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.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Phosphate is a specific signal for induction of osteopontin gene expression

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue.

Mice with a targeted mutation in the thyroid hormone β receptor gene exhibit impaired growth and resistance to thyroid hormone

Patients with mutations in the thyroid hormone receptor β (TRβ) gene manifest resistance to thyroid hormone (RTH), resulting in a constellation of variable phenotypic abnormalities. To understand the molecular basis underlying the action of mutant TRβ in vivo, we generated mice with a targeted mutation in the TRβ gene (TRβPV; PV, mutant thyroid hormone receptor kindred PV) by using homologous recombination and the Cre/loxP system. Mice expressing a single PVallele showed the typical abnormalities of thyroid function found in heterozygous humans with RTH. Homozygous PV mice exhibit severe dysfunction of the pituitary–thyroid axis, impaired weight gains, and abnormal bone development. This phenotype is distinct from that seen in mice with a null mutation in the TRβ gene. Importantly, we identified abnormal expression patterns of several genes in tissues of TRβPV mice, demonstrating the interference of the mutant TR with the gene regulatory functions of the wild-type TR in vivo. These results show that the actions of mutant and wild-type TRβ in vivo are distinct. This model allows further study of the molecular action of mutant TR in vivo, which could lead to better treatment for RTH patients.

Identification of two novel transmembrane γ-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

The proline-rich γ-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20–24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.

Prevention of development of autoimmune disease in BXSB mice by mixed bone marrow transplantation

Transplantations of fully allogeneic, autoimmune-resistant T-cell-depleted marrow (TCDM) plus syngeneic, autoimmune-prone TCDM into lethally irradiated BXSB mice were carried out to investigate the ability of the mixed bone marrow transplantation (BMT) to prevent development of autoimmune disease and, at the same time, to reconstitute fully the immunity functions of heavily irradiated BXSB recipients. Male BXSB mice were engrafted with mixed TCDM from both allogeneic, autoimmune-resistant BALB/c mice and syngeneic, autoimmune-prone BXSB mice. BMT with mixed TCDM from both resistant and susceptible strains of mice (mixed BMT) prolonged the median life span and inhibited development of glomerulonephritis in BXSB mice. BMT with mixed TCDM also prevented the formation of anti-DNA antibodies that is typically observed in male mice of this strain. Moreover, mixed BMT reconstituted primary antibody production in BXSB recipients, so that no annoying immunodeficiencies that are regularly observed in fully allogeneic chimeras were present in the recipient of the mixed TCDM. These findings indicate that transplanting allogeneic, autoimmune-resistant TCDM plus syngeneic, autoimmune-prone TCDM into lethally irradiated BXSB mice prevents development of autoimmune disease in this strain of mice. In addition, this dual BMT reconstitutes the immunity functions and avoids the immunodeficiencies that occur regularly in fully allogeneic chimeras after total-body irradiation.

Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo−/− and EpoR−/− mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210BCR-ABL and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR−/− mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

Testatin: A cystatin-related gene expressed during early testis development

To isolate genes involved in morphogenic aspects of testis development, and which may act in cell signaling pathways downstream of the testis-determining gene Sry, we have developed a modified mRNA differential display method named signal peptide differential display. It was used to target those genes that encode proteins having a signal peptide sequence. By using this method, we isolated a gene named testatin. This gene was found to be related to a group of genes that encodes cysteine protease inhibitors known as cystatins. Cystatins and their target proteases have been associated with tumor formation and metastasis, but also are involved in natural tissue remodeling events such as bone resorption and embryo implantation. We show that testatin expression is restricted to fetal gonads and adult testis. Furthermore, testatin is expressed during testis cord formation in pre-Sertoli cells, believed to be the site of Sry action, at a time immediately after the peak of Sry expression. This finding suggests that testatin might be activated by transcription factors that are known to orchestrate the early testis development pathway. This gene therefore represents one of the putative downstream targets likely to have an essential role in tissue reorganization during early testis development.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

We have generated RANK (receptor activator of NF-κB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK−/− mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1−/− (recombinase activating gene 1) mice, indicating that RANK−/− mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK−/− mice without inducing hypercalcemia, although tumor necrosis factor α treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK−/− mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Increased expression of preprotachykinin-I and neurokinin receptors in human breast cancer cells: Implications for bone marrow metastasis

Neuropeptides are implicated in many tumors, breast cancer (BC) included. Preprotachykinin-I (PPT-I) encodes multiple neuropeptides with pleiotropic functions such as neurotransmission, immune/hematopoietic modulation, angiogenesis, and mitogenesis. PPT-I is constitutively expressed in some tumors. In this study, we investigated a role for PPT-I and its receptors, neurokinin-1 (NK-1) and NK-2, in BC by using quantitative reverse transcription–PCR, ELISA, and in situ hybridization. Compared with normal mammary epithelial cells (n = 2) and benign breast biopsies (n = 21), BC cell lines (n = 7) and malignant breast biopsies (n = 25) showed increased expression of PPT-I and NK-1. NK-2 levels were high in normal and malignant cells. Specific NK-1 and NK-2 antagonists inhibited BC cell proliferation, suggesting autocrine and/or intercrine stimulation of BC cells by PPT-I peptides. NK-2 showed no effect on the proliferation of normal cells but mediated the proliferation of BC cells. Cytosolic extracts from malignant BC cells enhanced PPT-I translation whereas extracts from normal mammary epithelial cells caused no change. These enhancing effects may be protein-specific because a similar increase was observed for IL-6 translation and no effect was observed for IL-1α and stem cell factor. The data suggest that PPT-I peptides and their receptors may be important in BC development. Considering that PPT-I peptides are hematopoietic modulators, these results could be extended to understand early integration of BC cells in the bone marrow, a preferred site of metastasis. Molecular signaling transduced by PPT-I peptides and the mechanism that enhances translation of PPT-I mRNA could lead to innovative strategies for BC treatments and metastasis.

A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter resulted in a loss of β-galactosidase expression in the telencephalon and mesencephalon. Collectively, our experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development.

Requirement for membrane lymphotoxin in natural killer cell development

Development of natural killer (NK) cells is thought to depend on interactions between NK progenitors and the bone marrow (BM) microenvironment; however, little is known about the molecular signals involved. Here we show that lymphotoxin (LT) provides an important signal for the development of both NK cells and NK/T cells. LTα−/− mice show marked reduction in splenic and BM NK and NK/T cell numbers and dramatically impaired NK and NK/T cell function. Mice deficient in either tumor necrosis factor receptor (TNFR)-I or TNFR-II have normal numbers of NK and NK/T cells, implying that neither of the TNFRs nor soluble LTα3 is required for development of these cell types. Reciprocal BM transfers between LTα−/− and wild-type mice suggest that close interactions between membrane LT-expressing NK cell precursors and LT-responsive radioresistant stromal cells are necessary for NK cell development. When LT-deficient BM cells are incubated with IL-15, NK cells are formed. In addition, LT-deficient BM cells produce IL-15 after activation. Thus, membrane LT appears to deliver a signal for NK cell development that is either independent of IL-15 or upstream in the IL-15 pathway. These results reveal a novel function for membrane LT in NK and NK/T cell development. They also support a cellular and molecular mechanism by which NK cell precursors themselves deliver essential signals, through the membrane ligand, that induce the microenvironment to promote further NK cell and NK/T cell development.

Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide

Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat α1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP−/−). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP−/− mice and allowed their prolonged survival. “Rescued” animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP−/− mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.