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Faculty of Medicine-Endocrinology, Memorial University of Newfoundland (C.S.K., L.L.C., N.J.F.), St. John’s, Newfoundland, Canada A1B 3V6; Department of Biochemistry and Pediatrics, Memorial University of Newfoundland (J.F.K.), St. John’s, Newfoundland, Canada A1B 3X9; and Institute of Molecular Medicine and Genetics, Department of Pediatrics, Medical College of Georgia (N.R.M.), Augusta, Georgia 30912-2640
Address all correspondence and requests for reprints to: Dr. Christopher S. Kovacs, Faculty of Medicine-Endocrinology, Memorial University of Newfoundland, 300 Prince Philip Drive, St. John’s, Newfoundland, Canada A1B 3V6. E-mail: ckovacs{at}mun.ca
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Introduction |
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N-Terminal PTHrP activates the PTH1 receptor and mimics the actions of PTH in that respect, but PTHrP also has actions on receptors that are not shared with PTH (3). PTHrP is an important regulator of mineral ion homeostasis, particularly in fetal life, where its absence (Pthrp null) results in reduced fetal ionized calcium concentrations (4), reduced rate of placental calcium transfer (4), and chondrodysplasia characterized by shortened and misshaped long bones and accelerated terminal differentiation of cartilage (5). PTHrP is produced locally within the perichondrium and growth plate; together with Indian hedgehog and other factors, it directs the proliferation and differentiation of the chondrocytes in the cartilaginous structure that will later be destroyed and replaced by endochondral bone (6).
The relative contributions of PTH and PTHrP to the regulation of fetal calcium homeostasis and skeletal development have not been fully elucidated. The observation that PTH circulates in the fetus at lower levels than in the adult (and at levels lower than PTHrP) has been interpreted to mean that PTH might not be required for normal skeletal development or the regulation of fetal calcium homeostasis (7). As chondrodysplasia similar to that observed in Pthrp-null fetuses is evident in fetuses that lack the PTH1 receptor (Pthr1-null) (8), at first glance it might appear that the skeletal effects of the PTH1 receptor during development are entirely accounted for by PTHrP and not by PTHrP and PTH together. However, Pthr1-null fetuses have a more severe phenotype than Pthrp-null fetuses, suggesting a role for PTH in fetal development. These differences include the facts that Pthr1-null fetuses have an even lower blood calcium (4), reduced fetal size (8), embryonic lethality in some genetic backgrounds (8), decreased expression of osteoblast-specific genes (osteocalcin, osteopontin, and interstitial collagenase) (9), and differences in microscopic aspects of the chondrodysplasia (mineralization pattern and vascular invasion) (9, 10).
It has previously been shown that intact fetal parathyroid glands are required for normal skeletal development, as fetal thyroparathyroidectomy in lambs caused decreased ash content and rachitic changes in the fetal skeleton by term (11, 12). These effects could be partly reversed or prevented by fetal calcium and phosphate infusions; thus, much of the effect of fetal parathyroidectomy appeared to be the consequence of decreased blood levels of calcium and phosphate (12). However, although bone formation parameters were corrected by the calcium and phosphate infusion, bone resorption parameters remained abnormal in those lambs (reduced resorption cavities, reduced osteoclast numbers). Therefore, that study demonstrated that functioning fetal parathyroids are required for normal fetal bone resorption and mineralization, but it did not determine whether that requirement was for PTH, PTHrP, or both.
The fetal parathyroids contain abundant PTH mRNA, but evidence that the parathyroids make PTHrP as well is less certain (13). Thyroparathyroidectomy of fetal lambs caused a reduction in the rate of placental calcium transfer that could be reversed by infusion of midmolecular fragments of PTHrP, but not by PTH (14, 15). That result suggested that the fetal parathyroids (at least in lambs) regulate placental calcium transfer through the production of PTHrP; however, measurements were not obtained to determine whether parathyroidectomy caused a reduction in the fetal circulating PTHrP level. More recently, we have determined that fetal mice lacking parathyroids (Hoxa3-null) have normal circulating PTHrP levels but are functionally aparathyroid, as evidenced by absent PTH, reduced blood calcium, elevated serum phosphate, reduced serum magnesium, and reduced calcium in amniotic fluid (16). This confirms that the circulating level of PTH in normal fetal mice, although low, does have functional importance in regulating fetal calcium and magnesium metabolism.
