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Division of Endocrinology and Metabolism, Departments of Medicine (J.Q., S.M., T.N., J.A.F., T.L.C.) and Molecular and Cellular Physiology (J.N.L., R.L.S., C.W., R.J.P., J.A.F., T.L.C.), University of Cincinnati, and the Department of Pediatrics, Children’s Hospital (M.C.C.), Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Thomas L. Clemens, Ph.D., Division of Endocrinology and Metabolism, University of Cincinnati, Room 5564, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0547. E-mail: clementl{at}uc.edu
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Introduction |
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PTHrP shares N-terminal sequence homology with PTH, so that both peptides activate a common G protein-linked receptor termed the PTH/PTHrP type 1 receptor. PTH produced in the parathyroid gland regulates calcium and phosphorous metabolism by activating the PTH/PTHrP receptor (PTHrP-R) in bone and kidney (11). The ability of PTHrP to activate the PTH/PTHrP type 1 receptor in these tissues accounts for its hypercalcemic actions in patients with pathological overexpression of the peptide due to unregulated production by cancer cells (12). However, in contrast to PTH, which acts as a classical endocrine hormone, PTHrP appears to exert its normal effects locally in an autocrine/paracrine fashion (1). The PTH/PTHrP type I receptor is expressed in rat vascular smooth muscle cells, and its activation by PTHrP-(1–141) and synthetic N-terminal PTHrP fragments stimulates adenylyl cyclase activity (13). However, there is evidence for additional PTH/PTHrP-R. For example, Usdin et al. (14) isolated a second PTH receptor, termed the PTH-2 receptor, which bears homology to the PTH/PTHrP type 1 receptor and other members of the secretin G protein receptor family. Studies to date in cells transfected with recombinant PTH type 2 receptors suggest that it preferentially binds PTH and is relatively unresponsive to N-terminal PTHrP fragments (14, 15). However, the PTH-2 receptor is expressed in vascular smooth muscle and endothelial cells (16). Other studies showed that a synthetic PTHrP peptide comprising amino acids 38–64, which did not activate the PTH/PTHrP type 1 receptor, stimulated a calcium transient in squamous carcinoma cells (17), suggesting the existence of a novel midregion PTHrP receptor. In addition, the recent observations (10) of nuclear localization of PTHrP in vascular smooth muscle cells suggest that PTHrP may also function intracellularly. Thus, it is likely that multiple PTH/PTHrP-R and effectors have evolved to mediate distinct functions of this protein in different cell types.
To examine the role of PTHrP in vascular smooth muscle in
vivo, we developed transgenic mice that selectively overexpressed
PTHrP in vascular smooth muscle (18). These mice developed hypotension
consistent with the predicted role of this protein as a local
vasodilator. However, the constitutive expression of ligand in these
mice led to desensitization of their vasculature to PTHrP, which
compromised our ability to define the full range of activity of PTHrP
in vascular smooth muscle. Therefore, in the present studies we
targeted the expression of the PTH/PTHrP type I receptor to smooth
muscle of transgenic mice using the SMP8 
-actin promoter. We
reasoned that if the cardiovascular actions of PTHrP were mediated by
the PTH/PTHrP type I receptor rather than by another receptor, then the
phenotype of the SMP8-PTHrP type I receptor
(SMP8-PTHrP-R)-overexpressing mice would recapitulate that seen in the
SMP8-PTHrP transgenic mice. Indeed, mice overexpressing SMP8-PTHrP-R
were also hypotensive compared with wild-type littermates.
Interestingly, whereas the SMP8-PTHrP-R transgenic mice developed
normally, double transgenic mice overexpressing both SMP8-PTHrP and
SMP8-PTHrP-R died at approximately embryonic day 9.5 with severe
abnormalities in the developing heart.
