Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.

Parathyroid hormone-related protein is required for normal intramembranous bone development.

It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.

Expression of parathyroid hormone-related protein mRNA in the rat before birth: demonstration by hybridization histochemistry.

The ontogeny of parathyroid hormone (PTH) and PTH-related protein (PTHrP) gene expression was studied by hybridization histochemistry in the rat at various stages between implantation and full term. PTHrP mRNA was demonstrable in the early postimplantation trophoblastic giant cells but disappeared from this site before 13.5 days. Localized gene expression, detectable by the in-situ technique, began between 12.5 and 15.5 days in embryonic tissues. The distribution of gene expression suggests that PTHrP may be concerned with the process of implantation. Its widespread, yet clearly localized, distribution in embryonic and fetal tissues is consistent with a paracrine or autocrine function which may relate to the transforming growth factor-beta family of growth factors. PTH expression occurred solely in the parathyroid and was detectable in the fetal parathyroid at 13.5 days of gestation.

Expression of IGF-II and H19 mRNA in the neonatal rat during normal maturation and after dexamethasone administration.

In this study, the expression of IGF-II and H19 was examined in the liver, skeletal muscle and choroid plexus of the neonatal rat during normal maturation and after the administration of dexamethasone. If the two genes share common regulatory elements as postulated by an enhancer competition system, their patterns of expression should remain similar throughout maturation and after treatment with dexamethasone. In the liver, down-regulation of IGF-II and H19 during maturation and after dexamethasone administration was shown. This is consistent with the hypothesis that IGF-II and H19 are regulated by common enhancers. In the secretory cells of the choroid plexus, where expression of IGF-II is known to be biallelic, IGF-II was expressed in both untreated and dexamethasone-treated animals, regardless of age, whereas H19 expression was not detectable. This is consistent with the postulate that only one gene from each allele can be engaged by the enhancers. In skeletal muscle, H19 continues to be expressed in the adult after IGF-II is switched off suggesting that IGF-II can also be regulated independently of H19.

The role of fetal parathyroid hormone-related protein in transplacental calcium transport.

During pregnancy, a placental calcium pump maintains the fetus in a hypercalcaemic state relative to the mother, a condition which has been thought to facilitate normal development of the fetal skeleton. Based on experiments performed in the sheep, parathyroid hormone-related protein (PTHrP) has been implicated as the hormone responsible for maintaining the placental calcium pump. In the present study on mice in which the PTHrP gene has been ablated by homologous recombination, we have measured both fetal and maternal circulating total and ionised calcium levels, as well as fetal total body calcium, in order to determine whether absence of PTHrP during fetal development has an effect on fetal calcium levels. Our results show that, in fetuses lacking PTHrP, circulating ionised calcium levels are significantly lower than those of heterozygote and wild-type littermates, but circulating total calcium levels show no difference. Total body calcium levels of null mutants are significantly higher than those of normal littermates. The role of PTHrP in maintaining the integrity of the transplacental calcium pump in the rodent thus remains unclear. It may be that the lower levels of fetal blood ionised calcium in mutant animals are due to disruption of the placental pump, but, if this is the case, compensatory mechanisms have operated to allow the excessive calcium deposition seen in the skeletons of these animals. Alternatively, the increased avidity of the bones for calcium may in itself have produced a lower equilibrium level of available ionised calcium.

The expression of parathyroid hormone and parathyroid hormone-related protein in developing rat parathyroid glands.

