and Immunity (2013) 14, 1–6 & 2013 Macmillan Publishers Limited All rights reserved 1466-4879/13 www.nature.com/gene

REVIEW Dual-specificity 2: surprising positive effect at the molecular level and a potential biomarker of diseases

WWei1,2, Y Jiao2,3, A Postlethwaite4,5, JM Stuart4,5, Y Wang6, D Sun1 and W Gu2

Dual-specificity phosphatases (DUSPs) is an emerging subclass of the superfamily, a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/ phosphothreonine residues within the one substrate. Recently, a series of investigations of DUSPs defined their essential roles in cell proliferation, cancer and the immune response. This review will focus on DUSP2, its involvement in different diseases and its potential as a therapeutic target.

Genes and Immunity (2013) 14, 1–6; doi:10.1038/gene.2012.54; published online 29 November 2012 Keywords: dual-specificity phosphatases; disease; -activated protein ; immune

INTRODUCTION extracellular stimuli. Inducible nucleuses MKPs include DUSP1, Mitogen-activated (MAPK) activation cascades DUSP2, DUSP4 and DUSP5, which originated from a common mediate various physiological processes, such as cell proliferation, ancestral gene. They were shown to dephosphorylate Erks, Jnk differentiation, stress responses, inflammation, and and p38 MAPKs to the same extent and to be induced by growth immune defense.1–4 Dysregulation of MAPK activation cascades or stress signals. DUSP6, DUSP7 and DUSP9 are cytoplasmic Erk- has been implicated in various diseases and has been the focus of specific MPKs, which can preferentially recognize Erk1 and Erk2 extensive research.5–7 MAPKs are grouped into three major classes in vitro. DUSP8, DUSP16 and DUSP10 are cytoplasmic and nuclear. based on their preferential activation by extracellular stimuli: They recognize Jnk and p38 and can inactivate p38 and Jnk 15,16 classic Erk1 and Erk2 and p38 (p38a, p38b, p38g and p38 MAPKs. They negatively regulate members of the MAPK d) and Jun N-terminal (Jnk1, Jnk2 and Jnk3). These are all superfamily (MAPK/Erk, SAPK/Jnk and p38), which are associated activated by dual of the and tyrosine with cellular proliferation and differentiation. As major residues in a conserved ‘TXY’ (Thr-Xaa-Tyr) motif (where X is E for regulators of MAPK signaling, DUSPs have been implicated as Erk, G for p38 or P for Jnk). major modulators of the critical signaling pathways that are Dual-specificity phosphatases (DUSPs) are an emerging dysregulated in various diseases. subclass of the protein tyrosine phosphatase gene superfamily, Recently, a series of investigation in DUSPs defined essential 17–23 a heterogeneous group of protein phosphatases that can roles in cell proliferation and cancer and their immune 4,24 dephosphorylate both phosphotyrosine and phosphoserine/ response. Although there are at least 11 MKPs (many of them phosphothreonine residues within the one substrate.8 They can were thought to have a similar role to regulate MAPK cascades), regulate activity of MAPK through TXY motif it is obvious that each of them has different substrate specificities as well as represent particularly important negative regulators.8,9 and physiological roles. This review will focus on one of the In addition to their dephosphorylating capacity, DUSPs serve DUSPs: DUSP2. We will present what is currently known about to anchor or shuttle MAPKs and control their subcellular its involvement in different diseases and potential as a therapeutic localization.10,11 target. The contains 30 putative DUSP genes, 11 of which are specific for the MAPKs Erk, Jnk and p38.12,13 Those genes have been referred to as MAP kinase phosphatases HISTORY AND BASIC FUNCTIONS OF DUSP2 13 (MKPs). Another subgroup of DUSPs contains 19 DUSPs, which DUSP2 has also been referred to as the phosphatase of activated 13 have been referred to as ‘atypical’ DUSPs. Some of them have cells 1. It is a 32-kDa protein, originally cloned in 1993 from human been shown to regulate MAPKs as they share some characteristic T cells as an immediate-early gene.25 DUSP2 localizes in the of the MKPs. They contain the DUSP catalytic domain but lack the nucleus and is predominantly expressed in hematopoietic tissues 14 N-terminal CH2 domain found in MKPs. with high T-cell content, such as thymus, spleen, lymph nodes, Different members of the family of MKPs show distinct peripheral blood and other organs (brain and liver).25–28 Its substrate, different tissue distribution and subcellular localization, expression is induced rapidly in mitogen-stimulated T and B cells and different modes of inducibility of their expression by after stimulation.25,29 As a member of the dual-specificity protein