The evidence, therefore, suggests that both PTH and PTHrP may act in the regulation of fetal mineral metabolism. However, the possibility that PTH might have actions distinct from those of PTHrP in regulating skeletal formation has not been explored. We hypothesized that PTH is a critical determinant of fetal blood calcium regulation and skeletal mineral accretion, independent of PTHrP. To test this, we first examined the skeletal morphology and mineralization characteristics of fetal mice lacking parathyroids and PTH (Hoxa3-null mice), and we contrasted the findings with the effects of loss of PTHrP (Pthrp-null) and loss of PTH1 receptor (Pthr1-null) in a similar genetic background. Second, to better clarify the effects of PTH and PTHrP on the regulation of the blood calcium and skeletal development, we created double mutants that lack both PTH and PTHrP (Hoxa3-null x Pthrp-null), and examined blood calcium regulation and skeletal mineral accretion among the double mutant and the single mutant siblings. Finally, we contrasted the phenotype of the double mutant with the Pthr1-null and Pthrp-null.
We found that although the skeletal growth plates and limb lengths are unaltered, loss of PTH in the Hoxa3-null significantly reduces the blood calcium and skeletal mineral content. The reduction in blood calcium and skeletal mineral content is of similar magnitude to that observed in Pthr1-null fetuses. Loss of PTH caused a greater decrease in fetal blood calcium over that of PTHrP, but the loss of both simultaneously led to the greatest fall in fetal blood calcium. Combined loss of PTH and PTHrP caused a further shortening of limb length and a reduction in overall fetal size compared with Pthrp-null. We conclude that PTH appears to play a more dominant role than PTHrP in regulating fetal blood calcium, that the blood calcium level (and not placental calcium transfer) is a rate-limiting step for skeletal mineral accretion, and that lack of both PTH and PTHrP will cause fetal growth restriction. Furthermore, although PTHrP clearly regulates the development of the cartilaginous precursor of endochondral bone from within, PTH plays a separate, external role to direct the accretion of mineral by the developing bone matrix.
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Materials and Methods |
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Genomic DNA was obtained from fetal tails, and genotyping was accomplished by PCR using primers specific to Hoxa3, Pthrp, and Pthr1 gene sequences (4, 18), in a single tube, 36-cycle PCR reaction using a PTC-200 Peltier Thermal Cycler (MJ Research, Inc., Cambridge, MA). For the double knockout (Hoxa3 x Pthrp) mice, genotyping of the Hoxa3 allele was performed by PCR, and the Pthrp allele was characterized by Southern blot (4).
Chemical assays
Serum PTH was measured on embryonic day (ED) 18.5 fetuses using
a rodent PTH-(1–34) immunoradiometric assay kit (Nichols Institute Diagnostics, San Juan Capistrano, CA); the stated
detection limit of the assay was 2.0 pg/ml. Three or four samples of
fetal mouse serum were pooled to obtain sample volumes of 100 µl.
These pooled samples were then diluted with zero standard to meet the
sample size requirements of the assay.
Tissue collection
For in situ hybridization and immunohistochemistry of
fetal bones, whole fetuses (ED 17.5 or 18.5) were placed in 10%
formalin after first incising the abdomen to prevent its gaseous
expansion. After 12–24 h in the fixative, the lower limbs were removed
and separately processed, embedded in paraffin, and cut into 5-µm
sections.
Fetal ash and skeletal mineral assay
Intact fetuses (ED 18.5) were weighed, placed into covered
crucibles, and reduced to ash in a furnace (500 C, 24 h). The ash
was weighed, transferred to acid-washed vials, and dissolved in 253
µl ultra pure nitric acid. After 5 d, 9.747 ml distilled water
were added to each vial for a total volume of 10 ml and a concentration
of 0.2 N nitric acid. Samples were assayed for calcium and
magnesium on a Perkin-Elmer Corp. 2380 atomic absorption
flame spectrophotometer (Norwalk, CT). Data for total ash weight, total
calcium, magnesium content, and mineral content corrected for ash
weight were collected. As fetal size varied from litter to litter and
would affect the individual measurements (large litter, smaller
fetuses; small litter, larger fetuses), the data were normalized to the
mean value of the heterozygotes within each litter. The heterozygotes
were chosen as the baseline for this comparison, because, on the
average, they accounted for 50% of the fetuses in a given litter.
