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Division of Endocrinology and Metabolism (F.d.M., N.F.-T., J.C.L.-T., K.K.T., A.F.S.) , University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and Renovascular Physiology and Pharmacology Laboratory (T.M., J.-J.H.), Université Louis Pasteur, Strasbourg, France
Address all correspondence and requests for reprints to: Andrew F. Stewart, M.D., Chief, Division of Endocrinology and Metabolism, Biomedical Science Tower E-1140, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, Pennsylvania 15213. E-mail: stewart{at}msx.dept-med.pitt.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) by prohormone
processing enzymes present within the regulated and constitutive
secretory pathways (5, 6). Entry into the secretory
pathway is initiated at the endoplasmic reticulum as a consequence of
the interaction of a conventional signal peptide with docking molecules
on the cytoplasmic side of the endoplasmic reticulum compartment.
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). Interestingly, this NLS constitutes one of the same multibasic
regions that serves as a prohormone convertase cleavage site when PTHrP
is contained within the secretory compartment (5). The
functional consequences of nuclear entry by PTHrP that have been
observed to date include the regulation of proliferation and/or
apoptosis in vascular smooth muscle (VSM) cells and chondrocytes,
respectively (8, 9, 10). These nuclear actions of PTHrP have
been termed intracrine to distinguish them from the paracrine and
autocrine effects described above. In A-10 VSM cells, for example,
endogenous PTHrP is present in the nucleus of a small minority (0.13%)
of cells, whereas in the remaining 99.87%, it is principally
extranuclear (10). Data from several laboratories document that nuclear targeting results from at least two structural features in the PTHrP molecule and may occur via several cellular pathways. First, as noted above, there is a requirement for the NLS. Henderson and coworkers (8) have demonstrated that the bipartite NLS of PTHrP is necessary for the nuclear/nucleolar appearance of PTHrP, and we have confirmed these observations (10). Moreover, the NLS of PTHrP is able to drive the nuclear localization of a heterologous protein, ß-galactosidase (8). Lam and collaborators (11) have indicated that the nuclear transport process is regulated in part by the phosphorylation status of the cyclin-dependent kinase site, Thr85, with phosphorylation preventing nuclear entry. Second, there must be a mechanism for the peptide to avoid entry into the secretory pathway after translation so that it is free to traffic to the nucleus. Increasing evidence indicates that the structural feature in the PTHrP molecule that permits this option is the availability of multiple translational initiation sites (7, 16, 17). A standard AUG codon drives the translation of a molecule with a full and normally functioning signal peptide that directs the precursor into the endoplasmic reticulum. However, alternative CUG and GUG translational initiation sites have been demonstrated downstream of the conventional AUG. These additional translational start sites are positioned within the signal peptide. Translational initiation at these sites thus appears to disrupt the function of the signal peptide, allowing the nascently translated peptide to remain within the cytosol, where, under the direction of the NLS, it enters the nucleus. Third, there is also evidence that PTHrP, once secreted, is able to reenter the cell, perhaps (although this is controversial) via one of the several PTH/PTHrP receptors (12, 14, 15, 18), and from there gain entry into the nuclear compartment through as yet undefined mechanisms.
Although it seems clear that the nuclear entry of PTHrP is associated with cellular proliferative and/or antiapoptotic responses, it remains unknown how PTHrP may accomplish these effects. One group has suggested that PTHrP may interact with mRNA in the nucleolus (14), perhaps influencing the cell cycle. It also is possible that PTHrP could interact directly with DNA as a transcription factor, although evidence for this is lacking. It is further possible that PTHrP may interact with as yet unidentified nuclear proteins that may mediate or transactivate its nuclear effects. This area is particularly interesting and as yet poorly understood, in part because there is little in the way of a normal paradigm or example that an investigator might use as a model.
