scholarly journals Selective knockdown of AT1 receptors by RNA interference inhibits Val5-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells

2007 ◽  
Vol 293 (1) ◽  
pp. C367-C378 ◽  
Author(s):  
Xiao C. Li ◽  
Jia L. Zhuo
Keyword(s):  
Proximal Tubule ◽  
Physiological Effect ◽  
Mitogen Activated Protein Kinase ◽  
Receptor Protein ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Western Blots ◽  
Receptor Mediated Endocytosis ◽  
Mitogen Activated Protein

Receptor-mediated endocytosis of extracellular ANG II has been suggested to play an important role in the regulation of proximal tubule cell (PTC) function. Using immortalized rabbit PTCs as an in vitro cell culture model, we tested the hypothesis that extracellular ANG II is taken up by PTCs through angiotensin type 1 receptor (AT1; or AT1a) receptor-mediated endocytosis and that inhibition of ANG II endocytosis using a selective AT1 receptor small-interfering RNA (siRNA; AT1R siRNA) or endocytotic inhibitors exerts a physiological effect on total and apical sodium and hydrogen exchanger isoform 3 (NHE-3) protein abundance. Western blots and live cell imaging with FITC-labeled ANG II confirmed that transfection of PTCs with a human specific AT1R siRNA for 48 h selectively knocked down AT1 receptor protein by 76 ± 5% ( P < 0.01), whereas transfection with a scrambled siRNA had little effect. In nontransfected PTCs, exposure to extracellular ANG II (1 nM) for 60 min at 37°C increased intracellular ANG II accumulation by 67% (control: 566 ± 55 vs. ANG II: 943 ± 160 pg/mg protein, P < 0.05) and induced mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (163 ± 15% of control, P < 0.01). AT1R siRNA reduced ANG II endocytosis to a level similar to losartan, which blocks cell surface AT1 receptors (557 ± 37 pg/mg protein, P < 0.05 vs. ANG II), or to colchicine, which disrupts cytoskeleton microtubules (613 ± 12 pg/mg protein, P < 0.05 vs. ANG II). AT1R siRNA, losartan, and colchicine all attenuated ANG II-induced ERK1/2 activation and total cell lysate and apical membrane NHE-3 abundance. The scrambled siRNA had no effect on ANG II endocytosis, ERK1/2 activation, or NHE-3 expression. These results suggest that AT1 receptor-mediated endocytosis of extracellular ANG II may regulate proximal tubule sodium transport by increasing total and apical NHE-3 proteins.

scholarly journals Adiponectin as Well as Compressive Forces Regulate in vitro β-Catenin Expression on Cementoblasts via Mitogen-Activated Protein Kinase Signaling Activation

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiawen Yong ◽  
Julia von Bremen ◽  
Gisela Ruiz-Heiland ◽  
Sabine Ruf
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Western Blots ◽  
Protein Levels ◽  
Mitogen Activated Protein ◽  
Gsk 3Β ◽  
Signaling Activation

We aimed to investigate the molecular effect that adiponectin exerts on cementoblasts especially in the presence of compressive forces. OCCM-30 cells (M. Somerman, NIH, NIDCR, United States) were used. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and western blots were employed to verify if the mRNA and protein levels of adiponectin receptors (AdipoRs), mitogen-activated protein kinase (MAPK), and β-catenin signaling were influenced by compressive forces or adiponectin. Moreover, siRNAs targeting P38α, JNK1, ERK1, ERK2, and AdipoRs as well as pharmacological MAPK inhibition were performed. We found that compressive forces increase the expression of AdipoRs. Adiponectin and compression up-regulate P38α,JNK1, ERK1, and ERK2 as well as β-catenin gene expression. Western blots showed that co-stimuli activate the MAPK and β-catenin signaling pathways. MAPK inhibition alters the compression-induced β-catenin activation and the siRNAs targeting AdipoRs, P38α, and JNK1, showing the interaction of single MAPK molecules and β-catenin signaling in response to compression or adiponectin. Silencing by a dominantly negative version of P38α and JNK1 attenuates adiponectin-induced TCF/LEF reporter activation. Together, we found that light compressive forces activate β-catenin and MAPK signaling pathways. Adiponectin regulates β-catenin signaling principally by inactivating the GSK-3β kinase activity. β-Catenin expression was partially inhibited by MAPK blockade, indicating that MAPK plays a crucial role regulating β-catenin during cementogenesis. Moreover, adiponectin modulates GSK-3β and β-catenin mostly through AdipoR1. P38α is a key connector between β-catenin, TCF/LEF transcription, and MAPK signaling pathway.

