Chemical activation of divergent protein tyrosine phosphatase domains with cyanine-based biarsenicals.

Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction. However, targeted protein activation is chemically challenging, and few broadly applicable activation strategies for signaling enzymes have been developed. Here we report that classical protein tyrosine phosphatase (PTP) domains from multiple subfamilies can be systematically sensitized to target-specific activation by the cyanine-based biarsenical compounds AsCy3 and AsCy5. Engineering of the activatable PTPs (actPTPs) is achieved by the introduction of three cysteine residues within a conserved loop of the PTP domain, and the positions of the sensitizing mutations are readily identifiable from primary sequence alignments. In the current study we have generated and characterized actPTP domains from three different subfamilies of both receptor and non-receptor PTPs. Biarsenical-induced stimulation of the actPTPs is rapid and dose-dependent, and is operative with both purified enzymes and complex proteomic mixtures. Our results suggest that a substantial fraction of the classical PTP family will be compatible with the act-engineering approach, which provides a novel chemical-biological tool for the control of PTP activity and the study of PTP function.

[Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages].

OBJECTIVE: To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages, and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity, thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs. METHODS: IPSCs derived from human gingival fibroblasts at passages 5, 10, 15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine, then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining, including bone related markers: osteocalcin (OCN), bone sialoprotein (BSP); periodontal ligament related markers: periostin, vimentin; and cementum related markers: cementum attachment protein (CAP), cementum protein 1 (CEMP1). The untreated spontaneous differentiation groups were set as negative controls respectively. RESULTS: iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindle-like shape similar to fibroblasts upon periodontal specific differentiation. All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5: t=2.125, P=0.003; P10: t=2.246, P=0.021; P15: t=3.754, P=0.004; P20: t=3.933, P=0.002), but no significant difference in their calcium deposition were detected within passages 5, 10, 15 and 20 (periodontal differentiation: F=2.365, P=0.109; spontaneously differentiation: F=2.901, P=0.067). Periodontal tissue related marker expressions of iPSCs at all passages had also been significantly upgraded under the treatment of GDF-5 (P<0.05), but still, no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP: F=0.926 7, P=0.450; vimentin: F=0.917 1, P=0.455; CEMP1: F=2.129, P=0.1367). CONCLUSION: Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs, as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging. Therefore, iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.

Structure-Based Virtual Screening of Protein Tyrosine Phosphatase Inhibitors: Significance, Challenges, and Solutions.

Phosphorylation, a critical mechanism in biological systems, is estimated to be indispensable for about 30% of key biological activities, such as cell cycle progression, migration, and division. It is synergistically balanced by kinases and phosphatases, and any deviation from this balance leads to disease conditions. Pathway or biological activity-based abnormalities in phosphorylation and the type of involved phosphatase influence the outcome, and cause diverse diseases ranging from diabetes, rheumatoid arthritis, and numerous cancers. Protein tyrosine phosphatases (PTPs) are of prime importance in the process of dephosphorylation and catalyze several biological functions. Abnormal PTP activities are reported to result in several human diseases. Consequently, there is an increased demand for potential PTP inhibitory small molecules. Several strategies in structure-based drug designing techniques for potential inhibitory small molecules of PTPs have been explored along with traditional drug designing methods in order to overcome the hurdles in PTP inhibitor discovery. In this review, we discuss druggable PTPs and structure-based virtual screening efforts for successful PTP inhibitor design.

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation.

Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multiprotein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the proinflammatory cytokines IL-1ß and IL-18, can be inhibited by ethanol, and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1ß and caspase-1 cleavage and secretion, as well as diminished apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow-derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of γ-aminobutyric acid A receptor activation or N-methyl-d-asparate receptor inhibition but were associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, whereas administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1ß secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols.

Chemogenomics approaches to rationalizing the mode-of-action of traditional Chinese and Ayurvedic medicines.

