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Biomedical subjects

H M Kronenberg

Publications and source records attributed to H M Kronenberg.

At least 37 records · Page 2Linked to original sources
PubMed1997

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 alpha1 (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.

Animals↗
PubMed1996

Parathyroid hormone-related peptide (PTHrP) regulates fetal-placental calcium transport through a receptor distinct from the PTH/PTHrP receptor.

To determine the role of PTHrP in fetal calcium metabolism, blood calcium was measured in mice homozygous (HOM) for deletion of the PTHrP gene. On day 18.5 of gestation, ionized calcium and the maternal-fetal calcium gradient were significantly reduced in HOM PTHrP-ablated fetuses compared with that of their littermates. To assess the placental contribution to the effect of PTHrP, 45Ca and 51Cr-EDTA (as a blood diffusional marker) were administered by intracardiac injection to pregnant, heterozygous dams on day 17.5 of gestation. Five minutes after the injection, whole fetal 45Ca accumulation was significantly decreased in HOM PTHrP-ablated fetuses compared with that of their littermates. Next, two fetuses from each litter were injected in utero with fragments of PTHrP, PTH, or diluent 1 h before administering 45Ca and 51Cr to the dam. PTHrP-(1-86) and PTHrP-(67-86) significantly increased relative 45Ca accumulation in HOM PTHrP-ablated fetuses, but PTHrP-(1-34), PTH-(1-84), and the diluent had no effect. Finally, similar studies were performed on fetal mice that lacked the PTH/PTHrP receptor gene. Ionized calcium was significantly reduced in HOM PTH/PTHrP receptor-ablated fetuses. However, 5 min after maternal injection of 45Ca and 51Cr, relative accumulation of 45Ca was significantly increased in these fetuses. It was concluded that PTHrP is an important regulator of fetal blood calcium and placental calcium transport. In addition, the bioactivity of PTHrP for placental calcium transport is specified by a mid-molecular region that does not use the PTH/PTHrP receptor.

Animals↗
PubMed1996

Full activation of chimeric receptors by hybrids between parathyroid hormone and calcitonin. Evidence for a common pattern of ligand-receptor interaction.

Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G protein-coupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wild-type" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.

Animals↗
PubMed1996

Identification of an osteoblastic silencer element in the first intron of the rat osteocalcin gene.

The osteocalcin gene has been used as a model for studying the regulation of gene expression by 1,25-dihydroxyvitamin D3, as well as for examining factors which contribute to osteoblast-specific regulation of gene expression. Most of these studies have focused on transactivation. We report the identification of a sequence in the first intron of the rat osteocalcin gene which suppresses the expression of osteocalcin-CAT fusion genes approximately 10-fold in ROS 17/2.8 and UMR 106 osteosarcoma cells. Mutation of a TTTCTTT motif in the first intron abolishes this suppression. The silencing effect of this motif is also observed after bone morphogenic protein-2 (BMP-2)-induced expression of the osteoblastic phenotype in the MLB13MYC clone 17 cell line. Mutation of the splice donor site does not affect suppression by these sequences in ROS 17/2.8 cells. When multimerized and placed upstream of the native osteocalcin promoter, these sequences retain their ability to mediate transcriptional repression. Electrophoresis mobility shift analysis demonstrates a specific protein-DNA interaction with the TTTCTTT motif in nuclear extracts from ROS 17/2.8, UMR 106, and MLB13MYC clone 17 cells but not those from COS-7 kidney cells. The mutation of this motif, which abolishes suppressing activity in the native context, also abolishes binding. The presence and activity of this suppressor in cells of the osteoblast lineage suggest that it is expressed with other cell-specific transcriptional regulators of the osteocalcin gene, coordinately regulating expression of this gene in bone cells.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Physiological roles for parathyroid hormone-related protein: lessons from gene knockout mice.