We have been particularly interested in the effects of PTHrP in VSM. In the arterial wall, N-terminal PTHrP species interact in a paracrine/autocrine manner with the PTH/PTHrP receptor to vasodilate arterial smooth muscle via cAMP and nitric oxide pathways (19, 20, 21, 22). Clemens and collaborators (20, 21) have demonstrated that targeted overexpression of either the PTHrP molecule or its receptor in transgenic mice leads to arterial hypotension. In addition, PTHrP is up-regulated after arterial injury, for example, after angioplasty and in atherosclerotic disease (23, 24, 25, 26). It has been suggested that the proliferative effects of PTHrP, mediated by the nuclear entry and action of PTHrP in an intracrine manner, may participate in the cascade of VSM cell proliferation, migration, and matrix synthesis characteristic of atherosclerosis and of vascular restenosis after angioplasty (27, 28).
In this report, we have used the VSM system as a model to further explore the mechanism of action through which PTHrP drives proliferation after nuclear entry. We were specifically interested in defining the other structural elements, in addition to the NLS, required to drive VSM proliferation. To accomplish this, we have prepared signal peptide deletion mutants that are unable to exit the cell via the secretory pathway, NLS deletion mutants that are unable to enter the nucleus, a variety of deletion mutants affecting other regions of the molecule, and combinations of these mutants, and introduced them into the VSM cell line, A-10, to examine the consequences on PTHrP intracellular trafficking and PTHrP-regulated VSM proliferation. Using these approaches, we demonstrate that the NLS is necessary and sufficient to drive the nuclear entry of PTHrP. In contrast, the NLS is necessary but not sufficient to induce mitogenesis in VSM cells, and it requires an additional component of the PTHrP molecule. Interestingly, and unexpectedly, we demonstrate that the highly conserved N-terminal and mid region portions of the PTHrP molecule are not important, whereas the less highly conserved C-terminal region 108–139) of the peptide is necessary, and with the NLS sufficient, for the activation of proliferation in VSM.
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Materials and Methods |
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were generated by in
vitro site-directed mutagenesis as described previously
(10) using the cDNA for human PTHrP(-36/+139) cloned into
plasmid pGEM-3 as an initial template. All of the constructs begin with
a codon encoding methionine to allow translation. Each has an epitope
tag at the C terminus corresponding to human influenza hemagglutinin
(HA) for immunocytochemical detection. Each contains the 3'
untranslated region (UTR) of human ß-globin for stabilization of the
mRNA (to replace the native PTHrP 3'-UTR AUUUA instability motif that
accelerates mRNA degradation) and to provide transcriptional
termination, polyadenylation, and splicing signals. Confirmation of the
sequences was accomplished by DNA sequencing. The constructs were then
subcloned in the pLJ vector (10) and transfected into A10
cells as described below.
In vitro transcription and translation
To assess the in vitro transcription and translation
efficiency of the different mutants of PTHrP, 1 µg of each construct
in pGEM-3 plasmid was transcribed and translated in a transcription-
and translation-coupled rabbit reticulocyte lysate system
(Promega Corp., Madison, WI) according to the
manufacturer’s instructions. Translation products, labeled with
[3H]lysine, were analyzed by SDS-PAGE in a 10
to 20% polyacrylamide Tris-glycine gel and then examined
using autoradiography.
Cell culture and transfections
The VSM cell line A10 derived from embryonic rat thoracic aorta
was purchased from the American Type Culture Collection
(Rockville, MD). Cells were cultured in DMEM containing 4.5 g/liter
glucose, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2
mM L-glutamine. Twenty-four hours before
transfection, A10 cells were plated in six-well plates at a density of
1.5 x 105/well. Transfections were carried
out in serum-free medium with 1 µg of each plasmid and 10 µl of
lipofectamine (Life Technologies, Inc., Gaithersburg, MD)
for 6 h at 37 C. For transient transfections, after 24 h of
recovery cells were replated on glass chamber slides (LabTek,
Nalge Nunc International, Naperville, IL) and
immunostained 48 h later (see below). Stably transfected clones
were selected by treatment with 250 µg/ml geneticin (G418, Life Technologies, Inc.). Five to 12 individual clones for each
construct were selected, expanded, and analyzed for PTHrP construct
expression as described below. Clones were grown continuously in the
presence of 250 µg/ml G418.
