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PTH-CALCITONIN-VITAMIN D-BONE |
Molecular Endocrinology, Max-Planck-Institute for Biochemistry, Martinsried 85152, Germany; Endocrine Unit, Massachusetts General Hospital and Harvard Medical School (E.S., H.J.), Boston, Massachusetts 02114; and Institute of Animal Physiology, Ludwig-Maximilian University of Munich (R.G.E.), Munich 80539, Germany
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Abstract |
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1(I) collagen promoter. Expression of the Jansen
transgene under the control of the type 1(II) collagen promoter was,
furthermore, shown to delay chondrocyte differentiation and to prevent
the dramatic acceleration of chondrocyte differentiation in
PTHrP-/- mice, thus rescuing the early lethality of these
animals. In the present study we demonstrated that the type 1(II)
collagen promoter Jansen transgene restored most of the bone
abnormalities in PPR-/- mice, but did not prevent their
perinatal lethality. These findings suggested that factors other than
impaired gas exchange due to an abnormal rib cage contribute to the
early death of PPR-/- mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many signaling molecules and their receptors are known to influence endochondral ossification, including PTHrP and the PTH/PTHrP receptor (PPR) (3). The PPR, a G protein-coupled receptor with seven membrane-spanning helices, mediates the actions of PTH and PTHrP, two distinct, but related, ligands. PTH is, besides 1,25-hydroxyvitamin D3, the most important endocrine regulator of calcium homeostasis that acts primarily on kidney and bone (4). PTHrP was first discovered as the most frequent cause of the humoral hypercalcemia of malignancy syndrome (5, 6). However, PTHrP is expressed in a large variety of different tissues and usually acts as an autocrine/paracrine regulator rather than as an endocrine hormone (7). Important insights into these roles were obtained through the generation of mice in which both copies of the PTHrP gene were ablated or PTHrP was overexpressed under control of the type II collagen promoter, which targets expression to growth plate chondrocytes (8, 9, 10). These and additional studies (11, 12, 13) showed that the actions of PTHrP on growth plate cartilage are mediated via the PPR, which is most abundantly expressed in chondrocytes residing in the zone between proliferation and hypertrophy. It was, therefore, not unexpected that PPR-ablated mice (PPR-/-) show skeletal abnormalities that are similar to but more severe than those observed in PTHrP-/- animals (11, 12, 13).
Consistent with these findings in mice, activating PPR mutations are
the most likely cause of Jansen’s metaphyseal chondrodysplasia, a rare
autosomal dominant disorder characterized by hypercalcemia,
hypophosphatemia, and short-limbed dwarfism (14, 15, 16, 17, 18). When
introduced into the wild-type human PPR (19) and expressed
in mammalian cells, each of the three Jansen mutations identified to
date leads to ligand-independent constitutive cAMP accumulation
(14, 15, 16, 17, 18), thus providing a reasonable explanation for
abnormal regulation of mineral homeostasis and chondrocyte
differentiation. Furthermore, to prove that the constitutively active
PPR is indeed responsible for the growth plate abnormalities observed
in Jansen’s disease (20), one of the PPR mutants,
HKrK-H223R, was expressed in mice under the control of the 1(II)
collagen promoter to target expression to chondrocytes. The resulting
transgenic mice were viable and showed growth plate abnormalities
mimicking those observed in the Jansen’s metaphyseal chondrodysplasia
(18), i.e. a delay in chondrocyte
differentiation, a delay in vascular invasion, and a reduction or
absence of mineralization of bone elements that are formed through the
endochondral process. Because of this delay in endochondral bone
formation, these mice were used to prevent the acceleration of
chondrocyte differentiation and thus the bone abnormalities during
fetal development and the early lethality of
PTHrP-/- animals (18).
The goal of the present study was to determine whether the targeted expression of HKrk-H223R in the growth plate can also normalize the skeletal phenotype of PPR-/- mice, and whether expression of the transgene can rescue, at least partially, the early lethality of these animals. For this purpose, we generated and analyzed mice that lack the endogenous PPR, but express a constitutively active PPR in growth plate chondrocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
The generation of PPR-ablated mice
(PPR-/-) (11) and transgenic lines
(Tg-A or Tg-B) expressing a constitutively active form of the human PPR
(HKrk-H223R mutant) under the control of the rat 1(II) collagen
promoter was described previously (18).