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Materials and Methods |
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RNA isolation and Northern blot analysis
Total RNA was isolated from tissues with RNASTAT-60
(Tel-Test, Inc., Friendswood, TX). Ten micrograms of
tissue total RNA were gel-separated, transferred to Nylon membrane, and
then hybridized with random primed mouse PTHrP-R cDNA. The ethidium
bromide-stained 18S ribosomal RNA was used as an index of equivalency
of RNA loading.
Tissue morphometry of transgenic mice
Transgenic mice and their wild-type littermates were killed by
CO2 asphyxiation. After obtaining body weights, organs were
dissected, tissue-blotted, and weighed. Microscopic images of aortic
sections of SMP8-PTHrP-R transgenic mice and their age-matched
wild-type controls were color-captured into the computer and analyzed
as previously described (19).
Cardiovascular hemodynamic and vascular contractility
measurements
Blood pressure measurements in conscious mice, measurement of
mean arterial pressure and cardiac output in anesthetized intact mice,
and vascular contractility measurements were performed exactly as
described previously (18). All data were analyzed using a two-factor
ANOVA, with repeated measures on the second factor. Comparisons of
individual means were performed using individual contrasts.
Creation of SMP8-PTHrP/SMP8-PTHrP-R double transgenic mice
F1 progeny from SMP8-PTHrP-overexpressing mice (line
375) (18) were mated with F1 offspring from
SMP8-PTHrP-R-overexpressing mice. Genotyping of live offspring and
embryos was performed using PCR. Primers for the mouse PTHrP-R were:
forward, 5'-CTTGAAGTCCAA TGCCAGTGTCCAG-3', corresponding to nucleotides
1460–1484 of the mouse PTHrP-R; and reverse,
5'-GACACCTACTCACACAATGC-3', corresponding to the bovine GH
polyadenylase sequence. The primers for the SMP8-PTHrP were: forward,
5'-CAGAATCCTGCAATATGTCC-3'; and reverse, 5'-CTGTGTCTGAACATCAGCTC-3',
corresponding to nucleotides within 1048–1442 bp in the PTHrP human
cDNA (22). Embryos collected from timed pregnant females were examined
as whole mounts. All animals received humane care in compliance with
the local institutional animal care and use committee.
Peptides
Synthetic PTHrP-(1–34)NH2 was purchased from Bachem
(Torrance, CA) and stored at -20 C in 0.01 N acetic acid.
Acetylcholine and phenlyephrine were purchased from Sigma Chemical Co. (St. Louis, MO).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The transgenic SMP8-PTHrP-R
mRNA was detected in smooth muscle-rich tissues and was expressed at
highest levels in bladder, stomach, and colon, but was not detected in
brain, liver, or thymus. Figure 1B shows a Northern blot subjected to
phosphorimaging for an extended period to compare the relative
expressions of transgenic SMP8-PTHrP-R mRNA in bladder and aorta among
F1 offspring derived from the 4 individual SMP8-PTHrP-R
founders. The level of transgene expression varied among lines and was
highest in lines 122 and 127. These mice were propagated for further
study.
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). The total body weight of the
transgenic mice was marginally, but significantly, lower than that of
wild-type controls. The wet weight of the stomach from transgenic mice
was significantly greater than that in controls after adjusting for
body weight. There were no major differences in the weights of any
other organs after controlling for body weight. Similar results were
observed in mice from line 127 SMP8-PTHrP-R (data not shown).
Histomorphometry was performed on transverse sections of aorta from
transgenic and wild-type mice. Their were no significant differences in
outer perimeter, luminal area, or medial area or thickness in
transgenic vs. wild-type mice (data not shown).
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, blood pressure
measured in awake mice over a 5-day period was significantly lower in
transgenic animals (line 122) than in wild-type animals (117 ± 4
vs. 133 ± 3 mm Hg; P < 0.05). Heart
rate was slightly lower in transgenic mice compared with controls, but
this difference did not reach significance. Analogous measurements in a
second line of SMP8-PTHrP-R-overexpressing mice (line 127) showed a
trend toward lower blood pressure compared with that in controls,
although this difference was not significant (121 ± 3
vs. 127 ± 2 mm Hg; P > 0.05).
