PubMed Health⌕ Search

Biomedical subjects

J M Moseley

Publications and source records attributed to J M Moseley.

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

PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line (HaCaT) during the cell cycle.

Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.

Aphidicolin↗
PubMed1997

Vascular effects of PTHrP (1-34) and PTH (1-34) in the human fetal-placental circulation.

The aim of this study was to examine the vasodilatory effects of parathyroid hormone-related protein (PTHrP) (1-34) and parathyroid hormone (PTH) (1-34) on the human fetal-placental circulation utilising an in vitro placental perfusion model. In all experiments, the vasculature of an isolated human placental cotyledon was pre-constricted with the thromboxane A2 mimetic U46619. A simple dose of PTHrP (1-34) or PTH (1-34) (1.7-300 nM) was then infused into the fetal-placental circulation of the cotyledon. In other experiments, cotyledons were repeatedly infused with PTHrP (1-34) or PTH (1-34) (51.3 nM). Vasodilatory responses were significantly reduced in response to repeated exposure to PTHrP (1-34) (P < 0.001), indicating that this peptide desensitizes the fetal-placental vasculature. PTHrP (1-34) and PTH (1-34) equipotently stimulated a significant vasodilation of the fetal-placental circulation (P < 0.0001). The PTHrP receptor antagonist [Asn10, Leu 11]PTHrP (7-34) (102 nM) was infused in U46619-constricted placentae in the presence and absence of PTHrP (1-34) (10.2 nM). The PTHrP antagonist alone had no significant effect in the fetal-placental circulation. The antagonist significantly attenuated the response to PTHrP (1-34) (P < 0.015). Based on the data obtained in this study it is suggested that locally produced PTHrP (1-34) may be involved in the regulation of normal human fetal-placental vascular tone in autocrine and/or paracrine fashion.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

The expression of parathyroid hormone-related protein mRNA and immunoreactive protein in human amnion and choriodecidua is increased at term compared with preterm gestation.

Parathyroid hormone-related protein (PTHrP) gene expression and/or immunoreactive protein have previously been identified in the uterus and intrauterine gestational tissues. The putative roles of PTHrP during pregnancy include vasodilatation, regulation of placental calcium transfer, uterine smooth muscle relaxation and normal fetal development. The aims of this study were 1) to determine the tissue-specific and temporal expression of PTHrP mRNA and immunoreactive protein in human gestational tissues collected at preterm and term; and 2) to determine the effect of labour on PTHrP expression by collecting these tissues from women undergoing elective caesarean section (before labour), intra-partum caesarean section during spontaneous-onset labour (during labour), and women with spontaneous labour and normal vaginal delivery (after labour). Total RNA and protein were extracted from placenta, amnion (over placenta and reflected) and choriodecidua for analysis by Northern blot (using a specific human PTHrP cDNA probe), and by N-terminal PTHrP RIA respectively. In amnion over placenta, reflected amnion and choriodecidua both PTHrP mRNA relative abundance and immunoreactive protein were significantly elevated at term compared with preterm (P < 0.01). At term, both PTHrP and its mRNA were significantly greater in amnion than in placenta and choriodecidua (P < 0.05). Also, both PTHrP and its mRNA were significantly elevated in amnion over placenta compared with reflected amnion (P < 0.05). The expression of PTHrP and its mRNA did not change in association with term labour or rupture of the fetal membranes, therefore this study provides no evidence for a specific PTHrP role in the onset and/or maintenance of term labour. However, the significant up-regulation of PTHrP mRNA and protein in the fetal membranes at term compared with preterm suggests an important role in late human pregnancy.

Amnion↗
PubMed1997

Parathyroid hormone-related protein: hormone and cytokine.

While PTHrP acts as a hormone when it is produced in excess by certain cancers, and perhaps also in lactating women, the foetus, and lower vertebrates, it seems clear that PTHrP is an important paracrine regulator of several tissue-specific functions. Its roles in smooth muscle relaxation, placental calcium transport, and bone development are becoming firmly established. However, it is likely to be an important player in the control of cellular growth and differentiation, although much of our understanding of this role to date comes from indirect evidence. The next decade will be an exciting time in defining further both the hormonal and paracrine actions of PTHrP.

Animals↗
PubMed1996

PC8 [corrected], a new member of the convertase family.

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23-11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

Amino Acid Sequence↗
PubMed1996

Expression of parathyroid hormone-related protein in cells of osteoblast lineage.

The expression of parathyroid hormone-related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria-derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106-01 and -06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT-PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106-01 and -06 cells. Treatment of UMR 201 cells with all-trans-retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time-dependent decrease in PTHrP mRNA levels as determined by RT-PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1-34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the "osteoblast-rich" populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrP expression in cuboidal osteoblasts located adjacent to bone and in spindle-shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Animals↗
PubMed1996

Effects of continuous calcitonin treatment on osteoclast-like cell development and calcitonin receptor expression in mouse bone marrow cultures.

