scholarly journals Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability

2003 ◽  
Vol 95 (5) ◽  
pp. 1889-1895 ◽  
Author(s):  
Fiemu E. Nwariaku ◽  
Patricia Rothenbach ◽  
Zijuan Liu ◽  
Xudong Zhu ◽  
Richard H. Turnage ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Endothelial Permeability ◽  
Kinase Assay ◽  
Western Blots ◽  
Mapk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Mapk Activity ◽  
Gtp Binding ◽  
Extracellular Signal

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells

2001 ◽  
Vol 280 (1) ◽  
pp. G75-G87 ◽  
Author(s):  
Wei Li ◽  
Aydin Duzgun ◽  
Bauer E. Sumpio ◽  
Marc D. Basson
Keyword(s):  
Protein Kinase ◽  
Cyclic Strain ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Kinase C ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Static Conditions

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 μM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 μM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH2-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the α2-, α3-, α6-, or β1-integrin subunits but not on a substrate of functional antibody to the α5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.

scholarly journals Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway

2007 ◽  
Vol 196 (2) ◽  
pp. 275-281 ◽  
Author(s):  
Christian K Tipsmark ◽  
Christina N Strom ◽  
Sean T Bailey ◽  
Russell J Borski
Keyword(s):  
Weight Control ◽  
Time Course ◽  
Dependent Manner ◽  
Cellular Mechanisms ◽  
Concentration Dependent Manner ◽  
Western Blots ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Time Course Experiment

Leptin was initially identified as a regulator of appetite and weight control centers in the hypothalamus, but appears to be involved in a number of physiological processes. This study was carried out to examine the possible role of leptin in regulating prolactin (PRL) release using the teleost pituitary model system. This advantageous system allows isolation of a nearly pure population of lactotropes in their natural, in situ aggregated state. The rostral pars distalis were dissected from tilapia pituitaries and exposed to varying concentrations of leptin (0, 1, 10, 100 nM) for 1 h. Release of PRL was stimulated by leptin in a potent and concentration-dependent manner. A time-course experiment showed that the strongest response in PRL release with leptin occurs within the first hour (approximately sixfold), and stimulation was sustained after 16 h (approximately twofold). Many of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2. Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2.

Overexpression of p62 Induces Autophagy and Promotes Proliferation, Migration and Invasion of Nasopharyngeal Carcinoma Cells through Promoting ERK Signaling Pathway

2020 ◽  
Vol 20 (8) ◽  
pp. 624-637 ◽  
Author(s):  
Qiong Wu ◽  
Manlin Xiang ◽  
Kun Wang ◽  
Zhen Chen ◽  
Lu Long ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Clinical Analysis ◽  
Carcinoma Cells ◽  
Immunofluorescent Staining ◽  
Clinicopathological Parameters ◽  
Migration And Invasion ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Potential Biomarker

Background: Increasing evidence has shown that p62 plays an important role in tumorigenesis. However, relatively little is known about the association between p62 and tumor invasion and metastasis; in addition, its role in NPC (nasopharyngeal carcinoma, NPC) has been rarely investigated. Objective: To investigate the effect of p62 on tumorigenesis and metastasis in nasopharyngeal carcinoma. Methods: Western blotting, immunofluorescent staining and immunohistochemistry were used to evaluate p62 protein expression. Subsequently, cell viability, colony formation, migration, invasion and autophagy assays were performed. anti-p62 autoantibodies in sera were detected by ELISA. These data were correlated with clinicopathological parameters. Results: We confirmed that p62 was significantly up-regulated in NPC tissues. Furthermore, high expression of p62 was observed in NPC cell lines, and especially in the highly metastatic 5-8F cells. In vitro, down-regulation of p62 inhibited proliferation, clone forming ability, autophagy, migration, and invasion in 5-8F cells, whereas p62 overexpression resulted in the opposite effects in 6-10B cells. Moreover, we confirmed that p62 promotes NPC cell proliferation, migration, and invasion by activating ERK (extracellular signal-regulated kinase, ERK). Clinical analysis indicated that high p62 expression correlates with lymph node and distant metastasis (P<0.05). Serum anti-p62 autoantibodies were increased in NPC patients and levels were associated with metastasis. Conclusion : Our data establish p62 targeting ERK as potential determinant in the NPC, which supplies a new pathway to treat NPC. Furthermore, p62 is a potential biomarker which might be closely related to the tumorigenesis and metastasis in NPC.

