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Background: Given the lack of empiric recommendations for vascular access for palliative chemotherapy, we aimed to analyze survival and complications after placement of central venous access ports for palliative chemotherapy., Methods: We performed a retrospective chart review of 135 patients undergoing port placement for palliative chemotherapy at a single institution from January 2015 - July 2020., Results: The median age was 68 (range 47-91). Median overall survival was 7.7 months (95% CI, 6.5-8.9 months). The rate of port-related complications was 11.1% (15 of 135). Patients who developed port-related complications required corrective surgery in 73.3% (11 of 15) of cases. Results were similar among all patients, regardless of their primary diagnoses or central venous access sites., Conclusions: Increased awareness about the limited survival of patients after port placement for palliative chemotherapy, and their significant complication risk could be used to help patients and their providers make value-aligned decisions about vascular access.

Objective: Our study evaluated the willingness of retired surgeons to mentor newly trained surgeons., Summary Background Data: Although mentoring is very important during the transition in practice, many novice surgeons are faced with inadequacy or lack of mentoring., Methods: A survey regarding mentorship of new surgeons was sent in April 2018 to retired general, colorectal, vascular, and cardiothoracic surgeons that are members of the American College of Surgeons. The analysis of the data was performed in September 2018 and October 2018., Results: A total of 2295 of 5282 surveys were completed (43.4% response rate). Mean age was 79.0 ± 0.8 years, mean retirement age was 63.9 ± 0.1 years, and mean interval since retirement was 15.2 ± 0.9 years. Most retired surgeons were in private practice (66.4%), with other practice environments, including academic teaching hospital (12%), academic/private combination (11.3%), employment by community hospital or health system (6.4%), veteran affairs institution (2.7%), military hospital (1%), and Indian Health Service (0.09%). Approximately a third (31.1%) of respondents were not mentored when they first entered practice. The vast majority (98.3%) of participants considered mentoring beneficial during transition in practice. More than half (51.2%) of retired surgeons are interested in mentoring recently trained surgeons, with most of them (81.8%) willing to mentor even for free., Conclusion: Our findings suggest that a significant number of retired surgeons are enthusiastic about mentoring young surgeons during their transition in practice. Specific programs are necessary to meet the needs of newly hired surgeons and better utilize the expertise of retired surgeons., Competing Interests: The authors report no conflicts of interest., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Phone triaging patients with suspected malignant pleural mesothelioma (MPM) within the Veterans Healthcare Administration (VHA) system offers a model for rapid, expert guided evaluation for patients with rare and treatable diseases within a national integrated healthcare system. To assess feasibility of national open access telephone triage using evidence-based treatment recommendations for patients with MPM, measure timelines of the triage and referral process and record the impact on "intent to treat" for patients using our service., Methods: A retrospective study. The main outcome measures were: (1) ability to perform long distance phone triage, (2) to assess the speed of access to a mesothelioma surgical specialist for patients throughout the entire VHA, and (3) to determine if access to a specialist would alter the plan of care., Results: Sixty veterans were screened by our phone triage program, 38 traveled an average of 997 miles to VA Boston Healthcare system. On average, 14 d elapsed from initial phone contact until the patient was physically evaluated in our general thoracic clinic in Boston. The treatment plan was altered for 71% of patients evaluated at VA Boston Healthcare system based on 2012 International Mesothelioma Interest Group guidelines., Conclusions: Our initial experience demonstrates that in-network centralized care for Veterans with MPM is feasible within the VHA. National open access phone triage improves access to expert surgical advice and can be delivered in a timely manner for Veterans using our service. Guideline-based treatment recommendations ("intent to treat") changed the therapeutic course for the majority of patients who used our service., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Forced migration puts families at risk of household food insecurity and economic hardship. We administered a questionnaire to examine household food insecurity in a sample of 49 recently legally resettled Sudanese refugees with at least one child under age 3 years. Of households polled, 37% had experienced household food insecurity and 12% reported child hunger within the previous month. Increasing severity of household food insecurity was associated with decreased consumption of high-cost, high-nutrient-density food items and increased consumption of some low-cost traditional Sudanese foods by adult caregivers of young children. Furthermore, household food insecurity was associated with decreased household and per capita food expenditure, indicators of more limited dietary change with migration, and indicators of increased social support.

