MKP-1 Protein Expression Research Articles

Panax notoginseng saponin R1 improves glucocorticoid-inhibited airway epithelium repair via glucocorticoid receptor β

BackgroundPanax notoginseng saponin R1(PNS-R1), derived from Panax notoginseng roots, promotes wound repair, whereas glucocorticoids can inhibit the repair of airway epithelial damage in asthma. ObjectiveThis study investigated whether PNS-R1 counteracts the inhibitory effects of glucocorticoids on the repair of airway epithelial damage in asthma. MethodsIn vivo, female C57BL/6 mice were sensitized, challenged with house dust mites (HDM), and treated with dexamethasone, PNS-R1, and/or adenovirus GRβ-shRNA. Airway epithelium damage was examined using pathological sections of the trachea and bronchi, markers of airway inflammation, epithelial cells in bronchoalveolar lavage fluid, and expression of the E-cadherin protein. In vitro, we treated 16HBE cells with dexamethasone, PNS-R1, and/or GRβ-siRNA and detected cell proliferation and migration. The expression of GRβ and key components of MKP-1 and Erk1/2 were detected by western blotting. ResultsIn vivo, PNS-R1 reduced airway inflammation, hyperresponsiveness, and mucus hypersecretion; the combination of PNS-R1 and dexamethasone promoted airway epithelial integrity and reduced cell detachment. In vitro, PNS-R1 alleviated the inhibition of bronchial epithelial cell growth, migration, and proliferation by dexamethasone; PNS-R1 promoted GRβ expression, inhibited MKP-1 protein expression, and activated MAPK signaling, thereby promoting airway epithelial cell proliferation and repair. ConclusionsPanax notoginseng saponin R1 alleviated the inhibitory effect of dexamethasone on the repair of airway epithelial damage in asthmatic mice, likely by promoting the proliferation of airway epithelial cells by stimulating GRβ expression and activating the MAPK pathway.

Glutamine ameliorates hyperoxia-induced hippocampal damage by attenuating inflammation and apoptosis via the MKP-1/MAPK signaling pathway in neonatal rats.

Glutamine (Gln) is an immunomodulatory protein that mediates oxidative stress, inflammation, and apoptosis, but has not been reported in the treatment of hyperoxia (Hyp)-induced brain injury. The aim of this study was to determine whether Gln could improve hyp-induced brain injury in neonatal rats to and later learning and memory dysfunction, and to explore its possible mechanisms. We prepared a model of neonatal rat brain injury caused by normobaric hyperoxia while administered with Gln for 7days for evaluation. Learning memory function was assessed with the Morris water maze test. Histological analysis, protein expression analysis, oxidative stress and inflammation level analysis were performed using hippocampal tissue. Gln treatment significantly reduced brain tissue water content, oxidative stress levels, microglia activation and inflammatory factor expression, and attenuated tissue damage and apoptosis in the hippocampal region. Gln ameliorates hyp-induced learning, memory impairment in neonatal rats in water maze test. It also increased MKP-1 protein expression and decreased p-p38, p-ERK and p-JNK. Therefore, it is hypothesized that Gln may exert neuroprotective effects by increasing MKP-1 expression to negatively regulate MAPK signaling, with potential cognitive improvement in hyp-induced brain injury.

Oxidative Stress Impairs Glucocorticoids Sensitivity of Allergic Asthma Animal Model

