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.     

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate,but Not FAK

Background

Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.

Principal Findings

Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.

Conclusions

These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.     

Bi-phasic activation of eNOS in response to uni-axial cyclic stretch is mediated by differential mechanisms in BAECs

We investigated the signaling mechanism of stretch-induced NO (Nitric oxide) production in bovine arterial endothelial cells (BAECs). BAECs cultured on an elastic silicone chamber coated with fibronectin were subjected to uni-axial cyclic stretch (1 Hz, 20% in length) and the amount of produced NO was measured by a cGMP assay. NO production increased in a bi-phasic manner and peaked at 5 min and 20 min after stretch onset. Correspondingly, the activities of endothelial nitric oxide synthase (eNOS) and Akt/PKB (measured by phosphorylation at serine 1,177 and serine 473, respectively), showed two peaks over time. Application of Gd(3+), a potent SA channel blocker, and depletion of external Ca(2+) exclusively inhibited the first peaks of eNOS and Akt activity, but exerted little effect on the second peak. On the other hand, the PI3K inhibitors, Wortmannin, LY294002, almost completely inhibited the second peak but not the first. These results suggest that up-regulation of eNOS in response to cyclic stretch was mediated by two distinct pathways, [Ca(2+)](i) increases via the SA channel in an early phase (partially Akt/PKB), and PI3K-Akt/PKB pathways in a late phase.     

A novel mechanism for mitogen-activated protein kinase localization

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.     

Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium

Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.     

Suppressed nuclear envelope proteins activate autophagy of vascular smooth muscle cells during cyclic stretch application

Dysfunctions of vascular smooth muscle cells (VSMCs) play crucial roles in vascular remodeling in hypertension, which correlates with pathologically elevated cyclic stretch due to increased arterial pressure. Recent researches reported that autophagy, a life-sustaining process, was increased in hypertension. However, the mechanobiological mechanism of VSMC autophagy and its potential roles in vascular remodeling are still unclear. Using renal hypertensive rats in vivo and FX5000 stretch application Unit in vitro, the autophagy of VSMCs was detected. The results showed that LC3II remarkably enhanced in hypertensive rats and 15% cyclic stretch (mimic the pathologically increased mechanical stretch in hypertension), and the activity of mammalian target of rapamycin (mTOR) was suppressed in 15% cyclic stretch. Administration of autophagy inhibitors, bafilomycin A1 and chloroquine, repressed VSMC proliferation efficiently, but did not affect the degradation of two important nuclear envelope (NE) proteins, lamin A/C and emerin. Using RNA interference to decline the expression of lamin A/C and emerin, respectively, we discovered that autophagy was upregulated under both static and 5% cyclic stretch conditions, accompanying with the decreased mTOR activity. During 15% cyclic stretch application, mTOR inhibition was responsible for autophagy elevation. Chloroquine administration in vivo inhibited the expression of PCNA (marker of proliferation) of abdominal aorta in hypertensive rats. Altogether, these results demonstrated that pathological cyclic stretch suppresses the expression of lamin A/C and emerin which subsequently represses mTOR pathway so as to induce autophagy activation. Blocking autophagic flux may be a practicable way to relieve the pathological vascular remodeling in hypertensive.     

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1β. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.     

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.     

NO inhibits stretch-induced MAPK activity by cytoskeletal disruption

Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at -27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization.     

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.     

Nuclear translocation of lymphoblast glucocorticoid receptors in childhood leukemia does not predict steroid responsiveness

Nuclear translocation of glucocorticoid receptors (GR) was measured in lymphoblasts from 45 children with acute lymphoblastic leukemia (ALL). The percent of total GR translocated to the nucleus ranged from 0 to 100% (mean, 59%) and was independent of the total GR level. Neither the extent of GR nuclear translocation nor the absolute amount of GR translocated at 4 or 25 degrees C correlated with glucocorticoid response in 13 patients with single-agent glucocorticoid trial. These results indicate that defects in nuclear translocation of GR are not a common cause for steroid resistance in childhood ALL.     

NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway

4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-kappaB) activation in endothelial cells. HHE induced NF-kappaB activation by inhibitor of kappaB (IkappaB) phosphorylation via the IkappaB kinase (IKK)/NF-kappaB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH(2)-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-kappaB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IkappaB phosphorylation, and NF-kappaB luciferase activity. These findings strongly suggest that HHE induces NF-kappaB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells.     

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.