A null mutation in the PTHrP gene produces profound abnormalities in endochondral bone formation in vivo. The role of PTHrP as a modulator of the chondrocytic proliferation and differentiation program is evident in the alterations that occur in its absence in the temporal and spatial sequence of chondrocyte development and subsequent endochondral bone formation that is necessary for normal bone elongation. These actions of PTHrP are probably responsible for the delay in chondrocyte development seen in Jansen osteochondrodystrophy, a disease caused by ligand-independent activation of the PTH-PTHrP receptor (Schipani et al., 1995). Furthermore, these conclusions have been corroborated by the observation that chondrocyte-specific overexpression of PTHrP causes a profound delay in the developmental program of chondrocyte differentiation and endochondral ossification (Weir et al., 1995). The morphological abnormalities in the knockout mice were limited to the skeletal system, despite the widespread production of PTHrP during fetal development. At this point, one can only speculate about the limited tissue distribution of the abnormalities. It is possible, for example, that other gene products, such as PTH, can compensate for the loss of PTHrP in some tissues. Alternatively, possible abnormalities in proliferation and differentiation may be present but morphologically subtle. A molecular assessment of these possible actions may well reveal more widespread effects of PTHrP.

Animals↗
PubMed1996

Regulation of HSP70 by PTH: a model of gene regulation not mediated by changes in cAMP levels.

Parathyroid hormone (PTH) activates both adenylate cyclase and phospholipase C in target cells, and cloned PTH/PTH-related protein (PTHrP) receptor can mediate both responses when expressed in host cells such as LLC-PK1 renal epithelial cells. Because calcitonin (CT) is known to augment 70-kDa heat shock protein (HSP70) mRNA by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism in LLC-PK1 cells, we examined regulation of HSP70 transcription by PTH in these cells. Like CT, human PTH-(1-34) [hPTH-(1-34); 10(-10) to 10(-7) M)] increased porcine HSP70 mRNA and human HSP70 promoter-chloramphenicol acetyltransferase (CAT) expression within 4 h in LLC-PK1 cells that stably express > or = 100,000 PTH/PTHrP receptors per cell. The effect of PTH on HSP70 mRNA was not mimicked by cAMP analogues, forskolin, phorbol esters, Ca2+ ionophores, or alpha-thrombin; was insensitive to pertussis toxin; and was not due to increased mRNA stability. The upregulation of HSP70 gene transcription by hPTH (and CT) was clearly observed even after deletion of the functional heat shock consensus element in the promoter region of the human HSP70/CAT reporter. Upregulation of HSP70 transcription via endogenous PTH receptors also was observed in the osteoblastic cell lines SaOS-2 and ROS 17/2.8. Regulation of HSP70 gene transcription by PTH may be a common cellular response to the hormone, which, in some cells, may not be mediated by activation of adenylate cyclase or protein kinase C.

Animals↗
PubMed1996

Parathyroid hormone-related peptide delays terminal differentiation of chondrocytes during endochondral bone development.

To test the hypothesis that PTH-related peptide (PTHrP) is a paracrine regulator of endochondral bone development, we localized PTHrP and its cognate receptor during normal skeletal development at both messenger RNA (mRNA) and protein levels and compared the growth plate phenotypes of PTHrP-deficient [(PTHrP(-/-)] mice to those of normal littermates [PTHrP(+/+]. PTHrP mRNA was expressed adjacent to uncavitated joints, in the perichondrium of long bones and to a lower level in proliferating chondrocytes. In contrast, PTHrP protein was most evident at the interface of proliferating and hypertrophic zones, where it colocalized with PTH/PTHrP receptor mRNA and protein. Most strikingly, the proliferating zone was dramatically shorter in PTHrP(-/-) cartilage, although the percentage of cells in S-phase of the cell cycle in the proliferating zone was indistinguishable between PTHrP(+/+) and PTHrP(-/-) mice. Terminal differentiation of chondrocytes, which was characterized by cell hypertrophy, apoptosis (DNA fragmentation and decreased bcl-2 mRNA expression), and matrix mineralization, was more advanced in growth cartilage of PTHrP(-/-), compared with PTHrP(+/+) animals. These data demonstrate that PTHrP acts principally as a paracrine factor, which promotes elongation of endochondral bone by restraining or delaying the pace of chondrocytic development and terminal differentiation of growth-plate chondrocytes.

Animals↗
PubMed1996

Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor.