Parathyroid hormone-related protein (PTHrP) is required for mammary gland development and promotes the growth of breast cancer metastases within bone. However, there are conflicting reports of the prognostic significance of its expression in primary breast cancers. To study the role of PTHrP in early breast cancer, the effect of conditional deletion of PTHrP was examined in the context of neu-induced mammary tumorigenesis. Loss of PTHrP resulted in a higher tumor incidence. Transcriptional profiling of the tumors revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Immunohistochemical analysis of human breast cancers revealed that PTHrP expression was associated with both HER-2/neu expression and macrophage infiltration in preinvasive ductal carcinoma in situ. The gene expression signature associated with loss of PTHrP expression in vivo correlated with poorer outcome in human breast cancer. Together, these data indicate that loss of PTHrP accelerates mammary tumorigenesis possibly by a non-cell-autonomous tumor suppressor pathway.

We inoculated pasteurized whole milk with Escherichia coli strains GC4468 (intact marRAB locus), JHC1096 (Delta marRAB), or AG112 (Delta marR), and incubated each overnight at 37 degrees C. All strains were then recovered from the milk cultures, and susceptibilities to antimicrobial agents were determined by the E-test strip method (CLSI). Cells of strain GC4468, prior to culturing in milk, were susceptible to trimethoprim, gatifloxacin, cefotaxime and tetracycline. After culturing GC4468 in pasteurized milk, however, the minimal inhibitory concentrations (MICs) increased 1.4-fold for trimethoprim (P0.05), 1.5-fold for gatifloxacin (P0.05), 2.0-fold for cefotaxime (P=0.008), and 1.4-fold for tetracycline (P0.05). After culturing GC4468 on milk count agar the MICs were enhanced 3.4-fold for trimethoprim (P0.05), 10-fold for gatifloxacin (P=0.001), 7.1-fold for cefotaxime (P=0.011), and 40.5-fold for tetracycline (P=0.074), but exhibiting tetracycline resistance with a mean MIC of 74.7+/-18.47 microg/ml (CLSI). The MICs of the antimicrobial agents for JHC1096 cells after culturing in pasteurized whole milk were indistinguishable (P0.05) from baseline MICs measured before culturing in the same type of milk. Thus, Esch. coli cells harbouring the marRAB locus exhibit reduced susceptibilities to multiple antimicrobial agents after culturing in pasteurized whole milk.

Human intrauterine growth restriction is often associated with uteroplacental insufficiency and a decline in nutrient and oxygen supply to the fetus. This study investigated the effects of uteroplacental insufficiency and intrauterine growth restriction (Restricted) or reducing litter size for normally grown pups (Reduced Litter) on maternal mammary development and function, milk composition, offspring milk intake, and their resultant effects on postnatal growth. Uteroplacental insufficiency was surgically induced by bilateral uterine vessel ligation on day 18 of gestation in the Wistar Kyoto rat. At birth, a group of sham control rats had their litter size reduced to five (Reduced Litter) to match that of the Restricted group. Cohorts of rats were terminally anesthetized on day 20 of gestation or day 6 of lactation, and a third group was studied throughout lactation. Restricted pups had a lower birth weight (by 16%) and litter size (by 36%) compared with controls, as well as reduced mammary parathyroid hormone-related protein content and milk ionic calcium concentrations associated with reduced total pup calcium. Restricted dams with lower circulating progesterone experienced premature lactogenesis, producing less milk per pup with altered composition compared with controls, further slowing growth during lactation. Reducing litter size of pups born of normal birth weight (Reduced Litter) was associated with decreased pup growth, highlighting the importance of appropriate controls. The present study demonstrates that uteroplacental insufficiency impairs mammary function, compromises milk quality and quantity, and reduces calcium transport into milk, further restraining postnatal growth.