1Institute of Endemic Fluorosis Disease, Center for Endemic Disease Control, Center for Disease Control and Prevention, Harbin Medical University, Harbin, PRC; 2Departments of Orthopaedic Surgery and BME, Campbell Clinic, University of Tennessee Health Science Center (UTHSC), Memphis, TN, USA; 3Mudanjiang Medical College, Mudanjiang, PRC; 4Division of connective Tissue Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA; 5Department of Veterans Affairs Medical Center, Memphis, TN, USA and 6Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China. Correspondence: Professor D Sun, Institute of Endemic Fluorosis Disease, Center for Endemic Disease Control, Center for Disease Control and Prevention, Harbin Medical University, Harbin, 150081, PRC. or Professor W Gu, Department of Orthopaedic Surgery–Campbell Clinic, University of Tennessee Health Science Center, 956 Court Ave, Memphis, TN 38163, USA. E-mail: [email protected] or [email protected] Received 16 August 2012; revised 8 October 2012; accepted 10 October 2012; published online 29 November 2012 Dual-specificity phosphatases 2 effect in diseases W Wei et al 2

Figure 1. The DUSP2 phosphorylation signaling pathways in immune cells. MAPKs are activated by stresses and phosphorylated on Tyr and Thr residues. The three-tiered kinase dynamic cascade leads to downregulation of MAPKs (Jnk, Erk1/2 and p38) activation and in the nucleus to transcription factor activation for cellular responses, such as cytokine production, apoptosis, proliferation, differentiation and inflammatory. Simultaneously, the same pathways are triggered in macrophages and mast cells by TLR ligands and FceRI ligation, respectively. They induce the expression of the DUSP2 gene in the nucleus, resulting in the accumulation of DUSP2. DUSP2 may control Jun- Erk negative cross-talk in cells, inactivate and anchor Erk2, and it could dephosphorylate p38 in vivo and co-associates with another protein. Arrows indicate stimulation and red lines indicate inhibition within the pathway.

phosphatase subfamily, DUSP2 inactivates its target kinases by with DUSP243 that includes the amino-terminal domain.44 DUSP2 dephosphorylating both the phosphoserine/threonine and has two conserved, positively charged amino-acid residues phosphotyrosine residues.8 It is a member of MKPs with protein (Arg294 and Arg295), which are important for binding to the tyrosine phosphatase and CH2 domains.13,30 Similar to the other phosphothreonine of Erk2.44 Mutation of either residue alone or in members of DUSP family, the function of DUSP2 has been combination lead to a nearly complete loss of Erk2-induced described as a typically negative regulator of MAPK signaling. catalytic activation of DUSP2 and its ability to dephosphorylate DUSP2 can preferentially dephosphorylate Erk1/2 and p38 but not Erk2.43 DUSP2 contributes to the dephosphorylation of protein Jnk in vitro.31,32 Although there have been reports of low kinase C-activated Erk2 in the nucleus, inactivating and anchoring phosphatase activity of DUSP2 for Jnk in vitro,31 DUSP2 it. DUSP2, together with DUSP1 and DUSP4, regulates Erk2 deficiency has led to increased activity of Jnk and unexpected dephosphorylation and compartmentalization in response to impairment of the activity of extracellular Erk and p38.4 The sustained protein kinase C-mediated signaling. Overexpression DUSP2 signaling pathways are shown in Figure 1. of DUSP2 prevents Erk2 activation and causes Erk2 nuclear Erk1/2 is phosphorylated on Tyr and Thr residues and activated accumulation or immunoprecipitation with Erk2.45 by growth factors such as platelet-derived growth factor,33,34 Similarly, p38 MAPK is phosphorylated on Tyr and Thr residues epidermal growth factor,33 nerve growth factor,35 and in response and, generally, is more responsive to stress stimuli.46 The p38 can to insulin.36,37 Activation of Erk1/2 has a central role in many be strongly activated by various environmental stresses, such as cellular responses, such as cell motility, proliferation, diffe- oxidative stress, ultraviolet irradiation, hypoxia and ischemia.47 rentiation and survival.38,39 Activation of the Erk pathway can P38 MAPKs have also been shown to have roles in cell protect pancreatic tumor cells from apoptosis and regulate their proliferation and survival. The a-subtype of p38 can negatively progression in the .40 As the convergence point of most regulate cell-cycle progression and apoptosis.48,49 P38 also has an mitogenic signaling pathways, Erk is becoming an ideal target for important role in normal immune and inflammatory responses47 treatment of human cancer. In addition, the Erk subfamilies of and is activated in macrophages, neutrophils and T cells by MAPKs are essential in innate immunity.41,42 Notably, Erk2 is a numerous extracellular mediators of inflammation (including physiological substrate for DUSP2, and it forms a tight complex chemoattractants, cytokines, chemokines and bacterial

