Erratum to "Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development"

Proteoglycans, the molecules of extracellular matrix, carry a highly negative charge due to their glycosaminoglycan (GAG) chains and large volumes. They were considered to play a secondary role in activities like cell division, adhesion, blood coagulation, etc. until the importance of their sugar chains in the fibroblast growth factor (FGF) signalling was discovered (Science 252 (1991) 1705; Cell 64 (1991) 841). Studies of mutations in the genes sugarless(sgl) and sulfateless (sfl) have proved that the proteoglycans involved in Wg signalling contain heparan sulfate GAG chains (Development 124 (1997) 2623; Development 124 (1997) 3055; Development 124 (1997) 3565; Development 126 (1999) 3715). This has led to the attribution of specific functions to these molecules (J. Cell Biol. 148 (2000) 227). The Glypican family of heparan sulfate proteoglycans (HSPGs) is characterized by core proteins with conserved cysteine residues and attachment to the cell surface by a glycosylphosphatidyl inositol (GPI) anchor. This may lead to endocytic pathways that are different from other HSPGs, higher lateral mobility and possible apical localisation in a cell (Proc. Natl. Acad. Sci, USA 85 (1988) 9557). Variations in their HS contents may effect binding properties and localisation (J. Cell Biol. 124 (1994) 149; J. Cell Biol. 132 (1996) 487), thus specialising each member for a unique biological function. Glypicans play important roles in morphogenetic pathways, e.g. human glypican 3 (GPC3) is mutated in Simpson-Golabi-Behmel syndrome making an individual prone to tumours (Nat. Genet. 12 (1996) 241). Dally, the first Drosophila member of the family, is essential for the wingless and decapentaplegic signalling pathways (Development 121 (1995) 3687; Development 124 (1997) 4113). Here, we report a new Drosophila glypican, dally-like protein (dlp) with all the features of a glypican. Based on expression studies we report its colocalisation with Wg.     

Yes-associated protein is involved in proliferation and differentiation during postnatal liver development

It is known that the liver undergoes size increase and differentiation simultaneously during the postnatal period. Cells in the liver undergo a period of well-controlled proliferation to achieve the adult liver-to-body weight ratio. The postnatal liver growth is also accompanied by simultaneous hepatic differentiation. However, the mechanisms of liver size regulation and differentiation are not completely clear. Herein we report that yes-associated protein (Yap), the downstream effector of the Hippo Kinase signaling pathway, plays a role in liver size regulation and differentiation during the postnatal liver growth period. Postnatal liver growth was studied in C57BL/6 mice over a time course of postnatal days (PND) 0-30. Analysis of nuclear Yap by Western blot indicated peak Yap activation between PND15-20, which coincided with increased cyclin D1 expression and liver cell proliferation. Analysis of postnatal liver development in Yap(+/-) mice revealed a significant decrease in the liver-to-body weight ratio compared with Yap(+/+) mice at PND15 and -30. Yap(+/-) mice exhibited a significant decrease in postnatal liver cell proliferation, but no change in apoptosis was observed. Furthermore, global gene expression analysis of Yap(+/-) livers revealed a role of Yap in regulation of genes involved in bile acid metabolism, retinoic acid metabolism, ion transport, and extracellular matrix proteins. Taken together, these data indicate that Yap plays a role in both cell proliferation and possibly in hepatic differentiation during postnatal liver development.     

Expression of DPP6 in Meckel's cartilage and tooth germs during mouse facial development

Dipeptidyl peptidase-like protein 6 (DPP6), a member of the dipeptidyl aminopeptidase family, plays distinct roles in brain development, but its expression in embryonic Meckel's cartilage and tooth germs development is unknown. We analyzed the expression pattern of DPP6 in Meckel's cartilage and tooth germs development using in situ hybridization. DPP6 was detected in different patterns in Meckel's cartilage and tooth germs during mouse facial development from 11.5 to 13.5 days post-coitus (dpc) embryos. The expression pattern of DPP6 suggests that it may be involved in mandible and tooth development.     

Parathyroid hormone-related peptide mediates hypercalcemia in an islet cell tumor of the pancreas.

Hypercalcemia occurring in a patient with an islet cell carcinoma of the pancreas suggests the diagnosis of Multiple Endocrine Neoplasia Type I and associated hyperparathyroidism. We describe a patient with an islet cell carcinoma and hypercalcemia in whom low concentrations of PTH, the absence of skeletal metastases, hypophosphatemia, and elevated nephrogenous cAMP alternatively suggested the syndrome of humoral hypercalcemia of malignancy. The peptide PTHrP was measured in the patient's serum during the course of therapy by an immunoradiometric assay directed toward the midportion of the molecule. Hypercalcemia was treated with an investigational aminobisphosphonate. The concentration of PTHrP[56-86] increased over time and fell after the patient received chemotherapy directed toward the islet cell tumor.     

