Dehydroandrographolide facilitates M2 macrophage polarization by downregulating DUSP3 to inhibit sepsis-associated acute kidney injury.

BACKGROUND: Sepsis is perceived as lethal tissue damage and significantly increases mortality in combination with acute kidney injury (AKI). M2 macrophages play important roles in the secretion of anti-inflammatory and tissue repair mediators. We aimed to study the role of Dehydroandrographolide (Deh) in sepsis-associated AKI in vitro and in vivo through lipopolysaccharide (LPS)-induced macrophages model and cecal ligation and puncture-induced AKI mice model, and to reveal the mechanism related to M2 macrophage polarization. METHODS: Enzyme-linked immunosorbent assay kits were used to assess the levels of inflammatory factors. Expression of markers related to M1 macrophages and M2 macrophages were analyzed. Additionally, dual specificity phosphatase 3 (DUSP3) expression was tested. Cell apoptosis was evaluated by flow cytometry analysis and terminal-deoxynucleotidyl transferase-mediated nick end labeling staining. Moreover, renal histological assessment was performed by using hematoxylin and eosin staining. RESULTS: Deh reduced inflammation of THP-1-derived macrophages exposed to LPS. Besides, Deh induced the polarization of M1 macrophages to M2 and downregulated DUSP3 expression in THP-1-derived macrophages under LPS conditions. Further, DUSP3 overexpression reversed the impacts of Deh on the inflammation and M2 macrophages polarization of THP-1-derived macrophages stimulated by LPS. Additionally, human proximal tubular epithelial cells (HK-2) in the condition medium from DUSP3-overexpressed THP-1-derived macrophages treated with LPS and Deh displayed decreased viability and increased apoptosis and inflammation. The in vivo results suggested that Deh improved the renal function, ameliorated pathological injury, induced the polarization of M1 macrophages to M2, suppressed inflammation and apoptosis, and downregulated DUSP3 expression in sepsis-induced mice. CONCLUSION: Deh facilitated M2 macrophage polarization by downregulating DUSP3 to inhibit septic AKI.

l-Methionine potentiates anticancer activity of Sorafenib by epigenetically altering DUSP3/ERK pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most common cancer-related cause of death worldwide. Although Sorafenib is the standard systemic therapy for treating HCC, but it develops resistance very quickly, leading to poor prognosis. The current study was planned to explore the effect of l-methionine on the anticancer activity of Sorafenib in HCC. Ten millimolar of l-methionine treatment significantly reduced the IC50 of Sorafenib from 5.513 ± 0.171 to 0.8095 ± 0.0465 µM in HepG2 cell line. It also resulted in concomitant increase in oxidative stress and deactivation of ERK/AMPK/AKT pathway. Additionally, it also resulted in the increased expression of dual specificity phosphatase 3 (DUSP3). In a rat model of sorafenib-resistant HCC induced by diethylnitrosamine (DEN) (100 mg/L/day) and Sorafenib (10 mg/kg), l-methionine (300 and 500 mg/kg/day) supplementation overcame the drug resistance, as indicated by the reduced formation of surface tumor nodules, prevention of cellular hypertrophy, hyperplasia and inflammation, and improved animal survival. Furthermore, l-methionine in combination with Sorafenib also inhibited AMPK/AKT and ERK pathway. At chromatin level, l-methionine supplementation prevented global methylation of H3K27me3, an inactivation mark, and demethylation of H3K36me2, an activation mark. Interestingly, our findings suggest that inhibition of the ERK pathway via increased activity of DUSP3 is epigenetically regulated. Besides, chromatin immunoprecipitation data exhibited augmented H3K36me2 (an activation mark) levels on the DUSP3 promoter region. To the best of our knowledge, we are the first to report that l-methionine supplementation improves the chemosensitivity in Sorafenib-resistant HCC via modulating the epigenetic landscape and can be a potential therapeutic strategy.

DUSP3 regulates phosphorylation-mediated degradation of occludin and is required for maintaining epithelial tight junction.

