Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer

Dual-specificity phosphatases (DUSPs) dephosphorylate threonine/serine and tyrosine residues on their substrates. Here we show that DUSP1, DUSP4, and DUSP6 are involved in epithelial-to-mesenchymal transition (EMT) and breast cancer stem cell (CSC) regulation. DUSP1, DUSP4, and DUSP6 are induced during EMT in a PKC pathway signal-mediated EMT model. We show for the first time that the key chromatin-associated kinase PKC-θ directly regulates a subset of DUSP family members. DUSP1, DUSP4, and DUSP6 globally but differentially co-exist with enhancer and permissive active histone post-translational modifications, suggesting that they play distinct roles in gene regulation in EMT/CSCs. We show that nuclear DUSP4 associates with the key acetyltransferase p300 in the context of the chromatin template and dynamically regulates the interplay between two key phosphorylation marks: the 1834 (active) and 89 (inhibitory) residues central to p300’s acetyltransferase activity. Furthermore, knockdown with small-interfering RNAs (siRNAs) shows that DUSP4 is required for maintaining H3K27ac, a mark mediated by p300. DUSP1, DUSP4, and DUSP6 knockdown with siRNAs shows that they participate in the formation of CD44^hi/CD24^lo/EpCAM⁺ breast CSCs: DUSP1 knockdown reduces CSC formation, while DUSP4 and DUSP6 knockdown enhance CSC formation. Moreover, DUSP6 is overexpressed in patient-derived HER2⁺ breast carcinomas compared to benign mammary tissue. Taken together, these findings illustrate novel pleiotropic roles for DUSP family members in EMT and CSC regulation in breast cancer.     

Deregulation of DUSP activity in EGFR-mutant lung cancer cell lines contributes to sustained ERK1/2 signaling

Lung cancers demonstrate loss of cellular signaling control pathways. EGFR-mutant non-small cell lung cancer cell lines constitutively express active ERK1/2 and require ERK activity for survival. DUSP4 is a negative regulator of ERK activity and is up-regulated in EGFR-mutant lung cancer cell lines relative to K-ras mutant cells. Both DUSP4 and family member, DUSP1, can bind ERK in vitro. However, only DUSP1 has detectable binding to ERK in vivo in cell lines of either genotype. Depletion of DUSP4 in EGFR-mutant cells unexpectedly results in loss of pERK whereas loss of DUSP4 in K-ras mutant cells predictably yields increased pERK. These data support a role for DUSP4, and perhaps DUSP1, as a positive activator of ERK in EGFR-mutant lung cancer cell lines independent of the ability to bind to ERK.     

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4^?/? mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-β with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.     

DUSP1 is involved in the progression of small cell carcinoma of the prostate

Small cell carcinoma of the prostate (SCCP) is a rare and the most aggressive variant of prostate cancer. There is no effective cure or treatment for SCCP. Therefore, there is an urgent need for new therapy to improve the prognosis of patients with SCCP. DUSP1 is a dual specific phosphatase with an increasingly recognized in tumor biology. Altered expression of DUSP1 induced changes in the expression of genes involved in various biological pathways, including cell-cell signaling and angiogenesis. To understand more about the role of DUSP1 in SCCP, we evaluated the biological function and associated regulatory mechanism of DUSP1. In this study, DUSP1 was significantly down-regulated in human SCCP compared with the non-carcinoma tissues (P < 0.05). Overexpression of DUSP1 was found to suppress MAPK signaling and cell proliferation in PC-3 cells. Additionally, silencing of DUSP1 enhanced MAPK signaling and PC-3 cell proliferation. Moreover, it was observed that DUSP1 blocked the phosphorylation of p38 MAPK induced by anisomycin. Taken together, this investigation suggests that DUSP1 is involved in the progression of SCCP and may provide a new therapeutic target for SCCP treatment.     

Roles for the Mitogen-activated Protein Kinase (MAPK) Phosphatase,DUSP1, in Feedback Control of Inflammatory Gene Expression and Repression by Dexamethasone

Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.     

Role for DUSP1 (dual-specificity protein phosphatase 1) in the regulation of autophagy

Accumulating evidence suggests that mitogen-activated protein kinases (MAPKs) regulate macroautophagy/autophagy. However, the involvement of dual-specificity protein phosphatases (DUSPs), endogenous inhibitors for MAPKs, in autophagy remains to be determined. Here we report that DUSP1/MKP-1, the founding member of the DUSP family, plays a critical role in regulating autophagy. Specifically, we demonstrate that DUSP1 knockdown by shRNA in human ovarian cancer CAOV3 cells and knockout in murine embryonic fibroblasts, increases both basal and rapamycin-increased autophagic flux. Overexpression of DUSP1 had the opposite effect. Importantly, knockout of Dusp1 promoted phosphorylation of ULK1 at Ser555, and BECN1/Beclin 1 at Ser15, and the association of PIK3C3/VPS34, ATG14, BECN1 and MAPK, leading to the activation of the autophagosome-initiating class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Furthermore, knockdown and pharmacological inhibitor studies indicated that DUSP1-mediated suppression of autophagy reflected inactivation of the MAPK1-MAPK3 members of the MAPK family. Knockdown of DUSP1 sensitized CAOV3 cells to rapamycin-induced antigrowth activity. Moreover, CAOV3-CR cells, a line that had acquired cisplatin resistance, exhibited an elevated DUSP1 level and were refractory to rapamycin-induced autophagy and cytostatic effects. Knockdown of DUSP1 in CAOV3-CR cells restored sensitivity to rapamycin. Collectively, this work identifies a previously unrecognized role for DUSP1 in regulating autophagy and suggests that suppression of DUSP1 may enhance the therapeutic activity of rapamycin.     

Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK

DUSP5 is an inducible, nuclear, dual-specificity phosphatase, which specifically interacts with and inactivates the ERK1/2 MAP kinases in mammalian cells. In addition, expression of DUSP5 causes nuclear translocation of ERK2 indicating that it may act as a nuclear anchor for the inactive kinase. Here we show that induction of DUSP5 mRNA and protein in response to growth factors is dependent on ERK1/2 activation and that the accumulation of DUSP5 protein is regulated by rapid proteasomal degradation. DUSP5 is phosphorylated by ERK1/2 both in vitro and in vivo on three sites (Thr321, Ser346 and Ser376) within its C-terminal domain. DUSP5 phosphorylation is absolutely dependent on the conserved kinase interaction motif (KIM) within the amino-terminal domain of DUSP5, indicating that the same protein–protein contacts are required for both the inactivation of ERK2 by DUSP5 and for DUSP5 to act as a substrate for this MAPK. Using a combination of pharmacological inhibitors and phospho-site mutants we can find no evidence that phosphorylation of DUSP5 by ERK2 significantly affects either the half-life of the DUSP5 protein or its ability to bind to, inactivate or anchor ERK2 in the nucleus. However, co-expression of ERK2 results in significant stabilisation of DUSP5, which is accompanied by reduced levels of DUSP5 ubiquitination. These changes are independent of ERK2 kinase activity but absolutely depend on the ability of ERK2 to bind to DUSP5. We conclude that DUSP5 is stabilised by complex formation with its physiological substrate and that this may reinforce its activity as both a phosphatase and nuclear anchor for ERK2.     

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.     

Dual-specificity Phosphatase 9 protects against Cardiac Hypertrophy by targeting ASK1

The functions of dual-specificity phosphatase 9 (DUSP9) in hepatic steatosis and metabolic disturbance during nonalcoholic fatty liver disease were discussed in our prior study. However, its roles in the pathophysiology of pressure overload-induced cardiac hypertrophy remain to be illustrated. This study attempted to uncover the potential contributions and underpinning mechanisms of DUSP9 in cardiac hypertrophy. Utilizing the gain-and-loss-of-functional approaches of DUSP9 the cardiac phenotypes arising from the pathological, echocardiographic, and molecular analysis were quantified. The results showed increased levels of DUSP9 in hypertrophic mice heart and angiotensin II treated cardiomyocytes. In accordance with the results of cellular hypertrophy in response to angiotensin II, cardiac hypertrophy exaggeration, fibrosis, and malfunction triggered by pressure overload was evident in the case of cardiac-specific conditional knockout of DUSP9. In contrast, transgenic mice hearts with DUSP9 overexpression portrayed restoration of the hypertrophic phenotypes. Further explorations of molecular mechanisms indicated the direct interaction of DUSP9 with ASK1, which further repressed p38 and JNK signaling pathways. Moreover, blocking ASK1 with ASK1-specific inhibitor compensated the pro-hypertrophic effects induced by DUSP9 deficiency in cardiomyocytes. The main findings of this study suggest the potential of DUSP9 in alleviating cardiac hypertrophy at least partially by repressing ASK1, thereby looks promising as a prospective target against cardiac hypertrophy.     

Tumor necrosis factor-α-mediated suppression of dual-specificity phosphatase 4: crosstalk between NFκB and MAPK regulates endothelial cell survival

We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70–84 % and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.     

DUSP1 recuses diabetic nephropathy via repressing JNK-Mff-mitochondrial fission pathways

Excessive mitochondrial fission has been identified as the pathogenesis of diabetic nephropathy (DN), although the upstream regulatory signal for mitochondrial fission activation in the setting of DN remains unknown. In the current study, we found that dual-specificity protein phosphatase-1 (DUSP1) was actually downregulated by chronic hyperglycemia stimulus. Lower DUSP1 expression was associated with glucose metabolism disorder, renal dysfunction, kidney hypertrophy, renal fibrosis, and glomerular apoptosis. At the molecular level, defective DUSP1 expression activated JNK pathway, and the latter selectively opened mitochondrial fission by modulating mitochondrial fission factor (Mff) phosphorylation. Excessive Mff-related mitochondrial fission evoked mitochondrial oxidative stress, promoted mPTP opening, exacerbated proapoptotic protein leakage into the cytoplasm, and finally initiated mitochondria-dependent cellular apoptosis in the setting of diabetes. However, overexpression of DUSP1 interrupted Mff-related mitochondrial fission, reducing hyperglycemia-mediated mitochondrial damage and thus improving renal function. Overall, we have shown that DUSP1 functions as a novel malefactor in diabetic renal damage that mediates via modifying Mff-related mitochondrial fission. Thus, finding strategies to regulate the balance of the DUSP1-JNK-Mff signaling pathway and mitochondrial homeostasis may be a therapeutic target for treating diabetic nephropathy in clinical practice.