SESSION TITLE: Asthma III SESSION TYPE: Original Investigation Poster PRESENTED ON: Saturday, April 16, 2016 at 11:45 AM - 12:45 PM PURPOSE: To observe the influence of ozone inhalation on the typical therapeutic effects of glucocorticoids and further to explore the involvements of p38MAPK activation and MKP-1 expression upon a mouse asthma model. METHODS: Mice were sensitized and challenged with ovalbumin (OVA), dexamethasone were administrated ip 1 hr before each challenge (day24,25,26). Ozone expsoure (1.0 ppm 2hr) were performed on day 23, 25 and 27, and SB239063 were injected 1hr before and 4hr after each exposure. Enhanced pause (Penh) were measured at day 28 and followed by bronchoalveolar lavage (BAL). Lung tissues were collected for analysis of expression of inflammatory genes, MKP-1 and p38MAPK. RESULTS: Ozone inhalation significantly increased the AHR and airway inflammation of OVA model. Dexamethasone significantly decreased the AHR and airway inflammation of mice from OVA model. These inhibitory effects were significantly decreased after the ozone exposure. Administration of SB239063 partly restored the impaired effects of dexamethasone, parallel with inhibitions of the AHR and airway inflammation, especially the lung expression of IL-17 mRNA, in mice of O3-exposed OVA model. The activation of p38MAPK was promoted by ozone inhalation and inhibited by SB239063. There was no striking effect of dexamethasone on activation of p38MAPK except in O3-exposed OVA model group. Dexamethasone increased the mRNA and protein expression of MKP-1 on lungs of mice only from OVA group. This effects were diminished by ozone inhalation regardless of SB239063 administration. O3 inhalation promoted the expression of MKP-1 on mRNA but not on protein level. CONCLUSIONS: Ozone inhalation disturbs the inhibitory effects of glucocorticoids on allergic airway inflammation. This disturbance may involve the activation of p38 MAPK, induction of Th17 cytokines and negative regulation on MKP-1 protein expression. CLINICAL IMPLICATIONS: Glucocorticoid resistance is a common and hard-to-treat problem in chronic severe asthmatic patients. This study explored the underlying involvements of p38 MAPK and MKP-1 in the oxidative stress induced steroids resistance in a mouse asthma model, which may contribute to the knowledge accumulation to help understand the underlying mechanisms of Glucocorticoid resistance. DISCLOSURE: The following authors have nothing to disclose: Aihua Bao, Xin Zhou No Product/Research Disclosure Information

Parathyroid hormone-related peptide protects cardiomyocytes from oxidative stress-induced cell death: First evidence of a novel endocrine–cardiovascular interaction

Although there is a growing interest in the molecular cross-talk between the endocrine and cardiovascular systems, the cardiac effects of calcium-regulating hormones (i.e., parathyroid hormone-related peptide (PTHrP)) have not been explored. In this study, we examined the effect of PTHrP on the viability of isolated adult mouse cardiomyocytes subjected to oxidative stress. Myocytes from 19 to 22 week old male 129J/C57BL6 mice were exposed to oxidative insult in the form of H2O2 which led to more than 70% loss of cell viability. Herein we demonstrate, for the first time, that pretreatment with 100 nM PTHrP prior to 100 μM H2O2 incubation prevents H2O2 -induced cell death by more than 50%. Immunoblot analysis revealed H2O2 induction of MKP-1 protein expression while PTHrP decreased MKP-1 expression. Moreover, myocytes derived from MKP1 KO mice were resistant to oxidative injury. No added benefit of PTHrP treatment was noted in MKP-1 null cardiomyocytes. Using specific pharmacological inhibitors we demonstrated that P-p38, P-ERK and P-AKT mediated PTHrP's cardioprotective action. These data provide novel evidence that: i) down-regulation of MKP1 affords profound protection against oxidative stress; and ii) PTHrP is cardioprotective, possibly via down-regulation of MKP-1 and activation of MAPK and PI3K/AKT signaling.

Mitogen-activated protein kinase phosphatase-1 prevents lipopolysaccharide-induced apoptosis in immature rat intestinal epithelial cells.