BACKGROUND: Tight junctions (TJ) are multi-protein complexes that hold epithelial cells together and form structural and functional barriers for maintaining proper biological activities. Dual specificity phosphatase 3 (DUSP3), a suppressor of multiple protein tyrosine (Tyr) kinases, is decreased in lung cancer tissues. Here we demonstrated the role of DUSP3 in regulation of epithelial TJ. METHODS: Barrier functions of TJ were examined in wild-type or DUSP3-deficient lung epithelial cells. Animal and clinical data were analyzed for the association between DUSP3 deficiency and lung cancer progression. Proximity ligation assay, immunoblotting, and phosphatase assay were performed to study the effect of DUSP3 on the TJ protein occludin (OCLN). Mutations of Tyr residues on OCLN showed the role of Tyr phosphorylation in regulating OCLN. RESULTS: Compared to those of the DUSP3-expressing cells, we found the expression and distribution of ZO-1, a TJ-anchoring molecule, were abnormal in DUSP3-deficient cells. OCLN had an increased phosphorylation level in DUSP3-deficient cells. We identified that OCLN is a direct substrate of DUSP3. DUSP3 regulated OCLN ubiquitination and degradation through decreasing OCLN tyrosine phosphorylation directly or through suppressing focal adhesion kinase, the OCLN kinase. CONCLUSION: Our study revealed that DUSP3 is an important TJ regulatory protein and its decrease may be involved in progression of epithelial cancers.

UV Radiation-induced Impairment of Cellular Morphology and Motility is Enhanced by DUSP3/VHR Loss and FAK Activation.

DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability.

Regulation of signal transducer and activator of transcription 3 activation by dual-specificity phosphatase 3.

Since cancer is the leading cause of death worldwide, there is an urgent need to understand the mechanisms underlying cancer progression and the development of cancer inhibitors. Signal transducer and activator of transcription 3 (STAT3) is a major transcription factor that regulates the proliferation and survival of various cancer cells. Here, dual-specificity phosphatase 3 (DUSP3) was identified as a regulator of STAT3 based on an interaction screening performed using the protein tyrosine phosphatase library. DUSP3 interacted with the C-terminal domain of STAT3 and dephosphorylated p-Y705 of STAT3. In vitro dephosphorylation assay revealed that DUSP3 directly dephosphorylated p-STAT3. The suppressive effects of DUSP3 on STAT3 were evaluated by a decreased STAT3-specific promoter activity, which in turn reduced the expression of the downstream target genes of STAT3. In summary, DUSP3 downregulated the transcriptional activity of STAT3 via dephosphorylation at Y705 and also suppressed the migratory activity of cancer cells. This study demonstrated that DUSP3 inhibits interleukin 6 (IL-6)/STAT3 signaling and is expected to regulate cancer development. Novel functions of DUSP3 discovered in IL-6/STAT3 signaling regulation would help expand the understanding of cancer development mechanisms. [BMB Reports 2020; 53(6): 335-340].

Identification of Vaccinia-H1 Related Phosphatase as an Anticancer Target for 1,2,3,4,6-O-Pentagalloylglucose.

Protein tyrosine phosphatases are involved in diverse human diseases, including cancer, diabetes and inflammatory disorders. Loss of Vaccinia-H1 related phosphatase (VHR) has been shown to arrest at the G1-S and G2-M transitions of the cell cycle, and to increases cell death of prostate cancer cells through JNK activation, suggesting that VHR can be considered as an anticancer target. In this study, 658 natural products were screened through in vitro enzyme assay to identify VHR inhibitor. Among the VHR-inhibitory compounds, 1,2,3,4,6-O-pentagalloylglucose (PGG) was selected for further study as it has been reported to show antitumor effects against tumor model mice, but its direct target has not been identified. PGG inhibited the catalytic activity of VHR (Ki =53 nm) in vitro. Furthermore, the incubation of HeLa cervical cancer cells with PGG dramatically decreased cell viability and markedly increased the protein levels of the cleaved PARP, a hallmark of apoptosis. In addition, treatment of HeLa cells with PGG significantly reduced the protein levels of cyclin D1, Bcl-2 and STAT3 phosphorylation. Taken together, these results suggest that PGG could be a potential therapeutic candidate for the treatment of cervical cancer through VHR inhibition.

Dusp3 deletion in mice promotes experimental lung tumour metastasis in a macrophage dependent manner.

Vaccinia-H1 Related (VHR) dual-specificity phosphatase, or DUSP3, plays an important role in cell cycle regulation and its expression is altered in several human cancers. In mouse model, DUSP3 deletion prevents neo-angiogenesis and b-FGF-induced microvessel outgrowth. Considering the importance of angiogenesis in metastasis formation, our study aimed to investigate the role of DUSP3 in tumour cell dissemination. Using a Lewis Lung carcinoma (LLC) experimental metastasis model, we observed that DUSP3-/- mice developed larger lung metastases than littermate controls. DUSP3-/- bone marrow transfer to lethally irradiated DUSP3+/+ mice was sufficient to transfer the phenotype to DUSP3+/+ mice, indicating that hematopoietic cells compartment was involved in the increased tumour cell dissemination to lung tissues. Interestingly, we found a higher percentage of tumour-promoting Ly6Cint macrophages in DUSP3-/- LLC-bearing lung homogenates that was at least partially due to a better recruitment of these cells. This was confirmed by 1) the presence of higher number of the Ly6Bhi macrophages in DUSP3-/- lung homogenates and by 2) the better migration of DUSP3-/- bone marrow sorted monocytes, peritoneal macrophages and bone marrow derived macrophages (BMDMs), compared to DUSP3+/+ monocytes, macrophages and BMDMs, in response to LLC-conditioned medium. Our study demonstrates that DUSP3 phosphatase plays a key role in metastatic growth through a mechanism involving the recruitment of macrophages towards LLC-bearing lungs.