Traditional Chinese medicine (TCM) and Ayurveda have been used in humans for thousands of years. While the link to a particular indication has been established in man, the mode-of-action (MOA) of the formulations often remains unknown. In this study, we aim to understand the MOA of formulations used in traditional medicine using an in silico target prediction algorithm, which aims to predict protein targets (and hence MOAs), given the chemical structure of a compound. Following this approach we were able to establish several links between suggested MOAs and experimental evidence. In particular, compounds from the 'tonifying and replenishing medicinal' class from TCM exhibit a hypoglycemic effect which can be related to activity of the ingredients against the Sodium-Glucose Transporters (SGLT) 1 and 2 as well as Protein Tyrosine Phosphatase (PTP). Similar results were obtained for Ayurvedic anticancer drugs. Here, both primary anticancer targets (those directly involved in cancer pathogenesis) such as steroid-5-alpha-reductase 1 and 2 were predicted as well as targets which act synergistically with the primary target, such as the efflux pump P-glycoprotein (P-gp). In addition, we were able to elucidate some targets which may point us to novel MOAs as well as explain side effects. Most notably, GPBAR1, which was predicted as a target for both 'tonifying and replenishing medicinal' and anticancer classes, suggests an influence of the compounds on metabolism. Understanding the MOA of these compounds is beneficial as it provides a resource for NMEs with possibly higher efficacy in the clinic than those identified by single-target biochemical assays.

Xylitol and capsular gene expression in Streptococcus pneumoniae.

Xylitol is a sugar alcohol that inhibits the growth and adherence of Streptococcus pneumoniae. In clinical trials, xylitol has been shown to decrease the occurrence of acute otitis media in day-care children but did not decrease nasopharyngeal carriage of the pneumococci. It has also been shown that xylitol affects the ultrastructure of the pneumococcal capsule. Here, it was hypothesized that xylitol might affect the expression of pneumococcal capsular genes. Capsule gene expression levels were studied in 24 clinical pneumococcal isolates and one ATCC strain (49619) by using a real-time RT-PCR method targeting the mRNA of the second gene of the pneumococcal capsular locus, the cpsB gene. The isolates were exposed to 5 % glucose, 5 % xylitol and control medium (brain heart infusion medium containing 10 % fetal bovine serum) for 2 h. cpsB gene expression levels were measured by using a relative quantification method with calibrator normalization where the 16S rRNA gene of pneumococcus was used as a reference. Exposure to xylitol lowered cpsB gene expression levels significantly compared with those in the control (P=0.035) and glucose (P=0.011) media. This finding supports previous results where exposure to xylitol changed the ultrastructure of the pneumococcal capsule and could explain further the high clinical efficacy of xylitol in preventing otitis media.

Biologically relevant chemical properties of peroxymonophosphate (=O3POOH).

It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-derived regulator of cellular protein tyrosine phosphatase activity under physiological or pathophysiological conditions. To facilitate further consideration of the potential role of peroxymonophosphate in biological systems we present studies related to the preparation, characterization, stability, and fluorometric detection of this agent.

Targeting PTPs with small molecule inhibitors in cancer treatment.

Protein tyrosine phosphorylation plays a major role in cellular signaling. The level of tyrosine phosphorylation is controlled by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Disturbance of the normal balance between PTK and PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. A number of PTPs have been implicated in oncogenesis and tumor progression and therefore are potential drug targets for cancer chemotherapy. These include PTP1B, which may augment signaling downstream of HER2/Neu; SHP2, which is the first oncogene in the PTP superfamily and is essential for growth factor-mediated signaling; the Cdc25 phosphatases, which are positive regulators of cell cycle progression; and the phosphatase of regenerating liver (PRL) phosphatases, which promote tumor metastases. As PTPs have emerged as drug targets for cancer, a number of strategies are currently been explored for the identification of various classes of PTP inhibitors. These efforts have resulted many potent, and in some cases selective, inhibitors for PTP1B, SHP2, Cdc25 and PRL phosphatases. Structural information derived from these compounds serves as a solid foundation upon which novel anti-cancer agents targeted to these PTPs can be developed.