Riboprobe and DNA probe labeling
For in situ hybridization, the plasmids were
linearized with appropriate restriction enzymes and labeled with 125
µCi [35S]UTP using an SP6/T7 transcription
kit (Promega Corp./Fisher Scientific,
Burlington, Canada), and the appropriate polymerase. Unincorporated
nucleotides were removed with the NucTrap columns
(Stratagene, La Jolla, CA).
cDNAs included the pro-
1(I) chain of human type I collagen
(19), the pro-1(II) chain of rat type II collagen
(20), and mouse type X collagen (21) (gifts
from K. Lee); mouse osteocalcin (22) and rat osteopontin
(23) (gifts from B. Lanske); and murine interstitial
collagenase (24) and murine 92-kilodalton gelatinase (type
IV collagenase or MMP-9; gifts from S. M. Krane)
(25).
In situ hybridization
In situ hybridization was performed on 5-µm
tissue sections as described previously (9). Hybridization
was performed in a humidified chamber (16 h, 55 C) with the labeled
riboprobe diluted 1:20 in the hybridization solution. Sections were
successively washed, treated with ribonuclease, and dehydrated in
graded ethanol series. An overnight exposure of the slides to plain
x-ray film enabled an estimate of exposure time for the liquid emulsion
step. Slides were then dipped in NTB-2 liquid emulsion, dried, stored
in light-tight boxes, and kept at 4 C until developed (2–6 wk). The
emulsion was developed using standard developer and fixer, and the
sections were counterstained with hematoxylin-eosin.
All comparisons of wt to Hoxa3-null were made between tissues obtained from within the same litter that had been processed, embedded, and sectioned at the same time. Comparisons of Hoxa3-null to Pthrp-null and Pthr1-null were made on tissues that had been harvested and processed within a few days of each other. All comparative sections were always hybridized together with the same probe and washed together to validate the comparison and minimize artifacts. Assessments of signal intensity were determined in a blinded fashion (no knowledge of the genotype). The reproducibility of the results was confirmed independently on at least three separate litters of each knockout colony.
Histology
Five-micron sections were deparaffinized, rehydrated in a graded
ethanol series, and transferred to distilled water. For morphological
assessment of the growth plate, sections were stained with hematoxylin
and eosin or 1% methyl green, then dehydrated and mounted. For von
Kossa staining, the sections were transferred to 1% aqueous silver
nitrate solution and exposed for 45 min under a strong light. They were
then washed three times in distilled water, placed in 2.5% sodium
thiosulfate (5 min), and washed again three times in distilled water.
Finally, they were counterstained with methyl green, dehydrated in
1-butanol and xylene, and mounted.
Alizarin Red S and Alcian Blue preparations
Fresh fetuses (ED 18.5) were obtained, and skin, viscera, and
adipose tissue were carefully removed. In individual scintillation
vials, the fetuses were fixed in 95% ethanol for 5 d, followed by
acetone for 2 d to remove the remaining fat and firm up the
specimen. After this, the fetuses were stained for 3 d in 10 ml
freshly prepared staining solution at 37 C (1 vol 0.3% Alcian Blue 8GS
in 70% ethanol, 1 vol 0.1% Alizarin Red S in 95% ethanol, 1 vol
acetic acid, and 17 vol 70% ethanol). They were washed in distilled
water and then immersed in 1% aqueous KOH until the fetal skeleton was
clearly visible through the surrounding tissue (
12–48 h). They were
cleared in 1% KOH containing increasing concentrations (20%, 50%,
and 80%) of glycerine (7–10 d at each step). Finally, they were
transferred into 100% glycerine for permanent storage.
For fetuses obtained from the double knockout colony, the procedure was adapted for use on formalin-fixed specimens that had previously been genotyped, so that litters could be chosen in which wt, Pthrp-null, and double mutant fetuses were all present. They were first washed in tap water (six changes) for 24 h and then dehydrated in a graded series from distilled water to 95% ethanol (24 h at each step). After this, the protocol was unchanged, except for the duration of several steps, including 2 wk for 1% aqueous KOH and 2 wk for each step of 1% KOH with increasing glycerine concentrations.