Genes and Immunity (2013) 1 – 6 & 2013 Macmillan Publishers Limited Dual-specificity phosphatases 2 effect in diseases W Wei et al 3 lipopolysaccharide).50 It is critical for activation of some DUSP2-KNOCKOUT MODEL EXHIBITS IMMUNE AND transcriptions that control the expression of proinflammatory INFLAMMATORY RESPONSES cytokine genes (including interleukin-1, NF-kB and tumor necrosis It is known that dephosphorylated MAPKs have a negative 51–54 factor-a. Deletion of the p38 in epithelial cells can reduce regulatory role in MAPK signaling and cellular responses, similar 55 proinflammatory . It has been shown that to other members of the DUSPs. As in DUSP1 À / À and transfection of DUSP2 in NIH 3T3 and HeLa cells inhibits p38a- DUSP10 À / À mice, the immune system of DUSP2 À / À mice 31 mediated phosphorylation of its substrate, ATF2, suggesting that developed normally.62 However, contrary to the negative effects DUSP2 can dephosphorylate p38a effectively in vivo. In contrast, of other DUSP deficiency (such as DUSP1 and DUSP10), DUSP2 has in vitro results demonstrated that DUSP2 can form a stable an unexpected positive function in the induction and complex with p38a. However, this direct binding does not result in perpetuation of certain inflammatory responses in an dephosphorylation of p38a by DUSP2, nor cause any catalytic autoimmune model of rheumatoid arthritis.62 DUSP2 is the most 43 activation of DUSP2. This result is similar to another nuclear- highly induced DUSP transcript in activated human leukocytes, 56 43 specific MPK (MKP-2/DUSP4). Zhang et al speculated that the and DUSP2 proteins associate with human inflammatory tissue reason for the contrasting results could be because of another (rheumatoid arthritis synovium and tonsil).62 In addition, protein that co-associates with them in vivo. Dusp2 À / À mice showed greatly diminished clinical and histo- Alternatively, Jnk2 is not considered to be a physiological logical features of arthritis. DUSP2-deficient mast cells and substrate for DUSP2 because it neither stimulates the catalytic macrophages produce fewer inflammatory mediators, such as 43 activity of the phosphatases nor is dephophorylated by DUSP2. tumor necrosis factor and IL-6, after being stimulated by TLR and Another report showed that DUSP2 cannot inhibit the ability of FceR. The numbers of IL-12-producing cells and the amount of IL- 31 Jnk2 to phosphorylate the kinase’s substrate c-Jun in vivo. 12 per cell were reduced; also, there was a considerably reduced 4 However, Jeffrey et al. demonstrated that Jnk activity was production of prostaglandin E2 and nitric oxide after stimulation increased in DUSP2-deficient macrophages and mast cells. of DUSP2-deficient macrophages. Phosphorylation of Erk and p38 Moreover, SP600125, a specific inhibitor of Jnk, could completely decreased in DUSP2-deficient macrophages and mast cells. The restore the decline of Erk activity in DUSP2-deficient mast cells. gene deletion also reduced transcriptional Elk1 and NFAT–AP-1 DUSP2 may acts as a negative regulator of Jnk, therefore resulting activity, which are effectors downstream of MAPKs. Because of an 4 in negative cross-talk between Jnk and Erk. There is still a lack of increased rate of apoptosis, DUSP2-deficient mast cells exhibited direct evidence of DUSP2’s effect on Jnk. Further research is also decreased survival. In spite of the fact that there is no direct needed to confirm the complex of roles of DUSP2 on Jnk MAPK. evidence showing that DUSP2 has an obvious function in immune Although overexpression of DUSP2 appears to dephosphorylate cell development, overexpression of DUSP2 in cells enhanced 31 Erk and p38, deletion of DUSP2 unexpectedly impaired the inflammatory mediator production.4 In aggregate, these findings activity of Erk and p38 in macrophages and mast cells and suggest that DUSP2 has a critical and positive role in inflammatory 4 increased Jnk activity. It should be noted