Parathyroid hormone regulates the expression of the nuclear mitotic apparatus protein in the osteoblast-like cells, ROS 17/2.8

This study assessed the prevalence of tobacco and alcohol use in top-grossing American films from 1985 to 1995. The top ten money-making films for 1985 to 1995 were viewed to identify the prevalence of alcohol and tobacco use overall and by lead characters. While the use of tobacco and alcohol was stable across the study period, use of these substances was common. Most films (98%) had references that supported tobacco use and 96% had references that supported alcohol use. Discouraging the use of these substances was uncommon. Only one third of films had any references that discouraged the use of tobacco or alcohol (38% and 37%, respectively). At least one lead character used tobacco or alcohol in 46% and 79% of the films, respectively. The hazards of smoking and drinking are not reflected in the behaviors of film characters who are potential role models for youth facing the decision to smoke or drink.     

Plasma copolymer surfaces of acrylic acid/1,7 octadiene: surface characterisation and the attachment of ROS 17/2.8 osteoblast-like cells

The purpose of this study was: (a) to examine the effect of plasma-gas composition on plasma polymer oxygen/carbon (O/C) ratio, functional group composition and stability in water, and then (b) to examine cell attachment to surfaces containing different concentrations of O/C and functional groups. Oxygen-functionalised surfaces were deposited by means of the plasma copolymerisation of acrylic acid/1,7-octadiene. The use of a diluent hydrocarbon allowed the deposition of surfaces with a range of O/C concentrations. Plasma copolymer surfaces were characterised by X-ray photoelectron spectroscopy (XPS). Changes in functional group composition with % acrylic acid monomer and the non-dispersive and dispersive parts of the surface energy of these plasma copolymers were measured. The solubility of the plasma copolymers was assessed by means of XPS. The degree of attachment of ROS 17/2.8 osteoblast-like cells to plasma copolymer surfaces deemed to be 'stable' in aqueous medium was measured. Tissue culture polystyrene (TCPS) was included as a control. Attachment was found to be greatest to the plasma copolymer surface with an O/C of 0.11. This surface had a carboxylic acid concentration of ca. 3%. Attachment did not correlate with increased surface wettability (i.e. the non-dispersive component of the surface energy).     

Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens

We have reported that pretreatment with 1 alpha, 25(OH)2D3(1, 25) up-regulates responsiveness and sensitivity to 17 beta estradiol (E2) in osteoblast-like cells, as measured by parallel stimulation of [3H]thymidine incorporation into DNA and the specific activity of creatine kinase BB (CK). Increased responsiveness was correlated with increased E2 receptor concentration. In this study, we have extended these observations to new nonhypercalcemic analogs of 1,25. We compared the analogs hexafluoro vitamin D3 (FL), and the side chain modified derivatives: EB 1089 (EB), CB 1093 (CB) and MC 1288 (MC) with 1,25 and 25 (OH)D3(25 D3). Treatment with 30 nM E2 for 4 h stimulated CK activity in ROS 17/2-8 cells by 40%; there was no further increase after 3 daily additions of E2. Treatment by 3 daily additions, at 1 nM, of all analogs except 25 D3 caused a 2-3-fold increase in CK specific activity. This schedule of treatment also upregulated the response to 4 h exposure to 30 nM E2 by 30-70% above the response to vitamin D analogs alone, and by up to 2 fold compared to E2 without pretreatment. At 1 pM, the analogs doubled CK activity, and, except for 1,25, upregulated the response to E2 to levels characteristic of each analog. Pretreatment with vitamin D analogs also increased the sensitivity to E2 by lowering the dose for a comparable response to E2 by one or two orders of magnitude. Stimulation of specific activity of CK by the analogs was paralleled by increases in the steady state level of mRNA for CKB, but not in its half life. Whereas pretreatment by vehicle followed by E2 for 2 h was unable to increase CKB mRNA, pretreatment with the analogs made possible detection of mRNA responsiveness to E2. These results add to the evidence for the interaction of estrogens and antiestrogens with vitamin D metabolites in regulation of bone growth in vitro. They also strengthen the potential for treatment of bone loss, as occurs in postmenopausal osteoporosis, by a combination of nonhypercalcemic vitamin D analogs and estrogens.     

