Parathyroid Hormone-related Protein Receptor Research Articles

PTHrP promotes murine secondary trophoblast giant cell differentiation through induction of endocycle, upregulation of giant-cell-promoting transcription factors and suppression of other trophoblast cell types

The murine trophoblast cell lineage represents an intriguing experimental cell model as it is composed of four trophoblast stem (TS)-derived cell types: trophoblast giant cells (TGCs), spongiotrophoblast, syncytotrophoblast, and glycogen trophoblast cells. To investigate the role of parathyroid hormone-related protein (PTHrP) in TGC differentiation, we analyzed the effect of exogenous PTHrP on secondary TGCs of day 8.5 p.c. ectoplacental cone explant culture. Secondary TGCs expressed PTHrP and PTHR1 receptor in vivo and in vitro. TGCs treated with PTHrP had reduced proliferation and decreased apoptosis starting from day 2 in culture, and enhanced properties of giant cell differentiation: increased DNA synthesis, number of cells with giant nuclei and expression of placental lactogen-II (PL-II). The induction of TGC formation by PTHrP correlated with downregulation of cyclin B1 and mSNA expression, but upregulation of cyclin D1, thus allowing mitotic-endocycle transition. Moreover, PTHrP treatment influenced TGC differentiation by inducing the expression of transcription factors known to stimulate giant cell formation: Stra13 and AP-2γ, and inhibiting the formation of other trophoblast cell types by suppressing trophoblast progenitors and spongiotrophoblast-promoting factors, Eomes, Mash-2, and mSNA. Taken together with the spatial and temporal patterns of TGC formation and PTHrP synthesis in vivo, these findings indicate an important role for PTHrP in the differentiation of secondary TGCs during placentation.

A survey of haplotype variants at several disease candidate genes: the importance of rare variants for complex diseases

Background: The haplotype based association method offers a powerful approach to complex disease gene mapping. In this method, a few common haplotypes that account for the vast majority of chromosomes...

Uteroplacental restriction in the rat impairs fetal growth in association with alterations in placental growth factors including PTHrP

During pregnancy, parathyroid hormone-related protein (PTHrP) is one of many growth factors that play important roles to promote fetal growth and development, including stimulation of placental calcium transport. Angiotensin II, acting through the AT(1a) receptor, is also known to promote placental growth. We examined the effects of bilateral uterine artery and vein ligation (restriction), which mimics placental insufficiency in humans, on growth, intrauterine PTHrP, placental AT(1a), and pup calcium. Growth restriction was surgically induced on day 18 of pregnancy in Wistar-Kyoto female rats by uterine vessel ligation. Uteroplacental insufficiency reduced fetal body weight by 15% and litter size (P < 0.001) compared with the control rats with no effect on placental weight or amniotic fluid volume. Uteroplacental insufficiency reduced placental PTHrP content by 46%, with increases in PTHrP (by 2.6-fold), parathyroid hormone (PTH)/PTHrP receptor (by 11.6-fold), and AT(1a) (by 1.7-fold) relative mRNA in placenta following restriction compared with results in control (P < 0.05). There were no alterations in uterine PTHrP and PTH/PTHrP receptor mRNA expression. Maternal and fetal plasma PTHrP and calcium concentrations were unchanged. Although fetal total body calcium was not altered, placental restriction altered perinatal calcium homeostasis, as evidenced by lower pup total body calcium after birth (P < 0.05). The increased uterine and amniotic fluid PTHrP (P < 0.05) may be an attempt to compensate for the induced impaired placental function. The present study demonstrates that uteroplacental insufficiency alters intrauterine PTHrP, placental AT(1a) expression, and perinatal calcium in association with a reduction in fetal growth. Uteroplacental insufficiency may provide an important model for exploring the early origins of adult diseases.

