Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo.

Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1-34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.

Central nervous system inflammatory demyelinating disorders among Hong Kong Chinese.

Classical multiple sclerosis (CMS) and neuromyelitis optica spectrum disorders (NMOSD) are distinct central nervous system inflammatory demyelinating disorders (CNS IDD). Early diagnosis of CNS IDD is important as appropriate immunotherapies to optimize prognosis. We studied the diagnoses of CNS IDD among Hong Kong Chinese in a hospital-based setting. Consecutive Chinese patients who presented to our hospital with clinically isolated syndrome and subsequently diagnosed to have CNS IDD from 1980 to 2010 were reviewed. Patients with known diagnosis of CNS IDD referred for further care were excluded. Serial sera were assayed for aquaporin-4 autoantibodies (AQP4 Ab), at least 3 assays within 2-5years. A total of 210 patients diagnosed to have CNS IDD with disease duration of at least 2years were studied. Among 198 patients with serial sera available, 40 (20.2%, 20 had NMO and 20 other NMOSD) were AQP4 Ab-positive. Four patients who were AQP4 Ab-negative on the initial assay converted to AQP4 Ab-positive on repeated assays. The diagnoses of 210 patients were CMS in 88 (41.9%), NMOSD 47 (22.4%, 27 NMO, 20 other NMOSD), single attack of myelitis 23 (11.0%), single attack of optic neuritis 21 (10.0%), relapsing myelitis 10 (4.8%), acute disseminated encephalomyelitis (ADEM) 9 (4.3%), relapsing optic neuritis in 6 (2.9%), opticospinal multiple sclerosis 3 (1.4%) and single attack of brainstem encephalitis 3 (1.4%). Compared to CMS, NMOSD patients had older onset age, lower frequencies of brain MRI abnormalities and CSF OCB, higher frequency of LETM, higher CNS inflammation attack frequency in the first 2years, worse clinical outcome with higher EDSS score and mortality rate. This hospital-based study suggests that CMS (41.9%) and NMOSD (22.4%) are the most common CNS IDD among Hong Kong Chinese. NMOSD has worse clinical outcome than CMS. Detection of AQP4 Ab facilitates early diagnosis and prompts immunotherapies of NMOSD.

Plasma amyloid-ß oligomers level is a biomarker for Alzheimer's disease diagnosis.

Amyloid beta (Aß), especially Aß oligomers, is important in Alzheimer's disease (AD) pathogenesis. We studied plasma Aß(40), Aß(42), and Aß oligomers levels in 44 AD patients and 22 non-demented controls. Cognitive functions were assessed by Chinese version of mini-mental state examination (MMSE), Abbreviated Metal Test (AMT), Alzheimer's Disease Assessment Scale Cognitive Subscale (ADAS-cog). Plasma Aß monomers and oligomers levels were measured by ELISA. We found that the median plasma Aß(40) and Aß(42) levels were similar between AD and controls, and without significant correlation with cognition. Plasma Aß oligomers level was higher in AD than controls (642.54 ng/ml [range 103.33-2676.93] versus 444.18 ng/ml [range 150.19-1311.18], p=0.047), and negatively correlated with cognition. In multivariate logistic regression analysis, the highest tertile of Aß oligomers levels showed an increased risk of AD than the combined group of middle and lowest tertiles (OR=8.85, p=0.013), after adjustment of gender, age and APOE4 genotype. Increased plasma Aß oligomers level was associated with decreased MMSE and AMT scores (p=0.037, p=0.043, respectively) and increased ADAS-cog score (p=0.036), suggesting negative correlation with cognitive function. We concluded that plasma Aß oligomers level is an useful biomarker for AD diagnosis.

Detection of endocrine disruptors - from simple assays to whole genome scanning.

Endocrine disruptors frequently bear little structural relationship to the hormone whose actions they disrupt. Consequently, the threat of an uninvestigated chemical cannot easily be assessed. Here three different approaches to assessment are discussed. The first presumes an endocrine-disrupting property, following which a cell model capable of responding to such a hormone is used. Although simple and cheap, it provides limited data. A second approach involves multiple assays to detect multiple hormones. Increasing the amount of data increased the difficulty in assessing the significance of results. To meet this problem, cluster analysis based on a simple mathematical matrix was adopted. The matrix was used to determine (i) a limited number of assays to identify a maximum number of endocrine disruptors and (ii) the chemicals with the most wide-ranging effects. A third approach was a whole genome expression analysis based on expression of mRNAs in human TE671 medulloblastoma cells. Expression of individual mRNAs was assessed using the Affymetrix GeneChip(®) Human Genome U133 Plus 2.0 chip. The significance of differential expressed genes was assessed based on gene ontology and pathways analyses using DAVID and GenMaPP programs. The results illustrated the very wide-ranging effects of these chemicals across the genome.

