Phosphatase Specific Activity Research Articles

Identification of PP2A Complexes and Pathways Involved in Cell Transformation

The simian virus 40 small t (SV40ST) oncoprotein interacts with protein phosphatase 2A (PP2A), an abundantly expressed family of serine-threonine phosphatases. This interaction is essential for the transformation of human cells by SV40, and several PP2A subunits have been implicated as tumor suppressor genes. However, the pathways controlled by specific PP2A complexes involved in cell transformation remain incompletely understood. Using a comprehensive loss-of-function approach, we identified 4 PP2A regulatory subunits [B56α, B56γ, PR72/PR130, and PTPA (protein phosphatase 2A activator)], which when suppressed replaced the expression of SV40ST in human cell transformation. We found that manipulation of complexes containing PP2A B56α, B56γ, and PR72/PR130 activates the pathways regulated by c-Myc, Wnt, and PI3K (phosphoinositide 3-kinase)/Akt in a manner that depends on their specific phosphatase activity. In contrast, suppression of PTPA disrupts the assembly of PP2A heterotrimeric complexes, which leads to the activation of these same oncogenic pathways. These observations delineate the PP2A family members and pathways perturbed by SV40ST during human cell transformation.

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

Abstract 965: Mechanistic studies of a dietary combination in cancer prevention: The effects of manganese superoxide dismutase on tumor suppressor gene activation

Abstract Mechanistic Studies of a Dietary Combination in Cancer Prevention: The Effects of MnSOD on Tumor Suppressor Gene Activation Reactive oxygen species can act as signaling transduction molecules modulating various pathways, such as the PI3K/Akt pathway. It is known that this pathway is constitutively activated in tumorigenesis contributing to protein synthesis, cell cycle progression, migration and survival. However, the PI3K/Akt pathway is negatively regulated by the major tumor suppressor protein PTEN. PTEN has dual specificity phosphatase activity regulating Akt phosphorylation as a lipid phosphatase; as well as, regulating cyclin D1 expression as a protein phosphotase. However, these are the roles of cytoplasmic PTEN. Recently, the nuclear roles of PTEN have received significant attention. Nuclear PTEN plays a major role in maintaining cellular homeostasis and stability by contributing to chromosome stability, DNA repair and cell cycle arrest. Previous studies have shown that PTEN nuclear localization is mediated by oxidative stress and may play a role in p53 stabilization. Nevertheless, MnSOD is considered to be a novel tumor suppressor gene. MnSOD has been defined as a nuclear encoded primary antioxidant enzyme localized in the mitochondria. We have shown in previous studies that the dietary combination Protandim reduced tumor incidence and multiplicity via the synergistic induction of superoxide dismutase enzymes. It has been shown that overexpression of MnSOD in in vivo models can suppress tumor formation via modulation of AP-1 binding. Therefore, it is imperative to look at how MnSOD may affect the process of apoptosis and the activation of tumor suppressor genes involved (i.e. PTEN and p53). Using our in vivo models (wildtype, MnSOD knockout, non-transgenic and MnSOD transgenic mice), we investigated how modulation of MnSOD expression may affect PTEN-mediated p53 stabilization, and how this contributes to chemoprevention. In addition, we explored how mutations in p53 may affect the PTEN-p53 interaction and its effects on tumor promotion via a gene transfection approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 965.

Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana

Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase. LPA phosphatase gene has not been identified and characterized in plants so far. The BLAST search revealed that the At3g03520 is similar to phospholipase family, and distantly related to bacterial phosphatases. The conserved motif, (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases. In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity. These results suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.

Distribution and activity of Bacteria and Archaea in the different water masses of the Tyrrhenian Sea

