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To determine whether the action of the PTHrP nuclear localization sequence and C terminus is mediated through p27 in modulating dental and mandibular development, compound mutant mice, which are homozygous for both p27 deletion and the PTHrP1–84 knock-in mutation (p27−/−PthrpKI/KI), were generated. Their teeth and mandibular phenotypes were compared with those of p27−/−, PthrpKUK\ and wild-type mice. At 2 weeks of age, the mandibular mineral density, alveolar bone volume, osteoblast numbers, and dental volume, dentin sialoprotein-immunopo-sitive areas in the first molar were increased significantly in p27−/− mice and decreased dramatically in both PthrpKI/KI and p27−/− PthrpKI/KI mice compared with wild-type mice; however, these parameters were partly rescued in p27−/− PthrpKI/KI mice compared with PthrpKI/KI mice. These data demonstrate that the deletion of p27 in PthrpKI/KI mice can partially rescue defects in dental and mandibular development. Furthermore, we found that deletion of p27 in PthrpKI/KI mice partially corrected the dental and mandibular phenotype by modulating cell cyclin-regulating molecules and antioxidant enzymes. This study therefore indicates that the p27 pathway may function downstream in the action of PTHrP nuclear localization sequence to regulate dental and mandibular development. (Endocrinology 157: 1372–1384, 2016)

We examined the role of bone remodeling in the regulation of circulating concentrations of FGF23 using mouse models manifesting differing degrees of coupled and uncoupled bone turnover. Administration of the antiresorptive agent osteoprotegerin produced a profound reduction in bone resorption and formation in male and oophorectomized female mice, accompanied by an increase in serum levels of fibroblast growth factor 23 (FGF23) and a reduction in circulating 1,25-dihydroxyvitamin D [1,25(OH)2D]. In contrast, exogenous PTH(1-34) administration increased bone turnover and reduced circulating FGF23. In 1,25(OH)2D-deficient, 25-hydroxyvitamin D 1α-hydroxylase null mice on a high-calcium diet, endogenous PTH was elevated, bone formation but not resorption was increased, and serum FGF23 was virtually undetectable; on a rescue diet, serum calcium was normalized, PTH levels were reduced, bone formation was reduced, and serum FGF23 levels increased. After PTH treatment of wild-type mice, gene expression of dentin matrix protein 1 (DMP1) in bone was increased, whereas gene expression of FGF23 was reduced. In vitro studies in the osteoblastic cell line UMR-106 showed that externally added DMP1 could inhibit FGF23 gene expression and production stimulated by 1,25(OH)2D3. The results show that osteoblastic bone formation is a potent modulator of FGF23 production and release into the circulation, suggest that the biological consequences on mineral homeostasis of circulating FGF23 may also be dependent on the prevailing rate of bone turnover, and provide evidence that DMP1 may be a direct negative regulator of FGF23 production in osteoblastic cells.

To determine whether PTH and calcium (Ca) interact in neonatal bone formation, female lactating mice either heterozygous (PTH+/−) or homozygous (PTH−/−) for targeted deletion of the pth gene were fed either a normal (1% Ca, 0.6% phosphate) or high-Ca diet (2% Ca and 0.4% phosphate). Dietary effects on milk Ca content and Ca-regulating hormones were determined in dams, and the effects of milk content were assessed on bone turnover in 3-wk-old pups. On the normal diet, milk Ca and 1,25-dihydroxyvitamin D3 levels were lower, but milk PTH-related protein levels were higher in the PTH−/− dams compared with the PTH+/− dams. On the high-Ca diet, milk Ca levels were higher, but milk 1,25-dihydroxyvitamin D3 and PTH-related protein levels were lower in both PTH+/− and PTH−/− dams. In pups fed by PTH−/− dams compared with pups fed by PTH+/− dams on normal diets, bone mineral density, trabecular bone volume relative to tissue volume, and the number of osteoblasts were reduced in both PTH+/− (32.5 ± 1.2 vs. 39.6 ± 1.5 mg/cm2, P < 0.05; 23.3 ± 1.6 vs. 29.2 ± 2.8%, P < 0.01; and 94.2 ± 8.2 vs. 123.5 ± 3.5/mm2, P < 0.01, respectively) and PTH−/− (20.4 ± 0.9 vs. 27.0 ± 1.2 mg/mm2, P < 0.05; 16.8 ± 1.9 vs. 19.3 ± 2.1%, P < 0.05; and 48.6 ± 7.9 vs. 90.5 ± 8.6/mm2, P < 0.01, respectively) pups but were lower in the PTH−/− pups compared with the PTH+/− pups. In contrast, in pups fed by either PTH+/− or PTH−/− dams on the high-Ca diet, bone mineral density, bone volume/tissue volume, and osteoblast numbers were significantly higher, in both PTH+/− (50.5 ± 1.7 vs. 58.7 ± 2.0 mg/mm2, P < 0.05; 37.9 ± 5.2 vs. 46.1 ± 5.1, P < 0.05; and 120.5 ± 9.2 vs. 159.3 ± 14.7/mm2, P < 0.01, respectively) and PTH−/− (33.0 ± 1.2 vs. 47.5 ± 2.2 mg/mm2, P < 0.001; 23.8 ± 3.1 vs. 35.9 ± 2.0, P < 0.05; and 78.7 ± 10.1 vs. 99.8 ± 13.6/mm2, P < 0.05, respectively), and were highest in the PTH+/− pups fed by the PTH+/− dams on the high-Ca diet. These results indicate that PTH can modulate Ca content of milk, and that PTH and Ca can each exert cooperative roles on osteoblastic bone formation in the neonate. PTH can modulate calcium content of maternal milk, and PTH and calcium exert cooperative roles on osteoblastic bone formation in the neonate.