RNase protection assay
Three clones for each construct were selected on the basis of
similar expression of mRNA, assessed by RNase protection assay as shown
in Fig. 4. Briefly, 20 µg of total RNA from each clone, isolated with
Trizol reagent (Life Technologies, Inc.), was hybridized
overnight at 55 C with a 32P-labeled antisense
cRNA probe that protected a 230-bp EcoRI fragment of the 3'
UTR of human ß-globin mRNA. As an internal control, an antisense cRNA
probe recognizing rat cyclophilin was used (Ambion, Inc.,
Austin, TX). RNase-protected fragments were resolved on 6%
polyacrylamide-urea gels and autoradiographed.
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Fluorescent immunocytochemistry
Transfected or control A-10 cells growing in glass chamber
slides were fixed 48 h after plating with 4% paraformaldehyde in
PBS for 20 min at room temperature and permeabilized with 0.2% Triton
X-100 in PBS for 10 min at room temperature. After 1 h of exposure
to blocking buffer (3% normal goat serum, 0.1% BSA, 0.1% Tween-20 in
PBS), cells were incubated for 1 h at room temperature with mouse
monoclonal anti-HA (HA.11, Covance, Richmond, CA), diluted 1:250 in
blocking buffer and then for 1 h at room temperature with a
fluorescein isothiocyanate-conjugated antimouse IgG secondary antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and
diluted 1:100 in blocking buffer. After final washes, cells were mounted in
ProLong antifade mounting medium (Molecular Probes, Inc., Eugene, OR) and visualized with a Nikon
(Tokyo, Japan) FX fluorescence microscope.
Cell counting
Cells were plated into 24-well plates at a density of
104/well. They were trypsinized on the days
indicated in Fig. 6 and counted by hemocytometer (10).
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0.05 were
considered significant. |
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and examined these cells for evidence of nuclear or
secretory pathway presence of PTHrP. We used the influenza HA epitope
tag for detection, because the critical epitopes for many PTHrP
antisera had been deleted in many of the constructs. As is clear from
Fig. 2 (WT), and in confirmation of the
results of Massfelder, Henderson, and others (7, 8, 9, 10, 11, 12), in
the presence of an intact signal peptide the majority of PTHrP
immunoreactivity is found within the secretory pathway. In contrast,
when the signal peptide is deleted (
SP constructs in Fig. 2), there
is a marked and dramatic increase in the amount of PTHrP observed
within the nucleus. This nuclear appearance has a diffuse pattern.
Importantly, nuclear appearance does not require the presence of any
other region of the PTHrP molecule, because deletion of every other
region of the molecule—N-terminal, mid, and C-terminal regions—does
not negate the nuclear presence of PTHrP. The one exception to this
rule is the construct in which both the NLS and the signal peptide were
deleted (Fig. 2, SP/NLS). In this construct, there appears to be
some nuclear staining. The reasons for this are unclear.
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SP/(38–86)/(108–139) and
SP/(1–79)/(108–139), demonstrated no HA immunofluorescence
(Figs. 1 and 2). Given their marked truncation, it seemed possible that
these constructs were not translated. To test this possibility, the
complete panel of constructs was examined by in vitro
transcription and translation using a
[3H]lysine reticulocyte lysate system. As is
shown in Fig. 3, this presumption proved
to be correct: whereas each of the other constructs was efficiently
translated in vitro, these two constructs showed no evidence
of translation. These two constructs, therefore, were
noninformative.
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on cellular proliferation, the constructs were
stably transfected into A-10 VSM cells. Because most of the epitopes
for available PTHrP antisera had been deleted in these constructs,
selection of clones by PTHrP immunodetection methods was impossible.