Statistical analysis
Data were analyzed using SYSTAT 5.2.1 for Macintosh (SYSTAT,
Inc., Evanston, IL). ANOVA was used for the initial analysis; Tukey’s
test was used to determine which pairs of means differed significantly
from each other. Two-tailed probabilities are reported, and all data
are presented as the mean ± SE.
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Results |
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(A–D). In
the absence of PTH, the Hoxa3-null skeleton shows normal
crown-rump length, skull vault and mandible, and long bones (Fig. 1B).
There was slightly less intense Alizarin staining of the intact
Hoxa3-null skeleton compared with its wt littermate,
although this may not be obvious in Fig. 1B. In contrast to the
Hoxa3-null that lacks PTH, Pthrp-null fetuses had
the previously described (5) shortened mandible, rounded
vault of the skull, shortened and misshaped long bones of the upper and
lower limbs, and abnormally mineralized cartilaginous portions of the
ribs and sternum. Pthrp-null fetuses also had a slightly
shorter crown-rump length and body weight compared with their wt
siblings (Fig. 1C and Table 1), a finding
that has not previously been described. Finally, Pthr1
fetuses had the reported (8) markedly reduced crown-rump
length and overall skeletal size, with more severely shortened and
misshaped long bones; the fetal weight was significantly reduced
compared with wt (Fig. 1D and Table 1).
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) compared with wt (Fig. 1E) and the
shortened and misshaped long bones of Pthrp-null (Fig. 1G)
and Pthr1-null (Fig. 1H). Similar findings were present upon
detailed examination of the lower limbs (not shown).
Although the microscopic growth plate defects in Pthrp-null
and Pthr1-null have been well documented, the growth plates
of PTH-less Hoxa3-null fetuses have not been examined
previously. Figure 1 shows representative sections of tibias (ED 18.5)
that have been stained black for mineralized tissue (von Kossa stain)
and counterstained with methyl green (Fig. 1, I–L). In the von Kossa
method, silver displaces calcium to form silver phosphate and silver
carbonate complexes; as calcium is the only known cation that binds to
these insoluble anions in organic tissue, the method is considered to
be sufficiently specific for calcium (26, 27). Compared
with wt (Fig. 1I), Hoxa3-null shows a normal growth plate
length and a normal orderly progression of cartilaginous cells from the
proliferative to the hypertrophic zone (Fig. 1J). There is less black
staining in the region of trabecular bone, indicating that less mineral
is present. Thus, the Hoxa3-null limb appears to have normal
growth plate morphology, but to be undermineralized. In contrast, the
Pthrp-null section demonstrates the previously described
(5) shortened and disorganized growth plate, but
apparently normal mineral content, as shown by von Kossa stain (Fig. 1K). The Pthr1-null section demonstrates the previously
described (8) more marked shortening of the growth plate
and the entire limb length, such that almost the entire length of the
bone is visible in the section (Fig. 1L). The amount of mineral present
in Pthr1 bone appears to be reduced compared with that in wt
and Pthrp-null and similar to the reduced mineralization in
Hoxa3-null.