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia.

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.

GnRH-II agonist [D-Lys6]GnRH-II inhibits the EGF-induced mitogenic signal transduction in human endometrial and ovarian cancer cells.

The majority of human endometrial and ovarian cancers express receptors for GnRH type I (GnRH-I). Their proliferation is time- and dose-dependently reduced by GnRH-I and its analogs. GnRH-I analogs activate a phosphotyrosine-phosphatase (PTP) and inhibit EGF-induced mitogenic signal transduction. Recently we found that GnRH type II (GnRH-II) and its agonist [D-Lys6]GnRH-II also have antiproliferative effects on these tumor cells which are significantly greater than those of GnRH-I agonists. In a more recent study, we showed that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. The underlying signal transduction mechanisms of GnRH-II are still unknown. In this study we showed that the mitogenic effects of growth factors in endometrial and ovarian cancer cell lines were counteracted by GnRH-II agonist [D-Lys6]GnRH-II, indicating an interaction with the mitogenic signal transduction. We showed that [D-Lys6]GnRH-II reduces EGF-induced auto-tyrosine-phosphorylation of EGF-receptors via activation of a PTP and that EGF-induced activation of mitogen-activated protein kinase was blocked in cells treated with [D-Lys6]GnRH-II. Furthermore, EGF-induced expression of the immediate early gene c-fos was inhibited by treatment with [D-Lys6]GnRH-II. After knock-out of GnRH-I receptor expression, GnRH-II agonist [D-Lys6]GnRH-II still activated PTP and inhibited the EGF-induced mitogenic signal transduction. These data indicate, that the effects of GnRH-II are not due to a cross-reaction with the GnRH-I receptor. In conclusion these data suggest that the signaling of GnRH-II agonist [D-Lys6]GnRH-II is comparable to that of GnRH-I analogs.

Extracellular ATP counteracts the ERK1/2-mediated death-promoting signaling cascades in astrocytes.

Oxidative stress is the main cause of neuronal death in pathological conditions. Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species, activates many intracellular signaling cascades including src family and mitogen-activated protein kinases (MAPKs), some of which are critically involved in the induction of cellular damage. We previously showed that H(2)O(2)-induced cell death in astrocytes and adenosine 5(')-triphosphate (ATP), acting on P2Y(1) receptors, had a protective effect. Here, we examined the H(2)O(2)-induced changes in intracellular signaling cascades that promote cell death in astrocytes, showing the molecular mechanisms by which the activation of P2Y(1) receptors counteracts such signals. Although H(2)O(2) activated three MAPKs including ERK1/2, p38, and JNK, only the activation of ERK1/2 participated in the H(2)O(2)-evoked cell death. H(2)O(2) induced a sustained activation of ERK1/2 mainly in the nucleus region, which was well in accordance with the H(2)O(2)-induced cell death. H(2)O(2) also activated the src tyrosine kinase family, which was an upstream signal for ERK1/2. Activation of P2Y(1) receptors by 2methylthio-ADP (2MeSADP) inhibited the H(2)O(2)-evoked activation of src tyrosine kinase, resulting in the inhibition of the phosphorylated-ERK1/2 accumulation in the nucleus. 2MeSADP enhanced the gene expression and activity of protein tyrosine phosphatase (PTP), which was responsible for the inhibition of src tyrosine kinase. Thioredoxin reductase, another cytoprotective gene we previously showed to be upregulated by 2MeSADP, also controlled the activity of PTP. Taken together, ATP, acting on P2Y(1) receptors, upregulates the PTP expression and its activity, which counteracts the H(2)O(2)-promoted death signaling cascades including ERK1/2 and its upstream signal src tyrosine kinase in astrocytes.

Cyclophilin A is required for M-CSF-dependent macrophage proliferation.