VRK3-mediated nuclear localization of HSP70 prevents glutamate excitotoxicity-induced apoptosis and Aß accumulation via enhancement of ERK phosphatase VHR activity.

Most of neurodegenerative disorders are associated with protein aggregation. Glutamate-induced excitotoxicity and persistent extracellular signal-regulated kinase (ERK) activation are also implicated in neurodegenerative diseases. Here, we found that vaccinia-related kinase 3 (VRK3) facilitates nuclear localization of glutamate-induced heat shock protein 70 (HSP70). Nuclear HSP70 leads to enhancement of vaccinia H1-related phosphatase (VHR) activity via protein-protein interaction rather than its molecular chaperone activity, thereby suppressing excessive ERK activation. Moreover, glutamate-induced ERK activation stimulates the expression of HSP70 and VRK3 at the transcriptional level. Downregulation of either VRK3 or HSP70 rendered cells vulnerable to glutamate-induced apoptosis. Overexpression of HSP70 fused to a nuclear localization signal attenuated apoptosis more than HSP70 alone. The importance of nuclear localization of HSP70 in the negative regulation of glutamate-induced ERK activation was further confirmed in VRK3-deficient neurons. Importantly, we showed a positive correlation between levels of VRK3 and HSP70 in the progression of Alzheimer's and Parkinson's diseases in humans, and neurons with HSP70 nuclear localization exhibited less Aß accumulation in brains from patients with Alzheimer's disease. Therefore, HSP70 and VRK3 could potentially serve as diagnostic and therapeutic targets in neurodegenerative diseases.

Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis.

Therapeutic perspectives targeting angiogenesis in cancer stimulated an intense investigation of the mechanisms triggering and governing angiogenic processes. Several publications have highlighted the importance of typical dual-specificity phosphatases (DSPs) or MKPs in endothelial cells and their role in controlling different biological functions implicated in angiogenesis such as migration, proliferation, apoptosis, tubulogenesis, and cell adhesion. However, among atypical DSPs, the only one investigated in angiogenesis was DUSP3. We recently identified this DSP as a new key player in endothelial cells and angiogenesis. In this chapter we provide with detailed protocols and models used to investigate the role of DUSP3 in endothelial cells and angiogenesis. We start the chapter with an overview of the role of several DSPs in angiogenesis. We continue with providing a full description of a highly efficient transfection protocol to deplete DUSP3 using small interfering RNA (siRNA) in the primary human umbilical vein endothelial cells (HUVEC). We next describe the major assays used to investigate different processes involved in angiogenesis such as tube formation assay, proliferation assay and spheroids sprouting assay. We finish the chapter by validating our results in DUSP3-knockout mice using in vivo angiogenesis assays such as Matrigel plug and Lewis lung carcinoma cell subcutaneous xenograft model followed by anti-CD31 immunofluorescence and ex vivo aortic ring assay. All methods described can be adapted to other phosphatases and signaling molecules.

Design, synthesis and cytotoxicity of pyrano[4,3-b]indol-1(5H)-ones: A hybrid pharmacophore approach via gold catalyzed cyclization.

Reported herein is the gold(III)-catalyzed 6-endo-dig cycloisomerization of 2-alkynyl-indole-3-carboxylic acids to form pyrano[4,3-b]indol-1(5H)-ones, which are pharmaceutically important structural motifs. The hitherto unknown substrates required for this methodology were conveniently synthesized in five steps with good overall yields. The utility of this new cycloisomerization is demonstrated by the excellent regioselectivity obtained using a range of substrates. The mildness of the method allowed functional group compatibility towards hydroxyl tether, displaying exquisite chemoselectivity. All the synthesized compounds were screened for their tumor cell growth inhibitory activity against human cervix adenocarcinoma (HeLa). Compound 7d emerged as the most active (IC50=0.69µM) among the tested series compared to the standard cis-platin (IC50=0.08µM).