Background: Military recruits are at a higher risk of respiratory infection than their civilian counterparts. Continuous outbreaks of adenovirus (Ad)-associated acute respiratory disease were documented among US trainees before the implementation of serotype 4 (Ad4) and serotype 7 vaccines in 1971. The discontinuation of Ad vaccination programs in 1999 precipitated the reemergence of Ad in training sites, with Ad4 accounting for 98% of all diagnosed cases., Methods: A total of 724 Ad4 strains isolated from recruits presenting with febrile respiratory illness at 8 training sites nationwide between 1997 and 2003 were genome typed by restriction enzyme analysis., Results: Seven genome types were identified, all of which were distinct from the prototype Ad4p and the vaccine type 4p1. Results showed very different, and often stable, genome type distributions at different geographic sites, despite the homogeneity of the recruit source population., Conclusions: The data support the hypothesis that reservoirs for Ad outbreaks are within recruit training sites or in their immediate environments, not in the incoming recruit population. Molecular characterization beyond serotype is critical to understanding the transmission dynamics of Ad infection in these unique susceptible populations and to the implementation of effective prevention approaches.

Introduction: Studies in xenograft models and experimental models of metastasis have implicated several beta3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific alphavbeta3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear., Methods: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of alphavbeta3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of alphavbeta3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays., Results: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. alphavbeta3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, alphavbeta3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, alphavbeta3 increased 66cl4 tumor cell adhesion and alphavbeta3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro., Conclusion: These results demonstrate for the first time that tumor-specific alphavbeta3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors.

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.

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.

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.

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.

IL-18 was identified due to its ability to induce interferon-gamma (IFNgamma) production by T cells. It is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 has a role in T cell development, where it has been demonstrated to act cooperatively with IL-12 to regulate IFNgamma. In bone, IL-18 is mainly produced by macrophages, but is also expressed by osteoblasts and inhibits osteoclast formation through granulocyte-macrophage colony-stimulating factor (GM-CSF) and not IFNgamma production by T cells. We have investigated the effects of IL-18 on mature osteoclast activity and for potential actions on osteoblasts or chondrocytes. The effects of IL-18 on mature osteoclast activity were determined using two assays: isolated mature osteoclast cell culture and neonatal murine calvarial organ culture. IL-18 did not affect bone resorption in either assay system. The actions of IL-18 on osteogenic cells (primary cell cultures of fetal rat and neonatal mouse osteoblasts, as well as neonatal mouse calvarial organ culture) and primary chondrocytes (canine) were assessed by proliferation assays (quantification of cell numbers and thymidine incorporation). In each assay system, IL-18 acted as a mitogen to the osteogenic and chondrogenic cells. Since IL-18 signal transduction may involve IFNgamma or GM-CSF, we assessed their involvement in the IL-18 response. IL-18 did not induce IFNgamma production by primary osteoblasts, but, of greater significance, IFNgamma had the opposing action to IL-18 in that it inhibited the primary osteoblast cell proliferation. Although IL-18 rapidly induced GM-CSF production by primary osteoblasts, IL-18 was still mitogenic in osteoblast preparations established from GM-CSF-deficient mice. Combined, these studies indicate that IL-18 may have an autocrine/paracrine mitogen role for both osteogenic and chondrogenic cells, independent of the production of IFNgamma or GM-CSF.