Although increased circulating levels of PTH with mild hypocalcemia has been reported in Hyp mice, hyperparathyroidism in X-linked hypophosphatemic rickets is postulated to arise from the standard use of phosphate salts, which induce chronic stimulation of PTH secretion. In this study, we sought to examine the role of PTH in the metabolic derangements associated with Hyp by generating hemizygous hypophosphatemic (Hyp/Y) mice homozygous for the Pth-null allele (Pth(-/-);Hyp/Y). Early postnatal lethality was observed in the Pth(-/-);Hyp/Y mice. Within the first 6 h, postpartum serum phosphorus increased to levels comparable to those in the Pth(-/-) mice, whereas in Hyp mice, it decreased during the first 48 h after birth. Serum calcium concentration started low after birth and remained reduced in both Pth(-/-);Hyp/Y and Pth(-/-) mice although more profoundly so in the former group, whereas in Hyp/Y mice, the levels were initially lower than but reached wild-type levels by 24 h. Circulating PTH levels in Hyp/Y mice were higher than wild-type levels throughout the first 48 h after birth and continued to be so well into adulthood. Twice-daily administration of PTH 1-34 to Pth(-/-);Hyp/Y newborn mice increased serum calcium levels and prevented their early demise. The findings here indicate that the cause of death in the Pth(-/-);Hyp/Y mice is severe hypocalcemia. A potential role for fibroblast growth factor 23 in promoting secondary hyperparathyroidism by suppressing renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (Cyp27b1) activity while increasing that of renal 25-hydroxyvitamin D(3) 24-hydroxylase (Cyp24) is proposed. Hyperparathyroidism, therefore, is an integral component in the pathophysiology of Hyp, and likely X-linked hypophosphatemic rickets and serves to prevent severe hypocalcemia in mice and perhaps in patients afflicted with the disorder.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and PTH each modulate calcium and skeletal homeostasis. To identify 1,25(OH)(2)D(3)-mediated skeletal and mineral ion actions independent of PTH, double-knockout mice, which are homozygous for both the 1alpha-hydroxylase and PTH null alleles, were treated with 1,25(OH)(2)D(3), sc, from d 4 to 14 and compared with vehicle-treated animals. Serum calcium rose in 1,25(OH)(2)D(3)-treated double-knockout mice, and messenger RNA and protein levels of the renal calcium transporters TRPV5, calbindin-D(28K), calbindin-D(9K), and Na(+)/Ca(2+) exchanger 1 were up-regulated. Parameters of endochondral bone formation, including long bone length, epiphyseal volume, chondrocyte proliferation and differentiation, and cartilage matrix mineralization, were all increased by 1,25(OH)(2)D(3), Exogenous 1,25(OH)(2)D(3) also increased both trabecular and cortical bone; augmented both osteoblast number and type I collagen deposition in bone matrix; and up-regulated expression levels of the osteoblastic genes alkaline phosphatase, type I collagen, and osteocalcin. Furthermore, in 1,25(OH)(2)D(3)-treated double mutants, osteoclastic bone resorption appeared to decline. The results indicate that administered 1,25(OH)(2)D(3) used intestinal and renal but not skeletal mechanisms to elevate serum calcium and that this sterol can promote endochondral and appositional bone increases independent of endogenous PTH.

Fibroblast growth factor 23 (FGF23) is a recently characterized protein likely involved in the regulation of serum phosphate homeostasis. Increased circulating levels of FGF23 have been reported in patients with renal phosphate-wasting disorders, but it is unclear whether FGF23 is the direct mediator responsible for the decreased phosphate transport at the proximal renal tubules and the altered vitamin D metabolism associated with these states. To examine this question, we generated transgenic mice expressing and secreting from the liver human FGF23 (R176Q), a mutant form that fails to be degraded by furin proteases. At 1 and 2 months of age, mice carrying the transgene recapitulated the biochemical (decreased urinary phosphate reabsorption, hypophosphatemia, low serum 1,25-dihydroxyvitamin D3) and skeletal (rickets and osteomalacia) alterations associated with these disorders. Unexpectantly, marked changes in parameters of calcium homeostasis were also observed, consistent with secondary hyperparathyroidism. Moreover, in the kidney the anticipated alterations in the expression of hydroxylases associated with vitamin D metabolism were not observed despite the profound hypophosphatemia and increased circulating levels of PTH, both major physiological stimuli for 1,25-dihydroxyvitamin D3 production. Our findings strongly support the novel concept that high circulating levels of FGF23 are associated with profound disturbances in the regulation of phosphate and vitamin D metabolism as well as calcium homeostasis and that elevated PTH levels likely also contribute to the renal phosphate wasting associated with these disorders.

We examined the effect of PTH-related peptide (PTHrP) on modulating adipogenesis and osteoblastogenesis in the pluripotent mesenchymal cell line C3H10T(1/2). These cells express the type 1 PTH/PTHrP receptor, thereby allowing PTHrP to inhibit bone morphogenetic protein 2 (BMP2) from enhancing gene expression of peroxisome proliferator-activated receptor gamma and the adipocyte-specific protein aP2 and from augmenting the accumulation of lipid. In the presence of BMP2, PTHrP or a protein kinase C (PKC) stimulator (phorbol ester) increased the expression of indexes of the osteoblast phenotype, including alkaline phosphatase, type I collagen, and osteocalcin, whereas a PKC inhibitor (chelerythrin chloride) inhibited PTHrP action. PTHrP and a phorbol ester increased gene expression of the BMP IA receptor, and both enhanced BMP2-dependent increases in promoter activity of the signaling molecule SMAD6. Overexpression of the BMP IA receptor facilitated the capacity of BMP2 to increase osteoblastogenesis in the absence of PTHrP and a dominant negative BMP IA receptor variant inhibited this effect of BMP2. These results demonstrate that PTHrP can direct osteoblastic, rather then adipogenic, commitment of mesenchymal cells, implicate PKC signaling in this activity, and show that PTHrP action involves enhanced gene expression of the BMP IA receptor, which facilitates BMP2 action in enhancing osteoblastogenesis in pluripotent mesenchymal cells.

The PTH/PTH-related peptide (PTHrP) receptor (PTHR) is required for normal skeletal development, and a wide array of physiological responses mediated by PTH and PTHrP. We have previously identified three promoters, P1-P3, which control human PTHR gene transcription. P2 and P3 are (G+C)-rich, function in a number of tissues, lie within the same CpG island, and display many hallmarks of housekeeping promoters. However, they are differentially regulated during development as P2, but not P3, functions in fetal tissues. Here, we have used both stably and transiently transfected human osteoblast-like cells to delineate regions of P2 and P3 required for promoter activity. Deletion analyses performed in stably transfected cells indicated that sequences extending from -91 to -12 relative to the transcription start site were required for function of the P2 promoter. No negative regulatory elements were detected in P2. In contrast, deletion of an A-rich region of P3 extending from -147 to -115 was required for optimal basal activity, suggesting that this sequence acts as a repressor of P3. Strikingly, however, whereas the A-rich region also functioned as a negative element when inserted upstream of the (G+C)-rich P2 promoter, it enhanced expression from the thymidine kinase promoter, suggesting that its function depends on other transcription factors bound to promoter sequences. Fine deletion of P3 sequences proximal to -115 implicated Spl motifs and downstream initiation sites in P3 function. These studies indicate that function of P2 and P3 is controlled by ubiquitously expressed transcription factors and raise the possibility that P3 activity is repressed during fetal development.

The kidney is the major site of expression of the PTH/PTH-related peptide receptor (PTHR) gene. Previously we have shown that the PTHR gene is expressed from two promoters in kidney, an upstream kidney-specific promoter (P1) and a downstream promoter (P2) that is active in a wide variety of tissues. Here, we have used immunohistochemical and transcript-specific in situ hybridization techniques to map the expression of the PTHR gene and protein and to determine the distribution of P1- and P2-driven messenger RNAs in renal tissue. Immunohistochemical and immunoelectron microscopic analysis showed that PTHR protein is expressed on both basolateral and luminal membranes of proximal tubular epithelial cells, strongly suggesting a bipolar mode of action of PTH. Receptor protein also was detected on the surface of glomerular podocytes. Strikingly, immunoelectron microscopic analysis showed that endothelial cells of the peritubular vasculature, but not the glomerular vasculature, contain high levels of PTHR protein. We found that both P1 and P2 are expressed at moderate levels in both cortical and medullary epithelial cells of nephrons, correlating well with the immunohistochemical localization of PTHR protein. However, although abundant transcripts were detected in peritubular endothelial cells with P1-specific and coding sequence probes, P2-specific expression was not observed in these cells. These results provide evidence that the physiological effects of PTH- and/or PTH-related peptide on renal tubular function may be mediated not only through direct effects on epithelial cells but also indirectly through endothelial cell-based signaling. In addition to expression in vascular endothelial cells, high levels of P1-specific, but not P2-specific, PTHR messenger RNA were detected in vascular smooth muscle. Taken together, these experiments provide evidence for strong PTHR gene expression in renal vascular tissues. Moreover, given that previous studies have shown that P2, but not P1, is active in other tissues with an abundant vasculature, our results suggest that regulation of PTHR gene expression in renal vascular tissue is distinct from that of other organs.