Continuous treatment with calcitonin (CT) to inhibit osteoclastic bone resorption results in acquired resistance. The mechanisms of this "escape" phenomenon are not yet established. The aim of this study was to examine the effects of continuous treatment with CT on the generation of osteoclasts and calcitonin receptor (CTR) expression in mouse bone marrow cultures. This was done by daily CT treatment of mouse bone marrow cultures from day 0, when only undifferentiated mononuclear precursors of osteoclast-like cells were present, or commencing from day 6, when differentiated osteoclast-like cells were abundant. The response to CT treatment was determined by quantitation of cells positive for tartrate-resistant acid phosphatase (TRAP) and binding of 125I-salmon CT. Calcitonin receptor and TRAP mRNA levels were determined using semi-quantitative reverse transcription/polymerase chain reaction. When cultures were treated with CT from day 0, TRAP-positive multinucleated cells appeared. These cells expressed only very low levels of CTR or CTR mRNA and were morphologically indistinguishable from osteoclast-like cells formed in control cultures. They also displayed the ability to resorb bone. Continuous CT treatment of cultures from day 6 rapidly reduced the CTR mRNA levels, with a t1/2 of 6 to 12 h, and these levels remained low thereafter. 125I-salmon CT binding capacity, as determined by autoradiography, was lost in parallel. These effects were specific for the CTR since there was no consistent effect on TRAP mRNA levels. Based on these data, we suggest that the "escape" phenomenon may result from a prolonged CT-induced loss of CT responsiveness due, at least in part, both to reduced synthesis of CTR, and to the appearance in bone of CTR-deficient osteoclasts.

Acid Phosphatase↗
PubMed1995

Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein.

Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance. Cleavage products were identified by N-terminal sequencing. Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108. Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity. Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner.

Arginine↗
PubMed1995

Calcitonin receptor isoforms in mouse and rat osteoclasts.

Calcitonin receptors (CTRs) from several species have recently been cloned and shown to belong to the 7 transmembrane domain class of receptor. We have identified two CTR isoforms in the rat, termed C1a and C1b, identical except for a 37-amino-acid insert in the putative second extracellular domain of C1b. To examine the CTR isoforms expressed in rat and mouse osteoclasts and the time course of their appearance in culture, bone marrow cells were cultured from C57/Bl6J mice and osteoclasts were isolated from newborn rat long bones. CTR-bearing cells were detected by autoradiography of 125I-salmon CT binding, and cultures were stained for tartrate-resistant acid phosphatase (TRAP). RNA was extracted from parallel cultures, and CTR mRNA was detected by Northern blot analysis, using a rat digoxigenin-labeled riboprobe. Characterization of mRNA for the CTR isoforms was by reverse transcription-polymerase chain reaction (RT-PCR) using primer sets and oligonucleotide probes specific for the two rat receptor isoforms. In mouse marrow cultures, TRAP positive mononucleated cells were present by day 2 of culture at which time CTR positive cells were few. Multinucleated cells with both these markers were seen only from day 4 and later. By Northern analysis of total RNA, a band of approximately 4 kb could be detected in day 4 and later cultures. RT-PCR showed that mouse homologs of both C1a and C1b mRNA species were expressed early in cultures of mouse osteoclasts, although at each time C1a appeared to predominate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Evidence for the synthesis of parathyroid hormone-related protein (PTHrP) by nontransformed clonal rat osteoblastic cells in vitro.

Parathyroid hormone-related protein (PTHrP) is synthesized by a variety of tumors and is thought to be the main cause of the clinical syndrome of humoral hypercalcemia of malignancy (HHM). In addition to its parathyroid hormone (PTH)-like actions, novel actions of PTHrP on placental calcium transport and inhibition of in vitro osteoclast activity have been demonstrated. The fact that osteoblasts act as mediators of osteoclastic bone resorption prompted us to investigate whether nontranformed, osteoblastlike cells produce PTHrP. PTHrP has been detected in developing human fetal bones and in rat long bones in culture. For this study, osteogenic cells, CRP 5/4 and CRP 10/30, were employed. Both cell types represent clonal bone cell populations established from 1-day-old rats. While CRP 10/30 cells express the osteoblastic phenotype, CRP 5/4 cells resemble cells with preosteoblastic properties. With a radioimmunoassay (RIA), utilizing antiserum directed against the amino-terminal PTHrP(1-40), it was found that both cell types synthesize PTHrP constitutively. CRP 10/30 cells produce about twice as much as CRP 5/4 cells. Transforming growth factor-beta (TGF-beta 1) was shown to increase the synthesis of PTHrP in CRP 5/4 cells by about 2.5-fold, while in CRP 10/30 cells it caused an approximate 50% reduction of PTHrP. Employing the reverse transcriptase polymerase chain reaction (RT-PCR) technique it was found that both bone cell types express mRNA for PTHrP and that the modulation of the PTHrP mRNA levels by TGF-beta 1 in CRP 5/4, and to a lesser degree in CRP 10/30 cells, was reflected in a change in the level of PTHrP protein in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Short treatment of osteoclasts in bone marrow culture with calcitonin causes prolonged suppression of calcitonin receptor mRNA.

