rotated overnight at 4°C with 1 µg of mouse
anti-cyclin D1 (72-13G) and was precipitated with protein G+
agarose (Santa Cruz). Pellets were washed three times with IP buffer
and one time with 50 mM HEPES (pH 7.5). Pellets were analyzed by
SDS-PAGE as above.
In vitro protein kinase assays.
For cdk4 assays, immunoprecipitates of cell lysates were prepared using
15 µl of a rabbit anti-cdk4 antibody cross-linked to agarose (H-22)
in 1 mg of lysate. After the final wash with IP buffer,
immunoprecipitates were washed three times with Rb kinase buffer [50
mM HEPES (pH 7.5), 1 mM DTT, 10 mM MgCl2, and 5 mM
MnCl2], resuspended in 50 µl Rb kinase buffer containing 50 µM ATP, 10 µCi [-32P]ATP, 10 mM
-glycerophosphate, 0.1 mM Na3VO4, 1 mM NaF,
2.5 mM EGTA, and 1.5 µg GST-pRb fusion protein as a substrate, and incubated for 30 min at 30°C. The reactions were stopped by boiling for 5 min in Laemmli sample buffer. The samples were resolved on 10%
SDS-PAGE and transblotted to nitrocellulose, and the radioactive bands
were detected by phosphorimaging (Molecular Dynamics). All assays were
performed at least two times.
Statistical methods. A Student's t-test was used when comparisons were made between control and the treated groups at single time points. Comparison of the changes in cyclin D1 and p27kip1 levels at different times were analyzed using a one-way ANOVA with Dunnett's post hoc testing. Significance was defined as P < 0.05.
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RESULTS |
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PTHRP and cAMP induce G1 arrest of VSMC growth.
We have previously shown that PTHRP-(1---34)NH2 markedly
suppresses serum-stimulated DNA synthesis as measured by incorporation of tritiated thymidine in both primary rat aortic VSMC
(11) and in A10-14 VSMC stably expressing the
PTH/PTHRP receptor (18). Ligand-dependent activation of
the PTH/PTHRP receptor in A10-14 cells results in robust signaling
through adenylyl cyclase, with no detectable activation of
phospholipase C. Treatment of cells with forskolin or DBcAMP mimicked
the inhibitory effect of PTHRP, suggesting that the growth-inhibitory
effects of PTHRP were exerted though a cAMP-dependent mechanism. To
characterize the mechanism for the growth-inhibitory action of cAMP in
VSMC, we determined the effect of cAMP on the distribution of
A10-14 cells in the cell cycle by fluorescence-activated cell
sorter analysis. When serum-deprived cells were exposed to 5% serum
for 24 h, 57.9 ± 4.3% were in G1, 14.6 ± 3.5% in S,
and 18.2 ± 0.4% in G2/M phase (mean ± SE; Fig.
1B). Treatment of these cells
with DBcAMP for 24 h (Fig. 1C) significantly reduced
the proportion of cells in S phase and increased the proportion in G1
consistent with a G1 phase block. Similar results were obtained with
100 nM PTHRP-(1---34)NH2 (Fig. 1D). In three
separate experiments, the average change in S phase was 47.2 ± 3.7 and
48.5 ± 5.2% (mean ± SE, P < 0.015) with DBcAMP and PTHRP, respectively.
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Effects of cAMP on abundance of cyclins and cdks.
As indicated above, during mid-G1, D-type cyclins assemble with two
major catalytic partners, cdk4 or cdk6, to form a functional holoenzyme
that, upon phosphorylation by cyclin-associated kinase (CAK), executes
the rate-limiting phosphorylation of Rb at the restriction point. By
contrast, cyclin E is expressed later in G1 and, upon association with
cdk2, induces maximal kinase activity at the G1/S boundary
(29). The timing of the growth-inhibitory action of cAMP
(~16 h) suggested that cAMP inhibited growth by influencing the
abundance or activity of cyclin D1 with its associated kinases. To
determine whether cAMP influenced the abundance of cyclin D and its
associated cdks, we performed immunoblots on cell extracts harvested at
various times after exposure of quiescent cells to serum. As shown in
Fig. 3, exposure of cells to serum resulted in an increase in cyclin D1 that was significant at 4 and
9 h (P < 0.02). However, in the cells treated
with DBcAMP, there was no significant change in the cyclin D1 level at
any time point compared with the value at time 0. By
contrast, the levels of cdk4, cyclin E, and cdk2 remained relatively
constant after exposure to serum and were unaltered by cAMP.