that the decreased Erk responses. activity could be restored completely with a specific inhibitor of Jnk, suggesting that DUSP2 can increase activation of Erk by blocking Jnk. A negative cross-talk relationship between the DUSP2 OUTSIDE THE IMMUNE SYSTEM stress-activated JNK pathway and the mitogen-activated ERK 57 The MAPK signal pathway has an important role in not only the pathway has been previously described. The authors considered process of cell growth, proliferation and differentiation but also in that sustained Jnk activation uncouples Erk activation from MEK in cell-growth suppression and apoptosis, which is often dysregu- a manner that requires Jun-mediated gene transcription. Junttila 58 lated in cancers. The upstream components of the MEK/Erk et al. stated that Jnk/p38-mediated mechanisms negatively pathway are associated with the oncogenic process. P38 and Jnk regulate the Erk pathway; therefore, the function of the Erk may also have a role as tumor suppressors in some conditions.15,63 pathway was considered as survival promoting, contrasting with The dual-specificity MKPs are also essential for the initiation and the proapoptotic activity of the stress-activated c-Jun Jnk/p38 development of cancer. As one of the inducible nuclear MKPs, MAPK pathways. These results are consistent with the results of 4 nuclear-specific DUSP2 is controlled by immediately early genes Jeffrey et al. Moreover, it was postulated that DUSP2 interacts that are induced by activated MAPKs upon nuclear entrance. with p38, Jnk and Erk, providing a basis for the cross-talk between 59 The oxidative stress-dependent high level of DUSP2 induction MAPK. The importance of DUSP2 as a regulator of Erk, p38 and promotes apoptotic cell death.25 Jnk-dependent biological processes and specific functions is discussed below in detail. DUSP2 in human cancers—a potential biomarker of diseases DUSP2 was originally cloned from hematopoietic cells; therefore, studies of DUSP2 initially focused on the role in the immune ROLE OF DUSP2 IN IMMUNE REGULATION AND INFECTIOUS system. In recent years, the role of DUSP2 in pathological pro- DISEASE cesses of human cancers has been of great interest to researchers. There are three major MAPK family members (p38, Erk and Jnk) Initially, DUSP2 was described as a maximally expressed protein expressed in the immune system60 (Table 1). They are activated by tyrosine phosphatase in the hematopoietic tissue, which had a environmental stresses, and their main role is as inflammatory distinct role in the inactivation of the Erk signaling pathway. mediators.61 They are all activated downstream of innate Studies indicated that activation of the Erk cascade increases the immunity’s TLR to induce the production of cytokines and inflam- expression of DUSP2.25,32 However, the expression of DUSP2 is matory mediators. The early and robust DUSP2 transcription in unexpectedly downregulated in human acute leukemias, and this immune cells suggests that it is an important regulator of nuclear downregulation of DUSP2 significantly correlated with hyper- MAPK events after receptor activation. Expression of DUSP2, which expression of Erk activation.17 is different from the other members of its family, is limited to the Recently, Lin et al.18 reported that DUSP2 mRNA and protein are immune cells, highlighting its importance to immune-mediated significantly reduced in most solid cancers (such as cancerous inflammatory reactions. In addition, it also identifies DUSP2 to be breast, colon, lung, ovary, kidney and prostate), and suppression highly induced in T and B cells after activation.25,29 Furthermore, it of DUSP2 is associated with tumorigenesis and malignancy indicates the important and unique features of DUSP2, which is to (Table 1). Expression of DUSP2 is downregulated in cancer cells regulate immune cell effector functions via MAPK signaling. after inhibition by HIF-1a, whereas the expression of other nuclear