VHR/DUSP3 phosphatase: structure, function and regulation.

Vaccinia H1-related (VHR) phosphatase, also known as dual-specificity phosphatase (DUSP) 3, is a small member of the DUSP (also called DSP) family of phosphatases. VHR has a preference for phospho-tyrosine substrates, and has important roles in cellular signaling ranging from cell-cycle regulation and the DNA damage response to MAPK signaling, platelet activation and angiogenesis. VHR/DUSP3 has been implicated in several human cancers, where its tumor-suppressing and -promoting properties have been described. We give a detailed overview of VHR/DUSP3 phosphatase and compare it with its most closely related phosphatases DUSP13B, DUSP26 and DUSP27.

Dusp3 and Psme3 are associated with murine susceptibility to Staphylococcus aureus infection and human sepsis.

Using A/J mice, which are susceptible to Staphylococcus aureus, we sought to identify genetic determinants of susceptibility to S. aureus, and evaluate their function with regard to S. aureus infection. One QTL region on chromosome 11 containing 422 genes was found to be significantly associated with susceptibility to S. aureus infection. Of these 422 genes, whole genome transcription profiling identified five genes (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) that were significantly differentially expressed in a) S. aureus -infected susceptible (A/J) vs. resistant (C57BL/6J) mice and b) humans with S. aureus blood stream infection vs. healthy subjects. Three of these genes (Dcaf7, Dusp3, and Psme3) were down-regulated in susceptible vs. resistant mice at both pre- and post-infection time points by qPCR. siRNA-mediated knockdown of Dusp3 and Psme3 induced significant increases of cytokine production in S. aureus-challenged RAW264.7 macrophages and bone marrow derived macrophages (BMDMs) through enhancing NF-κB signaling activity. Similar increases in cytokine production and NF-κB activity were also seen in BMDMs from CSS11 (C57BL/6J background with chromosome 11 from A/J), but not C57BL/6J. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection.

DUSP3/VHR is a pro-angiogenic atypical dual-specificity phosphatase.

BACKGROUND: DUSP3 phosphatase, also known as Vaccinia-H1 Related (VHR) phosphatase, encoded by DUSP3/Dusp3 gene, is a relatively small member of the dual-specificity protein phosphatases. In vitro studies showed that DUSP3 is a negative regulator of ERK and JNK pathways in several cell lines. On the other hand, DUSP3 is implicated in human cancer. It has been alternatively described as having tumor suppressive and oncogenic properties. Thus, the available data suggest that DUSP3 plays complex and contradictory roles in tumorigenesis that could be cell type-dependent. Since most of these studies were performed using recombinant proteins or in cell-transfection based assays, the physiological function of DUSP3 has remained elusive. RESULTS: Using immunohistochemistry on human cervical sections, we observed a strong expression of DUSP3 in endothelial cells (EC) suggesting a contribution for this phosphatase to EC functions. DUSP3 downregulation, using RNA interference, in human EC reduced significantly in vitro tube formation on Matrigel and spheroid angiogenic sprouting. However, this defect was not associated with an altered phosphorylation of the documented in vitro DUSP3 substrates, ERK1/2, JNK1/2 and EGFR but was associated with an increased PKC phosphorylation. To investigate the physiological function of DUSP3, we generated Dusp3-deficient mice by homologous recombination. The obtained DUSP3-/- mice were healthy, fertile, with no spontaneous phenotype and no vascular defect. However, DUSP3 deficiency prevented neo-vascularization of transplanted b-FGF containing Matrigel and LLC xenograft tumors as evidenced by hemoglobin (Hb) and FITC-dextran quantifications. Furthermore, we found that DUSP3 is required for b-FGF-induced microvessel outgrowth in the aortic ring assay. CONCLUSIONS: All together, our data identify DUSP3 as a new important player in angiogenesis.

Unnatural amino acid mutagenesis reveals dimerization as a negative regulatory mechanism of VHR's phosphatase activity.

Vaccinia H1-related (VHR) phosphatase is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochemically well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR's biological mechanisms and roles but also to determine its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique "variable insert" region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chemical cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-association of VHR acts as a means for the negative regulation of its catalytic activity.

Proteomic, cellular, and network analyses reveal new DUSP3 interactions with nucleolar proteins in HeLa cells.

DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions.

Enteric commensal bacteria induce extracellular signal-regulated kinase pathway signaling via formyl peptide receptor-dependent redox modulation of dual specific phosphatase 3.

