Beate Lanske

Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein

Proper regulation of chondrocyte differentiation is necessary for the morphogenesis of skeletal elements, yet little is known about the molecular regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member of the conserved Hedgehog family of secreted proteins that is expressed during bone formation, has now been isolated. Ihh has biological properties similar to those of Sonic hedgehog (Shh), including the ability to regulate the conserved targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic chondrocytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. Misexpression of Ihh prevents proliferating chondrocytes from initiating the hypertrophic differentiation process. The direct target of Ihh signaling is the perichondrium, where Gli and Ptc flank the expression domain of Ihh. Ihh induces the expression of a second signal, parathyroid hormone—related protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (−/−) mutant mice indicated that the PTHrP protein signals to its receptor in the prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In vitro application of Hedgehog or PTHrP protein to normal or PTHrP (−/−) limb explants demonstrated that PTHrP mediates the effects of Ihh through the formation of a negative feedback loop that modulates the rate of chondrocyte differentiation.

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (−/−) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (−/−) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi‐2a. The phosphaturic effect was present in FGF23‐null mice, indicating a direct action distinct from Klothos known role as a coreceptor for FGF23. Direct inhibition of NaPi‐2a by Klotho was confirmed in cultured cells and in cell‐free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant β‐glucuronidase and is associated with proteolytic degradation and reduced surface NaPi‐2a. The inhibitory effect of Klotho on NaPi‐2a was blocked by β‐glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi‐2a from the apical membrane.—Hu, M. C., Shi, M., Zhang, J., Pastor, J., Nakatani, T., Lanske, B., Shawkat Razzaque, M., Rosenblatt, K. P., Baum, M. G., Kuro‐o, M., Moe, O. W. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J. 24, 3438–3450 (2010). www.fasebj.org

Molecular insights into the Klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

ABSTRACT Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/βKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between β strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the β1-β2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/βKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.

Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death.

Parathyroid hormone-related peptide (PTHrP) is a mediator of cellular growth and differentiation as well as a cause of malignancy-induced hypercalcemia. Most of the actions of PTHrP have been attributed to its interaction with a specific cell surface receptor that binds the N-terminal domain of the protein. Here we present evidence that PTHrP promotes some of its cellular effects by translocating to the nucleolus. Localization of transiently expressed PTHrP to the nucleolus was dependent on the presence of a highly basic region at the carboxyl terminus of the molecule that bears homology to nucleolar targeting sequences identified within human retroviral (human immunodeficiency virus type 1 and human T-cell leukemia virus type 1) regulatory proteins. Endogenous PTHrP also localized to the nucleolus in osseous cells in vitro and in vivo. Moreover, expression of PTHrP in chondrocytic cells (CFK2) delayed apoptosis induced by serum deprivation, and this effect depended on the presence of an intact nucleolar targeting signal. The present findings demonstrate a unique intracellular mode of PTHrP action and a novel mechanism by which this peptide growth factor may modulate programmed cell death.

Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process

Fibroblast growth factor 23 null mice (Fgf‐23−/−) have a short lifespan and show numerous biochemical and morphological features consistent with premature aging‐like phenotypes, including kyphosis, severe muscle wasting, hypogonadism, osteopenia, emphysema, uncoordinated movement, T cell dysregulation, and atrophy of the intestinal villi, skin, thymus, and spleen. Furthermore, increased vitamin D activities in homozygous mutants are associated with severe atherosclerosis and widespread soft tissue calcifications; ablation of vitamin D activity from Fgf‐23−/− mice, by genetically deleting the 1α(OH)ase gene, eliminates atherosclerosis and ectopic calcifications and significantly rescues premature aging‐like features of Fgf‐23−/− mice, resulting in prolonged survival of Fgf‐23−/−/1α(OH)ase−/− double mutants. Our results indicate a novel role of Fgf‐23 in developing premature aging‐like features through regulating vitamin D homeostasis. Finally, our data support a new model of interactions among Fgf‐23, vitamin D, and klotho, a gene described as being associated with premature aging process.