Necrotizing enterocolitis is characterized by intestinal inflammation and epithelial barrier dysfunction. Mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 plays a pivotal role in the feedback control of MAPK signaling, which regulates inflammation and apoptosis. We hypothesized that MKP-1 prevents lipopolysaccharide (LPS)-induced apoptosis in intestinal epithelial cells. Western blot analysis and qPCR were used to assess MKP-1, MAPK (p38, extracellular signal-regulated kinase (ERK), and c-Jun N terminal kinases (JNK)), caspase 3, caspase 9, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 expression levels in rIEC-6 enterocytes. MKP-1 expression was inhibited using small interfering RNA (siRNA) methodology. Viable cell number was determined using trypan blue exclusion. LPS stimulation led to activation of p38, JNK, and ERK, and induction of MKP-1 mRNA and protein expression. The induction of MKP-1 was associated with a decrease in p38 phosphorylation, and knockdown of MKP-1 prolonged p38 phosphorylation. While LPS stimulation significantly attenuated proliferation of rIEC-6 cells transfected with scramble siRNA, LPS stimulation resulted in a net decrease in viable cell number in cells transfected with MKP-1 siRNA. Following LPS stimulation, MKP-1 knockdown resulted in greater caspase 3 and 9 activities and greater proinflammatory cytokine (TNF-α, COX-2) expression than in cells transfected with scramble siRNA. Our results demonstrate that MKP-1 has a central role in preventing inflammation-induced apoptosis in rIEC-6 enterocytes.

NOD2 Mediates Odontoblast Differentiation and RANKL Expression

The precise regulation of odontoblast differentiation and osteoclastogenic cytokine expression in human dental pulp cells (HDPCs) is crucial for the pathology of bacteria-related pulpitis. Although the up-regulation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been reported in inflamed human dental pulps, the role of NOD2 in the differentiation of HDPCs remains unclear. Here, we show the involvement of NOD2 in odontoblast differentiation together with osteoclastogenic cytokine expression in HDPCs. Treatment with muramyl dipeptide (MDP), a known NOD2-agonist, significantly inhibited odontoblast differentiation of HDPCs, as revealed by reduced ALP activity, osteoblast/odontoblast marker expression, and mineralized nodule formation. Importantly, the forced down-regulation of NOD2 by small interfering RNA (siRNA) recovered MDP-down-regulated odontoblast differentiation. MDP-elicited suppression of odontoblast differentiation resulted from the increased expression of MKP-1 protein and the subsequent decline of MAPKs phosphorylation, which is a prerequisite for odontoblast differentiation. Furthermore, we found that MDP treatment elevated the expression of osteoclastogenic cytokines in HDPCs, which was also reversed by NOD2 silencing. Analysis of these data, taken together, suggests that the regulation of NOD2 expression upon MDP challenge might serve as an intrinsic mechanism that underlies the hindered dentin formation and accelerated dentin resorption in bacterial infection-mediated pulpitis. Abbreviations: HDPCs, human dental pulp cells; NOD2, nucleotide-binding oligomerization domain-containing protein 2; MDP, muramyl dipeptide; siRNA, small interfering RNA; MAPKs, mitogen-activated protein kinases; MKP-1, MAPK phosphatase-1; BMMs, bone-marrow-derived macrophages; and CM, conditioned medium.

The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of PP2Ac formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.

Abstract 157: MAP Kinase Phosphatase-1 Inhibitor Injection into the Solitary Tract Nucleus Impairs Cardiovagal Baroreflex Function in Transgenic ASrAOGEN Rats