Effects of acid challenges on type 2 angiotensin II receptor-sensitive ammonia production by the proximal tubule

2014 ◽  
Vol 307 (1) ◽  
pp. F53-F57 ◽  
Author(s):  
Glenn T. Nagami ◽  
Alexandria K. Plumer ◽  
Raymond M. Beyda ◽  
Oran Schachter
Keyword(s):  
Angiotensin Ii ◽  
Cell Surface ◽  
Proximal Tubule ◽  
Time Dependent ◽  
Receptor Protein ◽  
Cell Surface Expression ◽  
Ang Ii ◽  
Angiotensin Ii Receptor

Angiotensin II (ANG II) acting through its type 1 (AT1) receptor stimulates total ammonia (tNH3) production by the proximal tubule. The present studies explored the role of ANG II type 2 (AT2) receptors in modulating the stimulatory effects of ANG II on tNH3 production. Mouse S2 proximal tubule segments derived from 18-h and 7-day acid-loaded mice, and non-acid-loaded controls were dissected and microperfused in vitro. Adding ANG II to the luminal perfusion solution resulted in different increments in tNH3 production rates in tubules derived from 18-h vs. 7-day acid-loaded mice such that the increase in tNH3 production with ANG II was higher in tubules derived from 18-h acid-loaded mice compared with those derived from control and 7-day acid-loaded mice. Adding the AT2 receptor blocker PD123319 with ANG II increased ANG II-stimulated tNH3 production in S2 segments from control and 7-day acid-loaded mice but not in those from 18-h acid-loaded mice, and this increased effect of PD123319 was associated with higher AT2 receptor protein levels in brush-border membranes. Studies in cultured proximal tubule cells demonstrated that 2-h exposure to pH 7.0 reduced the modulating effect of PD123319 on ANG II-simulated tNH3 production and reduced cell surface AT2 receptor levels. We concluded that AT2 receptors reduce the stimulatory effect of ANG II on proximal tubule tNH3 production and that the time-dependent impact of AT2 receptor blockade on the ANG II-stimulated tNH3 production corresponded to time-dependent changes in AT2 receptor cell surface expression in the proximal tubule.

scholarly journals Effective Treatment of Metastatic Melanoma by Combining MAPK and PI3K Signaling Pathway Inhibitors

2019 ◽  
Vol 20 (17) ◽  
pp. 4235 ◽  
Author(s):  
Aasen ◽  
Parajuli ◽  
Hoang ◽  
Feng ◽  
Stokke ◽  
...  
Keyword(s):  
Brain Metastases ◽  
Combined Therapy ◽  
Combined Treatment ◽  
Mitogen Activated Protein Kinase ◽  
Western Blots ◽  
Mitogen Activated Protein ◽  
Treatment Experiment ◽  
Phosphoinositide 3 Kinase

Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases. Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies. In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance. Thus, combined treatment targeting both pathways is a promising strategy to overcome this. Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines. Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis. Finally, an in vivo treatment experiment was carried out on NOD/SCID mice. We show that combined therapy was more effective than monotherapy. Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo. This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

scholarly journals Role for the Ran Binding Protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 Signal Transduction

2004 ◽  
Vol 3 (6) ◽  
pp. 1544-1556 ◽  
Author(s):  
Jade Mei-Yeh Lu ◽  
Robert J. Deschenes ◽  
Jan S. Fassler
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
Osmotic Stress ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Osmotic Response ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

ABSTRACT Yeast Sln1p is an osmotic stress sensor with histidine kinase activity. Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions. Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood. To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors. Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen. The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo. mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor. The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes.

Proximal tubule microvilli remodeling and albuminuria in the Ren2 transgenic rat

2007 ◽  
Vol 292 (2) ◽  
pp. F861-F867 ◽  
Author(s):  
Melvin R. Hayden ◽  
Nazif A. Chowdhury ◽  
Shawna A. Cooper ◽  
Adam Whaley-Connell ◽  
Javad Habibi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Proximal Tubule ◽  
Structural Damage ◽  
Kidney Tissue ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Sprague Dawley Rats

TG(mRen2)27 (Ren2) transgenic rats overexpress the mouse renin gene, with subsequent elevated tissue ANG II, hypertension, and nephropathy. The proximal tubule cell (PTC) is responsible for the reabsorption of 5–8 g of glomerular filtered albumin each day. Excess filtered albumin may contribute to PTC damage and tubulointerstitial disease. This investigation examined the role of ANG II-induced oxidative stress in PTC structural remodeling: whether such changes could be modified with in vivo treatment with ANG type 1 receptor (AT1R) blockade (valsartan) or SOD/catalase mimetic (tempol). Male Ren2 (6–7 wk old) and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Systolic blood pressure, albuminuria, N-acetyl-β-d-glucosaminidase, and kidney tissue malondialdehyde (MDA) were measured, and ×60,000 transmission electron microscopy images were used to assess PTC microvilli structure. There were significant differences in systolic blood pressure, albuminuria, lipid peroxidation (MDA and nitrotyrosine staining), and PTC structure in Ren2 vs. Sprague-Dawley rats (each P < 0.05). Increased mean diameter of PTC microvilli in the placebo-treated Ren2 rats ( P < 0.05) correlated strongly with albuminuria ( r2 = 0.83) and moderately with MDA ( r2 = 0.49), and there was an increase in the ratio of abnormal forms of microvilli in placebo-treated Ren2 rats compared with Sprague-Dawley control rats ( P < 0.05). AT1R blockade, but not tempol treatment, abrogated albuminuria and N-acetyl-β-d-glucosaminidase; both therapies corrected abnormalities in oxidative stress and PTC microvilli remodeling. These data indicate that PTC structural damage in the Ren2 rat is related to the oxidative stress response to ANG II and/or albuminuria.