Abstract This study examines the abundance of the Bacteria, Crenarchaeota and Euryarchaeota and bulk activities (phosphatase and aminopeptidase activities, heterotrophic prokaryotic production and dark CO2 fixation) in the major water masses of the Tyrrhenian Sea (from surface to bottom: Modified Atlantic Water (MAW); Levantine Intermediate Water (LIW) and Tyrrhenian Deep Water (TDW)) in July and December 2005. Data from the catalyzed reporter deposition coupled with fluorescence in situ hybridization (CARD-FISH) analyses indicate that the percentage of Bacteria was always higher than the percentage of Crenarchaeota and Euryarchaeota throughout the water column. While the percentage of Euryarchaeota was relatively homogeneous (∼10%) through the water column, the percentage of Crenarchaeota increased with depth (from 5% to 14% in July and from 7% to 17% in December in MAW and TDW, respectively). Regarding differences between July and December 2005, the percentage of Bacteria in the MAW was lower in July than in December (25% versus 43%, respectively) while quite constant (∼40%) in the TDW. The pattern of phosphatase and aminopeptidase activity varied according to the stations considered, but both ectoenzyme activities showed higher maximum velocity rates in July than in December in the deep-sea waters. Particularly, specific activity of phosphatase in the deep-sea waters (TDW) was 7 times higher (median value) than in surface waters (MAW). Prokaryotic production, aminopeptidase and phosphatase activity measurements were always higher under in situ pressure conditions than after decompression. For the first time, the measurement of the dark CO2 fixation was investigated under in situ pressure conditions and its decompressed counterparts. These data give new information to understanding the role of prokaryotes (Bacteria and Archaea) in biogeochemical cycles of the meso- and batypelagic waters of the oceans.

A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

The production of reactive oxygen species (ROS) exerts an additional tier of control over tyrosine phosphorylation-dependent signal transduction by transiently inhibiting the catalytic activity of specific protein tyrosine phosphatases (PTPs). Hence, the ability to detect reversible oxidation of PTPs in vivo is critical to understanding the complex biological role of ROS in the control of cellular signaling. Here, we describe an assay for identifying those PTPs that are reversibly oxidized in vivo, which utilizes the unique chemistry of the invariant catalytic Cys residue in labeling the active site with biotinylated small molecules under mildly acidic conditions. We have applied this cysteinyl-labeling assay to the study of platelet-derived growth factor (PDGF) receptor signaling in an angiomyolipoma cell model. Doing so has allowed us to detect reversible oxidation of several proteins in response to sustained PDGF stimulation. As in other cell systems, we have observed the reversible oxidation of the classical PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation. Furthermore, we detected reversible oxidation of members of two other subclasses of PTPs, the receptor PTP LAR and the dual-specificity phosphatase MKP1. These data demonstrate the broad selectivity of the assay, allowing us to detect representatives of all of the major subgroups of the PTP superfamily. We anticipate that this cysteinyl-labeling enrichment strategy can be applied broadly to study reversible oxidation as a mechanism of harnessing PTP catalytic activity in a variety of signaling pathways.

The N Terminus Controls Sterol Binding while the C Terminus Regulates the Scaffolding Function of OSBP

Previously we reported that when cell cholesterol is acutely lowered with beta-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP(408-809), it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway.

Short-term variation in extracellular phosphatase activity: possible limitations for diagnosis of nutrient status in particular algal populations

Species-specific extracellular phosphatase activity (PA) was studied in natural phytoplankton with an emphasis on short-term variation in PA and its consequences for the diagnosis of inorganic phosphorus (P) deficiency. Extracellular PA was measured over 3 days at 4-h intervals to compare variations in algal PA in two distinct aquatic environments: in the Řimov reservoir and acidified Plesne Lake. PA was directly detected in an epifluorescence microscope using fluorescently labelled enzyme activity; the fluorescence signal was quantified using image analysis. High short-term variation was found both in the total PA of the samples and specific PA of Ankyra ancora (G. M. Smith) Fott, Monoraphidium dybowski (Wolosz.) Hind. and Kom.-Legn., and Chlorogonium fusiforme Matwienko. The specific PA of these species varied between zero and 157 fmol cell−1 h−1. The PA of Chlorogonium fusiforme showed a significant relationship with the time of day. During the study period, populations of the certain species were present that differed in physiological status and activity due to diurnal or random changes caused by mixing in the reservoir or lake, or both. Therefore, species-specific extracellular PA should be interpreted carefully, as an indicator of P deficiency in freshwater phytoplankton if based on limited number of samplings.