Hypotensive transgenic ASrAOGEN rats (AS) with low brain angiotensinogen resulting from glial overexpression of an antisense oligonucleotide to angiotensinogen, have enhanced baroreflex sensitivity (BRS) relative to hypertensive transgenic (mRen2)27 rats (mRen) that overexpress the murine Ren2 gene or normotensive Sprague Dawley rats (SD). The AS rats exhibit elevated protein expression and activity (less phosphorylated-ERK) of the MAP Kinase Phosphatase-1 [MKP-1], a negative regulator of MAP Kinase signaling in the brain dorsal medulla compared with either mRen or SD rats at 26 wks of age. To determine if MKP-1 plays a role in maintenance of BRS, we gave acute bilateral microinjections of an irreversible MKP-1 inhibitor {2,3-bis-[(2-Hydroxyethyl)thiol]-1,4-naphthoquinone, NSC 95397; 200 nM/120 nL} into the solitary tract nucleus (NTS), an important site in the dorsal medulla for modulation of BRS in urethane/chloralose anesthetized AS, mRen and SD rats at 21 wks of age. The MKP-1 inhibitor impaired the BRS for bradycardia in response to increases in pressure by 48 % at 10 min (1.01 ± 0.16 baseline vs. 0.49 ± 0.1 msec/mm Hg at 10 min), 56 % at 60 min and 64 % at 120 min in the AS rats (p = 0.02, n = 8), but not in the mRen or SD rats. In addition, NSC 95397 reduced resting pressure (91 ± 3 at baseline vs. 79 ± 3 at 60 min vs. 80 ± 3 mm Hg at 120 min) in the SD rats, with a similar trend in the other strains, without effects on resting heart rate in any strain. Thus, up-regulated expression of MKP-1 contributes to the higher resting BRS function in AS rats. AS animals are spared age-related declines in BRS and they maintain higher expression of MKP-1 protein in the dorsal medulla vs. either mRen or SD rats (AS: 1.6 ± 0.2, mRen: 0.8 ± 0.1, SD: 1 ± 0.1 arbitary units; p = 0.01, n = 6 per group) at 66 wks of age. Thus, higher MKP-1 may be involved the preservation of BRS in older AS rats, suggesting the phosphatase be a novel therapeutic target to improve BRS function in aging and hypertension.

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway

AimsCytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. Main methodsMale, 10–12week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5ng/mL or 5ng/mL, for 1 or 24h), and isometric contraction was recorded. Protein expression was determined by Western blotting. Key findingsConstrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1h with chemerin. Chemerin incubation for 24h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. SignificanceChemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases.

Differences in endoplasmic reticulum stress signalling kinetics determine cell survival outcome through activation of MKP-1

Endoplasmic reticulum (ER) stress signalling pathways are involved in various alterations of the central nervous system such as neurodegenerative diseases or ischemia. The current mechanisms linking ER stress activation to neuronal cell fate upon chronic or acute stresses remain however to be fully understood. Recent studies have associated ER stress severity and the relative activation levels of certain output pathways to influence cell-fate decisions. In the present report, to further test the impact of ER stress severity on neuronal survival, we designed an experimental system recapitulating acute and chronic stress in cerebellar granule neurons (CGNs) and c17.2 mouse neural stem cells (NSCs). Two well characterized ER stress inducers, tunicamycin (TM) and dithiothreitol (DTT), were used to induce “slow motion” and “fast motion” stresses, respectively. We show that the duration of JNK activation is critical for cell survival upon ER stress. TM-induced transient JNK activation is a protective event in CGNs and c17.2 NSCs via the phosphorylation of BAD, while DTT-induced prolonged JNK activation mediates pro-apoptotic signalling. In addition, we demonstrate that ER stress mediated MKP-1/DUSP1 expression regulates JNK activation kinetics. MKP-1 phosphorylation and protein expression level are differentially altered upon TM and DTT treatment. Increased MKP-1 protein stability via its phosphorylation on ser359 induced by TM accounts for transient JNK activation and the resulting cell survival in CGNs and c17.2 NSCs subjected to ER stress. These results suggest that MKP-1 plays a pivotal role in ER stress-induced cell apoptosis through regulating JNK-BAD signalling.