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PTHRP and cAMP impair cdk4 activity.
To examine the effect of cAMP treatment on Rb phosphorylation, lysates
were prepared from cells exposed to serum in the absence or presence of
100 nM PTHRP-(1---34)NH2 or 1 mM DBcAMP for 24 h and
were immunoblotted with an anti-Rb antibody (Pharmingin 14001A). As
shown in Fig. 4A, the increase
in abundance of the slower-migrating hyperphosphorylated form of Rb in
cell lysates from serum-stimulated cells was reduced by PTHRP and cAMP
treatment. Cyclin D-cdk4 kinase activity was determined by measurement
of GST-pRb phosphorylation. The cdk4 immunoprecipitates from lysates of
serum-deprived cells treated with serum in the absence or presence of
DBcAMP for 24 h were compared. Serum treatment increased the
activity of the cyclin D-cdk4 complex by ~75% (Fig. 4B),
whereas DBcAMP treatment reduced cdk4 activity by ~60% compared with
that observed in immunoprecipitates from the serum-treated cells. The
inhibitory effect of cAMP on cdk4 in mid-G1 phase was also associated
with a reduction in the activity of cdk2-cyclin E. In a similar
experiment, cdk2 was immunoprecipitated from lysates and assayed for
cdk activity using histone as the substrate. Figure 4B shows
that lysates from cells treated with DBcAMP had markedly reduced cdk2
activity compared with those assayed from serum-stimulated cells. The
more pronounced effect of serum on cdk2-mediated phosphorylation of
histone and the greater apparent inhibition by cAMP probably relate in
part to the greater sensitivity of this assay compared with the cdk4-Rb
kinase assay.
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Inhibition of cdk activity is accompanied by an induction of
p27kip1.
cdk Inhibitory proteins bind and inactivate cdks to negatively regulate
G1 phase progression in response to antiproliferative signals. We
therefore determined the effect of PTHRP and cAMP on the expression of
cdk inhibitors in serum-treated VSMC. As shown in Fig.
5, treatment of cells with serum resulted
in a progressive decrease in the abundance of p27kip1.
Interestingly, whereas neither PTHRP nor cAMP had any discernible effect on the abundance of p21cip1 (data not shown), they
significantly (P < 0.05) increased the level of
p27kip1 (Fig. 5). This observation suggested that PTHRP and
cAMP induced p27kip1, which inhibited cdk4 kinase activity
by associating with the cyclin D-cdk4 complex. To investigate this
possibility, lysates from cells grown for 24 h in the absence or
presence of cAMP were immunoprecipitated with an anti-cyclin D1
antibody, denatured, and then immunoblotted with an
anti-p27kip1 antibody. As can be seen in Fig.
6, the amount of p27kip1
associated with the cyclin D-cdk4 complex immunoprecipitated from
cAMP-treated cells was increased threefold (P < 0.015).
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DISCUSSION |
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PTHRP is a potent vasodilator and inhibitor of mitogen-activated DNA synthesis in VSMC (11, 14, 18). These effects are mediated through activation of the type 1 PTH/PTHRP receptor, which in VSMC exclusively activates adenylyl cyclase to increase intracellular cAMP. The growth-inhibitory effects of PTHRP in VSMC are mimicked by exposure to analogs of cAMP or by treatment with forskolin (18). The ability of cAMP to inhibit cell proliferation was first demonstrated by Burk (2) in baby hamster kidney and appears to represent a common mechanism operating in cells of mesenchymal origin, including VSMC (4, 13, 18). However, the precise mechanisms by which this cyclic nucleotide induces growth arrest have been difficult to ascertain and remain controversial. With the identification of several key cell cycle-regulatory molecules, and the clarification of their roles in cell cycle progression, we initiated the current studies, which sought to identify the mechanisms by which PTHRP inhibits VSMC proliferation.