scholarly journals Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization

2011 ◽  
Vol 301 (3) ◽  
pp. F554-F564 ◽  
Author(s):  
Sierra Delarosa ◽  
Julie Guillemette ◽  
Joan Papillon ◽  
Ying-Shan Han ◽  
Arnold S. Kristof ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Gel Filtration ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
Fk506 Binding Protein ◽  
Induced Apoptosis ◽  
Injury And Repair

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1–373) and a modified FK506 binding protein, Fv (Fv-SLK 1–373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1–373. In an in vitro kinase assay, the dimeric Fv-SLK 1–373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1–373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1–373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1–373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

scholarly journals Different Protein Tyrosine Kinases Are Required for B Cell Antigen Receptor–mediated Activation of Extracellular Signal–Regulated kinase, c-Jun NH2-terminal Kinase 1, and p38 Mitogen-activated Protein Kinase

1998 ◽  
Vol 188 (7) ◽  
pp. 1297-1306 ◽  
Author(s):  
Aimin Jiang ◽  
Andrew Craxton ◽  
Tomohiro Kurosaki ◽  
Edward A. Clark
Keyword(s):  
B Cell ◽  
P38 Mapk ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
B Cell Antigen Receptor ◽  
Mapk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Erk2 Activation

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate–sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.

Abstract 10246: Hydroxychloroquine Pretreatment Reduces Myocardial Ischemia-Reperfusion Injury: Role of Cardiac Extracellular-Signal-Regulated Kinase 5 and Autophagy

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Feiyan Yang ◽  
Chang Yin ◽  
Lei Xi ◽  
Rakesh C Kukreja
Keyword(s):  
Infarct Size ◽  
Ischemia Reperfusion Injury ◽  
Myocardial Infarct ◽  
Ischemia Reperfusion ◽  
Beclin 1 ◽  
Myocardial Infarct Size ◽  
Western Blots ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Related Proteins

Background: Hydroxychloroquine (HCQ) is an antimalarial drug, which is also widely used to treat chronic rheumatologic diseases. Since HCQ was reported to inhibit cell autophagy and to activate extracellular-signal-regulated kinase 5 (ERK5) in vascular endothelial cells, we designed the current study to determine the effects of HCQ on cardiac ischemia-reperfusion (I-R) injury and post-I-R expression of ERK5 and autophagy marker proteins. Methods: Adult C57BL/6J mice of both genders were pretreated with HCQ (50 mg/kg, i.p.) 1 hour prior to isolation of the hearts, which were subjected to 30 min of no-flow global ischemia followed by 60 min of reperfusion in Langendorff mode. Ventricular function was continuously assessed and myocardial infarct size was determined at the end of I-R. Heart samples were collected following normoxic perfusion (no-ischemic controls), I-R, or I-R with HCQ for assessing ERK5 and autophagy-related proteins with Western blots. Results: HCQ pretreatment reduced infarct size significantly in the female hearts (P<0.05) as compared with the male hearts (Fig. A). Post-I-R cardiac function was better in HCQ-treated males (Fig. B). I-R resulted in a robust increase in total ERK5 (Fig. C) and phosphorylated ERK5 (Thr218/Tyr220) in both genders, which was abolished in HCQ-treated groups. Conversely, either I-R or HCQ did not affect the post-I-R cardiac expression of autophagy-related proteins (e.g., Atg5, Beclin-1, LC3II/LC3I ratio), except Beclin-1 phosphorylation was inhibited in HCQ-treated male hearts, but not females (Fig. D). Conclusions: Acute HCQ pretreatment affords cardioprotection against I-R injury in both genders. Interestingly, cardioprotective effects of HCQ are associated with a strong inhibitory effect on the induction of ERK5 following I-R in the heart, indicating a novel molecular mechanism underlying the HCQ-induced cardioprotection. However, the cardioprotective dose of HCQ has no major impact on cardiac autophagy.