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Vaccinia H1-related phosphatase is a phosphatase of ErbB receptors and is down-regulated in non-small cell lung cancer.

Vaccinia H1-related phosphatase (VHR) is classified as a dual specificity phosphatase. Unlike typical dual specificity phosphatases, VHR lacks the MAPK-binding domain and shows poor activity against MAPKs. We found that EGF receptor (EGFR) was a direct substrate of VHR and that overexpression of VHR down-regulated EGFR phosphorylation, particularly at Tyr-992 residue. Expression of VHR inhibited the activation of phospholipase Cγ and protein kinase C, both downstream effectors of Tyr-992 phosphorylation of EGFR. Decreasing VHR expression by RNA interference caused higher EGFR phosphorylation at Tyr-992. In addition to EGFR, VHR also directly dephosphorylated ErbB2. Consistent with these results, suppression of VHR augmented the foci formation ability of H1299 non-small cell lung cancer (NSCLC) cells, whereas overexpression of VHR suppressed cell growth in both two- and three-dimensional cultures. Expression of VHR also suppressed tumor formation in a mouse xenograft model. Furthermore, VHR expression was significantly lower in NSCLC tissues in comparison to that in normal lung tissues. Collectively, this study shows that down-regulation of VHR expression enhances the signaling of ErbB receptors and may be involved in NSCLC pathogenesis.

PTP1B suppresses prolactin activation of Stat5 in breast cancer cells.

Basal levels of nuclear localized, tyrosine phosphorylated Stat5 are present in healthy human breast epithelia. In contrast, Stat5 phosphorylation is frequently lost during breast cancer progression, a finding that correlates with loss of histological differentiation and poor patient prognosis. Identifying the mechanisms underlying loss of Stat5 phosphorylation could provide novel targets for breast cancer therapy. Pervanadate, a general tyrosine phosphatase inhibitor, revealed marked phosphatase regulation of Stat5 activity in breast cancer cells. Lentiviral-mediated shRNA allowed specific examination of the regulatory role of five tyrosine phosphatases (PTP1B, TC-PTP, SHP1, SHP2, and VHR), previously implicated in Stat5 regulation in various systems. Enhanced and sustained prolactin-induced Stat5 tyrosine phosphorylation was observed in T47D and MCF7 breast cancer cells selectively in response to PTP1B depletion. Conversely, PTP1B overexpression suppressed prolactin-induced Stat5 tyrosine phosphorylation. Furthermore, PTP1B knockdown increased Stat5 reporter gene activity. Mechanistically, PTP1B suppression of Stat5 phosphorylation was mediated, at least in part, through inhibitory dephosphorylation of the Stat5 tyrosine kinase, Jak2. PTP1B knockdown enhanced sensitivity of T47D cells to prolactin phosphorylation of Stat5 by reducing the EC(50) from 7.2 nmol/L to 2.5 nmol/L. Immunohistochemical analyses of two independent clinical breast cancer materials revealed significant negative correlations between levels of active Stat5 and PTP1B, but not TC-PTP. Collectively, our data implicate PTP1B as an important negative regulator of Stat5 phosphorylation in invasive breast cancer.

Dual-specificity phosphatases: critical regulators with diverse cellular targets.

DUSPs (dual-specificity phosphatases) are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. DUSPs have been implicated as major modulators of critical signalling pathways that are dysregulated in various diseases. DUSPs can be divided into six subgroups on the basis of sequence similarity that include slingshots, PRLs (phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is cell division cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10), myotubularins, MKPs (mitogen-activated protein kinase phosphatases) and atypical DUSPs. Of these subgroups, a great deal of research has focused on the characterization of the MKPs. As their name suggests, MKPs dephosphorylate MAPK (mitogen-activated protein kinase) proteins ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 with specificity distinct from that of individual MKP proteins. Atypical DUSPs are mostly of low-molecular-mass and lack the N-terminal CH2 (Cdc25 homology 2) domain common to MKPs. The discovery of most atypical DUSPs has occurred in the last 6 years, which has initiated a large amount of interest in their role and regulation. In the past, atypical DUSPs have generally been grouped together with the MKPs and characterized for their role in MAPK signalling cascades. Indeed, some have been shown to dephosphorylate MAPKs. The current literature hints at the potential of the atypical DUSPs as important signalling regulators, but is crowded with conflicting reports. The present review provides an overview of the DUSP family before focusing on atypical DUSPs, emerging as a group of proteins with vastly diverse substrate specificity and function.

High intracellular Zn2+ ions modulate the VHR, ZAP-70 and ERK activities of LNCaP prostate cancer cells.

Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 microg/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.