Secretion of parathyroid hormone-related protein (PTHrP) by sheep fetal parathyroid glands is reported to be an important factor in the maintenance of a placental calcium pump. The aim of the present study was to determine whether the developing rat parathyroid glands express PTHrP or parathyroid hormone (PTH), or both. Hybridisation histochemistry was used to detect transcription of PTHrP and PTH in serial paraffin sections through the 12.5- and 13.5-day rat embryo parathyroid anlage, as well as in sections through the 17.5-day embryonic and adult parathyroid glands. Results show strong expression of PTH in the 13.5-day embryonic parathyroid anlage, as well as in the parathyroid gland of the 17.5-day embryo and adult. Transcription of the PTHrP gene was not detected. The more sensitive technique of reverse transcription PCR was then performed. The pharyngeal region of 11.5-, 12.5- and 13.5-day rat embryos was dissected out and, at each stage, RNA was extracted from these tissues, as well as pooled tissues from the rest of the embryo. RNA that had been extracted from adult thyroid/parathyroid tissue was also tested. After reverse transcription, the resulting cDNAs were amplified by PCR (50 cycles) using specific PTH and PTHrP primers. The results show an abundance of PTH mRNA, specific to the pharyngeal region of the 13.5-day embryo, as well as to adult thyroid/parathyroid tissue. PTHrP expression was detected at very low levels in both parathyroid and extraparathyroid tissues. The presence of immunoreactive PTHrP and immunoreactive PTH in the pharyngeal region and rest of the body of 12.5- and 13.5-day rat embryos was assessed by specific RIAs. Whilst immunoreactive PTHrP was not detected in any of the tissues assayed, immunoreactive PTH was detected only in the pharyngeal region of the 13.5-day embryo. This confirms the results obtained from the gene expression studies. We conclude then that, in the developing rat embryo, PTH rather than PTHrP is more likely to play a role in calcium regulation. This is in contrast with the reported situation in the sheep, and suggests that fundamental species differences in fetal calcium regulation exist in mammals.

The ontogeny of peroxisome-proliferator-activated receptor gene expression in the mouse and rat.

The expression of the gene coding for peroxisome-proliferator-activated receptor (PPAR), a novel transacting factor belonging to the steroid superfamily, has been determined in the mouse and rat throughout development using hybridization histochemistry. Messenger RNA is demonstrable in the liver and brown fat from the fetal period onwards and, additionally, in the heart, kidney and gut post-natally. It is proposed that the upregulation of transcription of peroxisomal beta oxidation genes in specific tissues follows binding of the receptor to its natural ligand. Thus PPAR may have an important role in cold adaptation and non-shivering thermogenesis as well as in detoxification.

Mandibular deformities in parathyroid hormone-related protein (PTHrP) deficient mice: possible involvement of masseter muscle.

Previous studies using parathyroid hormone-related protein (PTHrP) null mutant mice have indicated severe abnormalities in the endochondral ossification, suggesting that PTHrP affects chondrocyte differentiation. In this study, we found in newborn PTHrP-deficient mice some deformities in the mandible that is formed via intramembranous ossification. The mandibular ramus was bent downwards and a prominent bone crest to which the deep layer of masseter muscle was tendinously attached was observed in the mandibular body. Transmission electron microscopic studies showed that active bone formation was progressing along the tendon fibers of the masseter muscle. The examination of 3-D reconstruction models indicated that the mandibular ramus was bent at the site of muscle attachment, which was shifted in the direction of the muscle fibers. Muscle fiber type analysis using myosin ATPase staining showed that the masseter muscle in the newborn PTHrP-deficient mice contained numerous type 2B fibers, demonstrating premature maturation of this muscle. Based on these findings, we speculated that premature maturation of the masseter muscle leads, probably due to increased tensile forces, to accelerated bone crest formation and subsequent bending of the mandibular ramus. These results further suggest that PTHrP is involved in the regulation of muscle development in normal animals.

Induction of renal tumours in rats by the administration of 1,2 dimethylhydrazine.

Administration of dimethylhydrazine (DMH) to adult rats by two subcutaneous injections each of 120 mg(base)/kg body weight and spaced 10 days apart resulted in the development of renal tumours in over 90 per cent. of treated animals at 30 weeks after injection. The tumours were frequently bilateral and multiple, and had the structure of the "mesenchymal" tumours which occur in a high incidence following treatment with nitrosamines. The incidence of tumours both in the small intestine and in the colon was lower than that experienced using a weekly DMH treatment schedule.