The immunosuppressor sanglifehrin A (SfA) is a member of a family of immunophilin cyclophilin A-binding molecules and does not inhibit calcineurin activity. Sanglifehrin A inhibits M-CSF-dependent macrophage proliferation by arresting the G1 phase of the cell cycle but does not affect cell viability. This immunosuppressor exerts its action on proliferation by inactivating cyclin-dependent kinase 2 (Cdk2) activity. Moreover, c-myc expression is also repressed. In the early steps of M-CSF signaling, SfA inhibits the phosphorylation of Raf-1 and the external regulated kinases (ERK)1/2 and mitogen-activated protein kinase phosphatase-1, which are required for proliferation. The effects of SfA are not related to a block of the proteosome activity. These data show that immunophilin contributes to M-CSF-dependent proliferation through activation of the Raf-1/MEK/ERK pathway and the regulation of Cdk activities, which is required for cell cycle progression.

Redox regulation of MAP kinase phosphatase 3.

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation.

Quinolinic acid modulates the activity of src family kinases in rat striatum: in vivo and in vitro studies.

Quinolinic acid (QA) has been shown to evoke neurotoxic events via NMDA receptor (NMDAR) overactivation and oxidative stress. NMDARs are particularly vulnerable to free radicals, which can modulate protein tyrosine kinase (PTK) and phosphotyrosine phosphatase (PTP) activities. The src family of tyrosine kinases are associated with the NMDAR complex and regulate NMDA channel function. Because QA is an NMDAR agonist as well as a pro-oxidant agent, we investigated whether it may affect the activity of PTKs and PTPs in vivo and in vitro. In synaptosomes prepared from striata dissected 15 min, 30 min or 15 days after bilateral injection of QA we observed modulation of the phosphotyrosine pattern; a significant decrease in PTP activity; and a sustained increase in c-src and lyn activity at 15 and 30 min after treatment with QA, followed by a decrease 2 weeks later. Striatal synaptosomes treated in vitro with QA showed time- and dose-dependent modulation of c-src and lyn kinase activities. Moreover, the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl ester, the NMDAR antagonist d-2-amino-5-phosphonovaleric acid and pyruvate suppressed the QA-induced modulation of c-src activity. These findings suggest a novel feature of QA in regulating src kinase activity through the formation of reactive radical species and/or NMDAR overactivation.

Parathyroid hormone induces mitogen-activated kinase phosphatase 1 in murine osteoblasts primarily through cAMP-protein kinase A signaling.

BACKGROUND: Parathyroid hormone (PTH) regulates osteoblast function by binding to the PTH receptor 1 (PTHR1) to activate downstream signaling to induce expression of primary response genes (PRGs), which affect various aspects of the osteoblast phenotype. We previously identified PTH-induced PRGs in MC3T3-E1 cells, including mitogen-activated protein kinase (MAPK) phosphatase 1 (mkp1), which dephosphorylates members of the MAPK family. The aim of this study was to explore the molecular mechanisms of PTH's induction of mkp1 in primary mouse osteoblasts. METHODS: Northern and Western analyses were used to determine mkp1 mRNA and protein expression. In vivo experiments were also performed to determine PTH's effect on mkp1 in mouse calvariae and long bones. RESULTS: A total of 10 nM PTH and PTH-related protein (PTHrP) maximally induced mkp1 mRNA levels after 1 hour in osteoblasts. PTH also increased mkp1 protein expression, and induced mkp1 mRNA independent of new protein synthesis. PTHR1 triggers protein kinase A (PKA), PKC, and calcium pathways. Although PKA and PKC agonists induced mkp1 mRNA levels, only cyclic adenosine 3':5'-monophosphate (cAMP)-PKA inhibition blocked PTH-induced mkp1 mRNA levels. These data suggest that PTH-induced mkp1 mRNA levels are primarily mediated through the cAMP-PKA pathway. Further, prostaglandin E2 (PGE2), which activates cAMP-PKA and PKC, induced mkp1 mRNA to a greater extent than PGF2alpha and fluprostenol, which activate PKC signaling only. Finally, PTH maximally induced mkp1 mRNA levels in mouse calvariae and long bones in vivo at 0.5 hours. CONCLUSIONS: mkp1's in vitro and in vivo induction in PTH-target tissues suggests its involvement in some of the effects of PTH on osteoblast function. mkp1 may be an important target gene in the anabolic effect of PTH on osteoblasts.