C3G mediated suppression of malignant transformation involves activation of PP2A phosphatases at the subcortical actin cytoskeleton

In previous work, we demonstrated that C3G suppresses Ras oncogenic transformation by a mechanism involving inhibition of ERK phosphorylation. Here we present evidences indicating that this suppression mechanism is mediated, at least in part, by serine/threonine phosphatases of the PP2A family. Thus: (i) ectopic expression of C3G or C3GΔCat (mutant lacking the GEF activity) increases specific ERK-associated PP2A phosphatase activities; (ii) C3G and PP2A interact, as demonstrated by immunofluorescence and co-immunoprecipitation experiments; (iii) association between PP2A and MEK or ERK increases in C3G overexpressing cells; (iv) phosphorylated-inactive PP2A level decreases in C3G expressing clones and, most importantly, (v) okadaic acid reverts the inhibitory effect of C3G on ERK phosphorylation. Moreover, C3G interacts with Ksr-1, a scaffold protein of the Ras–ERK pathway that also associates with PP2A. The fraction of C3G involved in transformation suppression is restricted to the subcortical actin cytoskeleton where it interacts with actin. Furthermore, the association between C3G and PP2A remains stable even after cytoskeleton disruption with cytochalasin D, suggesting that the three proteins form a complex at this subcellular compartment. Finally, C3G- and C3GΔCat-mediated inhibition of ERK phosphorylation is reverted by incubation with cytochalasin D. We hypothesize that C3G triggers PP2A activation and binding to MEK and ERK at the subcortical actin cytoskeleton, thus favouring ERK dephosphorylation.

Expression and characterization of HSPC129, a RNA polymerase II C-terminal domain phosphatase

Phosphorylation status of RNA polymerase (RNAP) II's largest subunit C-terminal domain (CTD) plays an important role during transcription cycles. The reversible phosphorylation mainly occurs at serine 2 and serine 5 of CTD heptapeptide repeats and regulates RNAP II's activity during transcription initiation, elongation and RNA processing. Here we expressed and characterized HSPC129, a putative human protein bearing a CTD phosphatase domain (CPD). PCR analysis showed that it was ubiquitously expressed. HSPC129DeltaTM, the truncate HSPC129 with first 156 N terminal amino acids deleted, exhibited Mg(2+) dependent phosphatase activity at pH 5.0. Its specific CTD phosphatase activity was verified in vitro. Our research suggests that HSPC129 may regulate the dynamic phosphorylation of RNAP II CTD.

Dual Specificity Phosphatase (DUSP) activity regulates ERK activation and positive selection of thymocytes (87.16)

Abstract Regulation of MAP Kinase phosphorylation is thought to be important for proper translation of TCR signals into thymocyte cell fates. However, the role of phosphatase activity in thymic development has not been extensively characterized. DUSP-6, also known as Map Kinase Phosphatase-3 (MKP-3), is expressed in the thymus and can bind and dephosphorylate ERK. To explore the role of MKP in thymocytes, we expressed MKP-3 or a catalytically inactive form of MKP-3 (DM-MKP3) in an immature thymocyte cell line, 16610 D9. Lower amounts of phosphorylated ERK (p-ERK) were found in D9 cells that over express MKP-3, while D9 cells that over express DM-MKP3 exhibited higher amounts of p-ERK after TCR stimulation in comparison to vector control cells. Thus, over expression of a catalytically inactive ERK phosphatase results in inhibition of all ERK specific MKP activity, and acts as a dominant negative MKP. Utilizing real time PCR, we also observed elevated Egr-1 mRNA levels after TCR stimulation in D9 cells expressing DM-MKP3 compared to control D9 cells. Egr-1 is an ERK dependent immediate early gene known to support positive selection of thymocytes. Finally, we over expressed DM-MKP3 in vivo, and observed enhanced positive selection. Together these results indicate that MKP control of ERK activation after TCR stimulation is critical in regulation of thymocyte cell fate decisions. This work is funded by grants from the NIH and the ACS.

Vascular endothelial cell specific phospho-tyrosine phosphatase (VE-PTP) activity is required for blood vessel development

Vascular endothelial cell specific phospho-tyrosine phosphatase (VE-PTP) activity is required for blood vessel development

Phosphoinositide-Specific Inositol Polyphosphate 5-Phosphatase IV Inhibits Inositide Trisphosphate Accumulation in Hypothalamus and Regulates Food Intake and Body Weight