Cherubism – new hypotheses on pathogenesis and therapeutic consequences

The hereditary occurrence of cherubism indicates a probable genetic aetiology: a correlation with a mutation in the gene SH3BP2 has been demonstrated. A convincing concept of formal pathogenesis is not yet available. The study was aimed at advancing the understanding of the pathogenesis of cherubism by presenting a case study including genetic findings and an evaluation of the literature. Because of its association with the development of the second and third molars, cherubism could be defined as a genetically determined alteration of tooth development. In this context, disturbed PTHrP - PTHrP receptor interaction induced by the mutation in SH3BP2 is discussed. The temporal and spatial determination of the clinical symptoms is explained by an interaction of SH3BP2-dependent signal transduction pathways with jaw morphogenesis (e.g. Hox-gene Msx-1). Because of the disease-induced lack of determination of the cap phase of the second and third molar, a spatial compartmentation, which is necessary for normal dental development, does not take place. This leads to dysregulation of mesenchymal bone building tissue areas, and to the development of giant cell granulomas with high osteoclastic activity. Because of the genetic determination of cherubism and the associated dedifferentiation of the diseased tissue, a surgical removal should be exclusively restricted to specific indications. Therefore an attitude of wait and see is preferred.

Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor

Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1-34) or PTHrP(1-34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1-21), [M]PTH(1-15), and [M]PTH(1-14), and introduced substitutions at positions 1-3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)(1)-para-benzoyl-l-phenylalanine (Bpa)(2)-Deg(3). In HKRK-B7 cells, which express the cloned human PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21), [Deg(1,3),Bpa(2),M]PTH(1-15), and [Deg(1,3),Bpa(2),M]PTH(1-14) fully inhibited (IC(50)s = 100-700 nm) the binding of (125)I-[alpha-aminoisobutyric acid(1,3),M]PTH(1-15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) antagonized the cAMP-agonist action of PTH(1-34), as did PTHrP(5-36) (IC(50)s = 0.7 microm, 2.6 microm, and 36 nm, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg(1,3),Bpa(2), M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) inhibited the agonist actions of [alpha-aminoisobutyric acid(1,3)]PTH(1-34) and [M]PTH(1-14) (IC(50)s approximately 1 microm), whereas PTHrP(5-36) failed to inhibit. [Deg(1,3),Bpa(2),M]PTH(1-14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1-9), in which the PTH(1-9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR(cam)H223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.

Parathyroid Hormone-Related Protein Overexpression Decreases Blood Pressure in Spontaneously Hypertensive Rats

We have recently demonstrated that arterial PTHrP expression and cardiovascular responses to this protein are altered in SHR compared with normotensive animals, Wistar Kyoto (WKY) and Sprague-Dawley (SD) rats. To investigate whether the slightly, but significantly decreased, aortic PTHrP gene expression observed in SHR, compared to that of normotensive animals, may play a causative role in the maintenance of the elevated arterial blood pressure (ABP) of the SHR, we transfected a hepatic lobe with a PTHrP expression vector in a sense and antisense orientation. At 24 and 48 hours, sense pSV2neo-ECE induced a significant five-fold increase in PTHrP mRNA abundance with respect to antisense pSV2neo-ECE and vehicle. This increment in the PTHrP mRNA induced by the sense PTHrP expression vector was totally inhibited by the co-administration of the antisense PTHrP expression vector. At the same time, we observed a significant decrease of mean ABP (MABP) in SHR transfected with the sense pSV2neo-ECE to similar values as those obtained in the normotensive strain. Neither antisense PTHrP expression vector nor vehicle had any significant effect in any strain. Again, the effect of the sense PTHrP expression vector on MABP was blocked by the simultaneous treatment with the antisense PTHrP expression vector. At 48 hours, the hypotensive effect of the sense pSV2neo-ECE in SHR was reverted by the IV bolus injection of a specific competitive PTHrP receptor antagonist such as Nle8,18,Tyr34-bPTH(3–34)amide. We propose that a defect of this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in SHR. This defect can be ameliorated by transfecting tissues with protein-exporting capabilities, such as the liver. Finally, our work adds additional data to a cumulative body of evidence suggesting that it might be possible to design an effective gene therapy to treat the common polygenic and multifactorial form of hypertension by increasing the activity of potent and physiological vasodilators.