Abstract 360: Tamoxifen treatment induces mitogen-activated protein kinase phosphatase-1 expression in T47D breast cancer cells

Abstract Mitogen Activated Protein Kinase Phosphatase-1 (MKP-1) is a dual specificity phosphatase known to target the Extracellular Signal Regulated Kinases (ERK), c-Jun N-terminal Kinases (JNK) and the p38 Mitogen Activated Protein Kinases (p38), which make up the Mitogen Activated Protein Kinase (MAP Kinase) family. These pathways play vital roles in determining whether cells continue to survive or undergo apoptosis. ERK mainly contributes to cell survival while JNK and p38 signaling can be involved in both cell survival and apoptosis. The function of MKP-1 is to attenuate the signals perpetuated by ERK, JNK and p38. Previous research in our lab has shown that knockdown of either MKP-1 or ERK-2 leads to increased cisplatin sensitivity in ovarian cancer. ERK activation has also been linked to tamoxifen resistance and poor prognosis in breast cancer, but the effects of MKP-1, JNK and p38 in this area are not well studied. A previous study by Cui et al has shown that overexpression of MKP-3, which mainly targets ERK, increased tamoxifen resistance in breast tumors. We chose to focus this investigation on the contribution of MKP-1 to tamoxifen resistance because of its ability to dephosphorylate all three MAP Kinase family members. In our study, MKP-1 protein expression was shown to be elevated in MDA-MB-231, T47D and MCF7 breast cancer cells as compared to non-malignant MCF10A cells. After four and six hours of treatment with 1 μM tamoxifen, MKP-1 protein expression was increased in T47D cells compared to untreated parental control cells. Very little change in phosphorylated ERK protein levels was observed at these same time points. There were, however, decreases in phosphorylated JNK protein levels after four and six hours of 1μM tamoxifen treatment when compared to untreated parental control cells. Since some chemotherapeutic agents induce apoptosis in a JNK mediated manner, a decrease in JNK expression in tamoxifen treated breast cancer cells could contribute to decreased therapeutic efficacy. In conclusion, by examining changes in protein expression levels in tamoxifen treated breast cancer cells we have identified that increased MKP-1 expression leads to a decrease in phosphorylated JNK expression. This finding could constitute a mechanism contributing to tamoxifen resistance in estrogen receptor positive breast cancer cells and suggests that modulation of MKP-1 expression could increase tamoxifen sensitivity in these cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 360.

The inhibitory effect of ghrelin on sepsis-induced inflammation is mediated by the MAPK phosphatase-1

Hepatocellular dysfunction occurs early in sepsis and this appears to be caused by Kupffer cell-derived TNF-alpha production from the liver as a result of the increased release of the sympathetic neurotransmitter, norepinephrine, from the gut. Ghrelin, a novel stomach-derived peptide, is down-regulated in sepsis and administration of ghrelin into rodents decrease pro-inflammatory cytokines, attenuates hepatic and other organ injuries and improves survival. Ghrelin's beneficial effect in sepsis is mediated by the inhibition of the sympathetic nervous system (SNS), as evidenced by the reduced gut-derived norepineprine (NE) release in sepsis after ghrelin treatment. Recent data suggest that MKP-1, the MAPK phosphatase-1, is involved in the innate immune responses. To determine that the beneficial effect of ghrelin in sepsis is mediated by MKP-1, rats were subjected to sepsis by cecal ligation and puncture (CLP) alone, or treated with ghrelin, beginning at 5-h post-CLP and liver tissues were harvested and examined for MKP-1 mRNA and protein expression. CLP alone produced a significant decrease in MKP-1 gene expression in liver tissues at 20 h after CLP (P<0.05). MKP-1 mRNA was decreased by 30-40% at 2 and 5 h after CLP, but not statistically significant. MKP-1 protein expression was significantly decreased as early as 2 h after CLP and remained low at 5-20 h after CLP. While septic rats treated with vehicle produced significant decreases from sham rats, ghrelin treatment improved both mRNA and protein from vehicle group (0.58+/-0.069 vs. 0.91+/-0.16, P<0.05; 0.14+/-0.027 vs. 0.22+/-0.017, P=0.013), respectively. Since ghrelin's inhibitory effect is mediated by the SNS, we hypothesized that NE treatment in Kupffer cells may downregulate MKP-1. Kupffer cells were treated with NE and examined for MKP-1. Treatment with NE for 60 min showed an average of 46.9% decrease in MKP-1 mRNA expression compared to untreated cells (P<0.001). Likewise, NE treatment in RAW264.7 cells produced significantly lower MKP-1 mRNA than that of control cells. To further confirm the effect of NE on MKP-1, normal rats were infused with NE for 2 h through the portal vein and MKP-1 mRNA from the liver was examined. Infusion with NE produced a significant 73.7% decrease in MKP-1 mRNA. Therefore, ghrelin's inhibitory effect on gut-derived NE release in sepsis leading to the downregulation of pro-inflammatory cytokines is mediated by MKP-1.