Characterization and cloning of the E11 antigen, a marker expressed by Rat Osteoblasts and Osteocytes

A new marker for cells of the osteoblastic lineage was identified by raising monoclonal antibodies against an immortalized rat osteoblastic cell line. Among the different antibodies one was selected which, on tissue sections, strongly reacts with osteoblasts, preosteocytes, and osteocytes. This antibody, designated E11, recognizes an antigen localized at the cell surface. The cDNA encoding the E11 antigen was cloned from a cDNA library prepared from ROS 17/2.8 cells, using a eukaryotic expression system. The E11 cDNA sequence revealed homology with the murine OTS-8/gp38 sequence. In situ hybridization confirmed that E11 mRNA expression in bone is restricted to osteoblasts and osteocytes. The tissue specificity of the E11 expression was studied by immunohistochemistry and Northern blot analysis. Apart from bone, E11-positive cells were also found in lung: namely, the alveolar cells of type I. Epithelial cells of the choroid plexus and endothelial cells of lymphatic vessels were also labeled with mAb E11. These results were confirmed by Northern blot, as the 1.8 kb E11 mRNA transcript was detected in bone and also in lung, brain, and skin. In conclusion, we describe a novel osteoblastic product which is expressed by mature osteoblasts and newly formed osteocytes.

Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to and activate the same PTH/PTHrP receptor. Deletion of either the PTHrP gene or the PTH/PTHrP receptor gene leads to acceleration of differentiation of growth plate chondrocytes. To explore further the functional relationships of PTHrP and the PTH/PTHrP receptor, bones of knockout mice were analyzed early in development, and the phenotypes of double-knockout mice were characterized. One early phenotype is shared by both knockouts. Normally, the first chondrocytes to become hypertrophic are located in the centers of long bones; this polarity is greatly diminished in both these knockouts. The PTH/PTHrP receptor-deficient (PTH/PTHrP-R(-/-)) mice exhibited 2 unique phenotypes not shared by the PTHrP(-/-) mice. During intramembranous bone formation in the shafts of long bones, only the PTH/PTHrP-R(-/-) bones exhibit a striking increase in osteoblast number and matrix accumulation. Furthermore, the PTH/PTHrP-R(-/-) mice showed a dramatic decrease in trabecular bone formation in the primary spongiosa and a delay in vascular invasion of the early cartilage model. In the double-homozygous knockout mice, the delay in vascular invasion did not occur. Thus, PTHrP must slow vascular invasion by a mechanism independent of the PTH/PTHrP receptor.

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Indian hedgehog stimulates periarticular chondrocyte differentiation to regulate growth plate length independently of PTHrP

In the developing growth plate, periarticular chondrocytes proliferate, differentiate into columnar chondrocytes, and then further differentiate into postmitotic hypertrophic chondrocytes. Parathyroid hormone-related (PTH-related) protein (PTHrP), regulated by Indian hedgehog (Ihh), prevents premature hypertrophic differentiation, thereby maintaining the length of columns. Ihh regulates cartilage development through PTHrP-independent pathways as well. Here we show that Ihh stimulates differentiation of periarticular to columnar chondrocytes (periarticular chondrocyte differentiation) and thereby regulates the length of columns independently of PTHrP. Mosaic ablation of the PTH/PTHrP receptor in the growth plate caused upregulation of Ihh action, PTHrP upregulation, acceleration of periarticular chondrocyte differentiation, and elongation of the columnar region. Decreasing Ihh action in these mice reduced elongation of columns, whereas decreasing PTHrP showed only a modest effect on column length. Overexpression of Ihh caused PTHrP upregulation, elongation of columns, and acceleration of periarticular chondrocyte differentiation. PTHrP heterozygosity in this model had a minimal effect on the elongation of columns. Moreover, the elongation of columns and stimulation of periarticular chondrocyte differentiation in these models were still observed when PTHrP signaling was maintained so that it remained constant. These results demonstrate that Ihh acts on periarticular chondrocytes to stimulate their differentiation, thereby regulating the columnar cell mass independently of PTHrP.