Biochemical, Genetic, and Molecular Interactions in Development - part 8 pps

Fetal Mineral Homeostasis 297 had a greater impact on the fetal blood calcium than lack of PTHrP; the combined loss of both PTH and PTHrP resulted in the lowest blood calcium level, a level that is equal to that observed in fetal mice lacking the PTH/PTHrP receptor (Pthr1-null fetuses) in the same genetic background. Fetal parathyroids also are required for normal regulation of serum magnesium and phosphate concentrations, as observed in thyroparathyroidectomized fetal lambs that had a reduced serum mag- nesium concentration (26) and an increase in the serum phosphate level (27). Similarly, absence of PTH and parathyroids in Hoxa3-null fetuses also causes hypomagnesemia and hyperphosphatemia (4). Absence of PTHrP alone causes hyperphosphatemia, but the serum magnesium concentration is unaltered (unpublished data; ref. 4). Although a Pth-null model has now been published, the effects of lack of PTH on fetal calcium, phosphate or magnesium concentrations has not been reported (28). As discussed (see section titled Fetal Regulation), there is consistent evidence that PTHrP regu- lates placental calcium transfer, but conflicting evidence between sheep and mouse models that para- thyroids are required for this process or that parathyroids make PTHrP. A detailed examination of normal fetal rat parathyroids found no detectable PTHrP mRNA by in situ hybridization or rt-PCR, and no detectable PTHrP by immunohistochemistry (29). The fetal parathyroids are required for normal accretion of mineral by the fetal skeleton, as discussed in section entitled Fetal Skeleton. CALCIUM-SENSING RECEPTOR Postnatally, the CaSR sets the ionized calcium concentration in the peripheral circulation through its actions to regulate PTH. Homozygous ablation of the CaSR results in severe hyperparathyroidism and hypercalcemia (30), analogous to the human condition of neonatal severe primary hyperparathy- roidism. However, in fetal life, the role of the CaSR is less clearly established. The normal elevation of the fetal serum calcium above the maternal calcium concentration is dependent on PTHrP but not the CaSR; instead, the CaSR is likely suppressing PTH in response to the elevated fetal serum cal- cium concentration (Fig. 1A). In the absence of PTHrP (Pthrp-null fetuses), the fetal serum calcium falls to the normal adult level and the serum PTH is increased, likely indicating the responsiveness of the CaSR to this situation (Fig. 1B). The serum calcium increases above the normal fetal level in response to ablation of the CaSR in fetal life, and the serum PTH increases in a stepwise fashion from wt to Casr +/< to Casr null (8). However, the serum calcium of Casr-null fetuses is no higher than that Fig. 1. Fetal blood calcium regulation. A, Normal high fetal calcium level, which is dependent on PTHrP, activates the parathyroid CaSR, and PTH is suppressed. B, In the absence of PTHrP, the fetal calcium level falls to a level that is now set by the parathyroid CaSR; PTH is stimulated to maintain the ionized calcium at the normal adult level (= maternal). Reproduced with permission from ref. 1. 298 Kovacs of the heterozygous (Casr +/< ) siblings (8), indicating that (unlike in postnatal life) some aspect of the intrauterine environment prevents Casr-null fetuses from achieving a higher blood calcium level. The CaSR is clearly dependent on PTH but not PTHrP in order to contribute to the regulation of the fetal blood calcium level. Loss of PTHrP does not impair the effect of ablation of the CaSR to increase the fetal calcium level (Casr/Pthrp double mutant; ref. 8). However, if the effect of PTH is blocked by simultaneous deletion of the PTH/PTHrP receptor (Casr/Pthr1 double mutant), then ablation of the CaSR does not affect the fetal blood calcium level (8). Similarly, in the absence of PTH (Hoxa-null), ablation of the CaSR has no effect on the fetal blood calcium (Casr/Hoxa3 double mutant; ref. 4). Ablation of the CaSR has also been noted to decrease the rate of transfer of calcium across the pla- centa (8), and we have noted that the CaSR is expressed in murine placenta (31). The reduction in placental calcium transfer may be a consequence of the loss of calcium sensing capability within the placenta; alternatively, it may be that downregulation of calcium transfer is in response to the ele- vated serum calcium concentration or the elevated PTH concentrations that occur in these null mice. In addition to raising the blood calcium and increasing PTH secretion, ablation of the CaSR would be expected to decrease renal calcium clearance as it does in the adult (30). However, Casr +/< and