The enzyme phosphatidylinositol 3-kinase (PI3-kinase) exerts an important role in the transduction of the anorexigenic and thermogenic signals delivered by insulin and leptin to first-order neurons of the arcuate nucleus in the hypothalamus. The termination of the intracellular signals generated by the activation of PI3-kinase depends on the coordinated activity of specific inositol phosphatases. Here we show that phosphoinositide-specific inositol polyphosphate 5-phosphatase IV (5ptase IV) is highly expressed in neurons of the arcuate and lateral nuclei of the hypothalamus. Upon intracerebroventricular (ICV) treatment with insulin, 5ptase IV undergoes a time-dependent tyrosine phosphorylation, which follows the same patterns of canonical insulin signaling through the insulin receptor, insulin receptor substrate-2, and PI3-kinase. To evaluate the participation of 5ptase IV in insulin action in hypothalamus, we used a phosphorthioate-modified antisense oligonucleotide specific for this enzyme. The treatment of rats with this oligonucleotide for 4 d reduced the hypothalamic expression of 5ptase IV by approximately 80%. This was accompanied by an approximately 70% reduction of insulin-induced tyrosine phosphorylation of 5ptase IV and an increase in basal accumulation of phosphorylated inositols in the hypothalamus. Finally, inhibition of hypothalamic 5ptase IV expression by the antisense approach resulted in reduced daily food intake and body weight loss. Thus, 5ptase IV is a powerful regulator of signaling through PI3-kinase in hypothalamus and may become an interesting target for therapeutics of obesity and related disorders.

Vascular endothelial cell–specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

AbstractVE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na+,K+)-ATPase α-subunit expression and K+-phosphatase activity

The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21 per thousand salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6+/-4.9 U mg-1 and K0.5=1.31+/-0.05 mmol L-1. Stimulation of K+-phosphatase activity by magnesium (Vmax=125.3+/-7.5 U mg-1; K0.5=2.09+/-0.06 mmol L-1), potassium (Vmax=134.2+/-6.7 U mg-1; K0.5=1.33+/-0.06 mmol L-1) and ammonium ions (Vmax=130.1+/-5.9 U mg-1; K0.5=11.4+/-0.5 mmol L-1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI=304.9+/-18.3 micromol L-1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the approximately 2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar alpha-subunit expression in gill tissue from shrimps acclimated to 21 per thousand salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation.

Alteration of adenoid tissue alkaline and acid phosphatase in children with secretory otitis media

The role of pharyngeal lymphoid tissue in etiopathogenesis of secretory otitis is not yet defined. The influence of tonsillar and adenoid mass, weight, obstruction of naspharyngeal orrifitium, bacterial reservoire or some immunological events are of scientific interest. Tissue nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) are enzymes detected in lymphoid tissue, TNAP as characteristic of B cells, ACP as a characteristic of macrophages and folucullardentritic cells. These enzymes interfere in cell metabolism by removing 5' phosphate group from nucleotides and proteins. Specific activity and kinetic properties were studied in palatinal tonsils and adenoids of children with secretory otitis (OME) and compared with children with recurrent tonsillitis without ear involvement. Adenoid and tonsillar tissue of l7 children with OME and 30 children with recurrent tonsillitis were subjected to biochemical investigation using method of releasing of p-nitrophenol from p-nitrophenylphosphate (pNPP). Kinetic parameters as Michaelis-Menten constant were calculated by non-linear regression estimation method. Specific activity of adenoid alkaline phosphatase was lower in children with OME in relation to children with recurrent tonsillitis (t=5.733507, p<0.01). Specific activity of adenoid acid phosphatase was also lower in children with OME (t=3.655456, p<0.01). pH optimum for both enzymes was the same in these two groups of children. Michaelis-Menten constant for both enzymes was significantly higher in adenoid of children with OME than in children with recurrent tonsillitis suggesting lower enzyme affinity for the substrate. Differences in specific activities and kinetic properties of adenoid alkaline and acid phosphatases between children with OME and children with recurrent tonsillitis without OME were verified in this study. The results of the study are not able to explain the alteration of alkaline and acid phosphatase characteristics but could point to some possible and specific role of nasopharyngeal lymphoid tissue in pathogenesis of secretary otitis.

Identification of an ITPase/XTPase in Escherichia coli by Structural and Biochemical Analysis

Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.

The C. elegans lethal gut-obstructed gob-1 gene is trehalose-6-phosphate phosphatase

