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Endocrine Unit (C.B., H.K., K.L., D.R., H.J.), Pediatric Service (H.J.), and Anesthesiology (S.A.F.), Departments of Medicine and Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and the Department of Molecular and Cellular Biology, Harvard University (P.K.), Cambridge, Massachusetts 02138
Address all correspondence and requests for reprints to: Dr. Harald Jüppner, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
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Using ribonuclease protection analysis, significant xPPR-A messenger RNA expression was first detected in neurula stage embryos, which subsequently increased approximately 30-fold during tadpole development. Expression reached a maximum at the metamorphotic climax, when isoform B also became detectable at significant levels, and subsequently declined in postmetamorphotic froglets. In the adult frog, xPPR-A was prominently expressed in lung, brain, small bowel, and skin, whereas isoform B was highest in lung, heart, and brain. Using an xPPR-A antisense riboprobe for in situ hybridization, expression appeared during metamorphosis at all sites of chondrogenesis, specifically in the maturing zone of the amphibian growth plate. xPPR-A expression was also seen in a subpopulation of mononuclear cells, possibly representing osteoblasts that line perichondral bone and diaphyseal bone trabeculae. Our findings suggest that xPPRs serve a prominent role in amphibian skeletal development and possibly other functions during embryonal and early larval development.
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Introduction |
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The endocrine regulation of calcium and phosphate homeostasis in tetrapods relies mainly on the actions of PTH and 1,25-dihydroxycholecalciferol. In contrast, several other hormones, such as calcitonin, PRL, somatolactin, and stanniocalcin, have been implicated in the control of mineral metabolism in fish (11, 12, 13). Furthermore, PTH-like substances have previously been identified in the pituitary gland and the hypothalamus, and in the corpuscles of Stannius in fish, but their contribution to the control of calcium homeostasis remains uncertain (11). Parathyroids are not found in fish. These glands first appear with the origin of terrestrial vertebrates when bones and kidneys became relevant for calcium storage and homeostasis. Amphibia develop parathyroid glands when entering metamorphosis, but also retain some diencephalic control on calcium regulation through the pituitary gland and the paraphysis (11, 14). This animal class therefore, occupies an intermediate position in the evolution of vertebrate calcium homeostasis. Although the biological action of PTH is similar in amphibia and higher vertebrate species, little is known about function of PTHrP in nonmammalian vertebrates (15, 16). To date, no structural data on amphibian PTH or PTHrP are available, but antibodies directed against both mammalian peptides were able to provide immunological evidence for either ligand in the circulation of fish. Furthermore, these antibodies showed cross-reactivity with PTH- or PTHrP-like peptides in the pituitary glands of fish and amphibia, and PTHrP immunoreactivity was observed in a variety of tissues from the frog Rana temporaria (17, 18, 19).
To examine the evolutionary history of the PPR and to characterize its homolog(s) in an animal model widely used in developmental biology, we isolated cDNAs encoding PPRs from the African clawed frog Xenopus laevis (Daudin).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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UniZAP (Stratagene, La Jolla, CA) and contained approximately 2
x 106 plaque-forming units/µl [PCR buffer: 10
mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5
mM MgCl2; 0.001% gelatin; 200 µM
deoxy (d)-ATP, dTTP, dGTP, and dCTP; and 5 U/100 µl Ampli-Taq
polymerase (Perkin-Elmer, Norwalk, CT)]. Primers based on the
nucleotide sequence X1
(5'-AACTACTACTGGATC/TCTGGTG-3') were used
either as forward or reverse primer, in combination with the T7
promoter primer (5'-TAATACGACTCACTATAGGGAGA-3') that anneals to the
Bluescript SK- phagemid. The thermal cycler (M. J.
Research, Watertown, MA) profile was denaturation at 95 C for 5 min (45
sec for each of 45 subsequent cycles), annealing at 60 C for 45 sec,
and extension at 72 C for 2 min (final extension, 10 min). This
reaction was followed by a nested amplification using the forward
primer X2 (5'-CACAGCCTCATC/TTTCATGGC-3') and
the reverse primer X3
(5'-CCTCA/T/GCCATTGCAGAAACAG-3') at an
annealing temperature of 60 C for 35 cycles. The reaction products were
evaluated by Southern blot analysis at low stringency conditions (see
below) using a random-primed labeled probe (Boehringer Mannheim,
Indianapolis, IN) encoding a portion of the rat PPR
(AccI-NotI fragment; 750 bp; accession no.