Cells exhibiting osteoclast characteristics of calcitonin receptors (CTRs) and tartrate-resistant acid phosphatase (TRAP) histochemistry are formed in murine bone marrow cultures treated with 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We have previously demonstrated that CTR mRNA is highly expressed in these cultures. The aim of this study was to investigate homologous regulation of the CTR, and regulation of TRAP expression in osteoclast-like cells after brief treatment with salmon CT (sCT). Murine bone marrow cells were cultured in 9 cm dishes in the presence of 10 nmol/L 1,25-(OH)2D3. On day 6 of culture, when multinucleated cells were abundant, the cells were treated with 1 nmol/L sCT for 1 h. Both control and treated cells were then harvested at intervals up to 72 h posttreatment, and both CTR and TRAP mRNA levels assessed by reverse-transcription PCR (RT-PCR). In parallel cultures, cells with CTR expression detectable by autoradiography, and TRAP positivity by histochemistry, were counted. The effects of brief sCT treatment could be seen 6 h after treatment when the CTR RT-PCR product was markedly reduced. Total recovery of CTR mRNA levels had not occurred even after 72 h. Calcitonin treatment had little effect on TRAP mRNA levels. There was no difference in the numbers of multinucleated TRAP(+) osteoclast-like cells between treated and control cells. These results indicate that brief sCT treatment, while not influencing multinucleated osteoclast-like cell number, causes specific, acute reduction of CTR mRNA in bone marrow culture-derived osteoclasts. The prolonged decrease in CTR mRNA levels suggests that recovery may require new osteoclast formation, and indicates that regulation of the CTR in cells of the osteoclast lineage is different from that in nonosteoclastic cells and tissues.

Acid Phosphatase↗
PubMed1995

Parathyroid hormone-related protein (PTHrP) concentrations in human amniotic fluid during gestation and at the time of labour.

To establish the changes associated with gestational age and labour status in parathyroid hormone-related protein (PTHrP) concentrations in the amniotic fluid, human amniotic fluid was collected from non-labouring and labouring women at < 37 weeks of gestation (preterm) and at term (> or = 37 weeks). PTHrP was assayed by a specific N-terminal radioimmunoassay. PTHrP concentrations in amniotic fluid obtained from non-labouring women were significantly lower at preterm (15-36 weeks; 14.1 +/- 2.5 pmol L(-1); n = 11) than at term (37-42 weeks; 39.3 +/- 7.6 pmol L(-1); n = 16; P < 0.0009). Concentrations of PTHrP in amniotic fluid obtained from labouring women were also significantly lower at preterm (27-36 week; 12.2 +/- 4.7 pmol L(-1); n = 4; P < 0.01) than at term (37-42 weeks; 63. 8 +/- 19.6 pmol L(-1); n = 9). There were no significant changes in concentration associated with labour status, either at preterm or at term. The physiological significance of elevated amniotic fluid concentrations of PTHrP has yet to be established, but the data are consistent with the suggestion that PTHrP plays a role in fetal membrane function during late gestation.

Amniotic Fluid↗
PubMed1995

Heterogeneity of the calcitonin receptor: functional aspects in osteoclasts and other sites.

The recent cloning of the calcitonin receptor reveals it as a member of a new family of 7-transmembrane, G protein-linked receptors. Data from rat, mouse and human receptor cloning reveal that in each of these species the receptor exists in more than one form, most likely the result of alternate, splicing. In the rat, the two forms are C1a and C1b, the latter differing from C1a in that it contains a 37-amino acid insert in the second extracellular domain. The two receptor isoforms differ in their distribution in vivo, with the C1b predominantly in the central nervous system and C1a in other tissues. Both forms have been shown to be present in mature and developing osteoclasts, with the C1a isoform predominating. The two isoforms differ in their kinetics and pharmacological properties, with C1b being virtually unable to bind the human and rat calcitonins but readily binding salmon calcitonin. It will be important to elucidate the physiological significance of the structural heterogeneity of the calcitonin receptor.