In previous work we showed that the chondrodysplastic phenotype of mice homozygous for a null mutation of the PTH-related peptide (PTHrP) gene was due in part to reduced proliferation and aberrant differentiation of growth plate chondrocytes. In the present study we have extended those observations by examining chondrocytes for evidence of PTH/PTHrP receptor expression, proliferation, and programmed cell death. Receptor messenger RNA and protein were expressed in chondrocytes in the resting and proliferative zones of both wild-type and mutant mice. In normal animals, expression was abundant in the area of transition between proliferative and hypertrophic chondrocytes and absent from cells in the lower hypertrophic region. On the other hand, the hypertrophic zone in mutant mice contained nonhypertrophic chondrocytes, which exhibited characteristics of proliferating cells, including PTH/PTHrP receptor expression, [3H]thymidine incorporation, and expression of proliferating cell nuclear antigen. In contrast to the situation in normal animals, some cells adjacent to the zone of vascular invasion in mutant growth plates showed biochemical and morphological evidence of programmed cell death. In addition to these alterations in the maturation of growth plate chondrocytes, homozygous mutants demonstrated signs of aberrant differentiation of periosteal precursor cells. In some specimens, clusters of chondrocytes embedded in a cartilaginous matrix were observed between the layers of periosteal osteoblasts and the bony collar in the sterna and tibiae of mice homozygous for a null mutation of the PTHrP gene. Taken together, these observations indicate that PTHrP plays a pivotal role in the orderly progression of chondrocytes through stages of proliferation, differentiation, and programmed cell death in the epiphyseal growth plate and may also facilitate the commitment of precursors to cells of the chondrocytic or osteoblastic lineages.

PTH-related peptide (PTHrP) has been shown to be the major mediator of hypercalcemia of malignancy, but may also exert effects on cell growth and differentiation. The Leydig cell tumor H-500, when implanted in Fischer rats, produces abundant PTHrP and eventually causes the death of the host animal. In the present study we have used antisense RNA technology to block the effects of PTHrP in H-500 Leydig tumor cells in vivo. The full-length rat PTHrP complementary DNA encoding amino acid -36-->141 was subcloned as an EcoRI-BglII insert in the antisense orientation into the mammalian expression vector pRc/CMV to produce the plasmid pRc-PAS. This plasmid was then stably transfected into the H-500 Leydig tumor cells with a Lipofectin reagent. After selection with the neomycin derivative G-418, a stable cell line, H-500-PTHrP-AS, was obtained which showed 80% inhibition of endogenous PTHrP messenger RNA compared to wild-type or vector-only transfected H-500 cells. Conditioned culture medium from these experimental cells showed a marked decrease in PTHrP immunoreactivity and in the ability of the medium to stimulate adenylate cyclase in UMR-106 rat osteosarcoma cells. Furthermore, inhibition of PTHrP production resulted in a significant increase in the doubling time of the H-500 cells. Transfection of the experimental plasmid into Rat-2 fibroblasts, which do not produce PTHrP, had no effect on cell growth. Control and experimental cells were then implanted sc into male Fischer rats. Animals were killed at timed intervals, and their tumor volumes were determined. Experimental animals receiving cells transfected with antisense PTHrP plasmid showed near-normal levels of plasma calcium and decreased expression of tumoral PTHrP messenger RNA. These animals also showed a 30-70% lower tumor volume during the course of the experiment compared to control animals. These studies have demonstrated that PTHrP can play a role as a promoter of tumor growth in vitro and in vivo.

The transplantable rat Leydig cell tumor H-500 is known to cause hypercalcemia in vivo by the release of abundant PTH-related peptide (PTHRP) and to closely reproduce the human syndrome of malignancy-associated hypercalcemia. In the rat only a single messenger RNA species of 1.4 kilobases is expressed which encodes a peptide of 141 amino acid as the sole molecular form. We have examined in cultured rat Leydig tumor cells H-500, the capacity of multiple factors to regulate PTHRP messenger RNA expression and secretion. Both fetal bovine serum and epidermal growth factor stimulated PTHRP gene expression and secretion into conditioned culture medium. Dexamethasone and 1,25-dihydroxyvitamin D3 produced inhibition of PTHRP gene expression and secretion. Furthermore, in these testicular cells, after 12 h or more of incubation, testosterone produced a dose-dependent (10(-9)-10(-7) M) inhibition of PTHRP production. No significant difference in this inhibitory response was seen between testosterone and its 5 alpha-reduced metabolite dihydrotestosterone whereas 17 beta-estradiol, progesterone, LH, FSH, and PRL were ineffective. An androgen receptor antagonist Win 49596 blocked the androgen-mediated inhibition of PTHRP gene expression and secretion, but not that due to dexamethasone. Epidermal growth factor caused an increase, whereas androgen caused a decrease in PTHRP gene transcription. These studies demonstrated that growth factors, dexamethasone, and 1,25-dihydroxyvitamin D3 are broadly active regulatory agents of PTHRP production which cross species and tissue barriers. Testosterone may be a more selective modulator which can regulate PTHRP in tissues such as Leydig cell neoplasms which express the androgen receptor.

We have identified and characterized receptors for the amino-terminal domains of PTH and PTH-like peptide (PLP) on an immortalized human keratinocyte cell line, RHEK-1. Binding of both PLP-(1-34) and PTH-(1-34) to the RHEK-1 cells was consistent with a two-site model; affinities and capacities for each site were similar for the two peptides. Both peptides also stimulated adenylate cyclase activity with an equal ED50 in this cell line. Pertussis toxin pretreatment enhanced this peptide-mediated enzyme activity, suggesting linkage of the receptor to an inhibitory guanyl nucleotide-binding protein (Gi). Adenylate cyclase activity was diminished by both homologous [PLP-(1-34)] and heterologous [epidermal growth factor (EGF)] effectors. Malignant conversion of the immortalized cells with an activated H-ras oncogene to produce the RHEK-ras cell line was associated with a reduction in binding at both PLP/PTH and EGF receptors as well as a postreceptor defect in PLP/PTH-stimulated adenylate cyclase activity. The defect in enzyme activity appeared to be due in part to a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs), but not to an increase in Gi activity. Activation of the keratinocyte amino-terminal PLP/PTH receptor resulted in a small increase in [3H]thymidine incorporation, which was associated with an increase in cell numbers. This mitogenic effect was enhanced in the presence of EGF and was markedly reduced when cells were cultured in a high extracellular calcium environment. These studies demonstrate that the amino-terminal region of PLP and PTH activates adenylate cyclase-linked receptors, which are associated with mitogenesis, in RHEK-1 cells and suggest that this cell line represents a suitable model in which to examine the actions of PLP in keratinocytes.