Differential cellular and molecular effects of bortezomib, a proteasome inhibitor, in human breast cancer cells.

The cellular and molecular effects of the proteasome inhibitor bortezomib on breast cancer cells are as yet poorly characterized. Here, in a panel of six breast cancer cell lines, bortezomib reduced viability in a concentration-dependent, time-dependent, and cell line-dependent manner. Proteasome activity was relatively high in two of the three more resistant cell lines. No relationship was observed between bortezomib effects on cell viability and expression/phosphorylation of HER-2, epidermal growth factor receptor (EGFR), AKT, or extracellular signal-regulated kinase 1/2 (ERK1/2). Molecular effects of bortezomib were further studied in SK-BR-3 and BT-474 cells because they share expression of EGFR and overexpression of HER-2 while, in contrast, SK-BR-3 cells were 200-fold more sensitive to this agent. Proteasome activity was inhibited to a similar extent in the two cell lines, and known proteasome substrates accumulated similarly. In SK-BR-3 cells, a marked inhibition of EGFR, HER-2, and AKT phosphorylation was observed at a clinically relevant concentration of bortezomib. In contrast, phosphorylation of Raf/mitogen-activated protein kinase kinase 1/2 (MEK 1/2)/ERK1/2 increased by bortezomib. In BT-474 cells, the effects were much less pronounced. Treatment of SK-BR-3 cells with bortezomib combined with pharmacologic inhibitors of EGFR, phosphatidylinositol 3'-kinase, or MEK resulted in modest or no enhancement of the effects on cell viability. Collectively, these results show that bortezomib has differential cellular and molecular effects in human breast cancer cells. The bortezomib-observed effects on signaling transduction molecules might be relevant to help to design mechanistic-based combination treatments.

Inhibition of protein tyrosine phosphatase 1B by ursane-type triterpenes isolated from Symplocos paniculata.

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been proposed as a therapy for treatment of type 2 diabetes and obesity. Bioassay-guided fractionation of the MeOH extract of the leaves and stems of Symplocos paniculata (Thunb.) Miq. (Symplocaceae), using an in vitro PTP1B inhibitory assay, resulted in the isolation of three ursane-type triterpenes, ursolic acid (1), corosolic acid (2) and 2alpha,3alpha,19alpha,23-tetrahydroxyurs-12-en-28-oic acid (3). Compounds 1-3 inhibited PTP1B with IC (50) values of 3.8 +/- 0.5, 7.2 +/- 0.8 and 42.1 +/- 1.5 microM, respectively. Kinetic studies suggest that 1 is a competitive inhibitor with a K(i) value of 2.0 microM, whereas 2 is a mixed-type inhibitor of PTP1B. Our results indicate that the substitution of hydroxy groups on the ursane-type triterpenes is responsible for the loss of activity, and thus 1 and 2 possessing only one or two hydroxy groups can be potential PTP1B inhibitors.

Divergent expression and function of glucocorticoid receptor beta in human monocytes and T cells.