Aquaporin-4 autoantibodies cause asymptomatic aquaporin-4 loss and activate astrocytes in mouse.

BACKGROUND: Neuromyelitis optica (NMO) is a central nervous system inflammatory demyelinating disorder. Up to 90% of patients are seropositive for aquaporin-4 autoantibodies (AQP4 Ab). The pathogenetic mechanisms underlying clinical onset and relapse of NMO are uncertain. OBJECTIVE: Study the pathogenicity of AQP4 Ab in the absence of complement activation. METHODS: Female C57BL/6N mice (human IgG cannot activate mouse complements) pretreated with complete Freund's adjuvant (CFA, day 0) and pertussis toxin (PTx, day 0 and day 2) were transferred with IgG isolated from serum of healthy subjects or NMO patients (AQP4 Ab-positive or negative) intraperitoneally (day 7-9). Mice were observed for signs of experimental autoimmune encephalomyelitis (EAE) by standard 6-grade EAE scores. Spinal cord was obtained at day 11 for immunohistochemistry. RESULTS: None of the mice had clinical signs of encephalomyelitis, inflammatory cells infiltration or demyelination of spinal cord. CFA and PTx induce BBB breakdown evidenced by leakage of human IgG into cord parenchyma. Patchy areas of AQP4 loss were observed in spinal cord of mice transferred with IgG from AQP4 Ab-positive NMO patients but not in mice transferred with IgG from AQP4 Ab-negative NMO patients or healthy subjects; but there was no loss of glial fibrillary acidic protein immunoreactivity in all mice. Markedly increased proliferation of astrocytic processes suggestive of astrocytic activation was observed in mice transferred with IgG from AQP4 Ab-positive patients. CONCLUSION: AQP4 Ab cause asymptomatic AQP4 loss and astrocytic activation but not myelitis, demyelination or astrocytic cytotoxicity in spinal cord of mouse in the absence of complement activation.

Brain involvement in neuromyelitis optica spectrum disorders.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSDs) are severe inflammatory demyelinating disorders of the central nervous system. Brain involvement is increasingly recognized. OBJECTIVE: To study brain involvement in NMOSDs among Hong Kong Chinese patients. DESIGN: Retrospective study of patients with NMOSDs. SETTING: Tertiary medical center in Hong Kong. Patients  Thirty-four Hong Kong Chinese patients with NMOSDs of 2 years or longer were recruited. INTERVENTIONS: Brain and spinal cord magnetic resonance imaging was performed during NMOSD attacks and was repeated yearly for the first 3 years. MAIN OUTCOME MEASURES: We evaluated clinical features of NMOSDs associated with brain involvement and brain lesions on magnetic resonance imaging. RESULTS: Among 34 patients with NMOSDs of 2 years or longer, 20 (59%) had brain involvement. The mean age at onset among these 20 patients was 45.6 years (age range, 19-67 years); 18 were women. Eleven patients (32% of all the patients with NMOSDs) had clinical manifestation of brain involvement, 19 patients (56%) had brain abnormalities on magnetic resonance imaging consistent with inflammatory demyelination, and 2 patients (6%) fulfilled criteria for multiple sclerosis. Clinical manifestation of brain involvement included the following: trigeminal neuralgia; vomiting, vertigo, ataxia, dysphagia, and tetraparesis from lesions around the third and fourth ventricles and aqueduct; homonymous hemianopia, aphasia, hemiparesis, and cognitive impairment from extensive hemispheric white matter lesions; and ataxia, diplopia, hiccups, facial sensory loss, internuclear ophthalmoplegia, hemisensory loss, and hemiparesis from other lesions in the midbrain, pons, cerebellar peduncles, and medulla. Eight patients (24%) developed brainstem encephalitis clinically, and brainstem encephalitis was the initial clinical manifestation in 6 patients (18%). Brain abnormalities on magnetic resonance imaging were detected in brainstem in 15 patients (44%), hemispheric periventricular white matter in 7 patients (21%), deep white matter in 7 patients (21%), corpus callosum in 4 patients (12%), subcortical white matter in 3 patients (9%), thalamus in 2 patients (6%), hypothalamus in 1 patient (3%), basal ganglia in 1 patient (3%), internal capsule in 1 patient (3%), periaqueductal gray matter in 1 patient (3%), and around the third and fourth ventricles in 1 patient (3%); large confluent lesions were detected in 2 patients (6%). CONCLUSION: Brain involvement manifesting clinically as brainstem encephalitis is common among Hong Kong Chinese patients with NMOSDs.