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. Baseline blood pressure, mean
velocity, and peripheral resistance were similar in both groups. The
reason why baseline blood pressure was reduced in awake transgenic mice
but not in anesthetized mice is unclear, but could relate to the
decreased sympathetic tone induced by the anesthesia, which may have
obscured any further reduction in pressure from local PTHrP. A similar
result was observed in transgenic mice overexpressing PTHrP (18). The
magnitude of the vasorelaxant response to increasing doses of PTHrP
administered iv was significantly greater in the transgenic animals
than in the controls. At the highest dose, blood pressure decreased to
36 ± 2 mm Hg in the transgenic mice and to only 45 ± 9 mm
Hg in the controls (P < 0.05).
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, A and C).
PTHrP-(1–34) (10 nM) produced a more pronounced relaxation
response in aortas from SMP8-PTHrP-R receptor mice than in those from
wild-type mice. In wild-type mice, PTHrP-induced relaxation was greater
in endothelium-intact aortas (Fig. 4, A and B), as we have reported
previously (23). Figure 4B shows concentration-response relationships
generated using a single concentration of PTHrP-(1–34) in
endothelium-intact or denuded aortas from transgenic and wild-type
mice. Interestingly, at the higher doses of PTHrP-(1–34), the
relaxation response observed in transgenic mice was similar in
endothelium-intact and denuded aortic preparations. Similar results
were obtained in aortic ring preparations from a second line of
SMP8-PTHrP-R-overexpressing mice (line 127), as summarized in Table 2.
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). To determine the time of death during
embryogenesis, timed pregnant females from matings of the receptor- and
ligand-overexpressing transgenic mice were killed, and embryos were
genotyped by PCR at various gestational time points. These studies
demonstrated that embryos expressing both transgenes died at
approximately embryonic day 9. Preliminary examination showed greatly
enlarged hearts, with pericardial effusion and vascular pooling (Fig. 5). Ventricular contractions also
appeared unsynchronized. Histological sections through double
transgenic embryos revealed a reduced thickness of the ventricular
wall, a lack of trabeculae, and prominent gaps between the
cardiomyocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice overexpressing the SMP8-PTHrP-R transgene in vascular smooth muscle demonstrated a significant reduction in systemic blood pressure. Therefore, overexpression of either PTHrP or the PTH/PTHrP type 1 receptor in vascular smooth muscle results in hypotension consistent with the postulated role of PTHrP as a local vasorelaxant. However, in these overexpression transgenic mouse models, the alterations in blood pressure probably represent an amplification of the physiological effects of PTHrP in the vasculature. Therefore, the hypotension seen in the receptor-overexpressing mice probably results from sustained activation of the overexpressed receptor by locally produced PTHrP. Theoretically, it is also possible that overexpression of the PTH/PTHrP-R leads to constitutive activation of adenylyl cyclase, which is known to result when other G protein-coupled receptors are overexpressed (26). However, such a scenario seems less likely in light of our previous studies that showed that massive overexpression of the PTH/PTHrP type 1 receptor in stably transfected vascular smooth muscle cells did not influence basal cAMP production (9).
Aortic vessel preparations from the SMP8-PTHrP-R mice exhibited a marked increase in sensitivity to the relaxant action of PTHrP-(1–34) NH2. Transgenic mice also had a greater reduction in blood pressure when infused with PTHrP-(1–34) NH2 than control mice. Therefore, the SMP8-PTHrP-R transgene appears to drive high level expression of functional receptors in vascular smooth muscle. In wild-type mice, the relaxant effects of PTHrP in aorta were much more pronounced when endothelium was present in the vessel wall. We have previously shown that the ability of PTHrP to relax mouse aorta is largely endothelium dependent, whereas an intact endothelium was not necessary for maximal relaxation of mouse portal veins (23). In these studies, the endothelium-dependent component of PTHrP-induced aortic relaxation was unaffected by pretreatment with either L-NNA or indomethacin, but was abolished by pretreatment with tetrabutyl ammonium, suggesting a requirement for an endothelium-derived hyperpolarizing factor. Interestingly, in the receptor-overexpressing mice, the pronounced relaxation responses induced by PTHrP were similar in both endothelium-denuded and endothelium-intact aortas, indicating that high level activation of this signaling pathway in smooth muscle masked the endothelium component of these responses.