M77184). A 439-bp PCR product was obtained that showed 70% nucleotide
identity compared with the known mammalian PPR species. This fragment
was then used as a probe for plaque hybridization screening of the same
cDNA library at high stringency (see below) to identify three clones:
the full-length clone C4 encoding xPPR-A and two identical, but
partial, clones, C3 and C13, that encode xPPR-B. Rescreening of the cDNA library did not lead to the isolation of clones containing the 5'-coding region of the xPPR-B receptor isoform. Therefore, this receptor portion was isolated by RT-PCR from 2.5 µg total adult Xenopus kidney RNA that was reverse transcribed using the antisense primer X31 (5'-TGGACTCCAAAGAGAGGC-3'); cDNA clones with the same nucleotide sequence were obtained from two independent PCRs. Forty-five cycles of PCR amplification were performed as described above, using the xPPR-A sense primer X34 that included the start codon (5'-GCCAGCGCCTTGGATATG-3') and the xPPR-B specific antisense primer X33 (5'-GGCATCAAGACGAGAGTG-3'). A single PCR product of about 1200 bp was cloned into pGEM-T (Promega, Madison, WI), and clone X64-1200.4 was identified as xPPR-B on the basis of mismatches between C4 and C3/13 in the overlapping region.
To identify the 5'-untranslated region of xPPR-B and to confirm
portions of the 5'-coding region, a third PCR was performed using a
random primed T3-induced Xenopus tail library in ZAP
(provided by D. D. Brown, Baltimore, MD). Approximately 2 x
106 plaque-forming units of the library were subjected to
45 cycles of PCR using the T3 promotor sense primer and the
xPPR-B-specific antisense primer X37 (5'-CCTCCCTTTCCTTGGTCTCG-3') and
using the same thermal cycler profile as that described above with an
annealing temperature of 55 C. This was followed by a nested PCR using
the sense Bluescript primer blue1 (5'-GCCGCTCTAGAACTAGTG-3') and the
xPPR-B-specific antisense primer X35 (5'-GGCAAACAAGAGCCACCA-3') at an
annealing temperature of 65 C for 45 cycles. A DNA product of about 515
bp was cloned into pGEM-T (Promega, Madison, WI). Clones X96-1-5 and
X96-1-8 contained the first 45 amino acids of the coding region and a
5'-untranslated region of 380 bp.
For expression in COS-7 cells, the cDNA encoding xPPR-A was excised from the Bluescript SK- phagemid C4 by SacI and XhoI digestion and ligated into corresponding sites in the expression vector pcDNAI (Invitrogen, San Diego, CA). The cDNA encoding the full-length xPPR-B isoform was constructed from a VspI-XhoI cDNA fragment of phagemid clone C3 that encodes the carboxyl-terminal receptor region and from the SphI-VspI fragment of the RT-PCR subclone X64-1200.4 that encodes the amino-terminal receptor region.
Southern and Northern blot studies
Xenopus liver DNA and polyadenylated RNA were
prepared and analyzed according to standard procedures (20). After
transfer onto nitrocellulose membranes (NitroPure, MSI, Westborough,
MA), hybridization was performed in the presence of 30% formamide (low
stringency condition) or 50% formamide (high stringency condition) at
42 C for 8–18 h using standard protocols. The probes were prepared by
random primed labeling (Boehringer Mannheim) using restriction
fragments of plasmid DNA or by PCR in the presence of
[
-32P]dCTP and reduced concentrations of dATP, dTTP,
dGTP, and dCTP (2 µM). Washes were performed for 30 min
at room temperature with 2 x SSC (standard saline citrate)-0.1%
SDS, followed by a wash at 42 C in 1 x SSC-0.1% SDS (low
stringency condition) or at 55 C in 0.5 x SSC-0.1% SDS (high
stringency) followed by autoradiography at -70 C for 5–8 days.