Mechanical Regulation of Terminal Chondrocyte Differentiation via RGD-CAP/β ig-h3 Induced by TGF-β

RGD-CAP (βig-h3), initially cloned as a transforming growth factor (TGF)-β inducible gene in human lung adenocarcinoma cells, was demonstrated to have a negative regulatory function in mineralization in hypertrophic chondrocytes, and the expression was shown to be associated with mechanical stimulation. We hypothesized that mechanical stimulation may regulate the terminal chondrocyte differentiation through the TGF-β pathway by enhancing the RGD-CAP expression. To test this hypothesis, we investigated the effects of mechanical strain on the terminal differentiation and mineralization of growth-plate chondrocytes and assessed the mechanical regulation of TGF-β and RGD-CAP expression. A cyclic mechanical strain of 12% elongation was applied to the cultured prehypertrophic chondrocytes isolated from the rib cartilage of 4-week-old male rats at 30 cycles/min (loading and relaxation on every alternate second). The terminal differentiation and mineralization of chondrocytes were assessed by alkaline phosphatase (ALP) activity assay and alizarin red staining. The gene expressions of TGF-β and RGD-CAP, as well as chondrocytic terminal differentiation markers such as type X collagen and ALP, were examined with real-time RT-PCR. Cyclic mechanical strain decreased the ALP activity and intensity of alizarin red staining in prehypertrophic chondrocytes, as well as the gene expressions of type X collagen and ALP. TGF-β and RGD-CAP were upregulated in the prehypertrophic chondrocytes subjected to mechanical strain, whereas the level of PTHrP receptor mRNA was not affected by the mechanical strain. The neutralizing antibody for TGF-β suppressed the reduction of the mineralization of chondrocyte cultures with the downregulation of RGD-CAP. These results suggest that mechanical strain negatively regulates the terminal differentiation of chondrocytes through the signal pathway of TGF-β with the induction of RGD-CAP.

The (GT)n polymorphism and haplotype of the COL1A2 gene, but not the (AAAG)n polymorphism of the PTHR1 gene, are associated with bone mineral density in Chinese

Collagen type I alpha2 (COL1A2) and parathyroid hormone (PTH)/PTH-related peptide receptor (PTHR1) are two prominent candidate genes for bone mineral density (BMD). To test their importance for BMD variation in Chinese, we recruited 388 nuclear families composed of both parents and at least one healthy daughter with a total of 1,220 individuals, and simultaneously analyzed population stratification, total-family association, and within-family association between BMD at the spine and hip and the (GT)n marker in the intron 1 of the COL1A2 gene and the (AAAG)n marker in the P3 promoter of PTHR1 gene. We also performed these association analyses with haplotypes of the MspI and (GT)n polymorphisms in the COL1A2 gene. Significant within-family association was found between the M(GT)12 haplotype and trochanter BMD (P<0.001). Individuals with this haplotype have, on average, 9.53% lower trochanter BMD than the non-carriers. Suggestive evidence of the within-family association was detected between the (GT)17 allele and BMD at the spine (P=0.012), hip (P=0.011), femoral neck (P=0.032), trochanter (P=0.023), and intertrochanter (P=0.034). The association was confirmed by subsequent permutation tests. For the association, the proportion of phenotypic variance explained by the detected markers ranged from 1.2 to 3.9%, with the highest 3.9% at the trochanter for the M(GT)12 haplotype. This association indicates that there is strong linkage disequilibrium between the polymorphisms (MspI and GT repeat polymorphism) in the COL1A2 gene and a nearby quantitative trait locus (QTL) underlying BMD variation in Chinese, or the markers themselves may have an important effect on the variation of BMD. On the other hand, no significant within-family association, population stratification and total-family association between the PTHR1 polymorphism and BMD were found in our Chinese population.

Haplotype frequencies and linkage disequilibrium analysis of four frequent polymorphisms at the PTH/PTH-related peptide receptor gene locus

The parathyroid hormone (PTH)/PTH-related peptide receptor is a critical component in the control of mineral ion metabolism and in bone development. This receptor is encoded by a single gene ( PTHR1) on chromosome 3p21.1–p24.2, and mutations in this gene have been found in several clinical disorders of bone and mineral metabolism. To facilitate future genetic studies of this important gene, we determined haplotype frequencies and performed linkage disequilibrium (LD) analysis of four different polymorphisms at the PTHR1 locus. Combined analysis of Caucasian, African–American and Asian individuals indicated that LD exists between all but one pair of the four polymorphisms. However, the pattern of LD differed substantially among the three subpopulations; for example, LD between two closely spaced (154-bp apart) single nucleotide polymorphisms appeared to be present only in Asians. Depending on the population under study, genetic association studies may need to test even more closely spaced polymorphic markers when screening the PTHR1 locus. These findings may thus affect the design and interpretation of future genetic studies involving PTHR1.

Deconvoluting lung evolution using functional/comparative genomics.