Parathyroid hormone-related protein is not required for normal ductal or alveolar development in the post-natal mammary gland

PTHrP is necessary for the formation of the embryonic mammary gland and, in its absence, the embryonic mammary bud fails to form the neonatal duct system. In addition, PTHrP is produced by the breast during lactation and contributes to the regulation of maternal calcium homeostasis during milk production. In this study, we examined the role of PTHrP during post-natal mammary development. Using a PTHrP-lacZ transgenic mouse, we surveyed the expression of PTHrP in the developing post-natal mouse mammary gland. We found that PTHrP expression is restricted to the basal cells of the gland during pubertal development and becomes expressed in milk secreting alveolar cells during pregnancy and lactation. Based on the previous findings that overexpression of PTHrP in cap and myoepithelial cells inhibited ductal elongation during puberty, we predicted that ablation of native PTHrP expression in the post-natal gland would result in accelerated ductal development. To address this hypothesis, we generated two conditional models of PTHrP-deficiency specifically targeted to the postnatal mammary gland. We used the MMTV-Cre transgene to ablate the floxed PTHrP gene in both luminal and myoepithelial cells and a tetracycline-regulated K14-tTA;tetO-Cre transgene to target PTHrP expression in just myoepithelial and cap cells. In both models of PTHrP ablation, we found that mammary development proceeds normally despite the absence of PTHrP. We conclude that PTHrP signaling is not required for normal ductal or alveolar development.     

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.     

The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5^−/− mice have increased trabecular bone mass. We used Dock5^−/− mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5^−/− mice. We demonstrate that Dock5^−/− mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5^−/− growing mice as compared to Dock5^+/+ animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5^−/− osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.     

Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption

We have examined the effects of inhibitors of proton transport systems on osteoclastic bone resorption using an in vitro bone slice assay, where osteoclasts (OCs) are free from the influence of other bone cells. Amiloride (AM) and dimethylamiloride (DMA), inhibitors of the Na+/H+ antiporter, were potent inhibitors of bone resorption (IC50 approximately 9 and 0.7 microM for AM and DMA, respectively). Omeprazole (OM), a potent inhibitor of parietal cell K+/H+(-)ATPase, was a poor inhibitor of OC bone resorption (IC50 approximately 100 microM). These results strongly suggest that the Na+/H+ antiporter is the primary proton system used by OCs during bone resorption.     

Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.     

Influence of iron upon the development of tetracycline-treated mouse tooth germs in vitro (1).

The inhibitory action of tetracycline on the development of embryonic mouse incisors cultured in vitro was examined. Explants exposed to tetracycline were severely inhibited in development. In contrast, tooth germs cultured in the presence of both tetracycline and iron escaped inhibition and attained a stage of development which compared favorably with the controls.     

The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress

The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.     

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

Ultrastructure of alveolar bone during tooth eruption in the dog

Previous work from our laboratories has established that eruption of the permanent mandibular premolars in dogs is dependent upon the presence of the dental follicle and that it involves resorption of alveolar bone and the roots of the deciduous predecessor above and formation of alveolar bone below the developing crown. This study illustrates the topography of the bone surfaces of the crypt by scanning electron microscopy and the ultrastructure of the cells on alveolar bone surfaces during tooth eruption. Above the developing crown where the eruption pathway forms, the bone surface is a pitted sheet, the characteristic topographic feature of bone resorption; between the crown and the mandibular canal, the bone surface has numerous interconnecting trabeculae. Transmission electron microscopy of bone cells lining the eruption pathway area of the crypt showed numerous osteoclasts with adjacent mononuclear cells. Both cell types contained specific, membrane-bound cytoplasmic vesicles shown by the work of others to be characteristic features of osteoclasts and their precursors. Basal trabeculated bone in the crypt was covered by plump osteoblasts. These data show that the metabolic events in alveolar bone associated with tooth eruption have the appropriate cellular and bony surface correlates and that the suspected control of alveolar bone resorption by the dental follicle may be mediated by its recruiting and directing to adjacent bone surfaces the mononuclear precursors of osteoclasts.     

Novel functions of the well-known protein--prothymosin alpha is involved in protection of cells against apoptosis and oxidative stress

Several novel functions of the well-known and intensively studied protein prothymosin alpha have recently been revealed. In addition to such traditional functions of this protein as immunomodulatory activity and stimulation of cellular proliferation, prothymosin alpha was shown to be involved in protection of cells against apoptosis and in regulation of expression of the oxidative stress-protective genes. Methods and approaches used for revelation of prothymosin alpha novel functions are described in this review.