Ribonuclease protection assays
Riboprobes were transcribed from linearized plasmids as
previously described (21, 22); the xPPR-A probe was transcribed from
pcDNAI containing the 5' SphI-NsiI fragment of
clone C4, linearized with Eco47III (nucleotides 419–582).
The xPPR-B probe was transcribed from Bluescript SK-
containing the EcoRI-AccI fragment of clone C3,
linearized with DraI (nucleotides 3132–3259). The xPPR-A
probe, therefore, comprised amino acids 47–100 of the coding region,
whereas the xPPR-B probe comprised the 3'-untranslated region of
xPPR-B. Somatic Xenopus elongation factor-1 (EF1)
served as an internal control; it was first detectable in midblastula
stages and then remained equally expressed throughout development and
in all tissues (23). EF1 was detected by a
PstI-SacI fragment (375 bp) in pGEM1 that had
been linearized with AccI.
Twenty to 50 µg total RNA (adjusted to a final concentration of 100
µg with Torula RNA) were coprecipitated, either separately
or in combination, with riboprobes encoding xPPR-A and xPPR-B (200,000
cpm; SA, 
5 x 108 cpm/µg) and a riboprobe
encoding EF1 (100 cpm; synthesized as a probe with low SA, 4.5
x 105 cpm/µg). After resuspension in 30 µl
hybridization buffer [80% formamide, 40 mM PIPES
(piperazine-N,N’-bis[2-ethanesulfonic acid];
1,4-piperazinediethane sulfonic acid) (pH 6.4), 400 mM
sodium acetate, and 1 mM EDTA] and denaturation at 85 C
for 5 min, hybridization was performed at 45 C for 8–18 h. Thereafter,
300 µl digestion mixture [200 U ribonuclease T1 (Sigma Chemical Co.,
St. Louis, MO) in 10 mM Tris-HCl (pH 7.5), 5 mM
EDTA, and 300 mM NaCl] were added, and incubation was
continued at room temperature for 120 min. After the addition of 330
µg/ml proteinase K in 0.7% SDS (final concentrations) and continued
incubation at 37 C for 15 min, RNA was precipitated. Protected RNA
species were resolved on a 5% polyacrylamide-8 M urea
field gradient sequencing gel and were detected by autoradiography at
-70 C for 1–8 days.
COS-7 cell transfections
COS-7 African green monkey kidney cells were cultured as
previously described (24, 25) in DMEM (Mediatech, Washington, DC)
supplemented with 10% FBS (Sigma), 50 U/ml penicillin G, and 50
µg/ml streptomycin sulfate (Life Technologies, Grand Island, NY) at
37 C in a humidified 95% air-5% CO2 atmosphere. COS-7
cells grown in 10-cm dishes were transfected with 5–25 µg plasmid
DNA/dish using the diethylaminoethyl-dextran method as previously
described (24, 25). The cells were subcultured into 24-well plates
18–24 h after transfection and were functionally evaluated after
72 h.
cAMP accumulation
Twenty-four-well plates containing transfected COS-7 cells
(200,000 cells/well) were stimulated (15 min at 37 C) in DMEM, 2
mM 3-isobutyl-1-methylxanthine, 0.1% BSA, and 20
mM HEPES (pH 7.4) with increasing concentrations of either
[Nle8,18,Tyr34]bovine
PTH-(1–34)NH2 (PTH) or [Tyr36]human
PTHrP-(1–36)NH2 (PTHrP) (24, 25). cAMP was determined by
RIA as previously described (24, 25).
Inositol trisphosphate turnover
Transfected COS-7 cells were plated in 6-well plates at 200,000
cells/well and were grown for 2 days before preloading them with 2
µCi/ml myo-[3H]inositol (New England Nuclear, Boston,
MA) in inositol-free DMEM (Life Technologies) supplemented with 0.1%
BSA (37 C for 8–12 h). The next day, plates were incubated with
various concentrations of PTH or PTHrP (40 min at 37 C) in the same
buffer containing 20 mM LiCl. Total inositol phosphates
were isolated using anion exchange chromatography as described
previously (24, 25), and the entire eluate was counted with a liquid
scintillation counter (model LS 6000IC, Beckman, Fullerton, CA).