Cytoprotective Role of Mitogen‐activated Protein Kinase Phosphatase‐1 in Light‐damaged Human Retinal Pigment Epithelial Cells

The role of the mitogen-activated protein (MAP) kinase phosphatases (MKPs) in light-damaged cells is unclear. Therefore we investigated the involvement of MKP-1 in the regulation of apoptosis and cell survival mediated by MAP kinase pathways in light-damaged human retinal pigment epithelial cells (ARPE-19). Light dose-dependent changes in the expression of MKP-1 and in the phosphorylation status of the MAP kinases, c-Jun-N-terminal kinase (JNK) and p38 were demonstrated. Low light doses up to 2 J cm(-2) led to an upregulation of MKP-1 which resulted in the prevention of cell death by inactivating JNK kinase. However, higher light doses (> or =3 J cm(-2)) significantly reduced MKP-1 protein expression and subsequently led to an increased JNK kinase activity followed by a significant increase in cell death. JNK kinase inactivation by the JNK inhibitor SP600125 significantly reduced light-induced cell death, suggesting that the cytoprotective properties of MKP-1 are mediated mainly by the JNK MAP kinase pathway. Physiological concentrations of ascorbic acid or taurine were seen to prevent apoptosis and cell death in light-damaged ARPE-19 cells by reducing oxidative stress within cells, thus maintaining MKP-1 at high levels, leading to an inactivation of the JNK kinase pathway which resulted in an increased cell viability.

Dexamethasone-induced cardioprotection: A role for the phosphatase MKP-1?

Aims Previous studies suggested that p38 MAPK activation during sustained myocardial ischaemia and reperfusion was harmful. We hypothesize that attenuation of p38MAPK activity via dephosphorylation by the dual-specificity phosphatase MKP-1 should be protective against ischaemia/reperfusion injury. Since the glucocorticoid, dexamethasone, induces the expression of MKP-1, the aim of this study was to determine whether upregulation of this phosphatase by dexamethasone protects the heart against ischaemia/reperfusion injury. Main methods Male Wistar rats were treated with dexamethasone (3 mg/kg/day ip) for 10 days, before removal of the hearts for Western blot (ip Dex − P) or perfusion in the working mode (ip Dex + P). Hearts were subjected to 20 min global or 35 min regional ischaemia (36.5 °C) and 30 or 120 min reperfusion. In a separate series, dexamethasone (1 µM) was added to the perfusate for 10 min (Pre + Dex) before or after (Rep + Dex) ischaemia. Key findings Dexamethasone, administered intraperitoneally or added directly to the perfusate, significantly improved post-ischaemic functional recovery and reduced infarct size compared to untreated controls ( p < 0.05). These were associated with enhanced up-regulation of MKP-1 protein expression (arbitrary units (mean ± SD): Untreated: 1; ip Dex − P: 2.59 ± 0.22; ip Dex + P: 1.51 ± 0.22; Pre + Dex: 4.11 ± 0.73, Rep + 15′Dex: 1.51 ± 0.14; untreated vs. all groups, p < 0.05) and attenuation of p38 MAPK activation ( p < 0.05) in all dexamethasone-treated groups, except for Rep + 10′Dex. ERK and PKB/Akt activation were unchanged. Significance Dexamethasone-induced cardioprotection was associated with upregulation of the phosphatase MKP-1 and inactivation of pro-apoptotic p38 MAPK.