We identified the gob-1 ( gut- obstructed) gene in a forward genetic screen for intestinal defects in the nematode Caenorhabditis elegans. gob-1 loss of function results in early larval lethality, at least in part because of a blocked intestinal lumen and consequent starvation. The gob-1 gene is first expressed in the 8E cell stage of the embryonic intestine, and the GATA factor ELT-2 is sufficient but not necessary for this early phase of gob-1 expression; gob-1 expression later becomes widespread in embryos, larvae, and adults. GOB-1 is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. Trehalose is a glucose disaccharide found in bacteria, fungi, plants, insects, and nematodes but not in mammals. Trehalose plays a number of critical roles such as providing flexible energy reserves and contributing to thermal and osmotic stress resistance. In budding yeast and in plants, the intermediate in trehalose synthesis, trehalose-6-phosphate, has additional critical but less well-defined roles in controlling glycolysis and carbohydrate metabolism. Strong loss-of-function mutants in the C. elegans tps-1 and tps-2 genes (which encode the two trehalose phosphate synthases responsible for trehalose-6-phosphate synthesis) completely suppress the lethality associated with gob-1 loss of function. The suppression of gob-1 lethality by ablation of TPS-1 and TPS-2, the upstream enzymes in the trehalose synthesis pathway, suggests that gob-1 lethality results from a toxic build-up of the intermediate trehalose-6-phosphate, not from an absence of trehalose. GOB-1 is the first trehalose-6-phosphate phosphatase to be identified in nematodes and, because of its associated lethality and distinctive sequence properties, provides a new and attractive target for anti-parasitic drugs.

The response to sex steroid hormones and vitamin D of cultured osteoblasts derived from ovariectomized mice with and without 17β-estradiol pretreatment

This study investigated whether 17beta-estradiol (E2) may have different effects on osteoblasts derived from estrogen-deficient ovariectomized (OVX) mice compared to sham-operated normal animals. We studied the specific effects of 17beta-estradiol on the differentiation and function of cultured osteoblasts derived from these groups of animals, with or without estrogen replacement treatment. One-month-old mice were ovariectomized or sham-operated, and treated (every second day) for 4 weeks with 0.5 mg/kg 17beta-estradiol or with vehicle alone. At the end of the experiment, bones were removed for primary osteoblast cultures or for morphological and chemical evaluation. In cells from untreated OVX animals, alkaline phosphatase (ALP) specific activity was reduced, while collagen production and mineralization were unchanged when compared to cells from controls. In vivo estrogen pretreatment of the OVX mice elevated ALP activity and mineralization of the cells, while collagen production was reduced. The addition of 17beta-estradiol to the culture medium increased ALP activity, collagen production, and mineralization by all cultured osteoblasts, except in those derived from sham-operated, estrogen-pretreated mice, where these features remained unchanged. Osteocalcin production was unchanged. Addition of testosterone or 1,25(OH)2D3 to the culture medium induced changes that differed among the groups depending on the source of the cultured cells. It seems that ovariectomy in mice prior to culture affected the phenotype of the cultured osteoblasts and their response to estradiol, testosterone, and 1,25(OH)2D3, depending on whether animals were pretreated with estradiol or not. These results imply that the animal's estrogen status prior to culture can influence the response to estrogens; this finding may have important implications for hormone replacement therapy (HRT) in postmenopausal women.

Macrophages Survive Hyperoxia via Prolonged ERK Activation Due to Phosphatase Down-regulation

Macrophages exposed to hyperoxia in the lung continue to survive for prolonged periods. We previously reported (Nyunoya, T., Powers, L. S., Yarovinsky, T. O., Butler, N. S., Monick, M. M., and Hunninghake, G. W. (2003) J. Biol. Chem. 278, 36099-36106) that hyperoxia induces cell cycle arrest and sustained extracellular signal-related kinase (ERK) activity in macrophages. In this study, we determined the mechanisms of hyperoxia-induced ERK activation and how ERK activity plays a pro-survival role in hyperoxia-exposed cells. Inhibition of ERK activity decreased survival of hyperoxia-exposed macrophages. This was due, at least in part, to down-regulation of the pro-apoptotic Bcl-2 family member, BimEL. In determining the mechanism of ERK activation by hyperoxia, we found that ERK activation was not associated with hyperoxia-induced activation of the upstream ERK kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2. When we examined the ability of whole cell lysates from hyperoxia-exposed cells to dephosphorylate purified phosphorylated ERK, we found decreased ERK-directed phosphatase activity. Two particular ERK-directed phosphatases (protein phosphatase 2A and MAPK phosphatase-3) demonstrated decreased activity in hyperoxia-exposed cells. Moreover, whole cell lysates from normoxia-exposed cells depleted of PP2A or MAPK phosphatase-3 were also less able to dephosphorylate ERK. These data demonstrate that, in hyperoxia-exposed macrophages, sustained activation of ERK due to phosphatase down-regulation permits macrophage survival via effects on the balance between pro- and anti-apoptotic Bcl-2 family proteins.