RRAs
Radioligand studies with transfected COS-7 cells were performed
in 24-well plates (200,000 cells/well) using radiolabeled PTH or PTHrP
and increasing concentrations of unlabeled competing peptides as
previously described (24, 25).
Oocyte injection and animal cap autoinduction studies
For injection of sense or antisense messenger RNAs (mRNAs)
encoding xPPR-A, PTH, or PTHrP, the respective cDNAs were cloned into
the vectors pSP64TEN and pSP64TNE between the noncoding flanking
sequences of Xenopus ß-globin (22). Using reticulocyte
lysates (Promega), the mRNAs encoding PTH and PTHrP were in
vitro translated, and the size of the predicted translation
product was confirmed by SDS-PAGE. Injections of mRNA (5 ng/one-cell
stage embryo) and animal cap autoinduction assays were performed as
described. The induction of different developmental markers was
assessed by RT-PCR as previously described (22, 26, 27, 28). RT-PCR
products were resolved on a 5% polyacrylamide-6 M urea
field gradient gel and detected by x-ray film autoradiography for
2 h.
Oocyte expression and electrophysiological studies
Oocytes were harvested by minilaparotomy from anesthetized
female hCG-primed X. laevis (Xenopus I, Ann Arbor, MI) and
treated with 1.5 mg/ml collagenase D (Boehringer Mannheim, Mannheim,
Germany) in Ca2+-free buffer (82 mM NaCl, 2
mM KCl, 1 mM MgCl2, and 5
mM HEPES, pH 7.6) for 2 h to remove follicles. Stage V
and VI oocytes were selected and microinjected with 25–50 nl mRNA at a
concentration of 2 µg/µl. After incubation in ND-96 (96
mM NaCl, 2 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, and 5
mM HEPES, pH 7.6) for 48–72 h, oocytes were placed in a
0.3-ml perfusion chamber for whole cell electrophysiology. Oocytes were
voltage clamped (model OC-725, Warner Instrument Corp., Hamden, CT) at
-70 mV via two electrodes filled with 3 M KCl (0.5–2
Mohm). Oocytes were continuously perfused with ND-96 at 2 ml/min, and
Ca2+-dependent Cl- currents (29) were elicited
by flowing ND-96 containing PTH-(1–34) and PTHrP-(1–36). Currents
were recorded on a two-channel strip-chart recorder.
In situ hybridization studies
Sections of different developmental stages of X.
laevis were obtained from paraffin-embedded specimens that were
deparaffinized, rehydrated, and hybridized with a
[35S]UTP sense or antisense riboprobe as previously
described (21). The riboprobes were derived from full-length xPPR-A in
Bluescript SK-, linearized at an XmnI site 130
bp downstream of the stop codon (nucleotides 1–2030), or from the
fragment EcoRI-BspEI from xPPR-A in pcDNAI,
linearized with SmaI 30 bp before the start codon
(nucleotides 1–1834), respectively.
Nucleotide and amino acid sequence comparisons
Amino acid sequences were compared with the branch and bound
search option of PAUP 3.1 (30) using the receptors for CRH (P34998) and
calcitonin (U18542) as outgroups. The pylogenetic analysis was based on
184 informative characters, the shortest tree length was 846 steps, the
confidence interval using only informative characters was 0.932, the
retention index was 0.891, and the rescaled consistency index was
0.860. The bootstrap confidence intervals indicate the percentage of
trials that support a given branch in 500 heuristic iterations. The WI
Package (version 9.0, Genetics Computer Group, University of Wisconsin,
Madison, WI) was used for nucleotide sequence comparisons.
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Results |
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). Only 51% amino acid sequence
identity was observed with the human PTH2 receptor, a recently
identified member of the secretin/calcitonin/PTH receptor family that
is activated by PTH [and a recently characterized, hypothalamic
PTH-like peptide (31)], but not by PTHrP (32). The full-length clone
C4 was designated Xenopus PTH/PTHrP receptor type A
(xPPR-A); it contained 768 bp of 3'-noncoding sequence. Clones C3 and
C13 (2.6 kb each) were identical and encoded the last four
transmembrane domains and the connecting loops, the xPPR tail, and 1642
bp of 3'-noncoding sequence. The cDNA comprising the partial clones
C3/13 and their 5'-end was named xPPR-B and had a total cDNA length of
3.6 kb. The encoded protein was six amino acids long and showed 19
amino acid differences; compared with xPPR-A, the overall amino acid
sequence identity was 95%. The nucleotide sequences of both cDNA
clones differed by 5% in the coding region and by 14% in the 3'- and
5'-untranslated regions. Furthermore, xPPR-A and xPPR-B lacked the
equivalent of the mammalian exon E2 (see below) and contained 10–20
adenosines in their 3'-noncoding region, but lacked, like the mammalian
PPR genes, a consensus AATAAA polyadenylation signal (33).