Skeletal mineral content
Both the Alizarin-stained skeletal preparations and the tibial
sections stained by von Kossa’s method suggested that the amount of
mineral may be reduced in the Hoxa3-null skeletons. In
contrast, at the gross level the abnormal cartilaginous mineralization
seen in the intact Pthrp and Pthr1-null skeletons
suggested that the amount of mineral may be increased, whereas at the
microscopic level, the von Kossa method suggested that mineralization
was normal in Pthrp-null and reduced in
Pthr1-null. As these are at best qualitative results, the
amount of mineral present in the fetal skeletons was more precisely
quantitated by reducing intact fetuses to ash in a furnace and
measuring the calcium and magnesium content of the ash by atomic
absorption spectroscopy. As shown in Table 1, the weight of
Hoxa3-null fetuses did not differ from their wt or
heterozygous siblings; however, as shown in Fig. 2, the total amount of ash, calcium, and
magnesium was reduced in Hoxa3-null fetuses, confirming that
the Hoxa3-null skeleton is undermineralized in the absence
of PTH. In contrast, although the weight of Pthrp-null
fetuses was modestly, but significantly, lower than that of wt and
heterozygous siblings (Table 1), the ash weight, calcium content, and
magnesium content did not differ among Pthrp-null fetuses
and their siblings (Fig. 2). After correcting for the reduced fetal
weight of Pthrp-null fetuses, the calcium and magnesium
content still remained not significantly different among
Pthrp-null fetuses and siblings (not shown). Thus,
Pthrp-null fetuses had a normal amount of mineral, although
it was distributed into areas that normally are not mineralized. On the
other hand, Pthr1-null fetuses had a significantly reduced
weight (Table 1), ash weight, calcium content, and magnesium content
(Fig. 2) compared with their siblings. As reduced fetal size would, in
turn, reduce the size of the skeleton and the amount of mineral, the
calcium and magnesium results were further corrected for the ash
weight. When analyzed as milligrams of calcium (or magnesium) per g ash
and normalized for litter size, Pthr1-null fetuses still had
significantly reduced skeletal calcium content (77.9 ± 5.7 in
Pthr1-null vs. 100.0 ± 3.7 in
Pthr1+/- and 107.7 ± 5.7 in wt;
P < 0.02) and skeletal magnesium content (88.3 ±
3.4 in Pthr1-null vs. 100.0 ± 2.2 in
Pthr1+/- and 93.5 ± 5.0 in wt;
see also dashed lines in Fig. 2). Thus, the
Pthr1-null fetus is also undermineralized even after
accounting for the reduced fetal size; the degree of
undermineralization is similar to that observed in the
Hoxa3-null fetus.
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and data not
shown), indicating that, unlike in Pthr1-null, these
indicators of osteoblast function are not altered in the absence of PTH
alone.
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). The increased PTH
level may contribute to blood calcium regulation (and other aspects of
the phenotype) of the Pthrp-null, but Pthr1-null
fetuses lack the receptor to be able to respond to N-terminal PTH. We
have previously demonstrated that Hoxa3-null fetuses lack
detectable PTH (16). Thus, lack of PTH (absolute in
Hoxa3-null, relative in Pthr1-null) is a shared
factor in the two null phenotypes that have both the lowest fetal blood
calcium level and have undermineralized skeletons. The absence of PTHrP
causes a modest fall in the fetal serum calcium and no impairment of
skeletal mineralization, whereas loss of PTH causes a greater fall in
fetal serum calcium and reduced skeletal accretion of mineral. This
suggests that PTH may play a greater role in the regulation of fetal
blood calcium and skeletal mineralization than PTHrP. The data that
support this conclusion (from this and our previous studies) are
summarized in Table 3.
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(A–C), double
mutant fetuses were smaller than their wt and Pthrp-null
siblings; however, the reduction in fetal size was modest compared with
the growth restriction observed in Pthr1-null fetuses (Figs. 1 and 4C, double-headed arrow, and data not shown). The
double mutant showed the typical features of Pthrp-null
chondrodysplasia, with rounded skull, shortened mandible, protruding
tongue, and foreshortened, misshaped long bones. The limbs of the
double mutant were foreshortened more than in the Pthrp-null
sibling. The Alizarin-stained skeletal preparations (Fig. 4, D–F)
confirmed that the double mutant has typical Pthrp-null
chondrodysplasia, including abnormally calcified ribs, but it is
smaller and has shorter limbs than the Pthrp-null animal.
Microscopic examination of the tibias (Fig. 4, G–I) showed that the
double mutant cartilaginous growth plate is of similar length to the
Pthrp-null growth plate and exhibits a similar
foreshortening of the cartilaginous precursors. The length of the tibia
is shorter (almost the entire length is seen in the image), and thus
the extra shortening of the double mutant limb is in the shaft of the
bone and not in the growth plate itself. von Kossa staining of tibial
sections (Fig. 4, lower panels) shows apparently normal
mineral content in wt and Pthrp-null, but reduced staining
in the double mutant, indicating that skeletal mineralization is
reduced. Thus, the double mutant has a phenotype similar to that of
Pthr1-null, including shortening of the long bones and
reduced mineralization. The long bone shortening appears to be more
pronounced in Pthr1-null than in the double mutant.
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that single mutant Pthrp-null fetuses had a modestly reduced
blood calcium (equal to the maternal calcium concentration), single
mutant Hoxa3-null fetuses had a markedly reduced blood
calcium (below the maternal calcium concentration), and double mutant
fetuses had the lowest blood calcium. Thus, lack of PTH had a greater
impact on fetal blood calcium than lack of PTHrP; the combined loss of
PTH and PTHrP resulted in the lowest blood calcium level.