Passive immunization using antisera raised against rat PTH-like peptide [PLP-(1-34)] and rat PTH-(1-84) was used to assess and compare the roles played by PLP and PTH in modulating mineral metabolism in hypercalcemic rats bearing the Rice-500 Leydig cell tumor and in normocalcemic control animals. After immunization, plasma calcium in the tumor-bearing animals rapidly normalized and remained within the normal range for several days, plasma phosphate rose, and urinary phosphate and cAMP fell. These changes were associated with increased longevity of the tumor-bearing rats. Reduction of plasma calcium was shown to be a function of early (5 h) neutralization of PLP bioactivity in the kidney, whereas the effect of immunoneutralization of PLP in bone appeared later (24-48 h) and was more prolonged. Immunization against PTH in normocalcemic animals resulted in a hypocalcemic episode of smaller magnitude and shorter duration than that achieved in hypercalcemic animals immunized against PLP and appeared to be unassociated with neutralization of distal tubular effects. Neutralization of proximal tubular and skeletal actions of PTH appeared to occur in a manner analogous to that seen for PLP in tumor-bearing animals, but were of shorter duration. These studies suggest that in normal animals the action of PTH in the skeleton and/or on vitamin D metabolism contributes considerably more than renal transport activity to the maintenance of normocalcemia and that compensatory mechanisms rapidly restore homeostasis. PTH appears to be the major modulator of calcium homeostasis in normal rats, with PLP playing a minor role, if any. In tumor-bearing rats, mechanisms to restore calcium levels to baseline are delayed after PLP immunoneutralization, and PLP appears to be both necessary and sufficient for the hypercalcemic state.

We have used both biochemical and morphological techniques to characterize PTH receptors on the clonal osteosarcoma cell line UMR-106, a widely used model of the osteoblast phenotype. 125I-labeled rat (r) PTH-(1-34) bound to a single class of specific saturable receptors on both whole cells and membranes prepared from UMR-106 cells in a time- and temperature-dependent manner. A decrease in PTH receptor affinity seen in the presence of guanine nucleotides demonstrated that PTH receptors on the UMR-106 cells are coupled to guanyl nucleotide-binding proteins. Although PTH is a potent stimulator of adenylate cyclase in the UMR-106 cells, comparison of PTH-stimulated adenylate cyclase and PTH binding curves indicated the presence of receptors that are not linked to the adenylate cyclase system. Our studies also demonstrated that 125I-labeled rPTH-(1-34) bound to UMR-106 cells is rapidly internalized at 22 C, whereas PTH bound at 4 C remains intact and on the cell surface. Internalization of 125I-labeled rPTH-(1-34) was associated with degradation and release of the hormone at 22 C. Three morphologically distinct cell types were identified in subconfluent cultures of UMR-106 cells. Autoradiographic analysis of 125I-labeled rPTH-(1-34) binding demonstrated differential PTH receptor expression in these cell types. The most abundant PTH binding was observed over a cell type with long cytoplasmic extensions. This cell was reminiscent of the predominant PTH target cell previously identified in the rat metaphysis in vivo, suggesting that the UMR-106 cell line may represent neoplastic transformation of the PTH target cell.

We have examined the mechanisms of homologous and heterologous regulation of PTH receptor binding and receptor-mediated adenylate cyclase activity in the osteosarcoma cell line UMR-106. Pretreatment with PTH resulted in a time- and dose-dependent decrease in PTH-stimulated adenylate cyclase which was maximal after 2 h and at a concentration of 10(-8) M rat (r)PTH-(1-34). PTH pretreatment over the same dose range also diminished receptor binding of 125I-labeled rPTH-(1-34); however, maximal loss of binding required 14 h and was greater than the loss of maximal adenylate cyclase activity. After 24 h pretreatment with rPTH-(1-34), cell surface receptors were decreased from 21,000 sites per cell to 2,700 sites per cell, and these down-regulated PTH receptors could not be detected in either vesicular or cytosolic subcellular fractions. Recovery from such homologous down-regulation appeared to require new receptor synthesis. Heterologous down-regulation of PTH receptors was demonstrated when UMR-106 cells were preincubated with prostaglandin E2 or (Bu)2cAMP. Heterologous desensitization was shown to be the result of a reversible modification of the PTH receptor which decreased binding affinity and decreased PTH-stimulated adenylate cyclase. Postreceptor components were also examined, and PTH but not prostaglandin E2 pretreatment was shown to decrease guanyl nucleotide binding (G) protein-mediated adenylate cyclase stimulation. This decrease in G protein function was associated with a loss of cholera toxin-catalyzed ADP ribosylation and was also detected by immunoblotting. These results indicate that PTH responses in osteoblastic cells are modulated by diverse mechanisms involving modifications both to the receptor and to postreceptor components of adenylate cyclase.

Previous studies have indicated that bisphosphonate pretreatment can inhibit the anabolic actions of PTH. We examined the capacity of two anticatabolic agents with different mechanisms of action, alendronate and osteoprotegerin (OPG), to influence the anabolic activity of PTH. Oophorectomized mice were pretreated for 30 d with alendronate or OPG and then treated for 30 d with the respective anticatabolic alone or the respective anticatabolic plus PTH(1-34). Bones were analyzed by bone mineral density (BMD), microcomputed tomography, histology and histomorphometry, and biochemical bone markers. OPG pretreatment produced a greater inhibition of bone turnover and a greater increase in bone than alendronate. Increases in bone were sustained during subsequent treatment with vehicle or continued administration of the anticatabolic. Pretreatment with each anticatabolic blunted the capacity of PTH to increase BMD and bone volume and continued treatment with each anticatabolic agent also reduced the effectiveness of PTH. Although both anticatabolics decreased the maximal PTH effect, BMD and bone volume increased more when PTH was added than when only anticatabolics were used. These results demonstrate that mechanistically distinct anticatabolics may reduce PTH efficacy, that the characteristics of this inhibition may reflect the different modes of action of the anticatabolics, but that the addition of PTH still provides a skeletal benefit even if the anabolic effect is submaximal.