Parathyroid Hormone-related Protein (PTHrP) is a highly evolutionarily conserved, stretch-regulated gene that is necessary for the embryonic transition from branching morphogenesis to alveolization of the lung. It is expressed throughout vertebrate phylogeny, beginning with its expression in the fish swim bladder as an adaptation to gravity; microgravity downregulates the expression of PTHrP by alveolar type II cells, and by bones from rats exposed to 0 x g, suggesting that PTHrP signaling has been exploited for adaptation to 1 x g. PTHrP/PTHrP receptor signaling is upregulated by stretching alveolar type II cells and intersitial lung fibroblasts, whereas overdistension downregulates PTHrP and PTHrP receptor mRNA, further suggesting an evolutionary adaptation. Both surfactant homeostasis and alveolar capillary perfusion are under PTHrP control, indicating that alveolization and ventilation/perfusion matching may have evolved under the influence of PTHrP signaling. Phylogenetic analysis of lung evolution reflects the concomitant increases in alveolar surface area and surfactant production by "amplifying" the PTHrP pathway signal. This mechanism is discussed as a function of increased evolutionary respiratory demand to keep up with the increased metabolic demand for oxygen, and the role of the PTHrP signaling mechanism in leveraging this process.

Important role for the V-type H(+)-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor.

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.

Ligand-selective dissociation of activation and internalization of the parathyroid hormone (PTH) receptor: conditional efficacy of PTH peptide fragments.

G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.

The complexity of parathyroid hormone-related protein signalling.

Our understanding of the mode of action of parathyroid hormone-related protein (PTHrP) has changed profoundly during the last decade. Most PTHrP activities are mediated by membrane receptors through autocrine/paracrine pathways. However, both endogenous and exogenous PTHrP also appear to have intracrine effects through translocation into the nucleus. The present review proposes unconventional PTHrP signalling, based on novel clues. First, PTHrP binding to its membrane receptor triggers internalization of the whole complex, mediated by beta-arrestin. There is growing evidence that the receptor and arrestin are the effectors of biological responses, rather than the ligand (or in addition to the ligand). Second, the existence of putative PTHrP targets within the cytoplasm is beginning to be supported. Recent findings of interactions between a COOH-terminus of PTHrP and beta-arrestin and between the PTHrP receptor and 14-3-3 proteins represent the starting point for identification of intracellular partners of both the hormone and its receptor.

Deficiency of insulin receptor substrate-1 impairs skeletal growth through early closure of epiphyseal cartilage.

Morphological analyses in and around the epiphyseal cartilage of mice deficient in insulin receptor substrate-1 (IRS-1) showed IRS-1 signaling to be important for skeletal growth by preventing early closure of the epiphyseal cartilage and maintaining the subsequent bone turnover at the primary spongiosa. IRS-1 is an essential molecule for intracellular signaling by IGF-I and insulin, both of which are potent anabolic regulators of cartilage and bone metabolism. To clarify the role of IRS-1 signaling in the skeletal growth, morphological analyses were performed in and around the epiphyseal cartilage of mice deficient in IRS-1 (IRS-1(-/-)), whose limbs and trunk were 20-30% shorter than wildtype (WT) mice. The epiphyseal cartilage and the primary spongiosa at proximal tibias of homozygous IRS-1(-/-) and WT male littermates were compared using histological, immunohistochemical, enzyme cytohistochemical, ultrastructural, and bone histomorphometrical analyses. In and around the WT epiphyseal cartilage, IRS-1 and insulin-like growth factor (IGF)-1 receptors were widely expressed, whereas IRS-2 was weakly localized in bone cells. Chronological observation revealed that height of the proliferative zone and the size of hypertrophic chondrocytes were decreased in WT mice as a function of age, and these decreases were accelerated in the IRS-1 (-/-) cartilage, whose findings at 12 weeks were similar to those of WT at 24 weeks. In the IRS-1(-/-) cartilage, proliferating chondrocytes with positive proliferating cell nuclear antigen (PCNA) or parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor immunostaining had almost disappeared by 12 weeks. Contrarily, TUNEL+ apoptotic cells were increased in the hypertrophic zone, at the bottom of which most of the chondrocytes were surrounded by the calcified matrix, suggesting the closure of the cartilage. In the primary spongiosa, bone volume, alkaline phosphatase (ALP)+ osteoblasts, TRACP+ osteoclasts, and the osteopontin-positive cement line were markedly decreased. Bone histomorphometrical parameters for both bone formation and resorption were significantly lower in IRS-1(-/-) mice, indicating the suppression of bone turnover. The IRS-1(-/-) epiphyseal cartilage exhibited insufficient proliferation of chondrocytes, calcification of hypertrophic chondrocytes, acceleration of apoptosis, and early closure of the growth plate. Thus, the data strongly suggest that IRS-1 signaling is important for the skeletal growth by preventing early closure of the epiphyseal cartilage and by maintaining the subsequent bone turnover at the primary spongiosa.

Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity.

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.

Parathyroid hormone-related protein is an essential growth factor for human clear cell renal carcinoma and a target for the von Hippel-Lindau tumor suppressor gene.

Clear cell renal carcinoma (CCRC) is responsible for 2% of cancer-related deaths worldwide and is resistant to virtually all therapies, indicating the importance of a search for new therapeutic targets. Parathyroid hormone-related protein (PTHrP) is a polyprotein derived from normal and malignant cells that regulates cell growth. In the current study, we show that blocking PTHrP with antibodies or antagonizing the common parathyroid hormone (PTH)/PTHrP receptor, the PTH1 receptor, dramatically blunts the expansion of human CCRC in vitro by promoting cell death. Importantly, in nude mice, anti-PTHrP antibodies induced complete regression of 70% of the implanted tumors by inducing cell death. In addition, we demonstrate that the von Hippel-Lindau tumor suppressor protein, which functions as a gatekeeper for CCRC, negatively regulates PTHrP expression at the post-transcriptional level. These studies indicate that PTHrP is an essential growth factor for CCRC and is a novel target for the von Hippel-Lindau tumor suppressor protein. Taken together, these results strongly suggest that targeting the PTHrP/PTH1 receptor system may provide a new avenue for the treatment of this aggressive cancer in humans.

Patterns of linkage disequilibrium and haplotype distribution in disease candidate genes

BackgroundThe adequacy of association studies for complex diseases depends critically on the existence of linkage disequilibrium (LD) between functional alleles and surrounding SNP markers.ResultsWe examined the patterns of LD and haplotype distribution in eight candidate genes for osteoporosis and/or obesity using 31 SNPs in 1,873 subjects. These eight genes are apolipoprotein E (APOE), type I collagen α1 (COL1A1), estrogen receptor-α (ER-α), leptin receptor (LEPR), parathyroid hormone (PTH)/PTH-related peptide receptor type 1 (PTHR1), transforming growth factor-β1 (TGF-β1), uncoupling protein 3 (UCP3), and vitamin D (1,25-dihydroxyvitamin D3) receptor (VDR). Yin yang haplotypes, two high-frequency haplotypes composed of completely mismatching SNP alleles, were examined. To quantify LD patterns, two common measures of LD, D' and r2, were calculated for the SNPs within the genes. The haplotype distribution varied in the different genes. Yin yang haplotypes were observed only in PTHR1 and UCP3. D' ranged from 0.020 to 1.000 with the average of 0.475, whereas the average r2 was 0.158 (ranging from 0.000 to 0.883). A decay of LD was observed as the intermarker distance increased, however, there was a great difference in LD characteristics of different genes or even in different regions within gene.ConclusionThe differences in haplotype distributions and LD patterns among the genes underscore the importance of characterizing genomic regions of interest prior to association studies.

Identification and characterization of two parathyroid hormone-like molecules in zebrafish.

Zebrafish (Danio rerio) have receptors homologous to the human PTH (hPTH)/PTHrP receptor (PTH1R) and PTH-2 receptor (PTH2R) and an additional receptor (PTH3R) with high homology to the PTH1R. To find natural ligands for zPTH1R and zPTH3R, we searched the zebrafish genomic database and discovered two distinct regions that, when translated (zPTH1 and zPTH2), showed high homology to hPTH. Isolation of cDNAs and determination of the intron/exon boundaries revealed genomic structures which were similar to known PTHs. Peptides consisting of the first 34 amino acids after the pre- and prosequences of the zebrafish PTHs (zPTHs) were synthesized and were shown to be fully active at the hPTH1R. zPTH2(1-34) was, however, approximately 30-fold less potent at the zPTH1R than hPTH(1-34), hPTHrP(1-36), and zPTH1(1-34). When tested with zPTH3R, zPTH1(1-34) and hPTHrP(1-36) showed similar potencies, whereas the potency of zPTH2(1-34) was moderately (3-fold) reduced. To determine whether other fishes have multiple PTHs, we searched the genomic database of the Japanese pufferfish (Takifugu rubripes) and identified zPTH1 and zPTH2 homologs. Phylogenetic analysis showed that PTHs from zebrafish and pufferfish are more closely related to each other than to known mammalian PTH homologs or to PTHrP and tuberoinfundibular peptide of 39 residues. This is consistent with evolution of two teleost PTH-like peptides occurring after the evolutionary divergence between fishes and mammals. Overall, the PTH system appears more complex in fishes than in mammals, providing evidence of continued evolution in nontetrapod species. The availability of multiple forms of fish PTH and their receptors provide additional tools for PTH ligand/receptor structure-function studies.