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Discussion |
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Lack of PTH in the Hoxa3-null impaired skeletal mineralization, as suggested by the Alizarin and von Kossa preparations and quantitatively determined by the measurement of the calcium and magnesium content of the ashed skeletal residue through atomic absorption spectroscopy. As blood calcium and magnesium were also significantly reduced, the effect of lack of PTH on bone may have been through its effect on maintaining blood calcium and magnesium. That is, by impairing the amount of mineral presented to the skeletal surface and to osteoblasts, lack of PTH thereby impaired mineral accretion by the skeleton. It should also be noted that lack of PTHrP in the Pthrp-null did not increase the amount of skeletal mineral, as might be assumed by the observation of abnormally calcified rib and sternal cartilages. Similarly, loss of PTH1 receptor did not increase the accretion of mineral, but significantly decreased it, even after accounting for decreased fetal size. The effects of the PTH1 receptor on skeletal mineralization appear to be through PTH, but not PTHrP, in contrast to the effects of the PTH1 receptor on chondrogenesis that appear to be through PTHrP and not PTH.
Apart from undermineralization of the skeleton, the lengths of the long bones and the growth plates of Hoxa3-null were normal at both the gross and microscopic levels, and the expression of several osteoblast- and osteoclast-specific genes was unaltered by loss of PTH. In other words, loss of PTH did not appear to affect the development of the cartilaginous scaffold or of the bone matrix that replaced it, but loss of PTH did impair the final mineralization of that bone matrix. It is therefore unlikely that abnormal osteoblast function can explain the reduced mineralization of the Hoxa3-null bones. However, it is clear that the PTH1 receptor influences osteoblast function and the normal regulation of osteopontin, osteocalcin, and interstitial collagenase in the fetal growth plate, because the expression of these genes is selectively reduced in Pthr1-null (9). As PTHrP is produced locally in the growth plate and periosteum, it is probably the ligand that normally acts on the PTH1 receptor to regulate these genes. On the other hand, as the expression of osteopontin, osteocalcin, and interstitial collagenase is not reduced in Pthrp-null (9), PTH may be able to penetrate the relatively avascular growth plate and compensate for the absence of PTHrP. The elevated PTH levels we have now noted in the Pthrp-null fetus are compatible with this observation. Therefore, osteopontin, osteocalcin, and interstitial collagenase may be down-regulated in Pthr1-null because neither PTH nor PTHrP can act in the absence of the PTH1 receptor; these genes are not down-regulated by absence of PTH or PTHrP alone. As only Pthr1-null shows evidence of impaired osteoblast function, but both Hoxa3-null and Pthr1-null show a similar degree of reduced mineralization, the undermineralization of both null phenotypes may be due to the reduced availability of mineral presented to the osteoblast surface (i.e. the reduced blood calcium and magnesium levels in both phenotypes); the availability of mineral is dependent upon the action of PTH.
The interrelation among blood calcium (and magnesium) regulation, skeletal mineralization, and placental calcium transfer is complex, as seen from the contrasted findings in Hoxa3-, Pthrp-, and Pthr1-null fetuses. The normal elevation of fetal blood calcium above the maternal calcium concentration was historically taken as the first evidence that placental calcium transfer was largely an active process, and the rate of placental calcium transfer has been considered to be the rate-limiting step for skeletal mineral accretion. However, we can now conclude that the fetal blood calcium level is not simply determined by the rate of placental calcium transfer, because placental calcium transfer is normal in Hoxa3-null and increased in Pthr1-null, but both null phenotypes have significantly reduced blood calcium levels. In Pthr1-null, much of the calcium transferred to the fetus may return to the mother, because it is neither accreted by the skeleton nor excreted into the amniotic fluid (4). We can also conclude that the rate of placental calcium transfer is not the rate-limiting step for skeletal mineralization, because the accretion of mineral was reduced in the presence of normal placental calcium transfer (Hoxa3-null) and reduced in the presence of increased placental calcium transfer (Pthr1-null). Furthermore, Pthrp-null showed normal skeletal mineral content in the presence of reduced placental calcium transfer and a modestly reduced blood calcium level. The rate-limiting step for skeletal mineralization appears to be the blood calcium level, which, in turn, is largely determined by PTH. The level of blood calcium achieved in Pthrp-null, that is, the normal adult level of blood calcium, is sufficient to allow normal skeletal accretion of mineral, whereas lower levels of blood calcium [Hoxa3-null, Pthr1-null, and double mutant (Hoxa3-null/Pthrp-null)] impair the rate of mineral accretion. Further, the level of blood calcium in normal fetuses, that is, greater than the adult level, may be a consequence of the additive effect of PTHrP on placental calcium transfer over that of PTH.