When cAMP was added with PDGF or serum to quiescent VSMC, DNA synthesis was blocked ~6-8 h before S phase. Thus the growth-inhibitory effect of cAMP corresponded to the time when cyclin D-cdk4 complexes are enzymatically active. Although cAMP attenuated the serum-induced rise in cyclin D1 in VSMC, the effect was relatively modest. Moreover, cAMP did not alter the abundance of cdk4 or cyclin E and its associated kinase cdk2. This suggested that cAMP exerted its growth-inhibitory actions by impacting other cell cycle regulatory steps in addition to inhibiting cyclin D1 levels. We found that treatment of cells with either PTHRP or DBcAMP markedly increased the abundance of the cdk inhibitor p27kip1. It would therefore appear that PTHRP increases cellular cAMP, which in turn causes G1 arrest by acting on at least two cell cycle regulatory components. The nucleotide blunts the serum-induced rise in cyclin D1 and markedly induces the abundance of p27kip1. p27kip1 then associates with the cyclin D1-cdk4 complex and reduces its ability to phosphorylate Rb. Interestingly, cAMP-induced G1 arrest in fibroblasts is also associated with a reduction in cyclin D1 and an increase in p27kip1. In these studies, forced expression of cyclin D1 did not abrogate the cAMP-induced decrease in p27kip1 levels (17), suggesting that cAMP can alter p27kip1 abundance independent of the level of cyclin D1. In our studies, the degree of inhibition of cyclin D1 expression in cAMP-treated cells was less pronounced than was the induction of p27kip1, suggesting that p27kip1 may represent the more critical target for cAMP-induced G1 arrest in VSMC.
p27kip1 is increasingly recognized as a pivotal regulatory molecule controlling G1 to S transition. In normal cells, p27kip1 levels increase as cells become quiescent and abruptly decline upon cell cycle reentry (36). The induction of p27kip1 also appears to coordinate cell cycle arrest in response to anti-mitogenic stimuli (6, 20, 26). Kato et al. (16) first showed that cAMP caused G1 growth arrest in colony-stimulating factor-1-stimulated macrophages by inducing p27kip1 without altering the levels of cyclin D1 or cdk4. In these studies, p27kip1 inhibited cell cycle progression by associating with the cyclin D-cdk4 complex and thus prevented the cyclin-associated kinases from phosphorylating the holoenzyme. These findings in macrophages are similar to our observations in VSMC and suggest that cAMP exerts its anti-mitogenic effects in both cells types at least partly by elevating the abundance of p27kip1 and increasing its association with the cyclin D-cdk4 complex. In UMR-106 osteosarcoma cells, cAMP also induced G1 arrest and elevated p27kip1 levels. However, in these cells, p27kip1 preferentially associated and inhibited the activity of the cyclin E-cdk2 complex, likely because these cells lacked functional Rb (22).
In general, the abundance of p27kip1 is not regulated at the transcriptional level but rather through posttranscriptional stabilization of the protein (10). Consistent with this, we found that cAMP had no effect on p27kip1 mRNA in VSMC (Maeda and Clemens, unpublished observations). Interestingly, studies by Sheaff et al. (30) provide evidence that the level of p27kip1 is controlled posttranslationally by the cyclin E-cdk2 complex itself. In these studies, the accumulation of cyclin E-cdk2 complexes promoted cell cycle progression by phosphorylation of p27kip, which increased its removal from the cell. It is conceivable, therefore, that a reciprocal mechanism might operate in growth-arrested VSMC cells, namely that a reduction in cyclin E-cdk2 complexes occurring in arrested (or cAMP-treated) cells would effectively increase abundance of p27kip1 and perhaps serve to amplify growth-inhibitory signals.