Expression of parathyroid hormone-related protein mRNA by uterine tissues and extraembryonic membranes during gestation in rats.

Hybridization histochemistry and solution hybridization studies were performed to localize the expression of parathyroid hormone-related protein (PTHrP) during implantation in rats. Parallel studies were performed on rat uteri bearing oil-induced deciduomata and on cultured blastocysts. PTHrP mRNA begins to be expressed at day 5.5 of gestation by the uterine epithelium in the anti-mesometrial crypts marking the sites of implantation and in comparable regions during induction of deciduoma. Trophoblastic giant cells express the gene as soon as they are phenotypically recognizable both in vivo and in culture, but PTHrP mRNA cannot be detected in the early blastocyst or in cells of the inner cell mass. Decidual cells produce PTHrP mRNA both in normal gestation and after the induction of deciduomata. In each case, the expression of the gene in decidual cells follows its expression in uterine epithelium by 48 h. The uterine topographical location and time of expression of the PTHrP gene suggests that it plays a part in the implantation of the blastocyst.

Isolation and characterisation of murine homologues of the Drosophila seven in absentia gene (sina).

The seven in absentia gene (sina) is required for formation of the R7 photoreceptor cell in the developing eye of Drosophila melanogaster. The sina protein contains a putative zinc finger domain and localises to the cell nucleus in Drosophila. We report here the identification of a family of genes in the mouse (designated Siah) with extensive sequence homology to Drosophila sina. The Siah genes fall into two main groups: Siah-1, which consists of four closely related members, two of which appear to be functional, and Siah-2, which contains a single functional member. The predicted Siah proteins show an unusually high degree of conservation with sina over the majority of their lengths, diverging significantly only at their amino terminal ends. The Siah-1 and Siah-2 genes are widely expressed at a low level in the embryo and adult. Analysis of Siah-2 by hybridisation histochemistry shows that it is expressed at a higher level in a restricted number of sites during development, including the olfactory epithelium, retina, forebrain and proliferating cartilage of developing bone. The striking degree of sequence homology observed between the Drosophila and murine genes implies strong conservation pressure on the Siah genes and suggests that they play a significant role in vertebrate development.

Expression of insulin-like growth factor II (IGF)-II) and H19 in murine teratocarcinomas derived from embryonic stem (ES) cells.

Insulin-like growth factor II (IGF-II) is expressed during embryogenesis in rodents and humans, but is not produced in most adult tissues. This pattern of expression is closely shared by the gene H19, which lies 3' to IGF-II. This, together with the fact that the genes are reciprocally imprinted, has led to the proposal that the genes are under common transcriptional control by the H19 enhancers during development. In the present study, embryonic stem (ES) cells have been used to generate teratocarcinomas in mice. These tumours generate a wide range of differentiated tissues which have been subjected to hybridisation histochemistry with RNA probes to H19 and IGF-II. Coexpression of the two genes was found in a range of tissues, a pattern consistent with the idea of common transcriptional control. However, there were some areas in which H19 was expressed strongly in comparison with IGF-II and vice versa suggesting the existence of further control elements other than the H19 enhancers.

Expression of the IGF-II/mannose-6-phosphate receptor mRNA and protein in the developing rat.

The insulin-like growth factor II (IGF-II) receptor is identical to the mannose-6-phosphate receptor (M-6-P), but its role as a somatomedin transducer is uncertain. IGF-II/M-6-P receptor expression was studied by in situ hybridization (ISH) in the developing rat. Expression occurs in extra-embryonic membranes at the time of IGF-II mRNA induction and later at paracrine/autocrine sites of IGF-II action (skeletal muscle and perichondrium) in the embryo. Highest levels of receptor mRNA occur in heart and major vessels. Postnatally transcription is strongly down-regulated. This suggests a role for the IGF-II/M-6-P receptor in IGF-II action or turnover during development distinct from its role in lysosomal transport.