Abstract 564: Differential Expression and Regulation of Angiotensinogen in Renal Proximal Tubule Cells From S1, S2 and S3 Segments

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Kathleen S Hering-Smith ◽  
L G Navar
Keyword(s):  
Proximal Tubule ◽  
Kidney Injury ◽  
Specific Cell ◽  
S2 Cells ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Protein Levels ◽  
Pathogenic Factors ◽  
Renal Proximal Tubule Cells

In angiotensin II (Ang II)-dependent hypertension, intrarenal angiotensinogen (AGT) augmentation induced by Ang II and associated pathogenic factors including interleukin 6 (IL-6) cause further elevation of intratubular Ang II production, leading to the progression of hypertension and kidney injury. Recent studies have suggested that renal proximal straight tubules (S3 segment) are the main source of intrarenal AGT and that S1 and S2 segments do not express AGT mRNA under normal conditions. However, AGT expression and its regulation by Ang II and/or IL-6 in each proximal tubule segment have not been demonstrated an in vitro setting. The availability of specific cell lines derived from mouse S1, S2 and S3 segments provided an opportunity to decisively determine each segments’ capability to express AGT and respond to stimuli. Thus, this study was performed to determine AGT expression and its response to stimulation with Ang II and IL-6 in S1, S2 and S3 cell line. Basal AGT mRNA and protein levels were detected by RT-PCR and western blot analysis. Basal levels of Ang II type 1 receptor (AT1R) and STAT3, which is a transcription factor in IL-6 signaling pathway, were also measured. In addition, the cells were incubated with 100 nM Ang II and/or 400 nM IL-6 for 24 h. Basal AGT levels in S1 and S3 cells were lower than in mouse whole kidney (0.09-fold and 0.33-fold compared with mouse whole kidney). S2 cells exhibited the highest basal AGT levels (4.15-fold) among these cells. In S1 cells, AGT expression was stimulated by IL-6 (1.89 ± 0.32, ratio to control) and co-stimulation with Ang II and IL-6 (1.85 ± 0.28) although Ang II alone did not alter AGT levels. In S2 cells, only the co-stimulation increased AGT expression (1.35 ± 0.01). No changes were observed by similar treatments in S3 cells. Basal AT1R levels were lower in S3 than in S1 and S2 cells (0.97 ± 0.09 in S2, 0.32 ± 0.07 in S3, ratio to S1). S1 cells showed the highest basal levels of STAT3. Basal STAT3 levels in S3 cells were lower than that in S1 and S2 cells. These results indicate that S2 cells are main source of intrarenal AGT which can be augmented by Ang II and IL-6 during the development of Ang II-dependent hypertension. Furthermore, low basal levels of AT1R and STAT3 in S3 cells explain why these cells do not respond to Ang II and IL-6.

The Effect of Wnt Family Member 5a Gene Silencing on the Proliferation of Achondroplasia Using a DNAzymes-CoOOH Nanocomposite

2021 ◽  
Vol 17 (7) ◽  
pp. 1426-1434
Author(s):  
Hairui Xie ◽  
Lili Zhou ◽  
Zhijiang Chen ◽  
Hong Zhao
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Family Member ◽  
Interaction Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Endoplasmic Reticulum Stress Pathway ◽  
Protein Kinase Signaling ◽  
Kinase Signaling Pathway

Achondroplasia is a kind of congenital dysplasia due to the defect of endochondral ossification. Achondroplasia is considered to be a protein folding disease leading to endoplasmic reticulum stress. Endoplasmic reticulum stress may lead to disease by affecting the function and survival state of chondrocytes, but the specific mechanism requires further study. In this study, bioinformatics methods, online database mining, screening of differentially expressed genes for pathway enrichment, and interaction analysis were conducted to detect the Wnt family member 5a (Wnt5a) gene. Additionally, we designed a novel DNAzymes-based nanocomposite that can simultaneously silence Wnt5a genes in chondrocytes. The nanocomposite was composed of amino-functionalized cobalt oxyhydroxide nanoflakes modified by DNAzymes that target the Wnt5a gene. Further, we conducted in vitro experiments to verify that Wnt5a can mediate the mitogen-activated protein kinase signaling pathway through the endoplasmic reticulum stress pathway to affect the proliferation of chondrocytes.

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