We evaluated the capacity of estradiol (E2) to regulate PTHrP production, cell growth, tumor growth, and metastasis to the skeleton in breast cancer. In estrogen receptor (ER)-negative human breast cancer cells, MDA-MB-231, and cells transfected with full-length cDNA encoding ER (S-30), E2 caused a marked decrease in cell growth and PTHrP production, effects that were abrogated by anti-E2 tamoxifen. E2 also inhibited PTHrP promoter activity in S-30 cells. For in vivo studies, MDA-MB-231 and S-30 cells were inoculated into the mammary fat pad of female BALB/c nu.nu mice. Animals receiving S-30 cells developed tumors of significantly smaller volume compared with MDA-MB-231 tumor-bearing animals. This change in tumor volume was reversed when S-30 cells were inoculated into ovariectomized (OVX) hosts. Inoculation of MDA-MB-231 cells into the left ventricle resulted in the development of lesions in femora and tibia as determined by x-ray analysis. In contrast, these lesions were significantly smaller in volume and number in animals inoculated with S-30, and this lower incidence was reversed in OVX animals. Bone histological analysis showed that the tumor volume to tissue volume ratio was comparable with that seen by x-ray. Immunohistochemical analysis showed that PTHrP production was inhibited in S-30 group and restored to levels comparable to that seen in MDA-MB-231 tumor-bearing animals when S-30 cells were inoculated in OVX animals. Collectively these studies show that E2 production is inversely correlated with PTHrP production and that the growth-promoting effect of PTHrP has a direct impact on tumor growth at both nonskeletal and skeletal sites.

We investigated the relative contributions of PTH and PTHrP to the skeletal phenotype of mice deficient in PTH (PTH−/−). PTH−/− mice and PTH−/− mice lacking one allele encoding PTHrP (PTH−/−PTHrP+/−) were compared. Both mutants displayed similar biochemical abnormalities of hypoparathyroidism, but skeletal PTHrP mRNA and protein were decreased in PTH−/−PTHrP+/ − mice. PTH−/− mice had increased trabecular bone volume with diminished bone turnover. PTHrP haploinsufficiency reduced trabecular bone of the PTH−/− mice to levels below those in wild-type animals by decreasing osteoprogenitor cell recruitment, enhancing osteoblast apoptosis, and diminishing bone formation. The results show that the increased trabecular bone volume in PTH-deficient mice is due to diminished PTH-induced osteoclastic bone resorption and persistent PTHrP-stimulated osteoblastic bone formation. They also illustrate the changing role of PTHrP during bone development, demonstrate its bone- forming function in the postnatal state, and support its pharmacological potential as an anabolic agent.

We have examined the role of PTH in the postnatal state in a mouse model of PTH deficiency generated by targeting the Pth gene in embryonic stem cells. Mice homozygous for the ablated allele, when maintained on a normal calcium intake, developed hypocalcemia, hyperphosphatemia, and low circulating 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels consistent with primary hypoparathyroidism. Bone turnover was reduced, leading to increased trabecular and cortical bone volume in PTH-deficient mice. When mutant mice were placed on a low-calcium diet, renal 25-hydroxyvitamin D 1 α-hydroxylase expression increased despite the absence of PTH, leading to a rise in circulating 1,25(OH)2D3 levels, marked osteoclastogenesis, and profound bone resorption. These studies demonstrate the dependence of the skeletal phenotype in animals with genetically depleted PTH on the external environment as well as on internal hormonal and ionic circulatory factors. They also show that, although PTH action is the first defense against hypocalcemia, 1,25(OH)2D3 can be mobilized, even in the absence of PTH, to guard against extreme calcium deficiency.

We examined the effect of PTH-related peptide (PTHrP) on modulating adipogenesis and osteoblastogenesis in the pluripotent mesenchymal cell line C3H10T(1/2). These cells express the type 1 PTH/PTHrP receptor, thereby allowing PTHrP to inhibit bone morphogenetic protein 2 (BMP2) from enhancing gene expression of peroxisome proliferator-activated receptor gamma and the adipocyte-specific protein aP2 and from augmenting the accumulation of lipid. In the presence of BMP2, PTHrP or a protein kinase C (PKC) stimulator (phorbol ester) increased the expression of indexes of the osteoblast phenotype, including alkaline phosphatase, type I collagen, and osteocalcin, whereas a PKC inhibitor (chelerythrin chloride) inhibited PTHrP action. PTHrP and a phorbol ester increased gene expression of the BMP IA receptor, and both enhanced BMP2-dependent increases in promoter activity of the signaling molecule SMAD6. Overexpression of the BMP IA receptor facilitated the capacity of BMP2 to increase osteoblastogenesis in the absence of PTHrP and a dominant negative BMP IA receptor variant inhibited this effect of BMP2. These results demonstrate that PTHrP can direct osteoblastic, rather then adipogenic, commitment of mesenchymal cells, implicate PKC signaling in this activity, and show that PTHrP action involves enhanced gene expression of the BMP IA receptor, which facilitates BMP2 action in enhancing osteoblastogenesis in pluripotent mesenchymal cells.

PTHrP is the major pathogenetic factor for hypercalcemia in several malignancies including prostate cancer. In the current study, we have assessed the ability of androgens to regulate PTHrP production in androgen-insensitive human prostate cancer cells PC-3 and cells transfected with androgen receptor (PC-3T). Androgen responsiveness caused a marked decrease in PC-3T cell growth, and treatment of these cells with dihydrotestosterone led to inhibition of PTHrP production. These inhibitory effects were readily reversed by androgen receptor antagonist flutamide. To determine the effect of androgens on tumor growth and PTHrP production in vivo, PC-3 and PC-3T cells were injected into the right flank of male BALB/c nu/nu mice. Animals inoculated with PC-3 and PC-3T cells developed palpable tumors at wk 2 and 4, respectively. Inoculation of PC-3T cells into castrated animals resulted in rapid tumor growth in PC-3T tumors, effects that were reversed in PC-3T tumors grown in castrated hosts. Using PTHrP promoter luciferase reporter, a 30% decrease in luciferase activity was seen following treatment with dihydrotestosterone. These results indicate that PC-3 cell growth correlates inversely with androgen sensitivity and directly with PTHrP production in vitro and in vivo, androgens can regulate PTHrP production, and the androgen effect on PTHrP is mediated at least in part by transcriptional regulation via the androgen receptor.

We examined the capacity of PTHrP to modulate the terminal differentiation of the preadipocytic cell line, 3T3-L1. These cells express endogenous PTHrP and its receptor, but expression levels were undetectable after differentiation into mature adipocytes. Cells stably overexpressing PTHrP failed to differentiate when induced to undergo adipogenesis and proliferated at a faster rate. MAPK activity was elevated in PTHrP-transfected 3T3-L1 cells, and treatment with the PKA inhibitor H-8 decreased this activity. Inhibition of MAPK kinase with PD098059 permitted terminal differentiation of PTHrP-transfected 3T3-L1 cells to proceed. Although PPAR gamma gene expression levels remained relatively constant in the PTHrP-transfected cells, PPAR gamma phosphorylation was enhanced. Furthermore, the capacity of PPAR gamma to stimulate transcription in the presence of troglitazone was diminished by PTHrP. Expression of the PPAR gamma-regulated adipocyte specific gene aP2 transiently rose and then fell in PTHrP-transfected cells. These results indicate that PTHrP can increase MAPK activity in 3T3-L1 cells via the PKA pathway, thereby enhancing PPAR gamma phosphorylation. This modification can inactivate the transcriptional enhancing activity of PPAR gamma and diminish the expression of adipocyte-specific genes. These studies therefore demonstrate that PTHrP may inhibit the terminal differentiation of preadipocytes and describe a molecular pathway by which this action can be achieved.