PPR+/- mice were intercrossed with each of the
Jansen transgenic lines to obtain mice that lack one copy of the PPR
gene and contain one copy of the transgene (Tg-A+
or Tg-B+). Matings of those double heterozygous
mice resulted in offspring with different genotypes, i.e.
wild-type, heterozygous, or homozygous for the receptor knockout
(PPR+/-, PPR-/-), and
heterozygous or homozygous for the Jansen transgene
(Tg-A/B+ or Tg-A/B++). All
mice were genotyped by Southern blot analysis of tail clip DNA. Animals
were maintained in the facilities operated by the Max-Planck-Institute
for Biochemistry (Martinsried, Germany) in accordance with the NIH
Guide for the Care and Use of Laboratory Animals and were employed
using protocols approved by the institution’s subcommittee on animal
care.
Skeletal analysis
The mineralization pattern of the skeleton was analyzed on
embryonic day 18.5 (E18.5) as described previously by McLeod
(29). Briefly, on E18.5 embryos were harvested by cesarean
section. Fetuses were skinned, eviscerated, and fixed in 95% ethanol.
Subsequently, acetone was used to remove fat. Then skeletons were
stained by Alizarin Red S and sequentially cleared in 1% potassium
hydroxide. Mineralized bones were visualized by the staining.
Histology and tissue preparation
For histological analyses, either paraffin or methylmethacrylate
sections of bones were produced on E18.5. For paraffin sections,
fetuses were fixed in 4% paraformaldehyde (PFA)/PBS, pH 7.4, at 4 C,
rinsed in PBS, dehydrated at room temperature through an ethanol series
(70% for 6 h, 80% for 1 h, 96% for 1 h, and 100% for
3 h), cleared twice in xylene for 1 h/step, embedded in paraffin,
sectioned at 6 µm with a Microm HM 355 microtome (Microm
International GmbH, Walldorf, Germany), and mounted on SuperFrost Plus
slides (Carl Roth GmbH & Co., Karlsruhe, Germany).
To obtain methylmethacrylate sections, samples were fixed in 4% PFA for 24 h at 4 C and washed overnight in PBS containing 10% sucrose at 4 C. Subsequently, the left and right hindlimbs of each embryo were dehydrated and embedded undecalcified in conventional methylmethacrylate (30) or using a modified methylmethacrylate embedding method suitable for histochemistry and immunohistochemistry, respectively (31). Three-micron-thick sections were prepared in the midsagittal plane of the knee joint with an HM 360 microtome and stained with von Kossa/toluidine blue (30).
Riboprobes and in situ hybridizations
Complementary [35S]UTP-labeled
riboprobes (cRNAs) were used for in situ hybridizations.
Plasmids encoding the cDNA were linearized with appropriate restriction
enzymes to transcribe either antisense or sense riboprobes in
vitro using the appropriate RNA polymerase. In situ
hybridization was carried out as described previously
(12), only antisense riboprobes showed specific
signals.