An important physiological control of PTH gene expression is its transcriptional repression by 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. The mechanism of this 1,25-(OH)(2)D(3)-mediated transcriptional repression is poorly understood. Previous investigations have identified a DNA sequence in the 5'-regulatory region of the human PTH (hPTH) gene that binds the vitamin D receptor (VDR) and mediates transcription repression in response to 1,25(OH)(2)D(3) in GH4CI cells. The hPTH gene sequence does not mediate transcriptional repression in ROS 17/2.8 cells, even though up-regulatory vitamin D response elements (VDREs) are active in these cells. The hPTH DNA sequence differs from the upregulatory VDREs in that it contains a single copy of a hexameric motif (AGGUC) homologous to those repeated in the up-regulatory VDREs. The protein-DNA interactions of this sequence were examined using nuclear extracts from bovine parathyroid, GH4CI, and ROS 17/2.8 cells. In bovine parathyroid nuclear extracts, the VDR binds the down-regulatory hPTH DNA sequence independently of the retinoid X receptor (RXR). In GH4C1 nuclear extracts, two VDR-containing complexes are observed: one lacking RXR and one containing RXR. In ROS 17/2.8 nuclear extracts, a single VDRdependent complex containing RXR is observed. When the up-regulatory rat osteocalcin VDRE is used as a probe, only VDR-RXR-containing complexes are generated using nuclear extracts from all three cell types. These results demonstrate that the sequence that mediates transcriptional repression in response to 1 ,25-(OH)(2)D(3) differs from the up-regulatory response elements both in sequence composition and in its ability to bind VDR independently of RXR.

Animals↗
PubMed1995

Truncation of the carboxyl-terminal region of the rat parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of adenylyl cyclase but not phospholipase C.

The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent Kd values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 +/- 0.19 nM; R513, 1.24 +/- 0.12 nM; R480, 0.48 +/- 0.05 nM, mean +/- S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with Gs.

Adenylate Cyclase Toxin↗
PubMed1995

Parathyroid hormone (PTH)-PTH-related peptide hybrid peptides reveal functional interactions between the 1-14 and 15-34 domains of the ligand.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to a common PTH/PTHrP receptor. To explore structure-function relations in these ligands, we synthesized and functionally evaluated PTH-PTHrP hybrid peptides in which the homologous 1-14 portions were exchanged. Hybrid-2, PTH-(1-14)-PTHrP-(15-34)NH2, bound to LLC-PK1 cells expressing the cloned rat PTH/PTHrP receptor with high affinity (IC50 approximately equal to 7 nM). In contrast, hybrid-1, PTHrP-(1-14)-PTH-(15-34)NH2, bound with much weaker affinity (IC50 approximately equal to 8,700 nM). Thus, the 1-14 region of PTHrP is incompatible with the 15-34 region of PTH. The carboxyl-terminal incompatibility site was identified as residues 19-21 (Glu-Arg-Val in PTH and Arg-Arg-Arg in PTHrP); extending the amino-terminal PTHrP sequence to residue 21 but not to 18 cured the hybrid's binding defect. The amino-terminal incompatibility site was identified as position 5 (Ile in PTH and His in PTHrP), because Ile5-hybrid-1 bound with high affinity (IC50 approximately equal to 20 nM). The importance of these identified residues in the native ligands was established by evaluating the effects of substitutions at these sites in a series of PTH and PTHrP analog peptides. Overall, the results are consistent with the hypothesis that, in both PTH and PTHrP, the 1-14 and 15-34 domains interact when binding to the receptor and that residues 5, 19, and 21 contribute either directly or indirectly to this interaction.

Amino Acid Sequence↗
PubMed1995

Inefficient membrane targeting, translocation, and proteolytic processing by signal peptidase of a mutant preproparathyroid hormone protein.

A preproparathyroid hormone allele from a patient with familial isolated hypoparathyroidism was shown to have a single point mutation in the hydrophobic core of the signal sequence. This mutation, changing a cysteine to an arginine codon at the -8 position of the signal peptide, was associated with deleterious effects on the processing of preproparathyroid hormone to proparathyroid hormone in vitro. To examine the biochemical consequence(s) of this mutation, proteins produced by cell-free translation of wild-type and mutant cRNAs were used in assays that reconstitute the early steps of the secretory pathway. We find that the mutation impairs interaction of the nascent protein with signal recognition particle and the translocation machinery. Moreover, cleavage of the mutant signal sequence by solubilized signal peptidase is ineffective. The consequence of this mutation on processing and secretion of parathyroid hormone is confirmed in intact cells by pulse-chase experiments following transient expression of the mutant protein in COS-7 cells. The inability of the mutant signal sequence, however, to interfere with the targeting and processing of other secreted proteins does not support obstruction of the translocation apparatus as the mechanism underlying the dominant mode of inheritance of hypoparathyroidism in this family.

Alleles↗