Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development.

Vertebrate skeletogenesis requires a well-coordinated transition from chondrogenesis to osteogenesis. Hypertrophic chondrocytes in the growth plate play a pivotal role in this transition. Parathyroid hormone-related peptide (PTHrP), synthesized in the periarticular growth plate, regulates the site at which hypertrophy occurs. By comparing PTH/PTHrP receptor(-/-)/wild-type (PPR(-/-)/wild-type) chimeric mice with IHH(-/-);PPR(-/-)/wild-type chimeric and IHH(-/-)/wild-type chimeric mice, we provide in vivo evidence that Indian hedgehog (IHH), synthesized by prehypertrophic and hypertrophic chondrocytes, regulates the site of hypertrophic differentiation by signaling to the periarticular growth plate and also determines the site of bone collar formation in the adjacent perichondrium. By providing crucial local signals from prehypertrophic and hypertrophic chondrocytes to both chondrocytes and preosteoblasts, IHH couples chondrogenesis to osteogenesis in endochondral bone development.

Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues.

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-microm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.

A novel method for designing and fabricating custom-made artificial bones.

Artificial bones are useful for tissue augmentation in patients with facial deformities or defects. Custom-made artificial bones, produced by mirroring the bone structure on the healthy side using computer-aided design, have been used. This method is simple, but has limited ability to recreate detailed structures. The authors have invented a new method for designing artificial bones, better customized for the needs of individual patients. Based on CT data, three-dimensional (3D) simulation models were prepared using an inkjet printer using plaster. The operators applied a special radiopaque paraffin wax to the models to create target structures. The wax contained a contrast medium to render it radiopaque. The concentration was adjusted to achieve easy manipulation and consistently good-quality images. After the radiopaque wax was applied, the 3D simulation models were reexamined by CT, and data on the target structures were obtained. Artificial bones were fabricated by the inkjet printer based on these data. Although this new technique for designing artificial bones is slightly more complex than the conventional methods, and the status of soft tissue should also be considered for an optimal aesthetic outcome, the results suggest that this method better meets the requirements of individual patients.

Trehalose inhibits oral dryness by protecting the cell membrane.

This study assessed the clinical efficacy and acceptability of trehalose solution for oral dryness caused by dental treatment. The efficacy of trehalose on oral dryness under drying conditions was assessed by measuring the surface area of the fungiform papillae and the moisture content of the tongue in seven healthy volunteers. Based on the data from this pilot study, a clinical study was performed, in which the efficacy of oral trehalose spray was evaluated on oral dryness in 10 patients undergoing root canal treatment. The effects of trehalose on cell viability were also assessed under drying conditions in vitro. Trehalose suppressed oral dryness and associated pain caused by dental treatment and protected cells from dryness-related damage. These results indicate that pretreatment application of trehalose solution on the oral mucosa is effective in preventing oral dryness caused by dental treatment.

Aquaporin-5 expression, but not other peripheral lung marker genes, is reduced in PTH/PTHrP receptor null mutant fetal mice.

Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor are important developmental regulators of cell growth and differentiation in some organs. In lung, both the peptide and the receptor are expressed early in development and in alveolar cells in adults. In adult alveolar cells, PTHrP appears to promote the alveolar type II cell phenotype in vitro. Mice carrying null mutations in genes for either receptor or ligand die at birth of respiratory failure. To determine if absence of the PTH/PTHrP receptor alters morphogenesis or cellular differentiation of the distal lung, we analyzed the morphology and gene expression patterns in PTH/PTHrP receptor null mutant mice right before birth and compared them with wild-type and heterozygous null littermates. Using semiquantitative Northern blots, we observed that messenger RNA (mRNA) for aquaporin-5, the type I cell-specific water channel, was markedly decreased. The abundance of other marker mRNAs for type I and type II cell phenotypes, including T1alpha, surfactant proteins, and others, was unaltered. Gross morphology and lung pattern, assessed by in situ hybridization for surfactant protein C, were normal. We conclude therefore that, although signaling through this receptor may influence expression of specific lung genes, it does not play a major role in the general regulation of lung development and growth.

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation.

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.

The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones.

In the growth plate of endochondral bones, parathyroid hormone (PTH)-related peptide (PTHrP) regulates the rate of chondrocyte maturation from prehypertrophic chondrocytes to hypertrophic chondrocytes. Using an antibody specific for Sox9 phosphorylated at serine 181 (S(181)), one of the two consensus protein kinase A phosphorylation sites of Sox9, we showed that the addition of PTHrP strongly increased the phosphorylation of SOX9 in COS7 cells transfected with both SOX9- and PTH/PTHrP receptor-expressing vectors. PTHrP also increased the SOX9-dependent activity of chondrocyte-specific enhancers in the gene for type II collagen (Col2a1) in transient transfection experiments. This increased enhancer activity did not occur with a Sox9 mutant harboring serine-to-alanine substitutions in its two consensus protein kinase A phosphorylation sites. Consistent with these results, PTHrP also increased Col2a1 mRNA levels in rat chondrosarcoma cells as well as 10T1/2 mesenchymal cells transfected with a PTH/PTHrP receptor expressing plasmid. No phosphorylation of Sox9 at S(181) was detected in prehypertrophic chondrocytes of the growth plate or any chondrocytes of PTH/PTHrP receptor null mutants. In contrast in wild-type mouse embryos, previous immunohistochemistry experiments indicated that Sox9 phosphorylated at S(181) was detected almost exclusively in chondrocytes of the prehypertrophic zone. Sox9, regardless of the phosphorylation state, was present in all chondrocytes of both genotypes except hypertrophic chondrocytes. Our results indicated that Sox9 is a target of PTHrP signaling in prehypertrophic chondrocytes in the growth plate. We hypothesize that Sox9 mediates at least some effects of PTHrP in the growth plate and that the PTHrP-dependent increased transcriptional activity of Sox9 helps maintain the chondrocyte phenotype of cells in the prehypertrophic zone and inhibits their maturation to hypertrophic chondrocytes.

Functional analysis of the PTH/PTHrP network of ligands and receptors.

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) are two related proteins that activate a common PTH/PTHrP receptor, yet have quite distinct physiologic missions. PTH is the major peptide regulator of blood calcium in higher vertebrates, while PTHrP predominantly acts as a paracrine regulator of differentiation and local intercellular signaling. To analyze the physiological roles of PTHrP and the PTH/PTHrP receptor, "knockout" mice missing either the PTHrP or the PTH/PTHrP receptor gene were developed. Both the PTHrP (-/-) mice and the PTH/PTHrP receptor (-/-) mice exhibit a growth plate chondrodysplasia that reflects accelerated differentiation of proliferating chondrocytes. Growth plate chondrocytes regulate the local production of PTHrP by secreting the protein, Indian hedgehog (Ihh), as they are leaving the proliferative pool. Ihh stimulates the production of PTHrP, which then slows the differentiation of chondrocytes, thereby delaying the production of Ihh. PTHrP also stimulates transport of calcium across the placenta. PTHrP (-/-) mice lack the normal elevation of fetal blood calcium (when compared to maternal levels) and have low placental transport of calcium. Fragments of PTHrP that do not bind to the PTH/PTHrP receptor can correct the defect of placental calcium transport in these mice. Thus, this action of PTHrP is not mediated by the PTH/PTHrP receptor. The "knockout" mice thus help delineate the roles of PTH. PTHrP, and the PTH/PTHrP receptor in an interacting network of ligands and receptors.