Casr- null fetal mice have increased amniotic fluid calcium levels, suggesting that renal calcium excretion is increased in proportion to the raised serum calcium concentration (8). The discrepancy between adult and fetal effects of CaSR ablation on renal calcium handling may be explained by the observa- tion that the kidneys express very low levels of CaSR mRNA until the first postnatal day (32). THYMUS PTH is not solely produced in the parathyroids; Gcm2-null mice lack the two parathyroids that are normally present in mice but have parathyroid tissue in the thymus that produces relatively normal amounts of PTH (33). In contrast, Hoxa3-null mice lack parathyroids and thymus and have completely absent PTH (4). Whether thymic PTH normally contributes to fetal calcium metabolism has not been determined. Rats and mice have two parathyroid glands, in contrast to humans, who have four (and occasionally more than four). Whether thymic parathyroid tissue in mice is the evolutionary equiva- lent of the lower parathyroids of humans has not been determined. FETAL KIDNEYS AND AMNIOTIC FLUID Fetal kidneys may partly regulate calcium homeostasis by adjusting the relative reabsorption and excretion of calcium, magnesium, and phosphate by the renal tubules in response to the filtered load and other factors, such as PTHrP and/or PTH (1). The fetal kidneys may also participate by synthe- sizing 1,25-D, but because absence of VDR in fetal mice does not impair fetal calcium homeostasis or placental calcium transfer (7), it appears likely that renal production of 1,25-D is relatively unim- portant for fetal calcium homeostasis. Renal calcium handling in fetal life may be less important as compared with the adult for the reg- ulation of calcium homeostasis because calcium excreted by the kidneys is not permanently lost to the fetus. Fetal urine is the major source of fluid and solute in the amniotic fluid, and fetal swallowing of amniotic fluid is a pathway by which excreted calcium can be made available again to the fetus. FETAL SKELETON The skeleton must undergo substantial growth and be sufficiently mineralized by the end of ges- tation to support the organism, but as in the adult, the fetal skeleton participates in the regulation of mineral homeostasis. Calcium accreted by the fetal skeleton may be subsequently resorbed to help maintain the concentration of calcium in the blood, as indicated by several lines of evidence. Mater- nal hypocalcemia caused by thyroparathyroidectomy or calcitonin infusion increases the basal level of bone resorption in subsequently cultured fetal rat bones (34,35). These effects were blocked by Fetal Mineral Homeostasis 299 previous fetal decapitation, which is thought to mimic the effects of thyroparathyroidectomy (34,35); thus, fetal hyperparathyroidism mobilized calcium from the skeleton. Further, in response to mater- nal hypocalcemia, fetal rat parathyroid glands enlarge (36,37), and fetal femur length and mineral ash content are reduced (38). Several recent observations in genetically engineered mice also support a role for the skeleton in fetal calcium homeostasis. The ionized calcium of PTH/PTHrP receptor- ablated fetal mice (Pthr1-null) is lower than that of Pthrp-null fetal mice despite the fact that placen- tal calcium transport is supranormal in Pthr1-null fetuses and subnormal in Pthrp-null fetuses (3). Lack of bone responsiveness to the amino-terminal portion of PTH and PTHrP may well, therefore, contribute to the hypocalcemia in mice without PTH/PTHrP receptors. Placement of a constitutively active PTH/PTHrP receptor into the growth plates of Pthrp-null fetuses not only reverses the chondro- dysplasia (39) but results in a higher fetal blood calcium level (unpublished data). Casr-null fetuses have a higher ionized calcium than normal, and this is maintained at least in part through increased PTH-stimulated bone resorption (8). As a consequence of this increased resorption, the skeletal cal- cium and magnesium content of Casr-null skeletons is significantly depleted as compared to their siblings (unpublished observations; ref. 8). Functioning fetal parathyroid glands are needed for normal skeletal mineral accretion because thy- roparathyroidectomy in fetal lambs caused decreased skeletal calcium content and rachitic changes (40,41). These effects could be partly reversed or prevented by fetal calcium and phosphate infusions; thus, much of the effect of fetal parathyroidectomy was caused by a decrease in blood levels of cal- cium and phosphate (41). Recent examination of the skeletons of the aparathyroid Hoxa3-null fetuses are consistent with these observations in fetal lambs because, despite a