Clinical outcome of relapsing remitting multiple sclerosis among Hong Kong Chinese.

BACKGROUND: Clinical outcome of Chinese relapsing remitting multiple sclerosis (RRMS) patients is uncertain. AIM: To study the long-term clinical outcome of Chinese RRMS patients. METHOD: RRMS patients with duration of 10 years or longer followed up in our hospital is retrospectively studied. RESULTS: 61 RRMS patients (75% female) were studied. Their mean symptom onset age was 25.9 years and mean duration was 20.6 years (range 10-33); 36% patients had received ß-interferon and 30% azathioprine. Their mean EDSS scores were 3.3 (range 1-7) and 4.7 (range 1-8) at 10 years and latest follow-up (mean duration 20.6 years) respectively. At 10 years, 30% patients had EDSS score ≤2, 34% EDSS 2.5-3.5, 20% EDSS 4.0-5.5 and 16% ≥6; 18% developed SPMS. At latest follow-up, 15% patients had EDSS ≤2, 20% EDSS 2.5-3.5, 19% EDSS 4.0-5.5 and 46% ≥6.0; 53% developed SPMS. The median time from symptom onset to EDSS 6 was 22 years. No differences were detected in demographic characteristics, presenting neurological features, number of attacks in first 2 years, neuroradiological findings and disease modifying therapies between patients with EDSS <6 and ≥6 at ten years. EDSS scores at 10 years and latest follow-up were similar for patients who had received ß-interferon and those who had not. CONCLUSION: Hong Kong Chinese RRMS patients may have worse long-term clinical outcome than Caucasian patients.

Aquaporin-4 water channel expression by thymoma of patients with and without myasthenia gravis.

BACKGROUND: Neuromyelitis optica (NMO) is a serious idiopathic inflammatory demyelinating disorder characterized by acute transverse myelitis and optic neuritis. A significant proportion of NMO patients are seropositive for NMO-IgG, an autoantibody targeting aquaporin-4 (AQP4) water channel. Paraneoplastic NMO associated various tumors were recently reported. AIM: We studied the expression of AQP4 by thymoma from patients with and without myasthenia gravis (MG). METHODS: Thymoma obtained from thymomectomy in patients with and without MG were studied by immunohistochemistry and western blot. RESULTS: Ten thymoma patients (9 with MG) and two control patients without thymoma or MG were studied. Immunohistochemistry revealed AQP4 immunoreactivity in cell membrane of thymoma cells from all ten thymoma specimens whereas thymic tissues from patients without thymoma or MG were negative for AQP4 immunoreactivity. Western blot revealed that lysates of nine of the ten thymoma specimens reacted with anti-human AQP4 antibody with a band of ~30 kDa compatible with the molecular weight of AQP4. Interestingly, immunofluorescence revealed that IgG isolated from 2 NMO patients seropositive for NMO-IgG bound to cell membrane of thymoma cells from all ten thymoma specimens while IgG from healthy control subject did not. CONCLUSION: Thymoma cells of patients with and without MG express AQP4. AQP4 autoantibodies from serum of NMO patients bound to AQP4 expressed on thymoma cell membrane.

Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes.

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

Communication between ephrinB2 and EphB4 within the osteoblast lineage.

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Neuromyelitis optica-IgG in idiopathic inflammatory demyelinating disorders amongst Hong Kong Chinese.

BACKGROUND: Idiopathic inflammatory demyelinating disorders (IIDD) affect the central nervous system. In classical multiple sclerosis (CMS), brain, optic nerves [optic neuritis (ON)] and spinal cord [acute transverse myelitis (ATM)] are affected. In neuromyelitis optica (NMO), optic nerves and spinal cord are predominantly affected. NMO-IgG, an autoantibody targeting aquaporin-4, is a marker for NMO. We studied the frequency and clinical relevance of NMO-IgG seropositivity in IIDD patients. METHODS: Neuromyelitis optica-IgG was detected by indirect immunofluorescence using primate cerebellum. RESULTS: Neuromyelitis optica-IgG was detected in six of 10 NMO patients (60%), six of 10 idiopathic relapsing transverse myelitis (IRTM) patients (60%), two of nine idiopathic relapsing ON patients (22%), one of 11 patients (9%) having single ON attack, one of 30 CMS patients (3%), and none of patients having single ATM attack or controls. Comparing NMO-IgG seropositive (n = 12) with NMO-IgG seronegative (n = 8) patients having NMO or IRTM, NMO-IgG seropositivity was associated with a higher relapse rate in first 2 years, 1.5 and 0.6 attacks/year for seropositive and seronegative groups respectively (P = 0.006), and non-significant trend towards more severe ON and myelitis with poorer clinical outcome. CONCLUSION: Neuromyelitis optica -IgG facilitates diagnosis of NMO spectrum disorders. NMO-IgG seropositivity is associated with higher relapse rate in first 2 years.