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The isolation of two different cDNAs encoding two distinct isoforms of
the xPPR is consistent with the idea that X. laevis is a
tetraploid frog species. However, to obtain further independent
evidence for the presence of two nonallelic PPR isoforms, Southern blot
analysis was performed with genomic DNA that had been digested with
three infrequently cutting restriction endonucleases (BamHI,
HindIII, and SacI). At least two hybridizing DNA
species were detected with either of two small DNA probes (83 and 135
bp, respectively) that correspond to a single mammalian exon and do not
contain recognition sites for either of the above three restriction
enzymes (Fig. 2, A and B). These findings
supported the conclusion derived from nucleotide sequence comparison of
both cDNA clones that the two PPR isoforms are the products of two
distinct genes. Sequence comparison of xPPR-A and xPPR-B with other
known PPR sequences identified the xPPR clade as the sister group of
the mammalian PPRs (Fig. 3).
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). Compared with
xPPR-A, the B isoform was more than 100-fold less efficiently expressed
and reached only approximately 250,000 copies/cell, even at saturating
concentrations of plasmid DNA (25 µg plasmid DNA/10-cm dish); the
reason for this difference is currently unknown.
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). PTHrP was slightly more potent than PTH in
activating phospholipase C when tested with cells transfected with the
xPPR-A plasmid (EC50, 52 ± 17 nM for PTH
and 14 ± 4 nM for PTHrP). In contrast, cells
expressing xPPR-B did not show any detectable increase in inositol
phosphate turnover when stimulated with either PTH or PTHrP. However,
when xPPR-A was expressed at levels comparable to those of xPPR-B, the
former receptor isoform also failed to activate this second messenger
pathway (data not shown). The carboxyl-terminal fragment human
PTH-(39–84) and several unrelated peptides showed no measurable
activity in either assay system (data not shown).
Expression of xPPRs in adult X. laevis tissues and during
development
To determine whether alternatively spliced mRNA forms exist that
include the equivalent of the mammalian exon E2, RT-PCR was performed
using total RNA from various tissues. The isoform-specific primers that
were used for these experiments encode portions of the amino-terminal,
extracellular domain of either xPPR-A or xPPR-B and are located
adjacent to the putative exon E2. For all investigated tissues, only
DNA products of the expected size were amplified, and no evidence for
splice variants was obtained (data not shown). These findings suggested
that the isolated cDNA clones represent the predominant receptor
isoforms and that the equivalent of the mammalian exon E2 is either
missing in the Xenopus genome or is spliced out.
Northern blot analysis using total RNA showed no hybridizing messages
when probed with the full-length xPPR-A. However, 5–10 µg
polyadenylated RNA of adult Xenopus kidney and skin revealed
three RNA species that were approximately 3, 4, and 8 kb in length
(Fig. 2C). Due to the low expression levels, ribonuclease protection
assays were performed to quantify the expression of both receptor
isoforms during X. laevis development and in several tissues
of adult frogs. Using this more sensitive technique, xPPR-A message was
first detectable in 20–40 µg total RNA from whole embryos or
tadpoles at Nieuwkoop stage 13 (NF St. 13), when embryos undergo
neurulation (Fig. 4). Its expression
peaked during metamorphosis (with 30-fold higher levels at NF St. 61
compared with NF St. 13) and decreased subsequently. xPPR-B mRNA was
detected during metamorphosis, but at 4- to 5-fold lower levels than
the message encoding xPPR-A. Low expression levels of both xPPR-A and
xPPR-B mRNA could be detected in unfertilized eggs and early embryos
after Southern blot analysis of RT-PCR products (data not shown).