Kruppel-like transcription factor 5 promotes breast cell survival through pERK-mediated MKP-1 protein stabilization.

Abstract Abstract #3019 Kruppel-like Factor 5 (KLF5) is a transcription factor promoting cell survival and tumorigenesis in multiple cancers. High level of KLF5 mRNA is associated with a shorter survival for breast cancer patients. However, the role of KLF5 and mechanism of KLF5 actions in breast cancer remain unclear. In this study, we found that KLF5 knockdown by siRNA in two basal type breast cell lines MCF10A and BT20 induces apoptosis, as indicated by loss of cell viability and cleaved PARP and caspase 3. Interestingly, a survival phosphotase, MKP-1, is downregulated at protein level by KLF5 ablation. Consistently, KLF5 over-expression increases the MKP-1 protein expression. However, MKP-1 is not a KLF5 direct target gene because the MKP-1 mRNA level is not regulated by KLF5. By the cycloheximide chase assays, we found that KLF5 decreases the MKP-1 protein degradation. However, KLF5 does not decrease the MKP-1 E3 ubiquitin ligase SKP2 expression. The ERK inhibitor U0126 specifically blocks the KLF5 induced MKP-1, suggesting that KLF5 up-regulates MKP-1 through activating ERK signaling. Finally, we demonstrated that MKP-1 over-expression blocks KLF5 knockdown induced apoptosis in MCF10A cells. These findings suggest that KLF5 is a survival factor which promotes breast cell survival partially through pERK-mediated MKP-1 stabilization. The KLF5-pERK-MKP-1 signaling axis may provide new therapeutic targets for invasive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3019.

Abstract 1376: The Nadph Oxidase Nox4 Controls Mkp1 Expression and Thereby the Development of White Adipose Tissue

Obesity is a cardiovascular risk factor. Insulin promotes the formation of adipocytes by promoting fibroblast into adipocyte differentiation. Insulin however also acts as a mitogen for fibroblasts and the balance of insulin-induced proliferation and differentiation is incompletely understood. We hypothesize that the NADPH oxidase Nox4 acts as a central switch between these two processes. In 3T3-fibroblasts Nox4 siRNA reduced the cellular radical formation and attenuated the insulin-stimulated differentiation from fibroblasts into adipocytes. Importantly, insulin-induced proliferation was enhanced by Nox4 siRNA. Accordingly, Nox4 overexpression enhanced differentiation and reduced insulin-stimulated proliferation. We generated Nox4 knockout mice to study the in vivo relevance of this observation: The amount of adipose tissue was reduced in the Nox4−/− animals as compared to their wildtype littermates suggesting indeed an attenuated adipocyte differentiation, whereas fibroblasts obtained from Nox4−/− mice presented with enhanced proliferation. To uncover the underlying mechanism, we focused on the Erk1/2 pathway. Erk1/2 signaling is prevented by dephosphorylation through the dualspecific phosphatase MKP1. Luciferase reportergene assays using the full-length MKP1 promotor revealed that Nox4 siRNA reduced, and overexpression of Nox4 as well as treatment of the cells with H 2 O 2 induced MKP1 promotor activity. Indeed, Nox4 siRNA reduced MKP1 protein expression and thus enhanced basal and insulin-induced activation of Erk1/2 in fibroblasts. Nox4 overexpression had the opposite effect. Importantly, MKP1 expression was also reduced in adipose tissue from Nox4−/− mice. Overexpression of MKP1 in fibroblastes increased insulin-induced differentiation and attenuated proliferation, whereas MKP1 siRNA had the opposite effect. We conclude that Nox4, by regulating MKP1, is essentially involved in adipocyte differentiation and development of obesity.