Aim: Parathyroid hormone-related protein (PTHrP) is one of the critical factors for the differentiation and growth of chondrocytes. We examined the correlation between the co-expression of PTHrP and PTH/PTHrP receptor and the grade of malignancy in cartilaginous tumours., Methods: We analysed PTHrP and PTH/PTHrP receptor expression in chondrosarcoma by immunohistochemistry and compared specific staining with the expression in benign cartilaginous tumours. There were 38 cartilaginous bone tumours consisting of 26 chondrosarcoma, six enchondroma and six osteochondroma. Chondrosarcoma were composed of 20 conventional chondrosarcoma (10 grade 1, seven grade 2, and three grade 3), two dedifferentiated chondrosarcoma, two clear cell chondrosarcoma, and two myxoid chondrosarcoma. We performed the standard peroxidase-labelled streptavidin-biotin detection method for immunohistochemistry using an antibody raised against PTHrP (1-14) and PTH/PTHrP receptor. The magnitude of receptor positivity of PTHrP and PTH/ PTHrP in each tumour was assessed as a percentage of PTHrP and PTH/PTHrP-positive cells per thousand tumour cells in the most histologically aggressive area of the tumour., Results: All chondrosarcoma, five of six enchondroma, and four of six osteochondroma showed PTHrP-positive cells, and all chondrosarcoma, five of six enchondroma and five of six osteochondroma showed PTHrP receptor-positive cells. The grade of malignancy correlated with the percentage of both PTHrP and PTH/PTHrP receptor-positive tumour cells (P < 0.0001, either). Each grade of chondrosarcoma showed statistically higher expression of both PTHrP and PTH/PTHrP receptor than benign cartilaginous tumour., Conclusion: This is the first report of the co-expression of PTHrP and PTH/PTHrP receptor in chondrosarcoma. PTHrP and PTH/PTHrP receptor positivity may be valuable for differentiating between benign and malignant cartilaginous tumours.

Intrauterine parathyroid hormone-related protein (PTHrP) concentrations are reduced in association with growth restriction in the spontaneously hypertensive rat (SHR) compared to those of its normotensive control, the Wistar Kyoto (WKY) rat, implicating PTHrP as a pivotal fetal growth factor. The aim of this study was to examine, by embryo cross-transplanation between SHR and WKY, whether the mother, fetus, or both, are responsible for the suppressed SHR amniotic fluid PTHrP. One-day-old SHR embryos were gestated in either an SHR (SHR-in-SHR) or WKY (SHR-in-WKY) surrogate, similarly one-day-old WKY embryos were gestated in either an SHR (WKY-in-SHR) or WKY (WKY-in-WKY) mother. At 20 days gestation, maternal plasma and amniotic fluid samples were collected and assayed for PTHrP concentrations. Data were analysed by two-way ANOVA (mean+/-sem, n=5-9 mothers/group). There were no differences in litter number or maternal plasma PTHrP concentrations. Fetal weight (P< 0.009), fetal/placental weight ratio (P< 0.004) and amniotic fluid PTHrP concentrations (P< 0.001) were lower and amniotic fluid volume (P< 0.0001) was higher with an SHR fetus compared to the WKY fetus irrespective of maternal strain. Thus, the SHR fetus is growth restricted and has suppressed amniotic fluid PTHrP, which are largely determined by the fetus or gestational tissues and are independent of maternal hypertension or maternal PTHrP. We suggest that the low SHR amniotic fluid PTHrP may play a role in the development of SHR growth restriction., (Copyright 2001 Harcourt Publishers Ltd.)

We used Hoxa3 knockout mice and other mouse models to study the role of the fetal parathyroids in fetal calcium homeostasis. Hoxa3-null fetuses lack parathyroid glands, and absence of parathyroid hormone (PTH) was confirmed with a rodent PTH immunoradiometric assay. The ionized calcium level of Hoxa3-null fetuses was significantly lower than that of wild-type or heterozygous littermates or of the mother. Both the rate of placental calcium transfer and the plasma PTHrP level were normal in Hoxa3 mutants and their heterozygous siblings. Because we had previously observed an increase in placental calcium transfer in PTH/PTHrP receptor 1-null (Pthr1-null) fetuses, we assayed plasma PTHrP in those mice. Pthr1-null fetuses had plasma PTHrP levels 11-fold higher than those of their littermates. Northern analysis, immunohistochemical, and in situ hybridization studies of Pthr1-null fetuses indicated that liver and placenta had increased expression of PTHRP: In summary, loss of fetal parathyroids in Hoxa3-null fetuses caused marked hypocalcemia but did not alter placental calcium transfer or the circulating PTHrP level. The findings in the Pthr1-null fetuses indicate that several tissues may contribute to the circulating PTHrP level in fetal mice.