& 2013 Macmillan Publishers Limited Genes and Immunity (2013) 1 – 6 Dual-specificity phosphatases 2 effect in diseases W Wei et al 4 Table 1. Role of DUSP2 in diseases

Diseases Type of cells Species Levels of expression, relation of the Substrate References transcriptional induction with expression and other findings

Immune and Hemopoietic cells Human mice DUSP2 are highly induced in T and B Erk2 Rohan et al.25 and inflammatory cells after activation Grumont et al.29 responses Seizures Microglial cells Rat Seizures can induce DUSP2 expression Boschert et al.27 in microglial cells Acute leukemias Leukemic cells, Human The expression of DUSP2 dowregulated Erk Kim et al.17 myelogenous in human acute leukemias leukemia line (K562 cells) Cancer Colon cancer cell line Human DUSP2 is a transcriptional target of p53 Erk Yin et al.20 (EB-1 cells) in signaling apoptosis and tumor suppression. Serous carcinoma Ovarian carcinoma Human DUSP2 expressed increased in ovarian Givant-Horwitz of the ovary cells carcinoma cells in effusions, and high et al.21 DUSP2 expression was significantly correlated with poor prognosis Inflammatory Leukocytes Human mice DUSP2 is a key positive regulator of Erk, p38, Jnk and Jeffrey et al.62 responses inflammatory and effector Erk cross-talk functions. DUSP2-deficient cells produce fewer inflammatory mediators after stimulated, such as TNF, IL-6, PGE2 and NO Cancer Breast cancer cell line Human DUSP2 is a direct transcription target of Erk Wu et al.23 (MB435), Saos-2 E2F-1 in signaling apoptosis. The E2F-1- osteosarcoma cell line DUSP2 cascade in cancer cell killing may provide a molecular basis for cancer therapeutic intervention Cancer Breast, colon, lung, Human DUSP2 mRNA and protein significantly Erk Lin et al.18 ovary, kidney and reduce in most solid cancers, and prostate cancer cells suppression of DUSP2 is associated with tumorigenesis and malignancy. DUSP2 is a key downstream regulator of HIF-1-mediated tumor progression and chemoresistance CLN3 disease , Human mice DUSP2 is considered to be a potential Erk Lebrun et al.65 cerebellar precursor biomarker of CLN3 disease cells, HeLa cell and SH-SY5Y neuronal cell lines Endometriosis Endometriotic Human Hypoxia, via downregulation of DUSP2, Erk and p38 Wu et al.64 stromal cells induces ERK and p38 MAPK phosphorylation and cyclooxygenase-2 overexpression Breast cancer Breast cancer cell Human Identify DUSP2 as one of reliable Sano et al.19 lines prediction of breast cancer Abbreviations: DUSP, dual-specificity phosphatase; NO, nitric oxide; PGE2, prostaglandin E2; TNF, tumor necrosis factor. See text for more details.