In contrast to our studies and those of others which show that PTHRP is growth-inhibitory in VSMC, a recent study by Massfelder et al. (19) reported that, under certain conditions, PTHRP does not enter the secretory pathway but instead localizes to the nucleus where it promotes VSMC growth. In these experiments, the exogenous addition of PTHRP peptides to A10 VSMC inhibited proliferation, confirming the studies by Maeda et al. (18). However, overexpression of PTHRP in stably transfected A10 cells was associated with increased DNA synthesis and an increase in nuclear localization of PTHRP. Nuclear localization of PTHRP has also been reported to occur in chondrocytes and appears to require a multibasic region within the midregion of the molecule (9). These investigators propose that the ability of PTHRP to either promote or oppose VSMC mitogenesis is dependent on whether it is secreted and acts via ligand activation of the receptor or is targeted directly to the nucleus where it impacts unknown effectors of cell proliferation.
A better definition of the mechanisms regulating VSMC proliferation by PTHRP and other agents that elevate intracellular cAMP is key to understanding the role of these factors in states of normal and abnormal VSMC proliferation (e.g., vascular development restenosis and coronary artery disease). Although numerous factors that stimulate VSMC proliferation have been identified, relatively few agents have been described that inhibit VSMC growth. Several recent studies provide circumstantial evidence for an in vivo role for cAMP and PTHRP as inhibitors of VSMC growth. For example, local administration of cAMP or the phosphodiesterase inhibitors aminophylline or amrinone inhibited neointimal formation after experimental balloon injury in rat carotid arteries in vivo (13). Moreover, studies by Ozeki et al. (23) showed that PTHRP protein and mRNA expression were markedly upregulated in neointimal smooth muscle cells in balloon-injured arteries. These investigators also observed immunoreactive PTHRP at higher than normal levels in human arterial tissue removed from patients undergoing angioplasty. Other studies using a similar model of arterial injury showed that high levels of p27kip1 expression in the media and neointima were correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27kip1 in balloon-injured arteries attenuated neointimal lesion formation (35). It is therefore plausible that PTHRP, through its ability to induce p27kip1, functions in a growth-inhibitory signaling pathway that is triggered in response to proliferative cues within the injured arterial wall.
The growth-modulatory actions of PTHRP could also signify a role for this peptide in vascular development. In support of this concept, we have found that double transgenic mice overexpressing both PTHRP and the PTH/PTHRP receptor in cardiac (transiently) and vascular smooth muscle die at embryonic day 9.5 with evidence of greatly enlarged hearts, pericardial effusion, and vascular pooling (27). Thus excessive stimulation of the adenylyl cyclase pathway disrupts cardiovascular development. On the other hand, mice with targeted disruption of the PTHRP gene survive until birth, suggesting that heart and blood vessel development can take place in the absence of the protein. However, the mitotic rate of aortic VSMC in 18 day-old PTHRP null embryos measured by BrDU incorporation was reported to be significantly reduced compared with PTHRP(+/+) fetuses (19). The authors interpreted this result as in vivo evidence that PTHRP may function to promote VSMC mitogenesis during aortic development. Further studies with genetically manipulated mice should help clarify whether or to what extent PTHRP functions to oppose or promote VSMC proliferation during the development of the cardiovascular system.
In summary, we have shown that PTHRP, by raising intracellular cAMP, induces VSMC arrest secondary to an induction in p27kip1. The increased association of p27kip1 with CD1/cdk4 appears to result in the impairment of cdk4 kinase activity and inhibition of Rb phosphorylation. We postulate that the growth inhibitory actions of locally produced PTHRP could represent an important antiproliferative mechanism to restrict VSMC proliferation in response to mitogenic signals within the vessel wall.
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ACKNOWLEDGEMENTS |
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We thank Dr. Erik Knudson for helpful discussion of this work.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-47811 and HL-43802.
Address for reprint requests and other correspondence: T. L. Clemens, Div. of Endocrinology and Metabolism, Rm. 5564, 231 Bethesda Ave., Cincinnati, OH 45267-0547 (E-mail: clementl{at}UC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 7 September 1999; accepted in final form 16 February 2000.
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