PTH-related peptide (PTHrP) has been implicated in a variety of developmental and homeostatic processes. Although mice homozygous for the targeted disruption of the Pthrp gene have greatly expanded our capacity to investigate the developmental roles of the protein, the perinatal lethality of these animals has severely hindered the analysis of Pthrp's postnatal physiological effects. To overcome this obstacle, we have generated mice homozygous for a floxed Pthrp allele, i.e. two loxP sites flanking exon 4 of the Pthrp gene, which encodes most of the protein, with the aim of accomplishing cell type- and tissue-specific deletion of the gene. The ability of the Cre enzyme to cause recombination between the loxP sites and excision of the intervening DNA sequence was tested in vivo by crossing this strain to mice carrying a cre transgene under the transcriptional control of the human beta-actin promoter. The ubiquitous deletion of the floxed allele in the cre/loxP progeny resulted in perinatal lethality as a consequence of aberrant endochondral bone formation, fully recapitulating all the phenotypic abnormalities observed in the conventional Pthrp knockout mouse. The availability of the floxed Pthrp mice will serve as a valuable tool in genetic experiments that aim to investigate the physiological actions of Pthrp in the postnatal state.

The osteogenic sarcoma cell line UMR 106-01 exhibits heterogeneous morphology and hormone response in subconfluent monolayer cultures. In these studies we have explored the correlation between morphological profiles and patterns of cytosolic calcium [Ca2+]i response to PTH and other agonists in single UMR 106-01 cells loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2. Realtime recording of [Ca2+]i revealed that PTH (10(-7) M) produced a transient [Ca2+]i rise in 19% of the cells studied. [Ca2+]i transients were also induced by prostaglandins E2 and F2 alpha, and fetal bovine serum, but with different response frequencies (20%, 12%, and 58%, respectively). Spatial resolution of changes in [Ca2+]i by video image analysis revealed that the response to PTH was more frequent in large polygonal cells with long cytoplasmic processes and less common in smaller cells growing in clusters, whereas there was no clear subtype specificity for the effects of epidermal growth factor and fetal bovine serum on [Ca2+]i. Autoradiographic analysis of cell monolayers demonstrated a higher density of PTH-binding sites on cells with cytoplasmic extensions, whereas epidermal growth factor-binding sites were largely on colony-forming cells. Thus, the [Ca2+]i response to hormonal stimulation is heterogeneous within UMR 106-01 cell populations and within single cells, and it correlates with receptor density. This suggests that osteoblastic cells respond to PTH by activation of changes in [Ca2+]i only at certain specific steps during osteoblast development or stages of the cell cycle.

The interaction of epidermal growth factor (EGF) and PTH with UMR 106 osteosarcoma cells was examined biochemically and morphologically. EGF inhibited PTH-stimulated adenylate cyclase activity in association with a reduction in PTH receptors and a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs). Universal inhibition of agonist-stimulated adenylate cyclase activity did not occur, inasmuch as EGF did not reduce prostaglandin E2-enhanced enzyme activity. The influence of EGF on PTH action correlated with its effect in the UMR 106 cell population of promoting entry into the cell cycle. Morphological analysis with radioautography indicated that both EGF and PTH receptors could be colocalized to certain UMR 106 cells, but that each were more abundantly distributed over discrete UMR 106 cell types. Based on the distribution of [3H]thymidine incorporation, EGF receptors were predominantly found over rapidly proliferating cells, whereas PTH receptors were most densely distributed over more quiescent cells. The results indicate that EGF and PTH receptors are localized over specific types within the heterogeneous population of UMR 106 cells and suggest that EGF may reduce PTH action in these cultures by enhancing the proliferation of progenitor cells lacking PTH receptors and reducing differentiation in this cell population, which leads to PTH receptor-enriched target cells. EGF and PTH receptors may, therefore, be useful as probes to examine both functional interactions and differentiation pathways among cells in osteoblast models in vitro and perhaps in vivo.

Target tissue responses were examined and compared in renal and skeletal tissues of two species, the rat and rabbit, employing human calcitonin [hCT-(l–32)] and the synthetic amino-terminal analogs [des-amino-Cys1]hCT-(l–32), [Na-acetyl-Cys1]hCT-(l–32), and [NMet1.7]hCT-(l–32) [NMet, normethionine (S-methylcysteine)] as probes of membrane binding and adenylate cyclase stimulation. Close concordance was observed within each species in the concentration of each peptide required for binding to specific sites on target tissue membranes and for stimulating adenylate cyclase. Each peptide was found to be more potent in rabbit than in rat tissue when assayed in the absence or presence of the synthetic guanyl nucleotide, guanyl-5′-ate imidodiphosphate; however the relative order of potency of the different peptides did not vary between species. Within each species the potency of each peptide was similar in both target tissues examined. The characteristics of membrane binding differed between the two species in th...

We compared the bioactivities of a synthetic truncated NH2-terminal fragment of the human (h) PTH-like peptide (PLP) associated with malignancies [hPLP-(3-34)], an intact NH2-terminal fragment [hPLP-(1-34)], and an NH2-terminal fragment of PTH [hPTH-(1-34)]. Although hPLP-(1-34) was less potent than hPTH-(1-34) in stimulating adenylate cyclase in rat renal membranes, hPLP-(1-34) and hPTH-(1-34) were equipotent in stimulating adenylate cyclase in OK renal cells as well as in UMR 108 osteosarcoma cells in vitro. In osteosarcoma cells, each of these peptides could desensitize adenylate cyclase responses to itself and to the other peptide, but could not reduce stimulation by prostaglandin E2. Renal membranes of vitamin D-deficient rats with secondary hyperparathyroidism had a reduced PLP-stimulated as well as PTH-stimulated adenylate cyclase response. The truncated analog hPLP-(3-34) was only a weak partial agonist and an antagonist in vitro, produced equivalent inhibition of hPLP-(1-34) and hPTH-(1-34) in renal and osseous cells, and could not desensitize agonist responses. In thyroparathyroidectomized rats in vivo, hPLP-(1-34) and hPTH-(1-34) increased cAMP excretion, enhanced phosphaturia, maintained plasma calcium, and reduced calciuria. Equimolar concentrations of hPLP-(3-34) produced no increases above control levels; however, high concentrations of this peptide mimicked PTH actions on renal and plasma ion handling while modestly augmenting cAMP excretion. These results demonstrate the importance of the first two residues of PLP for bioactivity, indicate that PLP and PTH interact at common receptor sites in vivo as well as in vitro, suggest that PLP may not be less potent than PTH in renal target cells, and indicate that the net result of interaction of these peptides with their common receptor in target tissues may reflect both activation and desensitization of receptor-mediated events.