Animals↗
PubMed1995

Parathyroid hormone-related protein.

We review the current state of knowledge of the molecular properties and actions of parathyroid hormone-related protein (PTHrP) both in cancer patients and in normal physiology. PTHrP is a common product of squamous cancers and is the major mediator of the syndrome of humoral hypercalcemia of malignancy (HHM) by its actions through parathyroid hormone receptors in bone and kidney. Recently developed radioimmunoassays and tissue localization techniques indicate that PTHrP is produced by many more cancers than was originally indicated by clinical studies and that it contributes significantly to malignancy-related hypercalcemia associated with other etiologies, for example, cancers metastatic to bone and hematological malignancies. The gene encoding PTHrP is complex, with multiple exons coding for up to 12 alternate transcripts and three different length proteins, potentially in a tissue-specific manner, by the use of three promoters. Its expression is regulated by hormones and growth factors, and the untranslated exons display features in common with many cytokine genes. Although potential endocrine actions of PTHrP are evident in fetal development, further evidence suggesting that the normal physiological role of PTHrP is predominantly as a locally produced regulator/cytokine comes from localization studies and investigations of its actions in a variety of tissues. Such studies indicate that in addition to its parathyroid hormone-like actions, PTHrP has multiple activities, including those in fetal development, placental calcium transfer, lactation, smooth muscle relaxation, and on epithelial cell growth. Although PTHrP was discovered because of its production by cancers, evidence for its actions as a local regulator highlights the importance of understanding its roles not only in the etiology of HHM in cancer patients but also in normal tissues.

Amino Acid Sequence↗
PubMed1994

Dexamethasone regulation of parathyroid hormone-related protein (PTHrP) expression in a squamous cancer cell line.

Dexamethasone regulation of PTHrP expression has been studied in an epidermal squamous cancer cell line COLO 16, which secretes immunoreactive PTHrP into conditioned medium. Dexamethasone was found to suppress PTHrP expression in a time- and dose-dependent manner, which was reversible upon removal of dexamethasone. The half-maximal effective concentration of dexamethasone was 1 nM and an effect of dexamethasone on PTHrP mRNA was first observed after 2 h of treatment, with maximal inhibition by 6 h. Dexamethasone action on PTHrP expression was steroid specific since progestin, 5alpha-dihydroxytestosterone and oestrogen did not regulate PTHrP expression in COLO 16 cells. The gluocorticoid/progesterone receptor antagonist RU486 inhibited the dexamethasone effect, indicating glucocorticoid receptor-mediated regulation of PTHrP expression. The half-life of PTHrP mRNA in COLO 16 cells was approximately 120 min and was not altered by treatment of cells with dexamethasone. Nuclear run-on assays revealed that dexamethasone reduced PTHrP gene transcription in COLO 16 cells. Transient transfection assays with a series of reporter gene constructs encompassing 3.5 kb of the 5' end of the PTHrP gene failed to identify a region of the gene responsible for glucocorticoid down-regulation. PCR of reverse-transcribed RNA from COLO 16 cells revealed that dexamethasone down-regulated transcripts driven from all three promoters (i.e., the TATA promoters 5' to exons I and IV and the GC-rich promoter 5' to exon III) of the human PTHrP gene.

Carcinoma, Squamous Cell↗
PubMed1994

Effect of parathyroid hormone-related protein (PTHrP) on the contractility of the myometrium and localization of PTHrP in the uterus of pregnant rats.

The effect of parathyroid hormone-related protein (PTHrP) on the contractility of uterine segments taken from pregnant rats and the localization of PTHrP in the uterus during pregnancy were investigated. PTHrP(1-34) had a potent inhibitory effect on spontaneous contractions of the longitudinal layer of uterine myometrium taken from rats at day 4 of pregnancy (IC50 1.6 nmol l-1). In low calcium De Jalon's solution, it also decreased base-line tension (IC50 1.5 nmol l-1) in a concentration-dependent manner. The effect of PTHrP(1-34) on uterine motility decreased as pregnancy progressed until day 13, after which PTHrP(1-34) had no measurable effect on uterine contractility. In contrast, PTHrP(1-34) had no effect on the contractions of the circular smooth muscle of the uterus at any stage of pregnancy. PTHrP(50-69) had no effect on the contractility of either muscle layer of the myometrium. A temporal pattern of staining for PTHrP in the uterus of pregnant rats was observed, and the changes in the staining patterns in the endometrium and myometrium were different in each layer. These data suggest that PTHrP may have at least two distinct roles in the uterus, a relaxing action on longitudinal muscle that depends on the hormonal state of the rat and a novel effect either intraluminally or within the endometrial layer.

Animals↗