Expression of cartilage growth plate signalling molecules in chondroblastoma.

Chondroblastoma (CB) is a rare benign tumour (<1% of all bone tumours) involving epiphyseal long bones (male:female 1.5:1). During development, and in the postnatal period, IHh/PTHrP and FGF signalling molecules control the space and timing of chondrocyte differentiation. Considering the close relationship of CB with the growth plate (age and location), the expression of proteins involved in epiphyseal growth regulation was studied. Twelve cases of CB were retrieved. Immunohistochemistry was performed using antibodies against fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR-1), FGFR-3, bcl-2, p21, parathyroid hormone-related peptide (PTHrP), and parathyroid hormone-related peptide receptor (PTHR1). Three observers evaluated haematoxylin and eosin (H&E)-stained and immunostained slides independently. Semi-quantitative estimation of the matrix, the type of matrix, and immunostaining was performed. Cellular and matrix-rich areas were evaluated separately. Diverse amounts and types of matrix were present in different tumours, as well as within individual tumours. Signalling molecules were expressed in 50-100% of the cases. Higher levels of expression were found in cellular areas than in matrix-rich areas, especially for PTHR1, bcl-2, and FGFR-3. CB is an unusual entity affecting specific sites, showing that both IHh/PTHrP and FGF signalling are active. Higher expression was found in cellular than in matrix-rich areas, as in the proliferating/pre-hypertrophic growth plate zone in comparison with the hypertrophic/calcifying zone. Previous studies have shown the same molecules to be expressed with a similar pattern in chondrosarcomas. The sum of the evaluated features indicates that CB is a neoplasm originating from a mesenchymal cell committed towards chondrogenesis via active growth plate signalling pathways.

THE ROLE OF FIBROBLAST TRANSDIFFERENTIATION IN LUNG EPITHELIAL CELL PROLIFERATION, DIFFERENTIATION, AND REPAIR IN VITRO

Parathyroid hormone-related protein (PTHrP) expression is necessary for differentiation of mesenchymal lipofibroblasts, which induce epithelial type II (TII) cell differentiation, both of which are necessary for alveolarization. PTHrP deficiency may be associated with bronchopulmonary dysplasia (BPD), characterized by truncation of alveolarization among preterm infants. This is supported by the baboon model of BPD (failure of alveolarization) that manifests PTHrP deficiency. We provide evidence that TII cell PTHrP expression is downregulated by alveolar overdistension, resulting in the transdifferentiation of lipofibroblasts to myofibroblasts, characterized by progressive loss of PTHrP receptor expression and triglyceride content, and sequential upregulation of &#102 -smooth muscle actin ( &#102 SMA), typifying fibrosis. PTHrP reverses the downregulation of the PTHrP receptor and upregulation of &#102 SMA, reverting myofibroblasts to a lipofibroblast genotype. When TII cells are co-cultured with lipofibroblasts, they proliferate and differentiate, expressing surfactant protein-B; in contrast, TII cells co-cultured with myofibroblasts fail to develop, mimicking the failed alveolarization associated with BPD. Treatment of myofibroblasts with 15-deoxy- &#106 12, 14 prostaglandinJ 2 (PGJ 2 ) stimulates ADRP expression, reconstituting the lipofibroblast phenotype. PGJ 2 -treated myofibroblasts promote TII cell growth and surfactant protein-B expression, indicating that failed alveolarization due to transdifferentiation is reversible. We conclude that alveolar overdistension can cause fibroblast transdifferentiation, resulting in failed alveolarization.