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). Conversely, xPPR-B mRNA was
relatively highly expressed in lung, heart, and brain, whereas only low
levels were detected in all other organs. Heart showed higher message
levels for xPPR-B than for xPPR-A; in all other organs, xPPR-A mRNA was
the predominant message, indicating that the type A receptor isoform is
more prevalent than the type B receptor isoform. Similar results were
obtained using isoform-specific RT-PCR (data not shown).
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To assess whether the PPR mRNA is also found in growth plate
chondrocytes of X. laevis and to thereby determine whether
certain aspects of bone development can be studied in this widely used
animal model, paraffin sections of animals from several different
developmental stages were examined by in situ hybridizations
using a 35S-labeled, full-length xPPR-A antisense
riboprobe. Specific hybridization signals were first detected in St. 57
tadpoles at sites of chondrogenesis, and expression was maintained
until postmetamorphotic life (Fig. 4). Overnight autoradiograms of
sagittal sections of a 2-week-old postmetamorphotic froglet showed
specific hybridization in the cartilaginous ends of the vertebral
bodies (Fig. 6, A–C). Paraffin-embedded
or fixed frozen sections of all other tissues and early stages of whole
embryos, which were examined with various type A and B riboprobes,
showed no hybridization.
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).
During later stages of limb development, hybridization was confined to
the zone of maturing chondrocytes. xPPR mRNAs were also detected in the
perichondrium and in cells lining the primary spongiosa of the growth
plate of the humerus of adult frogs.
Function of PTH and PTHrP during X. laevis embryogenesis
The PPR is expressed in embryonic stem cells and is thought to be
involved in mediating the PTHrP-stimulated differentiation into partial
endoderm, thus suggesting that both proteins may have an important role
in early embryonic development (34). However, the addition of high
doses of PTH-(1–34), PTHrP-(1–36), or the receptor antagonists
PTH-(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) and PTHrP-(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) (10-6–10-5
M) to fertilized Xenopus eggs did not
macroscopically influence whole embryo development (data not shown).
Similarly, injection of synthetic sense and antisense mRNAs (5
ng/one-cell stage embryo) encoding full-length rat prepro-PTHrP or
chicken prepro-PTH did not alter early embryogenesis, although the
expected translation products were obtained after in vitro
translation using reticulocyte lysates (data not shown).
A sense message encoding xPPR-A and the noncoding flanking
sequences of Xenopus ß-globin showed, when injected into
Xenopus oocytes (80 ng RNA/oocyte), PTH-(1–34)- and
PTHrP-(1–36)-dependent channel activity in voltage-clamp experiments
that desensitized with prolonged hormone treatment, as previously
described (29). However, overexpression of sense xPPR-A, alone or
together with the mRNA encoding either ligand (5 ng/one-cell stage
embryo), again had no effect on whole embryo development. When tested
in animal cap autoinduction assays, each of the synthetic mRNAs also
failed to show induction of the ventral marker ß-globin, the dorsal
marker cardiac actin, the neural marker neutral cell adhesion molecule,
and the anterior (cement gland) marker XAG-1 (Fig. 7) (27, 28). Whole embryos and
Xenopus elongation factor 1 served as positive and
quality controls.
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Different isoforms with more than 90% amino acid identity were reported for several other X. laevis genes (35, 36, 37, 38, 39, 40). Such different isoforms exist only in this frog species, which most likely evolved approximately 30 million yr ago when two diploid Xenopus species hybridized to form the tetraploid X. laevis group (41). In contrast, cDNAs encoding related, but more distinct, protein isoforms, i.e. type I and type II activin receptors (42), have a more ancient origin and are consequently found not only in Xenopus, but also in higher vertebrate species. As xPPR-A and xPPR-B share 95% amino acid sequence identity, both receptor isoforms are likely to be restricted to the Xenopus group. This was confirmed by maximum parsimony analysis of all known PPR amino acid sequences that placed both xPPR isoforms within the same clade, as a sister group to the mammalian PPR homologs, but not as a separate group. These findings predict that the X. laevis genome contains not only two distinct PPR genes, but also two different, albeit closely related, PTH2 receptor genes.