Abstract 17225: Extracellular Signal-Regulated Kinase 1/2 is Essential for Lipocalin-2 Signaling in Endothelial Cells

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Markus Theurl ◽  
Andrea Schroll ◽  
Igor Theurl ◽  
Daniela Lener ◽  
Wolfgang-Michael Franz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Function ◽  
Umbilical Vein ◽  
Brdu Incorporation ◽  
Lipocalin 2 ◽  
Vascular Disorders ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Stimulation Of

Introduction: Lipocalin-2 (Lcn2) is an acute phase protein and a marker of kidney injury. Recently, elevated Lcn2-levels have been reported in heart failure and myocardial infarction. Moreover, stimulation of breast cancer angiogenesis was observed. Thus, we hypothesized that Lcn2 may be a regulator of vascular function and a target for the treatment of ischemic vascular disorders. Methods/Results: In-vitro Lcn2 mediated proliferation of human umbilical vein endothelial cells (HUVEC; rel. proliferation Lcn2 10 nM vs. ctr.: 1.4±0.09, n=3, P<0.001) and human coronary artery endothelial cells (HCAEC; rel. proliferation Lcn2 10 nM vs. ctr.: 1.3±0.07, n=3, P<0.01) as determined by BrdU-incorporation. In the in-vitro matrigel assay stimulation of HUVEC (1.4 fold vs. ctr., n=3, P<0.01) and HCAEC (1.6 fold vs. ctr., n=3, P<0.001) with Lcn2 resulted in a significant induction of capillary like tube formation. All effects were similar to vascular endothelial growth factor (VEGF). Mechanistically these results can be traced back to phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Real-time PCR analyses revealed expression of Lcn2 and its receptor by endothelial cells (EC) as well as a hypoxia-dependent up-regulation (rel. Lcn2 mRNA hypoxia vs. normoxia 1.6±0.2, P<0.05; rel. Lcn2-receptor mRNA hypoxia vs. normoxia 2.6±0.2, P<0.001). In the mouse aortic ring assay Lcn2-treatment resulted in a significant outgrowth of EC similar to VEGF. In the hind limb ischemia (HLI) model Lcn2 -/- mice showed an impressive phenotype. After induction of HLI we detected significantly more tissue defects compared to wild type (WT) mice. The ischemia-related lesions were more severe as determined by necrosis score (necrosis score Lcn2 -/- 1.8±0.2 vs. WT 0.7±0.2, n=5, P<0.01) and amputation rate was significantly higher. In ischemic hind limbs of Lcn2 -/- mice ERK1/2-phosphorylation was almost abrogated which might be an underlying mechanism. Transplantation of WT-bone marrow to irradiated Lcn2 -/- mice didn’t influence the outcome suggesting that observed effects are rather endothelium-dependent than influenced by an inflammatory response. Conclusion: Lcn2 might be a promising therapeutic factor for the treatment of ischemic vascular disorders.

Role of membrane phosphotyrosine proteins in human spermatozoal function

1991 ◽  
Vol 99 (1) ◽  
pp. 157-165
Author(s):  
R.K. Naz ◽  
K. Ahmad ◽  
R. Kumar
Keyword(s):  
Physiological State ◽  
Human Sperm ◽  
Kinase Assay ◽  
Relative Molecular Mass ◽  
Sperm Cells ◽  
Western Blots ◽  
Tyrosine Residues ◽  
Sperm Extract ◽  
32P Incorporation

The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94,000 +/− 3000 and 46,000 +/− 3000 Mr on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94,000 +/− 3000, 46,000 +/− 3000, 25,000 +/− 7000 and 12,000 +/− 2000 Mr, respectively, were labeled with 32P when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher 32P incorporation into 94,000 +/− 3000 Mr proteins and 17% less incorporation into 12,000 +/− 2000 Mr proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94,000 +/− 3000 Mr proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead.(ABSTRACT TRUNCATED AT 250 WORDS)

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