Specific Activation of the Glucocorticoid Receptor and Modulation of Signal Transduction Pathways in Human Lens Epithelial Cells

Prolonged use of glucocorticoids (GCs) can lead to cataract formation. Lens GC responses have been difficult to elucidate. A previous study showed the presence of the glucocorticoid receptor (GR) in immortalized and primary human lens epithelial cells (hLECs) and GC-induced changes in gene expression. This study demonstrates specific GR activation and identifies the biological effect of GC-induced changes in gene expression in hLECs. HLE B-3 (B-3) and primary cultures of hLECs were transfected with pGRE.Luc and treated with or without dexamethasone (Dex), RU-486, spironolactone, or vehicle. mRNA and protein expression were examined by real-time PCR and Western blot analysis, respectively. Cell proliferation and apoptosis were examined by WST-1 and flow cytometry, respectively. Dex treatment of B-3 and primary cultures demonstrated specific GR, but not mineralocorticoid receptor (MR), activation and phosphorylation. Pathway analysis revealed GC-induced changes in expression of MAPK regulators. Increased expression of GILZ mRNA and MKP-1 mRNA and protein was observed in immortalized and donor hLECs. This corresponded with a decrease in the phosphorylated forms of RAF, ERK, p38, and AKT, but not in JNK. No net change in LEC proliferation or apoptosis was observed with Dex treatment. GC treatment of hLECs activates the GR to modulate the expression of MAPK and PI3K/AKT regulators. This is the first demonstration of GC signaling in hLECs. GCs, MAPK, and PI3K/AKT are involved in cell processes implicated in steroid-induced cataractogenesis. The absence of a net change in cell activity with acute steroid treatment is consistent with the possibility that chronic treatment leads to prolonged modulation of these pathways and steroid-induced cataract.

Phosphatase-Mediated Crosstalk Control of ERK and p38 MAPK Signaling in Corneal Epithelial Cells

To test the hypothesis that the protein phosphatases PP2A and MKP-1 are involved in controlling epidermal growth factor (EGF)-induced increases in rabbit corneal epithelial cell (RCEC) migration by mediating crosstalk between signaling pathways eliciting EGF receptor control of migration and proliferation. Western blot analysis was used to determine the phosphorylation status of Erk1/2, p38, and the mitogen-activated protein kinase (MAPK) kinase (MEK1/2) using inhibitors of Erk1/2 or p38 and dominant-negative (d/n) Erk1 or d/n p38 cell lines. Coimmunoprecipitation was used to evaluate protein phosphatase (PP)2A and Erk1/2 interaction. Short-interfering RNA (siRNA) transfection was performed to analyze the involvement of MAPK phosphatase (MKP)-1 in crosstalk. Scratch-wound assay was used to determine EGF-dependent effects on cell migration. EGF (10 ng/mL) induced changes in activation of Erk1/2 and p38, which were enhanced by inhibition with 10 microM SB203580 and 10 muM PD98059, respectively. PP inhibition with sodium orthovanadate (100 microM), okadaic acid (10 nM), or Ro 31-8220 (10 microM) resulted in larger and more prolonged increases in the phosphorylation status of Erk1/2 and p38. After 1 hour, EGF induced 14-fold increases in MKP-1 protein expression. After MKP-1 siRNA transfection, EGF had induced a similar pattern of changes in the phosphorylation status in Erk1/2 and p38 following PP inhibition. EGF-induced cell migration was enhanced by Erk1/2 pathway inhibition and was accentuated after PP inhibition. Conversely, p38 pathway inhibition eliminated this response. EGF-induced changes in Erk1/2 and p38 phosphorylation status are dependent on PP-mediated crosstalk. This control modulates the magnitude of growth factor-induced increases in corneal epithelial cell migration.

ERK1/2‐driven and MKP‐mediated inhibition of EGF‐induced ERK5 signaling in human proximal tubular cells

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.

Parathyroid Hormone Induces Mitogen‐Activated Kinase Phosphatase 1 in Murine Osteoblasts Primarily Through cAMP‐Protein Kinase A Signaling

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. 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. 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. 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.