Briefly, bone sections were deparaffinized in xylene and rehydrated in a decreasing ethanol series (100%, 90%, and 70%). After proteinase K treatment and postfixation in 4% PFA, sections were incubated in 0.2 N HCl. Sections were then acetylated with 0.25% acetic anhydride in triethanolamine buffer. Before hybridization was performed, sections were dehydrated in 70% and 95% ethanol and air-dried. Sections were hybridized with 35S-labeled antisense or sense riboprobes in a humidified chamber at 50 C for 16 h. After hybridization, unspecifically bound riboprobes were removed by washing the slides with 2 x SSC and 2 x SSC/50% formamide at 50 C and treating them with ribonuclease at 37 C for 20 min. The final wash steps were performed once in 2 x SSC and twice in 0.2 x SSC at 50 C for 20 min. To detect the hybridization of riboprobes on tissues, sections were dehydrated in 70% and 95% ethanol and air-dried. To estimate the intensity of bound riboprobes, slides were exposed to x-ray film (Kodak Biomax MR-1) overnight at room temperature. Sections were then coated with Kodak NTB2 emulsion diluted 1:1 with H2O, exposed for the time needed (determined by autoradiography), developed with Kodak Dektol developer, and fixed with Kodak fixer. After counterstaining with hematoxylin-eosin, tissue sections were analyzed with a Carl Zeiss microscope (New York, NY) using bright- and darkfield optics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(II) collagen
promoter (18) were previously generated. To ensure that
the transgene is specifically expressed in the growth plate and not in
bone, we performed in situ hybridizations using a riboprobe
specific for a vector-derived portion of the transgene. Although growth
plate chondrocytes revealed a clearly detectable signal, no expression
was observed in osteoblasts of calvariae (data not shown); the presence
of osteoblasts in calvaria was, however, confirmed by hybridization
with a probe encoding collagen type I (data not shown). These studies
confirmed earlier findings (18), making it possible to
determine whether expression of the transgene in chondrocytes alone
could rescue the early lethality of PPR-/-
embryos. To generate animals of the genotypes
PPR+/-/Tg-A+ or
PPR+/-/Tg-B+, we first
mated PPR-/- animals with one of the two Jansen
transgenic mice. These were then intercrossed to generate animals that
express the transgene and are homozygous for ablation of the PPR
(genotypes: PPR-/-/Tg-A+,
PPR-/-/Tg-A++, or
PPR-/-/Tg-B+,
PPR-/-/Tg-B++). Genotypes
were established by Southern blot analysis (data not shown).
Survival rate
Genotyping by Southern blot analysis of embryos on E18.5 verified
the expected Mendelian ratio; i.e. the genotypes wild-type
and PPR-/- were present in 25% of the
offspring, and 50% had the genotype PPR+/-.
However, all PPR-/- animals in the absence or
presence of the Jansen transgene died perinatally, indicating that
targeted expression of the constitutively active PPR failed to rescue
the early lethality of PPR-/- animals (data not
shown). This result was different from that in
PTHrP-/-/Tg+ mice that
survived for up to 3 wk (18). Due to the persistent
perinatal lethality of
PPR-/-/Tg+ animals,
histological analyses were performed on E18.5.
Gross and histological phenotype of
PPR-/-/Tg+ mice
expressing a constitutively active Jansen receptor in the growth
plate
PPR-/- embryos died at birth or earlier
and were at each developmental stage smaller than their normal
littermates (11). Heterozygous Tg-A+
and Tg-B+ mice were macroscopically
indistinguishable from their normal littermates, and only homozygous
Tg-B++ animals showed foreshortening of limbs and
tail (18). With the exception of their smaller size,
PPR-/-/Tg-A+ and
PPR-/-/Tg-B+ mice were
indistinguishable from the wild-type animals. In particular, the former
mice lacked abnormalities of the skulls and extremities, suggesting
that the Jansen transgene was able to prevent the skeletal phenotype of
the PPR-/- embryos (Fig. 1
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) or Tg-B mice (data not
shown) prevented, at least partially, the skeletal abnormalities
observed in PPR-/- animals. Figure 2A shows the
skull of a wild-type (left), PPR-/-
(middle), and
PPR-/-/Tg-A+
(right) embryo on E18.5. Excessive mineralization was
present in the bones forming the base of the skull of
PPR-/- animals. These abnormalities were
not observed in
PPR-/-/Tg-A+
animals, which were indistinguishable from their normal littermates.
Correction of the advanced mineralization was also achieved in
forelimbs (Fig. 2B) and vertebrae (Fig. 2D). The ribs, which were
completely mineralized in PPR-/- mice (Fig. 2C), in the presence of the Jansen transgene showed no evidence for
calcium deposition; in fact, there appeared to be a delay in
mineralization, as indicated by the nonmineralized sternum (Fig. 2C).