Interestingly, both xPPR isoforms lack the portion of the extracellular, amino-terminal region that corresponds to the mammalian exon E2, and RT-PCR using total RNA from various tissues revealed no evidence for splice variants that contain this receptor region. In rat and human PPRs, the E2 region can be deleted or modified without a detectable loss of function (43), and its equivalent is absent in the PTH2 receptor (32) and in all other members of this family of G protein-coupled receptors (1, 2). This suggests that mammalian PPRs evolved from an ancestor that lacks the E2 region in the extracellular, amino-terminal domain.
Functional characterization of xPPR-A and xPPR-B in COS-7 cells
When expressed in COS-7 cells, both receptor isoforms bound
amino-terminal PTH and PTHrP analogs with equally high affinity.
Furthermore, both receptors activated the adenylate cyclase signal
transduction pathway with the same potency when challenged with either
peptide, indicating that xPPR-A and xPPR-B are functional PTH/PTHrP
receptors. As mammalian homologs of PTH and PTHrP have similar
activities when tested with amphibian or mammalian PPRs (1, 2),
amphibian PTH and PTHrP homologs, which remain to be isolated, may
share considerable similarity with the corresponding mammalian
peptides.
xPPR-A was able to activate inositol phosphate turnover, whereas xPPR-B, which was less efficiently expressed, did not stimulate this signal transduction pathway. However, when the A receptor isoform was expressed at levels that correspond to those obtained with the B isoform, inositol phosphate turnover was equally impaired. Similar to recent observations with truncated rat PPRs that are also expressed at reduced levels (24), it, therefore, appears likely that impaired cell surface expression of xPPR-B, rather than structural differences between both Xenopus receptor isoforms, is responsible for the difference in inositol phosphate accumulation.
Expression and function of PTH/PTHrP receptors during X. laevis
development and in adult life
RT-PCR analysis indicated that both xPPR isoforms are expressed,
albeit at very low levels, maternally and during early development
(data not shown). Significant levels of xPPR-A mRNA transcripts were
detectable at early neurula stages (NF St. 13), increased subsequently
by approximately 30-fold, and reached a maximum during metamorphosis.
The isoform B was also detected during metamorphotic stages, however at
approximately 5-fold lower levels.
xPPR-A was detectable by in situ hybridization in the metamorphotic stages at all sites of chondrogenesis, in putative osteoblasts lining the primary spongiosa, and in the periosteum. PTH and/or PTHrP thus appear to have roles during amphibian chondrogenesis and bone remodeling that may be similar to those found in higher vertebrates (2, 5, 6, 44). In mammals, PTHrP is also present in the decidua surrounding the implanted embryo, and the infusion of the antagonist PTHrP-(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) prevents normal development (45, 46). PTHrP was shown to induce differentiation of embryonic carcinoma cells into parietal endoderm (34). The PPR is first detected in parietal endoderm and later in the gut, lung, skin, and all sites of bone formation (9, 10). Despite these findings in mammals, which suggested a significant role for the PTHrP/PPR system in early embryonic development, we were unable to obtain evidence for such a role in the developing frog. For example, the injection of fertilized eggs with either sense or antisense PTH or PTHrP mRNAs in the presence or absence of mRNA encoding xPPR-A had no obvious effect on whole embryo development or in animal cap autoinduction assays. Furthermore, although Indian hedgehog induces in this model system XAG-1, a marker of the anterior cement gland (27, 28), no XAG-1 induction was observed when overexpressing chicken PTH, rat PTHrP, or xPPR-A. Different from the recent findings in mammalian and avian growth plates (5, 6), Indian hedgehog thus appears to mediate its actions in the amphibian cement gland through mechanisms that do not involve PTHrP. Similarly, the expression of markers for ventral mesoderm (ß-globin), dorsal mesoderm (cardiac actin), and neuroectoderm (NCAM) were unaffected by chicken PTH, rat PTHrP, or xPPR-A overexpression, indicating that the role(s) of xPPRs during early embryogenesis is more specific or less significant.
Consistent with these findings in amphibia, PPR-ablated mice are small from at least day 9.5 and die after midgestation, but show no early developmental defects (5). Similarly, PTHrP-ablated animals survive without major developmental defects until the end of gestation (44), and their postnatal survival can be prolonged by directing the expression of PTHrP or a constitutively active PPR to the growth plate (47, 48). Taken together, these findings in amphibia and mice indicate that PTHrP and its receptor are unlikely to have a major role in early embryonic development of vertebrates.