DUSPs (DUSP1, -4 and -5) is upregulated under the same cyclophosphamide combination chemotherapy (EC). Ultimately, conditions.18 This suggests that DUSP2 is a key nuclear DUSPs they selected DUSP2 as one of the reliable prediction markers. As for negative regulation of Erk phosphorylation in hypoxic cells. In a transcriptional target of p53, DUSP2 is induced in response to addition, it is demonstrated that hypoxia-inhibited DUSP2 both ZnCl2 and serum deprivation, which activates p53 and expression is primarily controlled by HIF-1a in multiple cancer induces apoptosis in a human colon cancer cell line (EB-1 cells).20 cell types, thus enabling prolonged Erk phosphorylation. DUSP2 DUSP2 mRNA was reported to be downregulated in most cancers, induces apoptosis, inhibits tumor growth and abolishes hypoxia- but it was upregulated in cells of effusions from ovarian serous induced drug resistance.18 However, inhibition of DUSP2 carcinoma.21 Givant-Horwitz et al.21 detected mRNA expression of expression can increase drug resistance.18 There are five genes the DUSP1, -2, -9 and -10 in 39 malignant effusions from patients that are negatively regulated by DUSP2. Those genes are early diagnosed with serous ovarian carcinoma. The results showed that growth response protein 1, multidrug resistance, cysteine-rich DUSP1 and -2 are expressed in the vast majority of cells; however, angiogenic inducer 61, glucose-regulated protein 78 and DUSP9 and DUSP910 were uniformly absent. Furthermore, DUSP1 osteopontin, which exert distinct functions via different showed no association with clinicopathological parameters, but signaling pathways, causing drug resistance. Sano et al.19 high DUSP2 expression was significantly correlated with poor attempted to identify potent marker genes of breast cancer prognosis. This also suggests that DUSP2 is a true negative through a prediction model for individual response to epirubicin/ regulator of MAPK in ovarian carcinoma cells present in effusions.

Genes and Immunity (2013) 1 – 6 & 2013 Macmillan Publishers Limited Dual-specificity phosphatases 2 effect in diseases W Wei et al 5 The functions of DUSP2 in cancer cell apoptosis functions of this single protein may be because of: 1) expression There are two MAP kinase inhibitors, DUSP2 and DUSP4 (MKP-2), level-related functions; 2) tissue-specific expressions for different that are considered to be downstream targets of p53 function in functions; and 3) polymorphism of genes for variations of apoptosis signaling.20,22 DUSP2 is a transcriptional target of p53 in phenotypes in human populations. To utilize DUSP2 in therapeutic signaling apoptosis and tumor suppression in vivo. It is the first applications, it will be essential to understand the regulation of reported p53 downstream gene that is required for p53-mediated expression at transcriptional and translational levels, as well as the apoptosis in response to oxidative stress. P53 activates trans- potential impact of polymorphisms of DUSP2. cription of DUSP2 via binding to a palindromic site in DUSP2 promoter during apoptosis. DUSP2 has been shown to be transcriptionally induced by the human tumor-suppressor CONCLUSION protein p53 in the cell’s response to serum deprivation and The critical role exerted by DUSP2 has been shown by its multiple oxidative stress. Moreover, DUSP2 acts as a key downstream levels of regulation and its regulation in multiple diseases. effector of p53 function to suppress the MAPK cascade and to Understanding the physiological roles of DUSP2 may pave the induce cell-growth suppression and apoptosis.20 way for developing new therapies for immune diseases, inflam- DUSP2 has also been found to be a direct transcriptional target of mation, cancer or other diseases, and further understanding of E2F-1 in apoptotic signaling23 (Table 1). E2F-1 is expressed in cancer MAPK signaling in physiological responses. cells. E2F-1 mediates the activity of the RB tumor suppressor and has a crucial role in the control of the cell cycle through transcriptional regulation of its target genes. In breast cancer cells, CONFLICT OF INTEREST expression of DUSP2 can be increased by ectopic E2F-1 at both The authors declare no conflict of interest. transcriptional and translational levels. Furthermore, E2F-1 sup- presses Erk activity through DUSP2 to enhance the sensitivity of cancer cells to chemotherapeutic agent N-4-hydroxyphenylretina- ACKNOWLEDGEMENTS mide treatment, which leads to elimination of cancer cells by This study was partially supported by the NIAAA (1R01 AA016342), NIH, the Veterans apoptosis. As described above, these findings reveal a mechanistic Administration Medical Center, and DNA Discovery Core, University of Tennessee, link between hypoxia and ERK activation, the two critical factors in Memphis, TN. We thank Xuenan Yan for editing this manuscript. cancer progression and malignancy. 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