Taken together, these data suggested that the expression of a
constitutively active PPR prevented the accelerated formation of
mineralized cartilage in PPR-/- mice.
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, A and E). In
contrast, sternebrae of Jansen transgenic animals exhibited a delay in
chondrocyte differentiation and, therefore, no evidence for blood
vessel invasion (Fig. 3, B and F). The sternebrae of
PPR-/- mutants were completely occupied by
hypertrophic chondrocytes (Fig. 3C), resulting in sternebrae that
consisted mainly of type X collagen-positive cells (Fig. 3G). In
contrast, PPR-/- mutants expressing the
constitutively active PPR in chondrocytes
(PPR-/-/Tg-A+ or
PPR-/-/Tg-B+) exhibited
relatively normal sternebrae (Fig. 3, D and H), that is hypertrophic
cells were only present in the center of bones, where one would expect
chondrocyte differentiation to occur. Interestingly, blood vessel
invasion was delayed to a similar extent in sternebrae of Jansen
transgenic animals (Tg-A or Tg-B; Fig. 3, B and F) with or without the
endogenous PPR gene, i.e. Tg-A+ or
PPR-/-/Tg-A+.
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).
Tg-A+ or Tg-B+ animals
showed a slight reduction in the length of their bones, possibly caused
by the delayed replacement of cartilage by bone (Fig. 4B). In contrast,
limbs of PPR-/- mice were disproportionally
shorter, and their long bones were misshapen, with a diminution in the
size of the growth plates and lack of trabecular bone (Fig. 4C). The
disturbed chondrocyte differentiation process was also accompanied by
changes in osteoblast development. Intramembranous bones adjacent to
the hypertrophic chondrocytes of the PPR-/-
embryo showed a dramatic accumulation of matrix-producing osteoblasts.
Osteoid failed to mineralize, as indicated by the lack of von Kossa
staining (Fig. 4G). The tibia of
PPR-/-/Tg-A+ embryos
(Fig. 4D) were almost normal in appearance, although the bones remained
shorter, but proportionate to the smaller size of the animals. The
shape of the bones was restored, the growth plates were normal in size,
and the organization of the chondrocytes within the growth plates was
similar to that of wild-type animals. In addition, the mineralization
defect in hypertrophic chondrocytes was restored. In contrast,
trabecular bone remained absent (Fig. 4D), but this may have been
related to the delay in cartilage replacement, as observed in
Tg-A+ or Tg-B+ animals
(Fig. 4B). Surprisingly, the thickening of the bone collar and the
diaphyseal cortex of the long bones of PPR-/-
mutants (Fig. 4, E and G) had been normalized by the presence of the
transgene in the growth plate (Fig. 4, F and H).
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, A–D), high levels of the mRNA
encoding this metalloproteinase were detected in the diaphyseal region
of PPR-/-/Tg-A+ or
PPR-/-/Tg-B+ animals,
which is consistent with the continuous presence of hypertrophic
chondrocytes. Collagen type I gene expression was strongest in the
cortical bones of PPR-/- mice. These changes,
which were probably due to the excessive intramembranous bone
formation, were normalized in
PPR-/-/Tg-A+ or
PPR-/-/Tg-B+ mice (Fig. 5, E–J). Animals homozygous for the Jansen transgene exhibited no
detectable collagen type I expression, consistent with the observation
that their bones were still cartilaginous. All other probes, including
osteopontin, osteocalcin, Indian hedgehog, collagen type II, collagen
type X, and patched, revealed only minor changes in mRNA expression
(data not shown), and these alterations appeared to be related to
chondrocyte differentiation rather than altered expression by the same
cell population within equivalent areas of the bones.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice expressing a constitutively active human PPR mutant
under the control of the rat 1(II) collagen promoter display
phenotypic changes similar to those observed in mice overexpressing
PTHrP under control of the mouse 1(II) collagen promoter
(9), yet the opposite of those observed in
PPR-/- animals (11). Based on
these findings, expression of the Jansen transgene in
PPR-/- mutants was predicted to prevent the
skeletal abnormalities and the early lethality of
PPR-/- mice.