Finally, the double mutant lacked both PTH and PTHrP and showed generalized growth restriction, with reduced size and length compared with the wt and Pthrp-null siblings. This suggests that loss of both PTH and PTHrP will impair fetal growth. The growth restriction observed in the double mutant is not as pronounced as that seen in Pthr1-null fetuses, indicating that the lack of the two ligands (PTH and PTHrP) in the fetus does not equate with loss of the PTH1 receptor. It may be that maternal PTH is able to act on the early double mutant embryo before the placental barrier forms, whereas the Pthr1-null fetus would be completely resistant to the N-terminal actions of PTH and lack the effects of PTH throughout gestation.
In summary, loss of PTH (Hoxa3-null) impairs skeletal mineralization, but does not alter gross and microscopic morphology or osteoblast-specific gene expression in growth plates and endochondral bone. The effect of loss of PTH may be through the markedly decreased blood calcium; the blood calcium level (and not the rate of placental calcium transfer) appears to be a rate-limiting step in skeletal mineralization. Both PTH and PTHrP act to regulate the fetal blood calcium, but PTH seems to have the more significant effect. Loss of PTH has no effect on overall fetal growth (crown-rump length, weight); loss of PTHrP has a modest effect on fetal growth; loss of PTH and PTHrP has a more pronounced effect on fetal growth; loss of the PTH1 receptor causes the most marked fetal growth restriction.
These results suggest the following model. PTH normally acts systemically (i.e. outside of bone) to direct the mineralization of the bone matrix by maintaining the blood calcium at the adult level, but PTH is capable of directing certain aspects of endochondral bone development in the absence of PTHrP (e.g. regulation of expression of osteocalcin, osteopontin, interstitial collagenase within the growth plate). On the other hand, PTHrP acts both locally within the growth plate to direct endochondral bone development and outside of bone to affect skeletal development and mineralization by contributing to the regulation of blood calcium and placental calcium transfer. How fetal cells containing the PTH1 receptor selectively respond to one ligand and not the other remains to be elucidated.
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Acknowledgments |
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Footnotes |
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Abbreviations: ED, Embryonic day; wt, wild-type.
Received May 30, 2001.
Accepted for publication August 6, 2001.
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References |
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1(II) chain of cartilage collagen. J Biol Chem 259:13668–13673This article has been cited by other articles:
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  C. S. Kovacs, M. L. Woodland, N. J. Fudge, and J. K. Friel The vitamin D receptor is not required for fetal mineral homeostasis or for the regulation of placental calcium transfer in mice Am J Physiol Endocrinol Metab, July 1, 2005; 289(1): E133 - E144. [Abstract] [Full Text] [PDF]
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 M. E. Wlodek, K. T. Westcott, R. O'Dowd, A. Serruto, L. Wassef, K. M. Moritz, and J. M. Moseley Uteroplacental restriction in the rat impairs fetal growth in association with alterations in placental growth factors including PTHrP Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1620 - R1627. [Abstract] [Full Text] [PDF]
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K. R. McDonald, N. J. Fudge, J. P. Woodrow, J. K. Friel, A. O. Hoff, R. F. Gagel, and C. S. Kovacs Ablation of calcitonin/calcitonin gene-related peptide-{alpha} impairs fetal magnesium but not calcium homeostasis Am J Physiol Endocrinol Metab, August 1, 2004; 287(2): E218 - E226. [Abstract] [Full Text] [PDF]
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C. S. Kovacs, L. L. Chafe, M. L. Woodland, K. R. McDonald, N. J. Fudge, and P. J. Wookey Calcitropic gene expression suggests a role for the intraplacental yolk sac in maternal-fetal calcium exchange Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E721 - E732. [Abstract] [Full Text] [PDF]
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