Therefore, we selected clones based on levels of construct-derived RNA
expression. Because each construct contained an identical 3' end
containing a portion of the 3' PTHrP UTR spliced to the ß-globin 3'
end, we selected a single RNA probe encoding this PTHrP-ß-globin 3'
region. As shown in Fig. 4, three clones
representing each of the 11 constructs were selected based on
comparable levels of PTHrP-ß-globin mRNA expression. Confirmation
that the peptide was indeed translated and secreted was performed by
IRMA of conditioned medium derived from each of the constructs in Fig. 1. As expected, the signal peptide-intact constructs secreted easily
measurable quantities of PTHrP into their conditioned medium (Fig. 5), whereas the signal peptide-deletion
mutants secreted no measurable peptide. That these other, signal
peptide-deleted constructs did indeed produce PTHrP peptide is clear
from information described below.
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, we also examined
the cellular distribution of the HA epitope-tagged PTHrP constructs in
the stably transfected clones. Although less intense than that observed
in the transiently expressing cells, reflecting the lower level of
expression in the stably, compared with the transiently, transfected
A-10 cells, the cellular distribution of expression of the stable
constructs was comparable to that of the transiently transfected A-10
cells shown in Fig. 2. An example is shown in Fig. 6. As can be seen in this figure, the
distribution of PTHrP-HA immunoreactivity observed using the stable
SP construct was similar to that observed with the same construct
transfected transiently (Fig. 2). Moreover, the large proportion of
nuclear targeted cells, also observed with all of the other signal
peptide-deleted constructs, both stable and transient, is apparent
in this low-power photomicrograph.
Using these stably transfected A-10 clones, we were able to evaluate
growth patterns of multiple clones derived from each construct. As is
shown in Fig. 7, the growth rates of the
various clones were dramatically different. Fig. 7A shows the cell
numbers as a function of time for untransfected A-10 cells and for
vector-transfected A-10 cells. The growth rates of these two types of
controls were indistinguishable, and in the other three panels they
have been combined into a single control group. As shown in Fig. 7B, the A-10 cells expressing the constructs in which the NLS had been
deleted (NLS and SP/NLS) proliferated at a rate that was
approximately 50% slower than that of the control cells, represented
by the open symbols. These changes were significantly different, as
indicated by the asterisks. In Fig. 7C, the proliferation of cells
expressing each of the constructs with both an intact NLS and an intact
C terminus was brisk. Interestingly, the rate of proliferation of each
of these cell lines was very similar, despite very different apparent
quantities of PTHrP within the nucleus (Fig. 2). This may suggest that
the nuclear proliferative response is saturable. In Fig. 7D, the growth
curves of the C-terminal deletion constructs are shown. The growth
rates of these cell lines are strikingly slower than those of the C
terminus-intact constructs shown in Fig. 7C. These results clearly
demonstrate that although the NLS is required for nuclear localization,
the C terminus in addition to the NLS is required for the activation of
the nuclear machinery required for proliferation.
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were not efficiently
expressed. This seems unlikely, because the immunohistochemical nuclear
presence of HA for each of these constructs was comparable. To directly
confirm the overexpression of the SP/(108–139) construct in A-10
cells at the protein level, we prepared extracts of VSM cells
transfected with the various constructs and assayed the extracts for
PTHrP(1–36) immunoreactivity using the PTHrP(1–36) IRMA. As shown in
Fig. 8, the level of PTHrP production was
comparable in the slowly proliferating cells expressing the C
terminus-deleted construct compared with the rapidly proliferating
cells expressing the C terminus-intact PTHrP constructs. Thus, the
slower proliferation observed in the C terminus-deleted mutants cannot
be ascribed to underexpression of PTHrP in cells bearing this
construct. These findings make it clear that the C terminus is
essential, and together with the NLS sufficient, for the stimulation of
cell growth in A-10 cells.