The expression of IGF-II in the early post-implantation rat conceptus.

Insulin-like growth factor II (IGF-II) gene expression was investigated in the early rat conceptus using in situ hybridisation. IGF-II mRNA has been detected as early as 7 days post coitum in the extraembryonic ectoderm of the egg cylinder (including the ectoplacental cone but excluding the trophoblast bordering the implantation chamber). There was no expression either in the early embryo at this stage or in the implanting blastocyst (5.5 days). At 9.5 days transcription was also detected in extra-embryonic mesodermal derivatives and the visceral yolk sac endoderm, but not in the embryonic region. At 10.5 days transcription was detected in embryonic mesodermal derivatives. High levels were found in the primitive heart, hepatic diverticulum and septum transversum; lower levels were found in the early somites and other primitive mesenchymal derivatives and midgut endoderm. No expression was detected at any stage in derivatives of the embryonic ectoderm, including the neurectoderm. Recent studies of IGF-II, the IGF-II/M-6-P receptor and enzymic extracellular matrix remodelling suggest that the expression of IGF-II in the early rat conceptus may be related to hyaluronate breakdown (with, or without, sulphated glycosaminoglycan synthesis), linked with the aggregation and differentiation of previously migrating mesenchymal cells.

The localization of laminin mRNA and protein in the postimplantation embryo and placenta of the mouse: an in situ hybridization and immunocytochemical study.

In situ hybridization (ISH) and immunocytochemistry were used to localize sites of synthesis and deposition of the basement membrane glycoprotein laminin during development in the postimplantation mouse embryo and extraembryonic membranes. In addition, similar studies were performed on postnatal viscera during the first 20 days after birth. Up to 10 days post coitum, embryonic laminin synthesis was confined to parietal endoderm. In maternal tissue, intense laminin mRNA expression was detected in decidual cells in the mesometrial and antimesometrial endometrium at 5-7 days. At 10 days, uniform expression was still seen within the mesometrial endometrium, with higher levels around migrating trophoblast, but in the antimesometrial aspect expression was restricted to the basal zone. High levels of mRNA expression persisted in parietal endoderm throughout gestation but much lower levels were detected in visceral yolk sac. In the mature placenta, laminin mRNA expression was also found associated with fetal vessels in the labyrinth and giant cells at the fetal/maternal boundary. In the embryo, the external limiting membrane of the cerebral vesicles and spinal cord stained for laminin protein and detectable mRNA was found in the pia mater. Growing peripheral nerves and dorsal and ventral root fibres expressed laminin mRNA and stained for laminin protein. Laminin mRNA expression was found in ureteric buds and nephrogenic vesicles (but not in metanephric blastema) during early prenatal kidney development, and in glomeruli, Bowman's capsule, loops of Henle and collecting duct cells at later stages of development, and after birth. All these structures possessed laminin-rich basement membrane (BM). Laminin mRNA expression fell to below detectable levels in the kidney around weaning. In the gut, laminin expression and protein staining was confined to the muscularis externa and the lamina propria during embryogenesis. After birth, the muscularis externa, muscularis mucosa and lamina propria cells corresponding to fibroblasts had detectable laminin mRNA, but in adult gut no laminin mRNA could be demonstrated in any cell type. In liver, low levels of laminin mRNA were seen in the capsule and in periportal connective tissue. After birth, laminin mRNA was associated with intrahepatic bile channels; no laminin mRNA was detected in the parenchyma and protein deposition was restricted to blood sinus BM. In the adult liver, no laminin mRNA was detected in any cell type. The developing heart showed uniform expression of laminin mRNA from 12 days to before birth. Postnatally, labelling was restricted to connective tissue cells.

Biosynthesis of proteoglycan in bone and cartilage of parathyroid hormone-related protein knockout mice.