Opposing influences of dexamethasone and retinoic acid on adenylate cyclase activity in ROS 17/2.8 cells

Exposure of ROS 17/2.8 cells to dexamethasone (DEX) or retinoic acid (RA) increases and decreases, respectively, adenylate cyclase activity (ACA) in response to isoproterenol, forskolin, guanylylimidodiphosphate, or NaFl. Despite dramatic changes in ACA, there were no significant changes in levels of cholera toxin- or pertussis toxin (PT)-dependent ADP-ribosylation of membranes prepared from cells after DEX or RA exposure as compared to controls. Similarly, immunochemical detection of alpha S, alpha i1-3, and alpha O, as well as Northern blot analysis of messenger RNA for each of the respective GTP binding proteins, also failed to demonstrate an influence of DEX or RA when contrasted with controls. In a novel use of the cyc- reconstitution assay, wherein the influence of inhibitory guanine nucleotide binding proteins in the extracts of control, DEX-, and RA-treated membranes is removed by a previous 24-h incubation with PT in the intact cell, we demonstrate that this PT treatment markedly enhances ACA in the cyc- reconstitution assay for all three preparations, but that the fold-increase due to PT-treatment is greatest in RA-treated cells. The greater magnitude of the effect of PT on RA-treated ROS 17/2.8 cells, in the absence of any obvious quantitative changes in the levels of the PT substrates, suggests that the effect of RA on ROS 17/2.8 cells appears to be an augmentation of the influence of inhibitory guanine nucleotide binding proteins, ultimately leading to reduced ACA.     

Cholera toxin increases IL-6 synthesis and decreases TNF-alpha production by rat peritoneal mast cells

Mast cells have been traditionally associated with an acute allergic response. However, their role in regulating chronic inflammatory processes must also be considered in view of evidence that mast cells synthesize and release a number of cytokines. In this study, we have examined the effect of cholera toxin (CT) on peritoneal mast cell IL-6 and TNF-alpha production. Highly purified, freshly isolated, rat peritoneal mast cells from Brown Norway rats were cultured in the presence of CT or its B subunit (CTB) alone or in combination with anti-IgE or bacterial LPS. Histamine release was measured after 10 min; IL-16 and TNF-alpha production was assessed in supernatants after 18 h. We found that CT or CTB alone did not affect histamine release; however, mast cell IL-6 production was significantly enhanced by CT but not by CTB. In contrast, constitutive production of TNF-alpha was inhibited by CT. The effects of CT were similar to our previous observations of the actions of prostaglandin E2 on mast cells. We also examined the effects of CT in combination with other mast cell activating agents. CT had no significant effect on anti-IgE-induced histamine release. An additive effect on IL-6 production was observed in the context of LPS. Forskolin, an agent known to increase intracellular cAMP levels, also induced a significant increase in IL-6 production, whereas TNF-alpha production was decreased. These data have important implications for our understanding of the regulation of mast cell cytokine production and the effects of CT on local cytokine production.     

Regulation of the transcription of parathyroid-hormone/parathyroid-hormone-related peptide receptor mRNA by dexamethasone in ROS 17/2.8 osteosarcoma cells

The normal epithelial cell-specific 1 (NES1) gene is a recently identified novel serine protease-like gene which is down-regulated during breast cancer progression. The gene product has 34-42% identity with the members of three distinct serine protease families: the trypsin-like family, activators of kringle domain-containing growth factors, and the kallikrein family (X. L. Liu et al., (1996) Cancer Res 56, 3371-3379). Although the cDNA of this gene has been cloned, its genomic structure and chromosomal position are not as yet known. Here, we report the genomic characterization and mapping of the NES1 gene. By subcloning and sequencing a PAC clone containing the complete NES1 gene, we were able to characterize the structure of this gene. The NES1 gene spans 5.5 kb and is composed of five coding exons and one untranslated exon. The positions of the introns were similar to trypsinogen, prostate specific antigen (PSA), and tissue plasminogen activator (TPA). NES1 gene was also localized with somatic cell mapping, radiation hybrid mapping, and fluorescence in situ hybridization techniques to chromosome 19q13.3-q13.4, the same region where the human kallikrein gene family resides. Taken together, our results suggest that the NES1 gene originates from the same ancestor as trypsinogen, PSA, and TPA, but remains in close proximity to PSA.     

IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha

IL-17 is a novel T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated whether hapten-specific T cells isolated from patients with allergic contact dermatitis (ACD) to nickel produce IL-17 and the effects of IL-17 alone or in combination with IFN-gamma or TNF-alpha on the immune activation of keratinocytes. Skin affected with ACD to nickel and skin-derived, nickel-specific CD4+ T cell lines expressed IFN-gamma, TNF-alpha, and IL-17 mRNAs. Four of seven nickel-specific CD4+ T cell clones positive for the skin-homing receptor, cutaneous lymphocyte-associated Ag, were shown to corelease IL-17, IFN-gamma, and TNF-alpha. In contrast, two nickel-specific CD8+ T cell clones failed to synthesize IL-17. Normal human keratinocytes were found to express constitutively the IL-17 receptor gene. IL-17 specifically and dose-dependently augmented IFN-gamma-induced ICAM-1 expression on keratinocytes at both the mRNA and the protein level, whereas HLA-DR, MHC class I, and CD40 levels were not modulated by IL-17. On the other hand, IL-17 alone did not affect ICAM-1 or enhance TNF-alpha-induced ICAM-1. In addition, IL-17, both directly and in synergism with IFN-gamma and/or TNF-alpha, stimulated synthesis and release of IL-8 by keratinocytes. In contrast, IFN-gamma- and TNF-alpha-induced production of RANTES was markedly inhibited by IL-17, and the synthesis of macrophage chemotactic protein 1 was not changed. Taken together, the results suggest that IL-17 is an important player of T cell-mediated skin immune responses, with synergistic or antagonist effects on IFN-gamma- and TNF-alpha-stimulated keratinocyte activation.     