Gluteal compartment syndrome following abdominal aortic aneurysm repair: a case report.

Compartment syndromes occur when the elevated tissue pressure within a confined limb's myofascial compartment exceeds capillary pressure, with subsequent neurovascular compromise. In order to reduce disability and the consequences of ensuring ischemia, it is essential for early recognition and intervention. This is more commonly recognized in the calf. We report an unusual case of gluteal compartment syndrome after abdominal aortic aneurysm (AAA) repair.

Effects of plasticisers and related compounds on the expression of the soluble form of catechol-O-methyltransferase in MCF-7 cells.

Previously we have shown that E2 down regulates S-COMT expression. Here the effects of four phthalate esters and 4-(tert-octyl)phenol on the intra-cellular levels of S-COMT and COMT activity were studied in MCF-7 cells as a measure of estrogenic activity of these compounds. The four phthalate esters caused significant reductions in both S-COMT protein and COMT activity levels. These effects were inhibited by the ERalpha receptor antagonist ICI182780. 4-(tert-octyl)phenol also caused reductions in these parameters, but the effects were not abolished by ICI182780. Assay of S-COMT protein levels represents a simple and convenient method of assessing the estrogenic potential of a compound.

Estrogenic phenol and catechol metabolites of PCBs modulate catechol-O-methyltransferase expression via the estrogen receptor: potential contribution to cancer risk.

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

Uncoupling proteins: targets of endocrine disruptors?

The roles of uncoupling proteins (UCPs) are discussed. Particular attention has been paid to the roles of UCP2 to UCP5 as agents mediating thermogenesis, and to the concept of limited or "mild" uncoupling as a means of reducing oxidative stress. The role of the endocrine system, thyroid hormones and catecholamines, in regulating expression of UCPs is also discussed.

Effect of nuclear factor-kappa B inhibitors and peroxisome proliferator-activated receptor-gamma ligands on PTHrP release from human fetal membranes.

Parathyroid hormone-related protein (PTHrP) has been implicated in many processes during normal and pathological pregnancies. In the human fetal membranes, PTHrP exhibits cytokine-like actions. We have recently shown that inhibitors of the nuclear factor-kappa B (NF-kappaB) and activators of the peroxisome proliferator-activated receptor (PPAR)-gamma signalling pathways down-regulate cytokine release from human gestational tissues. Therefore, the aim of this study was to determine whether NF-kappaB and PPAR-gamma also regulate PTHrP release from human fetal membranes. Human amnion and choriodecidua explants were incubated in the absence (control) or presence of two known NF-kappaB inhibitors (1, 5 and 10 mM sulphasalazine (SASP) or 5, 10 and 15 mM N-acetyl-cysteine (NAC)), and two PPAR-gamma ligands (15 and 30 microM 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) or 15 and 30 microM troglitazone), under basal conditions. After 18 h incubation, the tissues were collected and NF-kappaB p65 DNA binding activity in nuclear extracts was assessed by ELISA, and the incubation medium was collected and the release of PTHrP was quantified by RIA. Treatment of amnion and choriodecidual tissues with SASP concentrations greater than 5 mM, 15 mM NAC, 30 microM 15d-PGJ(2) and 30 microM troglitazone significantly reduced the release of PTHrP (p < 0.05). This study demonstrates that PTHrP release from human fetal membranes is regulated by inhibitors of NF-kappaB, and ligands of PPAR-gamma.

PTH/PTHrP receptor and mid-molecule PTHrP regulation of intrauterine PTHrP: PTH/PTHrP receptor antagonism increases SHR fetal weight.