Glucocorticoid (GC) insensitivity is a significant problem in the treatment of immune-mediated diseases. The current study examined whether T cells and monocytes differed in their response to GC and the potential molecular basis for their variation in response to steroids. Functional studies revealed that dexamethasone (DEX) inhibited phorbol 12-myristate 13-acetate/ionomycin-induced tumor necrosis factor alpha and interleukin-6 production to a significantly lesser extent in monocytes than T cells. In parallel, a significantly longer period of time was required for DEX to induce the steroid-responsive gene, mitogen-activated protein kinase phosphatase-1 (MKP-1), in human monocytes as compared with T cells. It is interesting that such differences were not observed between murine T cells and monocytes. GC receptor beta (GCRbeta) is a splicing variant of the classic GCR, GCRalpha, which functions as a dominant-negative inhibitor of GCRalpha in humans, not mice (as mice do not express GCRbeta mRNA as a result of a difference in the murine GCR 9b exon sequence). It was found that human monocytes had a significantly higher level of GCRbeta than T cells. Furthermore, GCRbeta was found in the cytoplasm and nucleus of monocytes, and GCRbeta was localized to the nucleus of T cells. This raised the possibility that GCRbeta in the cytoplasm could affect GCRalpha cellular shuttling in response to DEX. Indeed, we found that DEX-induced nuclear translocation of GCRalpha was decreased in monocytes as compared with T cells. Specific RNA silencing of GCRbeta in human monocytes resulted in enhanced steroid-induced GCRalpha transactivation and transrepression. Our data suggest that GCRbeta contributes to variation in the GC responses of monocytes versus T cells.

Protein tyrosine phosphatase 1B negatively regulates macrophage development through CSF-1 signaling.

Protein tyrosine phosphatase 1B (PTP-1B) is a ubiquitously expressed cytosolic phosphatase with the ability to dephosphorylate JAK2 and TYK2, and thereby down-regulate cytokine receptor signaling. Furthermore, PTP-1B levels are up-regulated in certain chronic myelogenous leukemia patients, which points to a potential role for PTP-1B in myeloid development. The results presented here show that the absence of PTP-1B affects murine myelopoiesis by modifying the ratio of monocytes to granulocytes in vivo. This bias toward monocytic development is at least in part due to a decreased threshold of response to CSF-1, because the PTP-1B -/- bone marrow presents no abnormalities at the granulocyte-monocyte progenitor level but produces significantly more monocytic colonies in the presence of CSF-1. This phenomenon is not due to an increase in receptor levels but rather to enhanced phosphorylation of the activation loop tyrosine. PTP-1B -/- cells display increased inflammatory activity in vitro and in vivo through the constitutive up-regulation of activation markers as well as increased sensitivity to endotoxin. Collectively, our data indicate that PTP-1B is an important modulator of myeloid differentiation and macrophage activation in vivo and provide a demonstration of a physiological role for PTP-1B in immune regulation.

Expression analysis and modulation by HIV-Tat of the tyrosine phosphatase HD-PTP.

The human immunodeficiency virus type 1 Tat transactivates viral proteins and also affects the expression of eukaryotic genes. Since Tat is angiogenic, we assumed that the isolation of differentially expressed genes in Tat-treated endothelial cells would yield insights into the molecular mechanisms of the angiogenic process. By RNA fingerprinting, we found that Tat upregulates the tyrosine phosphatase HD-PTP mRNA in a human endothelial cell line. At the moment, little is known about HD-PTP. We here show that HD-PTP is highly conserved through evolution from yeast to man, and is ubiquitously distributed in adult and fetal tissues. HD-PTP is expressed in human cell lines derived from different tumors, but the mRNA levels do not appear to correlate with the malignant phenotype of the cells. HD-PTP mRNA was also detected in cell lines derived from tumors that develop in BKV/Tat-transgenic mice. Interestingly, a relation exists between the amounts of secreted Tat and the levels of HD-PTP mRNA. HD-PTP encodes a 185-kDa protein which is expressed in human endothelial from the umbilical cord and in human Kaposi-spindle cells. Tat-induction of HD-PTP mRNA parallels only with a slight increase of the protein, which occurs after 24 and 48 h of incubation in the presence of Tat. These results suggest that HD-PTP amounts might be regulated both at the transcriptional and post-transcriptional levels.