normal rate of placental cal- cium transfer, Hoxa3-null fetuses have skeletons that have accreted less calcium and magnesium by the end of gestation (5). Further comparative study of Pthrp-null, Pthr1-null, and Hoxa3-null fetuses has clarified the rela- tive role of PTH and PTHrP in regulation of the development and mineralization of the fetal skeleton. PTHrP produced locally in the growth plate directs the development of the cartilaginous scaffold that is later broken down and transformed into endochondral bone (42), whereas PTH controls the miner- alization of bone through its contribution to maintaining the fetal blood calcium and magnesium (5). In the absence of PTHrP, a severe chondrodysplasia results (18), but the fetal skeleton is fully miner- alized (5). In the absence of parathyroids and PTH (Hoxa3-null), endochondral bone forms normally but is significantly undermineralized (5). Because the blood calcium and magnesium were also sig- nificantly reduced in Hoxa3-null fetuses, the effect of lack of PTH on bone may have been through its effect on maintaining the blood calcium and magnesium. That is, by impairing the amount of min- eral presented to the skeletal surface and to osteoblasts, lack of PTH thereby impaired mineral accre- tion by the skeleton. When both PTH and PTHrP are deleted (Hoxa3/Pthrp double-mutants), the typical Pthrp-null chondrodysplasia results but the skeleton is smaller and contains less mineral (5). Simi- larly, in the absence of the PTH/PTHrP receptor, Pthr1-null skeletons are significantly undermineral- ized (5). Therefore, functioning fetal parathyroids are required for normal mineralization of the skeleton; the specific contribution may be through PTH alone. Whether that contribution is through direct actions of PTH on osteoblasts, or indirect through the actions of PTH to maintain the fetal blood calcium, remains to be clarified. Apart from undermineralization of the skeleton, the lengths of the long bones and the growth plates of the Hoxa3-null were normal at both the gross and microscopic level, and the expression of several osteoblast and osteoclast specific genes was unaltered by loss of parathyroids and PTH (5). 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 Hoxa3-null bones. However, it is clear that the PTH1 receptor influences osteoblast function in the fetal growth plate because Pthr1-null growth plates show a defect in osteoblast function and reduced 300 Kovacs expression of osteopontin, osteocalcin, and interstitial collagenase (43,44). Because PTHrP is pro- duced locally in the growth plate and periosteum it is likely the ligand that normally acts on the PTH1 receptor to regulate these genes. However, because the expression of osteopontin, osteocalcin, and interstitial collagenase is not reduced in the Pthrp-null fetus (43) and there is no evidence of impaired osteoblast function (44), PTH may be able to penetrate the relatively avascular growth plate and com- pensate for the absence of PTHrP. The elevated PTH levels observed in the Pthrp-null fetus are com- patible with this observation (5). Therefore, osteopontin, osteocalcin, and interstitial collagenase may be downregulated in the Pthr1-null fetus because neither PTH nor PTHrP can act in the absence of the PTH1 receptor; these genes are not downregulated by absence of PTH or PTHrP alone. Because only Pthr1-null shows evidence of impaired osteoblast function (44) but both the Hoxa3 null and the Pthr1 null show a similar degree of reduced mineralization (5), the undermineralization of both null pheno- types may be the result of the reduced availability of mineral presented to the osteoblast surface (i.e., the reduced blood calcium and magnesium level in both phenotypes); the availability of mineral is dependent on the action of PTH. The recently reported Pth-null mice also have undermineralized skeletons, but they differ from the phenotype of Hoxa3-null mice in that the long bones of the Pth-null mice are modestly shortened, and there is evidence of reduced osteoblast number and function in studies that were not been per- formed on Hoxa3-null mice (28). The Pth-null and Hoxa3-null models will need to be compared within the same genetic background to be certain which aspects of the respective phenotypes are caused by the loss of PTH and which might be caused by other confounding effects (e.g., aparathyroid and athy- mic in Hoxa3-null mice, marked parathyroid hyperplasia in Pth-null mice, lower blood calcium in C57BL6 background of studied Pth-null mice vs higher blood calcium in Black Swiss background of studied Hoxa3-null mice, etc). In summary, normal mineralization of the fetal skeleton requires intact fetal parathyroid