Parathyroid hormone-related protein (PTHrP) has important roles in fetal growth and development through stimulation of placental calcium transport, vasodilatation of the uteroplacental vasculature and regulation of cellular growth and differentiation. The growth restricted spontaneously hypertensive rat (SHR) has reduced fetal plasma, placental and amniotic fluid PTHrP concentrations compared to its progenitor, the Wistar Kyoto (WKY) rat. The aim of this study was to determine whether intrauterine PTHrP infusions can restore PTHrP levels and promote SHR fetal growth. PTHrP(1-34), midmolecule PTHrP(67-94), the PTH/PTHrP receptor antagonist [Asn(10), Leu(11)]-PTHrP(7-34) or vehicle were infused via a mini-osmotic pump between 10 and 20 days of gestation into the uterine lumen of SHR and WKY rats. Uterine, placental, amniotic fluid and plasma (fetal and maternal) PTHrP were measured via N-terminal radioimmunoassay. PTH/PTHrP receptor antagonism and mid-molecule PTHrP(67-94) induced endogenous intrauterine PTHrP production with receptor antagonism eliciting a greater and more wide spread effect. The PTH/PTHrP receptor antagonist [Asn(10), Leu(11)]-PTHrP(7-34) acting through a receptor other than the PTH/PTHrP receptor increased SHR fetal and placental weights above vehicle (P<0.05) to that of the WKY and restored SHR amniotic fluid volume (P<0.05). This was associated with a highly significant up regulation of placental, uterine and plasma (fetal and maternal) PTHrP (P<0.05). Modest increases in placental and uterine PTHrP (P<0.05) following intrauterine infusions of PTHrP(1-34) and PTHrP(67-94) had no effect on WKY and SHR fetal weight. Effective growth promoting actions of increased endogenous PTHrP were observed following PTH/PTHrP receptor antagonism rather than exogenous PTHrP administration. A novel finding was that mid-molecule PTHrP also up regulates endogenous intrauterine N-terminal PTHrP production supporting the existence of a mid-molecule receptor. This study highlights that an increase in endogenous uterine, placental and fetal plasma PTHrP following PTH/PTHrP receptor antagonism was associated with increased SHR fetal growth presumably by improving placental growth and function.

Impaired mammary function and parathyroid hormone-related protein during lactation in growth-restricted spontaneously hypertensive rats.

Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) during lactation, including stimulation of mammary and pup growth. As spontaneously hypertensive rat (SHR) pups are growth restricted compared with the control Wistar Kyoto (WKY), we examined the relative roles of pup suckling and maternal lactational environment on pup growth, mammary PTHrP, and milk PTHrP and calcium concentrations. SHR pups were lighter compared with the control from 6 days. SHR mammary PTHrP content and milk PTHrP were lower but maternal plasma PTHrP was raised compared with WKY. SHR mammary morphological development was also impaired compared with control. Cross fostering growth-restricted pups onto WKY mothers increased pup weight in association with normal mammary function and higher milk PTHrP and calcium. Control pups suckling on an SHR mother had reduced body weight. Both cross fostering groups were associated with increased maternal and milk PTHrP concentrations, indicating the importance of suckling, together with a functional mammary gland. The results suggested that impaired SHR mammary function and milk PTHrP are associated with a reduced SHR postnatal growth. Our data also indicated that milk and mammary PTHrP are regulated by different mechanisms but that they are influenced by the maternal lactational environment and the suckling pup.

Local secretion of parathyroid hormone-related protein by an osteoblastic osteosarcoma (UMR 106-01) cell line results in growth inhibition.

Parathyroid hormone-related protein (PTHrP) has been implicated as being important in the growth of tumor cells responsive to the peptide. We utilized a rat osteoblastic osteosarcoma cell line, UMR 106-01, which has PTHrP receptors and a PTHrP-responsive adenylate cyclase/cAMP messenger system, to produce a modified cell line that overexpresses PTHrP. The human PTHrP cDNA sequence was transfected by electroporation into UMR 106-01 cells and the stable cell lines UMR-36 and UMR-34 were established. The modified cell line, UMR-36, had increased levels of PTHrP mRNA compared with control cell lines and secreted PTHrP into the culture medium at levels of 0.01-0.1 pmol/10(7) cells in 12 h. The secreted peptide was biologically active as indicated by its ability to activate adenylate cyclase. The number of UMR-36 cells following 9 days in culture was reduced by up to 80% compared with control lines, which was associated with decreased (3)H-thymidine incorporation into genomic DNA. Addition of 1000-fold excess of the PTHrP antagonist, PTHrP(7-34), to UMR-36 cells resulted in the escape of growth inhibition and increased rate of growth. In vivo, tumors derived from UMR-36 cells were smaller in size compared with tumors derived from control cells. In conclusion, increased autocrine secretion of, and responsiveness to, PTHrP results in inhibited growth kinetics of an osteoblast-like bone tumor cell line in vitro and in vivo.