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Discussion |
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Lam and collaborators (11, 12) have previously demonstrated that in the absence of phosphorylation of Thr85, PTHrP can bind, through its NLS region, to importin ß and that both a dephosphorylated state of Thr85 and direct interaction with importin ß are required for nuclear entry. Thus, a novel paradigm describing the regulated access to, and transit through, the nuclear pore complex is emerging. In this regard, the constructs used in the current study in which the mid region was deleted conform nicely to the observations of Lam et al. (11, 12): in the mid region deletion mutants, there is no Thr85 and thus presumably no braking mechanism to prevent PTHrP from transiting the nuclear pore complex. Thus, this mid region-deficient construct not only entered the nucleus but was sufficient to drive cell proliferation, indicating that the mid region is not requisite for proliferation or for nuclear access.
Several investigators have suggested that the N terminus of PTHrP may be not only involved in the activation of the extracellular domain of the PTH/PTHrP receptor but also may play a role in guiding PTHrP from the extracellular domain into the nucleus (12, 14, 15, 18). The studies in the current report are surprising, then, in that they clearly demonstrate that the 1–37 region is not required for nuclear access or for the activation of proliferation in VSM cells. These findings do not directly conflict with the secretion/receptor-mediated internalization hypothesis, but they suggest that if it occurs it is not the only mechanism for nuclear access.
In marked contrast, the C terminus, although nonessential for nuclear entry, is absolutely essential for the stimulation of proliferation, at least in VSM cells. This is consistent with our earlier studies in which NLS deletion mutants failed to drive VSM proliferation, whereas overexpression of the intact wild-type molecule, including the C terminus, stimulated proliferation (10). The term proliferation is used advisedly here, for we previously demonstrated not only increases in cell number but also increases in [3H]thymidine incorporation in A-10 cells overexpressing wild-type PTHrP and a decline in [3H]thymidine incorporation in the NLS deletion mutants (10). Moreover, we have also demonstrated that, in aortic VSM cells of the PTHrP knockout mouse, proliferation in vivo using bromodeoxyuridine is reduced compared with that observed in wild-type aortic VSM cells (10).
These results are of interest for several reasons. First, although it
seems clear that the C terminus of PTHrP [e.g.
PTHrP(107–139) or PTHrP(107–111), so called osteostatin] is likely
to be a mature secretory form of the peptide, and although this peptide
appears to have biological effects in some cell systems such as
osteoblasts and osteoclasts when added to the culture medium of these
cells (32, 33, 34, 35), there is controversy regarding the
reproducibility and biological relevance of these findings. These
considerations, together with the current findings, raise the
possibility that the C terminus of PTHrP may be as important, or more
important, as a transactivating domain for nuclear effects of PTHrP
than as a discrete secretory form of the peptide. Second, they suggest
that there may be specific nuclear protein or nucleic acid targets for
this region of the peptide. Third, they suggest that the results of
experiments exploring the physiology of the NLS through the use of
premature termination codons upstream of the NLS (36) will
need to be interpreted in light of the deletion of the C terminus as
well. In support of the concept that the C terminus plays a regulatory
role in cell physiology, there appear to be potential phosphorylation
sites in the 107–139 region of the peptide that could be important in
regulating the cellular actions of the peptide (Fig. 9). Clearly, further fine mapping of this
region will be critical to identify specific residues or regions
required for the activation of proliferation. Similarly, this region
may be a reasonable bait for two-hybrid studies aimed at identifying
the potential nuclear protein partners of PTHrP. Finally, the studies
described here used human PTHrP constructs in murine cells. It will be
important to determine whether species-homologous PTHrP constructs
and cell lines display these same proliferative associations with the C
terminus. For example, does the human C terminus influence
proliferation in human VSM cells? Does the murine PTHrP C terminus
influence proliferation in murine VSM cells?