Our studies have now shown that the targeted expression of a constitutively active PPR in chondrocytes is able to almost completely prevent the skeletal abnormalities of homozygous PPR-ablated fetuses by slowing the abnormal acceleration of chondrocyte differentiation and by correcting osteoblast development. With the exception of the smaller body size, the macroscopic appearance of PPR-/-/Tg+ mice was, on E18.5, identical to that of their normal littermates. In addition to the correction of growth plates, the rescued animals (PPR-/-/Tg+) showed normalization of long bones and decreased thickening of the cortical bone. Furthermore, the presence of the Jansen transgene restored the abnormal bone collar in PPR-/- mice. Given that the transgene is not expressed in osteoblasts (see above), these findings indicate that through paracrine actions a cross-talk occurs between cartilage and bone (36).
On E18.5 PPR-/- mice were present at the expected Mendelian ratio (see Results). However, all of the rescued PPR-/-/Tg+ mice died postnatally despite their almost normal skeletal phenotype. In contrast to the transgene-induced survival of PTHrP-ablated mice, expression of the constitutively active PPR was unable to prevent the perinatal death of PPR-/- animals. These data are consistent with previous findings indicating that PPR-ablated mice exhibit a more severe phenotype than PTHrP-/- mice (11).
The early lethality could be related to the lack of functional PPRs leading in utero to PTH resistance in bone (and kidney) and an abnormal regulation of mineral ion homeostasis. However, in preliminary studies on E18.5, PPR-/- mice showed blood ionized calcium concentrations approximately 25% lower than those in wild-type animals, consistent with our previous findings (37); PPR-/-/Tg-A+ mice showed calcium levels approximately 11% below normal. These findings suggested that the intrauterine regulation of calcium homeostasis is not severely impaired, although postnatal calcium concentrations would be predicted to be as low as those in Gmc-2-ablated mice that had undergone thymectomy to remove extraparathyroidal PTH production (38). Thus, although fetal hypocalcemia may have contributed to the perinatal lethality of PPR-/-/Tg+ mice, it appears more likely that additional factors contribute to the perinatal death of PPR-/-/Tg+ mice. In fact, recent studies have demonstrated that the early lethality of PPR-/- mice that occurs around E10.5 in the C57BL/6 background may be due to cardiac dysmorphogenesis and subsequent vascular collapse or circulatory insufficiency (39, 40). However, a lack of functional PPR may also affect PTHrP-dependent functions in the central nervous system (41, 42).
In conclusion, we have demonstrated that the targeted expression of a constitutively active PPR in chondrocytes is able to almost completely rescue the bone phenotype of homozygous PPR-ablated fetuses by preventing the abnormalities in chondrocyte differentiation; this finding is similar to the observations in rescued PTHrP-/- mice. Furthermore, there was a correction of the anomalies in osteoblast development, but underlying mechanisms leading to this effect remain uncertain. In contrast to prolonged postnatal survival of rescued PTHrP-/- mice, the presence of the Jansen transgene did not improve the early lethality of PPR-/- animals, indicating that persisting abnormalities in mineral ion homeostasis and other PTH- and PTHrP-dependent actions, not only respiratory insufficiency due to the rib cage abnormalities, contribute to the perinatal lethality. Further investigations are needed to determine the cause of the lethality and to fully rescue the PPR-/- embryos.
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Acknowledgments |
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Footnotes |
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This work was supported by Roche Diagnostics and the Bavarian Ministry for Economic Affairs, Transport, and Technology, and by the NIH (DK 50708) (to H.J.).
Abbreviations: E18.5, Embryonic d 18.5; JMC, Jansen’s metaphyseal chondrodysplasia; PFA, paraformaldehyde; PPR, PTH/PTHrP receptor; Tg-A+ or Tg-B+, transgenic line A heterozygous or transgenic line B heterozygous; Tg-A++ or Tg-B++, transgenic line A homozygous or transgenic line B homozygous.
Received April 16, 2001.
Accepted for publication August 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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