Recently, several investigators have detected calcitonin (CT)-like material in pituitary tissues by immunochemical techniques. In the present study we examined CT biosynthesis in rat pituitaries using specific and sensitive hybridization assays which used a cloned CT complementary DNA (cDNA) to detect CT mRNAs. Rat CT cDNA encoding the entire CT precursor, was radiolabeled to a high specific activity with 32P-labeled nucleotide and hybridized with mRNA extracted from pituitaries of both normal and genetically obese Zucker rats, in which elevated levels of immunoreactive CT-like material have been detected. Using these assays, we failed to detect mRNA with size and hybridization characteristics similar to those of thryoidal CT mRNA in either normal or Zucker rat pituitaries. We conclude that a mRNA homologous in sequence to that encoding rat thyroidal CT is not synthesized in the pituitary gland. In previous immunochemical studies a CT-like peptide may have been detected which is synthesized in the pituitary but lacks sufficient structural homology for the mRNA to react with our cDNA probe to thyroidal CT mRNA. Alternatively, in these prior studies thyroidal CT bound to pituitary receptors may have been detected.

The specific binding of the amino-terminal region of parathyroid hormone (PTH) to liver has been assessed by in vivo radioautography employing a biological active 125I-labeled synthetic bPTH analog ([Nle-8,18, Tyr-34]bPTH-(1-34) amide) as a probe. Two minutes after intrajugular injection of the labeled analog into rats, free hormone was separated from that bound to cells by intracardiac perfusion with buffer (30 sec), followed by perfusion-fixation with glutaraldehyde. By light and electron microscope radioautography, the distribution of silver grains over the liver sections revealed localization over the periphery of hepatocytes as well as over endothelial cells. In simultaneous control experiments, the unlabeled synthetic active fragment bPTH-(1-34) and unlabeled intact native bPTH-(1-84) significantly inhibited binding of the labeled analog to liver hepatocytes but not to endothelial cells. Greater uptake of the 125I-labeled bPTH analog was found in rat liver (28% of the injected dose) than in kidney, bone, or other organs examined. In parallel experiments using purified plasmalemma fractions from hepatocytes, both bPTH-(1-34) and intact bPTH-(1-84) demonstrated a dose-dependent activation of adenylate cyclase which was less than that of glucagon but greater than that of epinephrine. The combined results support the concept of the liver as a target organ for the amino-terminal biologically active region of PTH.

The interaction of three guanyl nucleotides with parathyroid hormone (PTH)-sensitive adenylyl cyclase in canine renal cortical membranes was assessed. Results seen after mixing various proportions of the naturally occurring nucleotide GTP, and the nucleotide analogs 5′ -guanylylimidodiphosphate (Gpp(NH)p) and 5′ -guanylyl methylene diphosphonate (Gpp(CH2)p) suggest that the compounds share a common locus of action, probably distal to the hormonereceptor interaction. The nucleotide (Gpp(NH)p) exerted a marked influence on a variety of synthetic analogs of PTH, increasing the apparent affinity of all PTH derivatives tested and the intrinsic activity of all partial agonists. Most notably the potency of the NH2- terminal tetratriacontapeptide of bovine PTH (bPTH), which in assays using dog renal membranes is only 20% that of the native hormone, increased to 100% when assayed with the nucleotide, and the hormone analog (desamino-Ala-l)-bPTH-(1–34), shown to be active in vivo but inert in in vitro adenylyl cycl...

Previous studies have provided evidence that tumors associated with humoral hypercalcemia of malignancy produce a factor that shares certain biological properties with, but is not, native PTH. In the present study, media from Xenopus oocytes microinjected with polyadenylated RNA prepared from three human or animal tumors associated with humoral hypercalcemia of malignancy are shown to have activity in the cytochemical bioassay for PTH. This activity parallels that of the PTH standard in the assay and is completely inhibited by the PTH analog [Nle8,Nle18,Tyr34]bovine PTH-(3-34)NH2. Media from oocytes injected with mRNA from control tumors contain no such activity. The mRNA encoding this activity in one of the animal tumors has been enriched approximately 10-fold by preparative polyacrylamide gel electrophoresis. These results demonstrate that tumors associated with humoral hypercalcemia of malignancy have the capacity for directing the synthesis of a secretory protein that fulfills certain criteria for being the humoral mediator in question. These techniques may provide the basis for molecular cloning of this factor.

We have examined the capacity of calcitonin gene-related peptide (CGRP) to exert two biological actions in the central nervous system (CNS): modulation of GH release and alteration of food intake. These effects were compared with those of calcitonin (CT). Intracerebroventricular administration of both rat (r) CGRP (10 micrograms/10 microliter) and salmon (s) CT (250 ng/10 microliter) to chronically cannulated freely-moving rats reduced the frequency and amplitude of spontaneous GH secretory pulses; however, the suppressive effect was of longer duration with sCT than with rCGRP. Both peptides also significantly reduced spontaneous food intake over a 6-h period when administered centrally, although the decrease with sCT was again more extensive than that with rCGRP. The results demonstrate that CGRP can simulate two of the effects of CT in brain. Furthermore, the findings suggest that CGRP may function as an endogenous ligand for CT receptors in the CNS, and participate centrally in the modulation of GH release and the control of satiety.

Specific binding sites for calcitonin (CT) were sought in preparations of rat brain in vitro. Whole rat brains or discrete regions of the brain were homogenized, and the 10,000 × g fraction was tested for binding of 125I-labeled salmon CT ([125I]sCT). The highest total and specific binding occurred in the hypothalamus, and the least occurred in the cortex and cerebellum, relative to whole brain. Intermediate levels of binding of the radioligand were found in the pons-midbrain and medulla. More detailed characterization of the hypothalamusradioligand interaction revealed that the binding was saturable and linear with increasing concentrations of tissue protein from 25–800 μg/ml. Furthermore, it was both time and temperature dependent, reaching equilibrium by 1 h at 4, 23, and 37 C. After 1 h of incubation at 4 C, half-maximal inhibition of binding of the radioligand occurred at a sCT concentration of 1 × 10-9 M. Both porcine and human CT were less effective than sCT in inhibiting the binding of [125I]sCT t...

We examined the characteristics of PTH resistance in vitamin D-deficient rats employing renal membranes in vitro. Homologous desensitization was characterized by diminished PTH-stimulated adenylate cyclase activity and was associated with a reduction in PTH-binding capacity, but not affinity. Heterologous desensitization was also seen, as manifested by decreased calcitonin (CT)-stimulated adenylate cyclase activity with normal CT receptor binding. The reduced capacity of the nonhormonal effectors NaF and guanylylimidodiphosphate to stimulate adenylate cyclase indicated a postreceptor defect at the level of the guanyl nucleotide-binding protein (G protein), whereas a normal forskolin response was consistent with a fully functional catalytic component. The G protein deficiency was confirmed by demonstrating that the addition of extracts of vitamin D-sufficient membranes to preparations of vitamin D-deficient membranes restored the normal responses to NaF and guanylylimidodiphosphate. In addition, cholera toxin- and pertussis toxin-catalyzed labeling of vitamin D-deficient renal membranes with [32P]NAD revealed a decrease in both the stimulatory and inhibitory binding proteins. Experiments with testicular membranes in vitro indicated that the adenylate cyclase abnormality was absent in tissue lacking PTH receptors. The results suggest that a major contribution to PTH resistance in vitamin D-deficient animals is a postreceptor defect at the level of the G proteins and that this defect is manifest only in tissue expressing the PTH receptor.