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Henderson and collaborators (8, 9) as well as others have suggested that when PTHrP enters the nucleus, it is principally observed in the nucleolar compartment. In contrast, we and others have observed PTHrP principally in a diffuse nuclear pattern (10 and refs. therein). The reasons for the nuclear vs. nucleolar predominance in these various studies requires clarification. It does not seem to result from the use of different antisera, because PTHrP has been observed in both the nucleus and the nucleolus using HA epitope-tagged constructs (16) as well as the same commercially available PTHrP antisera used by several groups. It does not appear to be attributable to a species difference in the rat vs. the human cDNA sequences, because the sequences of these two constructs are so similar that this would seem unlikely a priori. Indeed, the nuclear and nucleolar patterns have been observed in studies using both the human and rat PTHrP cDNAs (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Our presumption, then, is that the pattern may be cell type specific, appearing principally in the nucleus in certain cell types and principally in the nucleolus in other cell types. Clarification of this issue again requires further study.
The observations that the NLS deletion mutants demonstrate slower
proliferation than that observed in untransfected and mock-transfected
A-10 cells merits attention. First, this independently confirms using
new constructs the earlier observations of Massfelder et al.
(10). In addition, because the constructs used in the
current study, including wild-type and NLS constructs, all contained
a C-terminal HA epitope tag, these findings indicate that the HA tag
does not interfere either with the ability of the C terminus to
stimulate proliferation or with nuclear targeting. One is left to
speculate as to the mechanisms underlying the reduction in the
proliferation rate induced by NLS deletion. One possibility is that the
deletion of the NLS does not prevent, and indeed may favor, the
secretion of the growth inhibitory, adenylyl cyclase-activating,
N-terminal portion of the molecule, as suggested by Ditmer et al.
(37). Alternatively, it may be that the proteins derived
from the NLS constructs interfere in some dominant-negative fashion
with nuclear access or nuclear function of the wild-type PTHrP.
Clarification of these issues will require further study. In either
case, however, these observations suggest that NLS and/or C-terminal
truncated forms of PTHrP may have therapeutic utility in disorders
associated with smooth muscle cell proliferation. Examples of
conditions in which excessive smooth muscle proliferation yields
pathological results are uterine fibroid tumors, prostatic hypertrophy,
bronchial asthma, portal hypertension in cirrhosis, pulmonary and
systemic arterial hypertension, arteriosclerosis, and vascular
restenosis after angioplasty. The possibility that delivery of modified
PTHrP constructs may ameliorate, at least in part, some or all of these
disorders deserves further investigation. Indeed, Ishikawa and
colleagues (26) have recently demonstrated that
application of PTHrP to injured arteries in a model of arterial
restenosis does indeed dramatically reduce the degree of restenosis
after arterial injury.
Finally, it should be noted that the studies described here used forced
overexpression of PTHrP in VSM cells. One must wonder whether the
results of such studies reflect events occurring when the protein is
expressed at physiological levels. Two observations provide
reassurance. First, the levels of overexpression are very low, as
described in Figs. 5 and 8, with only low picomolar concentrations
appearing in the medium of transfected cells. Second, VSM proliferation
in PTHrP null mice is slower than that observed in wild-type
littermates (10), suggesting that underexpression has the
reverse effect of the overexpression observed here.
In summary, these studies independently demonstrate that nuclear-targeted PTHrP activates proliferation in VSM cells. Importantly and surprisingly, these studies unequivocally indicate that the C terminus of the molecule is critically important in driving the intracrine stimulation of proliferation in VSM cells. This result is surprising because the C-terminal region is the least highly conserved region of the molecule, in contrast to the extremely highly conserved N-terminal and mid region portions of the molecule. These findings suggest that the C-terminal region may serve some sort of transactivating role for nuclear partners of PTHrP and provide an attractive target for two-hybrid strategies aimed at identifying such potential partners. Finally, these studies suggest possible therapeutic potential for NLS- or C terminus-deleted forms of PTHrP.
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Acknowledgments |
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Footnotes |
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1 F.d.M. and N.F.-T. contributed equally to this work. 
Abbreviations: HA, Hemagglutinin; IRMA, immunoradiometric assay; NLS, nuclear/nucleolar localization signal; UTR, untranslated region; VSM, vascular smooth muscle.
Received March 30, 2001.
Accepted for publication May 28, 2001.
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, P < 0.05 compared with