glands and adequate delivery of calcium to the fetal circulation. Although both PTH and PTHrP are involved, PTH plays the more critical role in ensuring full mineralization of the skeleton before term. MATERNAL SKELETON The maternal skeleton may accrete mineral early in gestation in preparation for the peak fetal demand later in pregnancy, such that the maternal skeleton contributes to the mineral ultimately accreted by the fetal skeleton (reviewed in detail in refs. 2 and 45). The contribution during pregnancy is much more modest than the 5–10% decline in bone density that occurs during lactation in humans and the 30% or greater decrease in maternal skeletal mineral content during lactation in rodents (2,45). Experi- mental calcitonin deficiency induced by thyroidectomy worsened the maternal calcium losses (reviewed in refs. 1 and 2), a finding that prompted the hypothesis that calcitonin normally protects the maternal skeleton from excessive resorption during pregnancy and lactation. The decline in bone mineral con- tent that occurs during pregnancy and (especially) lactation is normally reversed after weaning. FETAL RESPONSE TO MATERNAL HYPERPARATHYROIDISM In humans, maternal primary hyperparathyroidism has been associated in the literature with adverse fetal outcomes, including spontaneous abortion, stillbirth, and tetany (2). These adverse fetal outcomes are thought to result from suppression of the fetal parathyroid glands; because PTH cannot cross the placenta, the fetal parathyroid suppression may result from increased calcium flux across the placenta to the fetus, facilitated by the maternal hypercalcemia. Similar suppression of the fetal parathyroids occurs when the mother has hypercalcemia because of familial hypocalciuric hypercalcemia (2). Chronic elevation of the maternal serum calcium in Casr +/< mice (the equivalent of familial hypo- calciuric hypercalcemia in humans) results in suppression of the fetal PTH level as compared with fetuses obtained from wild-type sibling mothers (8), but fetal outcome is not notably affected by this. Fetal Mineral Homeostasis 301 FETAL RESPONSE TO MATERNAL HYPOPARATHYROIDISM Maternal hypoparathyroidism in human pregnancy has been associated with the development of intrauterine, fetal hyperparathyroidism. This condition is characterized by fetal parathyroid gland hyperplasia, generalized skeletal demineralization, subperiosteal bone resorption, bowing of the long bones, osteitis fibrosa cystica, rib and limb fractures, low birth weight, spontaneous abortion, still- birth, and neonatal death (2). Similar skeletal findings have been reported in the fetuses and neonates of women with pseudohypoparathyroidism, renal tubular acidosis, and chronic renal failure (2). These changes in human skeletons differ from what has been found in animal models of maternal hypocal- cemia (discussed previously), in which the fetal skeleton and the blood calcium is generally normal. INTEGRATED FETAL CALCIUM HOMEOSTASIS The evidence discussed in the preceding sections suggests the following summary models. Calcium Sources The main flux of calcium (and other minerals) is across the placenta and into fetal bone, but calcium is also made available to the fetal circulation through several routes (Fig. 2). Some calcium filtered by the kidneys is reabsorbed into the circulation; calcium that is excreted by the kidneys into the urine and amniotic fluid may be swallowed and absorbed by the intestine; calcium is also resorbed from the developing skeleton to maintain the circulating calcium concentration. Some calcium also returns to the maternal circulation (backflux). The maternal skeleton is a critical source of mineral (in addition to maternal dietary intake), and the maternal skeleton is compromised in maternal dietary deficiency states in order to provide to the fetus. Fig. 2. Calcium sources in fetal life. Reproduced with permission from ref. 1. 302 Kovacs Blood Calcium Regulation The fetal blood calcium is set at a level higher than maternal through the actions of PTHrP and PTH acting in concert (among other potential factors; Fig. 3). Although the parathyroid CaSR appears to respond appropriately to this increased level of calcium by suppressing PTH, the low level of PTH is critically required for maintaining a normal blood calcium and normal mineral accretion by the skeleton. 