Exposure of quiescent bovine parathyroid cells to serum or a serum substitute caused an elevation in [3H] thymidine incorporation, followed by an increase in cell number. This was preceded by a rapid transient rise in c-myc and c-fos proto-oncogene mRNA levels. Alterations in the medium calcium concentration had no effect on the growth state of quiescent parathyroid cells. In addition, varying the medium calcium concentration did not influence either the time course or the degree of induction of proto-oncogene expression or the subsequent increase in [3H] thymidine uptake or proliferation of stimulated parathyroid cells. In contrast, when 1,25-dihydroxycholecalciferol was added with serum or serum substitute to quiescent parathyroid cells, no increase in c-myc mRNA levels was observed, and the expected increase in parathyroid cell number failed to occur. The augmentation in c-fos mRNA in response to serum was not, however, altered by 1,25-dihydroxycholecalciferol. These results indicate that 1,25-dihydroxyvitamin D, but not extracellular calcium, may directly modulate parathyroid cell proliferation by altering the expression of specific replication-associated oncogenes.

Specific binding sites for blood-borne calcitonin were localized by means of quantitative radioautography to the circumventricular organs of the rat brain. By this method, using normal Long-Evans rats as controls, specific binding of blood-borne calcitonin in the median eminence region of the hypothalamus was reduced by one-third in homozygous Brattleboro rats, which are genetically deficient in vasopressin. Competitive binding analysis in vitro of the hypothalami from these animals confirmed the binding deficit in homozygous rats, and Scatchard analysis suggested a reduction in the number of binding sites. In homozygous rats daily vasopressin replacement therapy restored normal water balance but did not normalize the hypothalamic calcitonin binding deficit. These studies delineate for the first time specific sites within the central nervous system which could serve to mediate direct actions of blood-borne calcitonin on brain function. The deficit in the Brattleboro rat may provide a model for further investigation of the role of calcitonin within selective regions of the central nervous system.

We have shown that parathyroid hormone (PTH) stimulates 25-hydroxyvitamin D2-1 alpha-hydroxylase activity in mitochondrial preparations of guinea pig kidney in vitro.1,25-[3H]dihydroxyvitamin D3 produced from 25-[3H]hydroxyvitamin D3 was identified by both normal phase and reversed-phase high pressure liquid chromatography. Enzyme stimulation by PTH was dose-dependent, peaked after 15 minutes of incubation with hormone and was maximal at concentrations of approximately 10(-10) M PTH-(1-84) and PTH-(1-34). This guinea pig renal model therefore permits direct examination of hormonal regulation of the mammalian enzyme in vitro.

Previous studies in vitro and in vivo have demonstrated the presence of receptor sites for PTH on cells of the osteoblast phenotype. Nevertheless, it is unclear whether the diverse functions of this hormone in bone can all be attributed to its interaction with a single cell type. In this study, we have used a radioautographic method to examine the competitive binding of 125I-labeled rat PTH-(1-34) to the long bones of rats in vivo. Our studies confirm the presence of competitive binding to mature osteoblasts and the absence of significant competitive binding to multinucleated osteoclasts. However, by light and electron microscopic radioautographic analysis, the majority of specific competitive PTH binding was present over a cell in the intertrabecular space of the metaphyseal region, which was distinct from the mature osteoblast. This large mononuclear cell with multiple cytoplasmic extensions appeared to interface with both the bone matrix and the microvascular osseous circulation and may provide an additional target to mediate hormonal effects on the skeleton.

Previous studies have suggested that parathyroid hormone (1–84), the predominent glandular and secreted form of parathyroid hormone (PTH), might be cleaved to an active fragment before exerting its hormonal effect in target tissues. It was subsequently established that this form of the hormone is active without the requirement for proteolysis in stimulating adenylate cyclase in renal tissue. The present studies were undertaken to examine the necessity for cleavage of PTH (1–84) in the other major target tissue (bone) and thereby to determine if a tissue-specific enzyme might confer target organ specificity upon the PTH molecule. Unlabeled or 125I-labeled bovine PTH (1–84) was incubated for 10 min at 22 C or 37 C with fetal rabbit calvarial tissue, and the incubation mixtures were analyzed by gel filtration and gel electrophoresis. RIA, using antisera directed against the NH2- and COOHtermini, were employed for the detection of unlabeled hormone. Analysis of the metabolic fate of the hormone was correlated...

We have purified peptides with PTH-like bioactivity from a rat Leydig cell tumor (H-500) and a human squamous cell carcinoma, both associated with a syndrome of humor-induced hypercalcemia. Tumor extracts were shown to be active in an in vitro renal cytochemical bioassay and in an in vitro osteosarcoma cell (UMR 108) adenylate cyclase assay; activity in both assays could be reduced by the PTH antagonist [norleucine-8,18,tyrosine-34]bovine PTH-(3-34)-amide. Partially purified extracts of both tumors and of rat tumor-conditioned culture medium were active in vivo in thyroparathyroidectomized rats in preventing hypocalcemia and increasing fractional phosphorus excretion and cAMP excretion. Ion exchange chromatography demonstrated that active peptides were basic in character. Employing reverse phase HPLC and gel permeation HPLC, active peptides of approximately 9,000 and 9,500 daltons were purified from extracts of the human and rat tumors, respectively, which had similar but not identical compositions. Two additional bioactive peptides were detected in rat tumor extract, and the more active had a mol wt of approximately 28,000. The results demonstrate that peptides that mimic PTH in a variety of in vivo and in vitro bioassays can be extracted from malignancies associated with hypercalcemia, that multiple molecular species may be detected in tumors that demonstrate PTH-like activity, and that at least one of these peptides may be similar in two tumors of highly divergent cell and species origin.

We have examined in vivo binding of bovine (b) PTH-(1-84) and the analog [Nle8,18, Tyr34]bPTH-(1-34) amide to hepatic, skeletal, and renal rat tissues. Bioactive 125I-labeled bPTH-(1-84) or the 125I-labeled bPTH-(1-34) analog was injected alone into experimental animals or with either unlabeled PTH or unlabeled unrelated hormone into control animals. Corresponding tissues from experimental and control animals were then processed for light and electron microscope radioautography and analyzed quantitatively and qualitatively. Binding of both PTH forms occurred on hepatocytes and sinusoidal cells in liver, and hepatocyte binding was clearly specific and competitive. Intact PTH-(1-84), but not the amino-terminal fragment, bound to Kupffer cells, indicating a sequence-specific interaction. In bone, specific competitive PTH binding was seen over osteoblasts, but not osteoclasts. Skeletal PTH binding was also seen over connective tissue mononuclear cells, and sinusoidal endothelial cells. In kidney, specific competitive PTH binding was seen over glomerular podocytes, and over the antiluminal surface of cells of the proximal tubule, the thick ascending limb of Henle's loop, and the distal tubule. Noncompetitive binding was seen on the luminal surface of proximal convoluted tubule cells. We have, therefore, distinguished specific competitive binding sites, probably related to hormone action, from noncompetitive binding sites, presumably associated with hormone metabolism, on discrete cell types or cell regions within several tissues. Our approach provides morphological correlates to the biochemical interaction of PTH with its target tissues and should enhance our understanding of the relationship of target cell structure and function.