Silica exposure increases expression of pulmonary intercellular adhesion molecule-1 (ICAM-1) in C57Bl/6 mice

Although the pathogenic mechanisms underlying silica-induced lung damage are well described, few studies have examined the expression and role of adhesion molecules in lung injury induced by this particle. Here we report that intratracheal instillation of silica crystals (alpha quartz) (SI) into the lungs of C57Bl/6 mice results in a significant increase in levels of intercellular adhesion molecule-1 (ICAM-1) in lung tissue and in lung lavage fluid. This increased expression of ICAM-1 appeared to be associated with later (> or = 24 h) cell influx and lung injury rather than in the initiation of these events. Exposure of mice to the nontoxic particle titanium dioxide did not elicit increased expression of ICAM-1 in lung tissue or lavage fluid. Passive administration of rat anti-mouse ICAM-1 monoclonal antibody significantly decreased the influx of neutrophils (PMNs) into the alveoli and the levels of lung tissue ICAM-1 and yet had no effect on measures of lung injury or increased collagen deposition. These data suggest that increased ICAM-1 expression in lung tissue following exposure to silica plays a partial role in the trafficking of neutrophils into the airways.     

Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates

We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.     

IL-1-alpha and TNF-alpha differentially regulate CD4 and Mac-1 expression in mouse microglia

The regulatory effects of the proinflammatory cytokines, interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were investigated on CD4 and Mac-1 expression in mouse microglial cultures. The identity of the microglia in cultures was confirmed by multiple indices including morphology, uptake of acetylated low-density lipoprotein and lectin RCA 120 staining. Microglia growing on a monolayer of astrocytes (astrocyte-supported microglia) were both CD4- and Mac-1 positive (out of 94.5 % Mac-1-positive cells, 85.3% were also CD4 positive). When astrocyte-supported microglia were replated directly onto culture dishes (plate-supported microglia), the percentage of CD4- and Mac-1-positive cells decreased to 12-29 and 20-25% respectively. The addition of IL-1alpha or TNF-alpha to plate-supported microglia led to an upregulation of Mac-1 expression in a time- and dose-dependent manner with different EC50s (0.5 ng/ml for IL-1alpha and 2 ng/ml for TNF-alpha) but exhibited similar time-to-peak responses (over 12 h). The addition of IL-1alpha, but not TNF-alpha, also led to an increase in CD4 expression on plate-supported microglia with a similar dose response and time course. IL-1alpha treatment gave rise to an increase in the level of CD4 mRNA as assessed by RT-PCR. The possibility that cell proliferation was responsible for the observed effects on microglia was excluded by an analysis of 3H-thymidine incorporation. Our results suggest that cultured mouse microglia express CD4 molecules which can be upregulated by IL-1alpha while Mac-1 can be upregulated by both IL-1alpha and TNF-alpha.     

TNF-alpha induction of A1 expression in human cancer cells

A1, a member of the Bcl-2 gene family, was originally identified as a hemopoietic-specific early response gene. Later it was found that A1 was overexpressed in human stomach cancer tissues and was induced by tumor necrosis factor-alpha (TNF-alpha) in human vascular endothelial cells. However, its expression in human cancer cells has not been well characterized. In the present study, we examined the expression of A1, as well as the antioxidant manganous superoxide dismutase (MnSOD), in four human thyroid carcinoma cell lines, two human pancreatic carcinoma cell lines, and two human prostate carcinoma cell lines. A1 mRNA was expressed in all four thyroid carcinoma cell lines. TNF-alpha induced A1 in a time- and dose-dependent manner. In contrast, A1 mRNA was not detectable in the pancreatic and prostate carcinoma cell lines in the presence or absence of TNF-alpha. However, TNF-alpha induced manganous superoxide dismutase (MnSOD) mRNA in all the cell lines tested. Furthermore, an agonist antibody to the p55 TNF-alpha receptor induced A1, but the agonist antibody against p75 TNF-alpha receptor did not have this effect. The results indicate that A1 is expressed in human thyroid carcinoma cells and TNF-alpha induces A1 through the p55 TNF-alpha receptor-mediated pathway.