The SMP8-PTHrP-R-overexpressing mice developed normally and had no gross abnormalities of any organ, aside from a modest increase in stomach weight. This is in contrast to the severe abnormalities in bone and heart observed in several of the transgenic mouse lines overexpressing SMP8-PTHrP described in the accompanying report (18). The variation in phenotype between ligand- and receptor-overexpressing mice may relate to the fact that transgenic PTHrP can diffuse and activate receptors expressed in adjacent cells. Thus, the hypercellular bone marrow and grossly increased bone volume in two SMP8-PTHrP transgenic mouse lines probably resulted from local production of transgenic PTHrP by stromal cells and activation of adjacent osteoblasts. By contrast, the targeted expression of the PTHrP-R remains restricted to smooth muscle and would be activated only when ligand is available.
The bone and heart abnormalities observed in several of the
PTHrP-overexpressing transgenic mouse lines described in the companion
report (18) as well as the relatively low numbers of founder mice
obtained from multiple oocyte injections with the ligand expression
construct suggested that high levels of PTHrP receptor activation at
this gestational time point may have disrupted the development of these
organs. To investigate this possibility, we created double transgenic
mice that overexpressed both the ligand and receptor transgenes. All
double transgenic mice died on approximately embryonic day 9.5 with
evidence of major abnormalities in the developing heart. Although we
have not formally established temporal and spatial expression of the
SMP8 -actin promoter in the mouse during embryogenesis, the native
smooth muscle -actin gene is known to be expressed in smooth muscle
of the developing large blood vessels and in cardiac muscle on
approximately embryonic day 10.5 in both mouse (27) and rat (28).
Moreover, both PTHrP and the PTH/PTHrP type 1 receptor are expressed in
rat fetuses on approximately day 11 (29), coincident with the time of
heart and major blood vessel formation in the rat. We attribute the
developmental abnormalities in the double transgenic mice to the
effects of persistent activation of the PTH/PTHrP receptor at this
gestational time point. It remains to be determined whether and to what
extent this peptide plays a role in the normal development of the
cardiovascular system. 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. On the other hand,
Massfelder et al. (10) reported that the mitotic rate of
aortic vascular smooth muscle cells in 18-day-old PTHrP null embryos
was significantly reduced compared with that in PTHrP+/+
fetuses. These data together with the established importance of PTHrP
and its receptor in the development of mammary gland (30), skin (31),
and cartilage (32, 33) suggest that PTHrP also plays a role during the
development of the cardiovascular system.
In summary, overexpression of the PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure in awake mice, probably through sustained activation of the receptor by endogenous ligand. To our knowledge, this study and the one described in the accompanying report are the first to demonstrate the outcome of expression and function of any paracrine vasoactive agent and its receptor in vascular smooth muscle of transgenic mice. Moreover, the similar cardiovascular phenotypes (i.e. hypotension) in both the ligand- and receptor-overexpressing mice strongly suggest that locally elaborated PTHrP acts through the classical PTH/PTHrP type I receptor in vascular smooth muscle to modulate vascular tone. Finally, the embryonic lethality and morphological features observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP also participates in the morphogenesis of heart and perhaps the vasculature. Studies are now underway to formally characterize the defective cardiovascular abnormalities in these animals.
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Acknowledgments |
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Footnotes |
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Received August 17, 1998.
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-vascular but not the 
-enteric isoform in
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