Proteoglycans are suggested to regulate cell adhesion, differentiation and mineralization of hard tissues. In vitro studies have shown that many humoral and local factors regulate proteoglycan synthesis. Among them, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have potent stimulating effects on proteoglycan synthesis. However, the exact role of PTHrP on the biosynthesis and metabolism of proteoglycans during skeletal development is not clear. To clarify this point, we examined bony and cartilaginous explants of newborn mice with disrupted PTHrP alleles. Ribs of homozygous PTHrP-knockout mice and wildtype littermates were dissected into bony and cartilaginous regions and metabolically labeled with [35S]sulfate in culture. Radiolabeled proteoglycans were analyzed by column chromatography. The elution profiles of [35S]-labeled proteoglycan from cartilaginous explants did not differ between homozygous PTHrP-knockout mice and wild-type littermates. However, the amount of labeled proteoglycan in homozygous PTHrP-knockout mice was only 4%-5% that of wild-type littermates. In contrast with cartilaginous explants, the amount of labeled proteoglycans in bony explants did not differ between the two genotypes. Interestingly, besides the common major peak (Kd = 0.10-0.16) observed in the bony explants of both genotypes, a minor peak (Kd = 0.42) was specifically present in homozygous PTHrP-knockout mice. This minor peak was earlier than that of free glycosaminoglycan (GAG) chains, suggesting that the core protein, but not GAG chain, was cleaved in the bony explants of homozygous PTHrP. These findings demonstrate a crucial nonredundant role of PTHrP in the regulation of proteoglycan synthesis and metabolism during skeletal development.

Mechanisms of organ selective tumour growth by bloodborne cancer cells.

The sites of tumour development for 6 rat tumours injected into syngeneic rats via different vascular routes was determined. Xenografts of human tumours were also injected intra-arterially (i.a.) into immunosuppressed rats. Following intravenous (i.v.) and intraportal (i.ptl.) injection of cells tumour colonies localized in lung and liver respectively due to tumour cell arrest. Arterially injected radiolabelled cells disseminated and arrested in a similar distribution to cardiac output and did not 'home' to any organs. Following arterial injection of unlabelled tumour cells colonies grew in many organs. While the pattern of growth for a particular tumour varied with the cell dose, the 'arterial patterns' for all of the tumours studied followed a similar pattern. Some organs (eg adrenals, ovaries and periodontal ligament) were consistently preferred, others (eg skin and skeletal muscle) only supported tumour growth following the delivery of large numbers of cells, while in some tissues (eg spleen and intestines) tumour never grew. Viable tumour cells could be demonstrated by bioassay in many organs for up to 24h after i.a. injection. However tumour growth only occurred in certain organs and the pattern of this growth was not related to the number of tumour cells arrested or their rate of autolysis. This site preference could be expressed quantitatively as the probability of an arrested cell developing into a tumour and was considered a 'soil effect'. Site preference was not directly related to organ vascularity. Organ colonisation was promoted by steroid treatment but the mechanism was unclear and was not secondary to T-cell immunosuppression or prostaglandin synthesis suppression. The adrenal glands were preferred sites of tumour growth but pharmacological manipulation of adrenal function did not alter tumour growth to this organ. Sites of injury and healing were preferred sites of tumour colonisation and this could not be accounted for by increased delivery of tumour cells to these regions. The possibility that the macrophage component of the inflammatory response promoted tumour growth was suggested from studies in which the interval between trauma and inoculation of tumour cells was varied as well as by promotion of intraperitoneal (i.p.) tumour growth by a macrophage infiltrate.

Parathyroid-hormone-related protein in tumours associated with hypercalcaemia.

Parathyroid-hormone-related protein (PTHrP) messenger RNA was identified by in-situ hybridisation in seven tumours complicated by hypercalcaemia. In addition, among samples from normocalcaemic patients, it was commonly found in tumours of types frequently complicated by hypercalcaemia but was not found in tumours that are rarely complicated by hypercalcaemia. Positive results were obtained with both freshly frozen and archive paraffin-embedded material. These findings support the view that PTHrP is a common cause of hypercalcaemia in malignant disorders.