Breast cancer is the most common malignancy in women, with a worldwide prevalence of 1.5 million throughout the industrialized countries. Its mortality rate is second only to lung cancer in the USA and Europe. Its high incidence and prevalence makes it a major public health problem. Up to one-third of women with early stage breast cancer will eventually succumb to the disease, and most of these will develop bone metastases during the course of the disease. Approximately 70% of patients with breast cancer have bone metastases, with 27% having lung and liver metastases. Hypercalcemia also is very common in advanced breast cancer, manifesting itself in one-third of patients late in the disease. Breast cancer accounts for approximately 25% of the cases of hypercalcemia in cancer. The major skeletal complications of breast cancer--hypercalcemia and bone metastases--almost certainly share certain mechanisms and these will be discussed.

Objective: Parathyroid hormone-related protein has roles in normal fetal growth, placental calcium transport, and vascular tone regulation; these factors are compromised in growth-restricted fetuses. Our objective was to determine whether intrauterine parathyroid hormone-related protein expression was increased in association with fetal growth restriction., Study Design: The expression of parathyroid hormone-related protein was examined in intrauterine tissues from women with idiopathic fetal growth restriction with preterm (n = 8-10) and term (n = 8-10) gestations and from gestation-matched control subjects. The abundance and immunoreactive content of parathyroid hormone-related protein messenger ribonucleic acid were determined by Northern blot and radioimmunoassay, respectively, in the placenta, amnion, and chorion-decidua., Results: The expression of parathyroid hormone-related protein messenger ribonucleic acid was increased in the amnion (placental and reflected) in association with preterm fetal growth restriction (P <.05). Both parathyroid hormone-related protein messenger ribonucleic acid and protein expression were increased in chorion-decidua in association with preterm fetal growth restriction (P <.05). In term gestations both parathyroid hormone-related protein messenger ribonucleic acid and protein expression were greater in amnion over placenta than in reflected amnion (P <.05); these in turn were greater than those in chorion-decidua (P <.05). No significant changes were detected in parathyroid hormone-related protein messenger ribonucleic acid or in protein expression in association with term fetal growth restriction., Conclusion: Either parathyroid hormone-related protein messenger ribonucleic acid or protein expression, or both, was increased in the fetal membranes in association with fetal growth restriction in preterm but not term gestations, suggesting that parathyroid hormone-related protein may be involved in the pathogenesis of preterm fetal growth restriction.

The parathyroid glands are the only known source of circulating parathyroid hormone (PTH), which initiates an endocrine cascade that regulates serum calcium concentration. Glial cells missing2 (Gcm2), a mouse homologue of Drosophila Gcm, is the only transcription factor whose expression is restricted to the parathyroid glands. Here we show that Gcm2-deficient mice lack parathyroid glands and exhibit a biological hypoparathyroidism, identifying Gcm2 as a master regulatory gene of parathyroid gland development. Unlike PTH receptor-deficient mice, however, Gcm2-deficient mice are viable and fertile, and have only a mildly abnormal bone phenotype. Despite their lack of parathyroid glands, Gcm2-deficient mice have PTH serum levels identical to those of wild-type mice, as do parathyroidectomized wild-type animals. Expression and ablation studies identified the thymus, where Gcm1, another Gcm homologue, is expressed, as the additional, downregulatable source of PTH. Thus, Gcm2 deletion uncovers an auxiliary mechanism for the regulation of calcium homeostasis in the absence of parathyroid glands. We propose that this backup mechanism may be a general feature of endocrine regulation.

Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) in stimulating cell growth and differentiation, placental calcium transport, and placental vasodilatation. As spontaneously hypertensive rat (SHR) fetuses are growth restricted compared with those of its normotensive control, the Wistar Kyoto (WKY) rat, we examined intrauterine PTHrP and total and ionic calcium concentrations in these rats. Fetal plasma PTHrP concentrations, but not total calcium concentrations, were lower in the SHR compared with WKY (P < 0.05). SHR placental concentrations of PTHrP were lower than in WKY (P < 0.03) and failed to show the increase observed in WKY near term (P < 0.05). PTHrP concentrations in amniotic fluid from SHR were not raised near term and were lower compared with WKY (P < 0.0005). The increased ionic calcium concentrations in amniotic fluid in the WKY near term (P < 0.05) were not detected in the SHR. Thus SHR fetal plasma, placental, and amniotic fluid PTHrP concentrations were reduced and associated with fetal growth restriction. We suggest that PTHrP may play a role in the etiology of both growth restriction during pregnancy and hypertension later in life.

Parathyroid hormone (PTH)-related protein (107-139) (PTHrP(107-139)) and PTHrP(107-111) have been reported to be potent inhibitors of isolated osteoclast activity, and inhibition of bone resorption by PTHrP(107-139) occurs in vivo. However, the actions of C-terminal PTHrP on osteoblast activity has not been studied much. The present study addresses this issue by examining the effect of PTHrP(107-139), PTHrP(107-119), PTHrP(120-139), and PTHrP(107-111) on the proliferation of fetal rat osteoblasts. Treatment with PTHrP(107-139) for 24 h caused a dose-dependent increase in cell number, [3H]thymidine and [3H]phenylalanine incorporation in cultured osteoblasts. The effect was apparent at concentrations of 10-10 M and greater and was sustained over time. PTHrP(107-119) and PTHrP(107-111) had effects on cell number, DNA, and protein synthesis which were comparable to those of PTHrP(107-139), whereas PTHrP(120-139) was without effect. Retroverted PTHrP(107-111) also stimulated all three activities but was only one tenth as potent as PTHrP(107-139). PTHrP(107-139) had no effect on osteoblast apoptosis. It is concluded that PTHrP(107-139) is not only an inhibitor of osteoclastic bone resorption but that it also stimulates osteoblast growth. This activity resides within the pentapeptide fragment PTHrP(107-111). These findings support a possible role for C-terminal fragments of PTHrP in the normal regulation of bone cell function and, possibly, bone mass.

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.

Maternal hypertension, vasoconstriction and placental insufficiency are features of pre-eclampsia. Alterations in calcium homeostasis and in the production of calciotropic hormones and vasoactive agents have also been described in association with pre-eclampsia. Parathyroid hormone-related protein (PTHrP) is abundantly expressed in intrauterine tissues during normal pregnancy and has roles in fetal growth and calcium homeostasis, placental calcium transport and vascular tone regulation. Intrauterine PTHrP mRNA expression and tissue PTHrP content were determined by Northern blot analysis and radio-immunoassay, respectively, in preterm and term pre-eclamptic women. PTHrP mRNA expression and PTHrP content in placenta, amnion over placenta, reflected amnion and choriodecidua from preterm pre-eclamptic women (n=8-10) were not different from preterm controls (n= 10-12). PTHrP mRNA expression and content in amnion over placenta and reflected amnion were significantly greater in term compared to preterm pre-eclamptics (P<0.05). PTHrP mRNA expression was significantly lower in choriodecidua from term pre-eclamptic women (n=8) compared to term controls (n=28, P<0.05), but was not different in placenta or amnion. PTHrP content was not altered in term pre-eclamptic women (n=8) compared to controls (n=25) for any tissue. In summary, PTHrP expression in placenta and amnion was not increased in pre-eclamptic women in association with maternal hypertension, placental insufficiency and vasoconstriction. PTHrP mRNA expression was decreased in choriodecidua in association with term but not preterm pre-eclampsia, however, levels of the protein were not decreased. The data suggest that PTHrP is not involved in the placental pathophysiology of pre-eclampsia in late gestation.

Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.

Using immunohistology with two specific antisera raised against N-terminal parathyroid hormone-related protein (PTHrP) and in situ hybridization (riboprobe to common coding exon), evidence is provided for the expression of PTHrP by mouse, rabbit, and human osteoclasts derived from several in vitro and in vivo sources. In cocultures of mouse bone marrow and calvarial cells treated with 1,25-dihydroxyvitamin D3, the generated osteoclasts expressed both PTHrP messenger RNA (mRNA) and protein. In addition, PTHrP was detected in the majority of actively resorbing osteoclasts in sections of newborn and adult mouse long bones. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in osteoclasts at active bone resorption sites as well as in actively synthesizing osteoblasts and bone lining cells. Localization of PTHrP was also demonstrated in osteoclast-like cells of human giant cell tumors from bone. In some of these tumors, a small proportion of the multinucleated cells expressed tartrate resistant acid phosphatase (TRAP), but not PTHrP mRNA or protein. Finally, both mRNA and protein for PTHrP were expressed in osteoclasts in sections of bone or joints from patients with Paget's disease, rheumatoid arthritis, and osteoarthritis. These observations raise the possibility that PTHrP might participate in osteoclast function.

Parathyroid hormone-related protein (PTHrP) was detected at 32.8 +/- 3.9 pmol 1-1 in uterine luminal fluid from immature rats treated with oestradiol. As mRNA encoding PTHrP has previously been localized to implantation sites in pregnant rats, the role of luminal PTHrP during pregnancy was explored. Infusion of a parathyroid hormone (PTH)/PTHrP receptor antagonist, [Asn10,Leu11]PTHrP(7-34) amide, into the uterine lumen during pregnancy in rats resulted in excessive decidualization. This effect was also observed after intrauterine infusion of a monoclonal antibody raised against PTHrP. The effect of infusion of PTH/PTHrP receptor antagonist was dependent upon successful implantation, was dose-dependent and confined to the treated horn. A decrease in the number of apoptotic decidual cells in antagonist-infused uterine horns compared with vehicle or non-infused horns was detected immunohistochemically at day 13 of pregnancy, and this decrease is likely to contribute to the 'over-decidualization' observed. In pseudopregnant rats, infusion of PTH/PTHrP receptor antagonist into the uterine lumen resulted in an increase in uterine wet weight of the infused horn compared with the non-infused horn, indicating a direct effect on deciduoma formation. Thus, activation of the PTH/PTHrP receptor by locally produced PTHrP appears to be crucial for normal decidualization during pregnancy in rats.

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.

The structure of human parathyroid hormone (PTH) related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. This mutant retains quite high levels of adenylate cyclase activity based on slightly reduced PTH receptor binding capacity. Three segments of helix were revealed extending from His5 to Lys11, Lys13 to Arg19, and from Phe22 to Thr33/Ala34, with a decided kink between the first two helices around Gly12. N- and C-terminal helices were stabilized by charged and hydrophobic side chain interactions between His5 and Glu30, Asp17 and both His9 and His25, and between Leu8 and Ala29, resulting in a globular molecule occupying a single conformation. While the structure of the entire mid-molecule region differed greatly from the structure of the native peptide, the structure of both N- and C-terminal regions remains essentially unaltered. The residues responsible for initiating signal transduction in the mutant are located in the vicinity of the residues responsible for receptor binding. The C-terminal amphipathic helix forming the receptor binding site exhibits reduced binding as a result of the closely applied N-terminal signal transduction-activating region. Although not contributing directly to receptor binding, the N-terminal region can sterically affect hormone binding through modifications to certain N-terminal side chains.

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.

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.

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.

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.

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.

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.

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.

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.