1,25-D synthesis and secretion are, in turn, suppressed due to the effects of low PTH, and high blood calcium and phosphate. The parathyroids may play a central role by producing PTH and PTHrP, or may produce PTH alone whereas PTHrP is produced by the placenta and other fetal tissues. PTH and PTHrP, both present in the fetal circulation, independently and additively regulate the fetal blood calcium, with PTH having the greater effect. Neither hormone can make up for absence of the other: if one is missing, the blood calcium is reduced, and if both are missing, the blood calcium is reduced even further. How the PTH/PTHrP (PTH1) receptor can mediate the actions of these two ligands in the circulation, simultaneously and independently, is not clear. The contribution of PTHrP to the fetal blood calcium may not be through the PTH1 receptor at all, but perhaps only through the actions of mid-molecular PTHrP to regulate placental transfer of calcium (a process which has been shown to be independent of the PTH1 receptor). Thus, PTH may contribute to the blood calcium through actions on the PTH1 receptor in classic target tissues (kidney, bone), whereas PTHrP might contribute through placental calcium transfer and actions on other (non-PTH) receptors. The normal elevation of the fetal blood calcium above the maternal calcium concentration was historically considered as the first evidence that placental calcium transfer was largely an active pro- Fig. 3. Fetal blood calcium regulation. PTH has a more dominant effect on fetal blood calcium regulation than PTHrP, with blood calcium represented schematically as a thermometer (light gray = contribution of PTH; dark gray = contribution of PTHrP). In the absence of PTHrP, the blood calcium falls to the maternal level. In the absence of PTH (Hoxa3-null that has absent PTH but normal circulating PTHrP levels), the blood calcium falls well below the maternal calcium concentration. In the absence of both PTHrP and PTH (Hoxa3/Pthrp double mutant) the blood calcium falls even further than in the absence of PTH alone. Reproduced with permission from ref. 1. Fetal Mineral Homeostasis 303 cess. However, 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 mice, but both null phenotypes have significantly reduced blood calcium levels (3,4). Also, Casr-null fetuses have reduced placental calcium transfer but markedly increased blood calcium levels (8). Placental Calcium Transfer Placental calcium transfer is regulated by PTHrP but not by PTH (Fig. 4). Although the exact tissue source(s) of PTHrP that are relevant for placental calcium transfer remain uncertain, the pla- centa is one proven source of PTHrP that is likely involved in calcium transfer. Whether the parathy- roids produce PTHrP or not is uncertain; in contrast to fetal lambs, experiments in Hoxa3-null mice indicate that absence of parathyroids does not impair placental calcium transfer(4). Skeletal Mineralization PTH and PTHrP have separate roles with respect to skeletal development and mineralization (Fig. 5). 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, and possibly by direct actions on osteoblasts within the bone matrix. 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; ref. 5). In contrast, PTHrP acts both locally within the growth plate to direct endochondral bone development, and outside of bone to affect skeletal devel- opment and mineralization by contributing to the regulation of the blood calcium and placental cal- cium transfer. PTH has the more critical role in maintaining skeletal mineral accretion as compared to PTHrP. Fig. 4. Placental calcium transfer is regulated by PTHrP but not by PTH. Whether the parathyroids produce PTHrP or not is uncertain; experiments in Hoxa3-null mice indicate that absence of parathyroids does not impair placental calcium transfer. Reproduced with permission from ref. 1. 304 Kovacs The rate of placental calcium transfer has been historically considered to be the rate-limiting step for skeletal mineral accretion. However, it is now possible to conclude that the rate of placental calcium transfer is not the rate limiting step for skeletal mineralization since the accretion of mineral was reduced in the presence of normal placental calcium transfer (Hoxa3-null) and increased placen- tal calcium transfer (Pthr1-null mice; ref. 5). Furthermore, Pthrp-null fetuses showed normal skel- etal mineral content in the presence of reduced placental calcium transfer and a modestly reduced blood calcium (5). 