In adult X. laevis, xPPR-A was highly expressed in adult lung, brain, small bowel, and skin, whereas xPPR-B expression was most prevalent in lung, heart, and brain. In the emerging frog, R. temporaria, PTHrP was recently detected in similar locations using an antibody against human PTHrP-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) and a riboprobe encoding chicken PTHrP-(55–65), respectively (19). The identification of PTHrP and its receptor in the same or adjacent tissues could indicate that this peptide mediates its actions in lower vertebrate species through similar para-/autocrine mechanisms as those in mammals.
Interestingly, steady state levels of both receptor isoforms were relatively low in amphibian kidney and bone, whereas these tissues show the highest levels of PPR expression in mammals. Bovine parathyroid extracts were shown to increase blood calcium and decrease blood phosphate in amphibia, indicating that PTH may have actions similar to those in mammals. However, little if any direct effects of PTH on calcium and phosphate transport were documented in the amphibian kidney, which corresponds embryologically to the mammalian mesonephros (reviewed in Refs. 15 and 16). Similarly, the amphibian skeleton may not be involved in mineral ion balance to the same degree as that in mammals (11, 12). This could indicate that the PTH/PTHrP receptor in adult amphibian kidney and bone has biological roles distinct from those in mammals.
Additional specialized organs that influence the function of the amphibian parathyroid glands are the paravertebral endolymph sacs, which accumulate calcium (49), and the paraphysis (14, 50, 51). The latter organ is located in the choroid plexus, in which high levels of PTHrP immunoactivity or mRNA were reported in fish, frog, and rat (18, 19, 21); however, no evidence for xPPR expression was found in either tissue.
PTH/PTHrP receptors are expressed in mammals at sites other than kidney, bone, and cartilage (2, 7, 8, 9, 10). In X. laevis, some of these nonclassical tissues showed the highest xPPR-A and xPPR-B expression levels. For example, abundant PPR transcripts are found in the dermal layer of frog and mammalian skin, and at least in mammals, PTHrP expression in kerotinocytes has been implicated in their differentiation (2, 3, 52). Similar to the findings in this study, mice and rats express PPRs in small bowel and lung, at various sites in the brain, and in the heart (2, 53), where it may mediate positive chronotropic and inotropic effects of PTH (54, 55). In contrast to the findings in kidney and bone, mammalian and frog PPRs may, therefore, have similar functions in other tissues.
In summary, two nonallelic PTH/PTHrP receptor isoforms (xPPR-A and xPPR-B), both lacking the domain corresponding to the mammalian exon E2, were identified in X. laevis. Isoform A, when stimulated with either PTH or PTHrP, activated at least two signal transduction pathways, adenylate cyclase and inositol phosphate turnover, whereas the xPPR-B isoform was less efficiently expressed and stimulated only adenylate cyclase. During embryonic development, xPPR-A transcripts were first detected by ribonuclease protection in neurula stages. Maximal expression levels of both receptor isoforms were reached during metamorphosis, and isoform expression was tissue specific in the adult animal. Although the function of xPPR-A in neurula stages remains to be determined, the abundant xPPR expression in maturing chondrocytes and in osteoblasts during metamorphosis may be indicative of a role of PTH and/or PTHrP during amphibian cartilage differentiation, bone elongation, and bone remodeling.
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Footnotes |
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2 Present address: Abteilung für Endokrinologie, Medizinische
Hochschule Hannover, Carl Neuberg Strasse 1, 30625 Hannover, Germany.
Received August 12, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß during embryogenesis
and metamorphosis in Xenopus laevis. J Biol Chem 269:24459–24465 expressed during Xenopus development respond to retinoic
acid. Mech Dev 39:81–93[CrossRef][Medline]
-globin genes
and the amino acid sequece of the three major tadpole -globin
polypeptides. Nucleic Acids Res 13:5407–5421 in Xenopus
laevis. Nucleic Acids Res 18:3489–3493 of
Xenopus laevis (Daudin). North-Holland,
Amsterdam
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