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 the Pthrp- null—that is, the normal adult level of blood calcium—is sufficient to allow normal skeletal accre- tion of mineral, whereas lower levels of blood calcium (Hoxa3-null, Pthr1-null, and Hoxa3/Pthrp double-mutant mice) impair the rate of mineral accretion. ACKNOWLEDGMENTS Supported by a Scholarship and Operating Grants from the Canadian Institutes for Health Research (formerly Medical Research Council of Canada), in addition to support from Memorial University of Newfoundland. I gratefully acknowledge the support and advice of Dr. Henry M. Kronenberg, my collaborators (Drs. Marie Demay, James Friel, Robert Gagel, Andrew Karaplis, Gerard Karsenty, Nancy Manley, Jack Martin, Jane Moseley, Ernestina Schipani, and Peter Wookey), my research assistant Neva Fudge, and my students. REFERENCES 1. Kovacs, C. S. (2003) Fetal mineral homeostasis, Chapter 11, in Pediatric Bone: Biology and Diseases (Glorieux, F. H., Pettifor, J. M., and Jüppner, H., eds.), Academic Press, San Diego, CA, pp. 271–302. Fig. 5. Schematic model of the relative contribution of PTH and PTHrP to endochondral bone formation and skeletal mineralization. PTHrP is produced within the cartilaginous growth plate and directs the development of this scaffold that will later be broken down and replaced by bone. In the absence of PTHrP (Pthrp-null), a severe chondrodysplasia results but the skeleton is normally mineralized. PTH reaches the skeleton systemically from the parathyroids and directs the accretion of mineral by the developing bone matrix. In the absence of PTH (Hoxa3-null fetus), the bones form normally but are severely undermineralized. Reproduced with permission from ref. 1. Fetal Mineral Homeostasis 305 2. Kovacs, C. S. and Kronenberg, H. M. 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P., and Pols, H A (1992) Bidirectional regulation of the 1,25-dihydroxyvitamin D3 receptor by phorbol ester-activated protein kinase-C in osteoblast-like cells: interaction with adenosine 3',5'-monophosphate-induced up-regulation of the 1,25-dihydroxyvitamin D3 receptor Endocrinology 130, 2259–2266 235 Reinhardt, T A and Horst, R L (1994) Phorbol 12-myristate 13-acetate and 1,25-dihydroxyvitamin D3... I-mfa IP3R M-CSF MMP-9 MMP-13 Na+-Ca2+ exchange NGF Notch 1 and 2 Notch 4 NPR-C ODF OPG OPGL PDGF PTHrP VEGF a IL-1 b Species Reference mRNA mRNA + protein mRNA Aromatase Source Human Human Rat (293) (294) (37) mRNA Human ( 38) Protein mRNA mRNA mRNA Protein Protein mRNA mRNA + protein mRNA + protein Protein mRNA + protein Protein Protein Protein Protein mRNA + protein mRNA Protein mRNA mRNA + protein... Kassem, M (2001) 1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells Eur J Endocrinol 144, 549–557 175 Schmid, C., Schlapfer, I., Gosteli-Peter, M A., Hauri, C., Froesch, E R., and Zapf, J (1996) 1 alpha,25-dihydroxyvitamin D3 increases IGF binding protein-5 expression in cultured osteoblasts FEBS Lett 392,... by TGF- was not followed by an increased induction of both osteocalcin and osteopontin mRNA expression and osteocalcin protein synthesis In contrast, the upregulation of VDR by TGF- is coupled to a strong inhibition of osteocalcin and osteopontin expression (233) This inhibition was the result of a TGF- –induced block of the VDR–retinoid X receptor complex binding to the VDRE in the osteocalcin and osteopontin... VDR and the increase in data on a membrane receptor for 1,25(OH)2D3, a new class of 1,2 5-( OH)2D3 regulatory proteins has been identified: intracellular vitamin 316 van Leeuwen et al Table 1 1,2 5-( OH)2D3 Effects on Growth Factors and Other Osteoblast-Related Molecules Factor 1,2 5-( OH)2D3 effect Cbfa1 (1 h) ( 48 h) EGFR ETRA HCYR61 HLA-DR GM-CSF IL-1 IL-1R I IL-1R II IL-IRa IL-4R IL-6 IL-6R IL-11 IL-11R... synthesis in osteoblast-like cells Prostaglandins Leukot Essent Fatty Acids 51, 27–31 257 Kozawa, O., Tokuda, H., Kaida, T., Matsuno, H., and Uematsu, T (19 98) Effect of vitamin D3 on interleukin-6 synthesis induced by prostaglandins in osteoblasts Prostaglandins Leukot Essent Fatty Acids 58, 119–123 2 58 Menaa, C., Vrtovsnik, F., Friedlander, G., Corvol, M., and Garabedian, M (1995) Insulin-like growth... focussed on 1,2 5-( OH)2D3 regulation of IGF-I in bone cells and cultured bone tissue 1,2 5-( OH)2D3 increased IGF-I levels in human bone cell supernatants (166) and caused a small but not significant increase in the release of IGF-I in the supernatant of rat osteoblast-like cells (167) However, 1,2 5-( OH)2D3 inhibited production of IGF-I in mouse osteoblasts and mouse calvaria (1 68) Vitamin D and Osteoblasts . mRNA and protein, which are shown to be involved in cell differentia- tion and `-glycerophosphate-induced mineralization (70). Also in mice with impaired function of the 25-hydroxyvitamin D-24-hydroxylase. stimulate IGFBP-4 mRNA expression and secretion (1 68, 172). Another study also showed at mRNA and protein level the stimulation of IGFBP-2, -3 , and -4 but not -5 and -6 by 1,2 5- (OH) 2 D 3 in human. Metab. 86 , 2344–23 48. Vitamin D and Osteoblasts 307 307 From: The Skeleton: Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis Edited by: E. J. Massaro and J. M. Rogers