scholarly journals Cilia movement regulates expression of the Raf-1 kinase inhibitor protein

2011 ◽  
Vol 300 (5) ◽  
pp. F1163-F1170 ◽  
Author(s):  
Kelli M. Sas ◽  
Michael G. Janech ◽  
Elizabeth Favre ◽  
John M. Arthur ◽  
P. Darwin Bell
Keyword(s):  
Primary Cilia ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Collecting Duct ◽  
Duct Cell ◽  
Inhibitor Protein ◽  
Ciliary Movement ◽  
Renal Epithelial Cell ◽  
Adult Mice

Renal epithelial cell primary cilia act as mechanosensors in response to changes in luminal fluid flow. To determine the role of cilia bending in the mechanosensory function of cilia, we performed proteomic analysis of collecting duct cell lines with or without cilia that were kept stationary or rotated to stimulate cilia bending. Expression of the Raf-1 kinase inhibitor protein (RKIP), an inhibitor of the MAPK pathway, was significantly elevated in rotated cilia (+) cells. This was compared with RKIP levels in cilia (−) cells that were stationary or rotated as well as in cilia (+) cells that were stationary. This result was confirmed in cilia knockout adult mice that had lower renal RKIP levels compared with adult mice with cilia. Downstream of RKIP, expression of phosphorylated ERK was decreased only in cells that had cilia and were subjected to constant cilia bending. Furthermore, elevated RKIP levels were associated with reduced cell proliferation. Blockade of PKC abrogated ciliary bending-induced increases in RKIP. In summary, we found that ciliary movement may help control the expression of the Raf-1 kinase inhibitor protein and thus maintain cell differentiation. In terms of polycystic kidney disease, loss of cilia and therefore sensitivity to flow may lead to reduced RKIP levels, activation of the MAPK pathway, and contribute to the formation of cysts.

Polaris, a protein disrupted inorpkmutant mice, is required for assembly of renal cilium

2002 ◽  
Vol 282 (3) ◽  
pp. F541-F552 ◽  
Author(s):  
Bradley K. Yoder ◽  
Albert Tousson ◽  
Leigh Millican ◽  
John H. Wu ◽  
Charles E. Bugg ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Collecting Duct ◽  
Physiological Role ◽  
Duct Cell ◽  
Cystic Kidney Disease ◽  
Cystic Kidney ◽  
Apical Surface ◽  
Cystic Disease ◽  
Cortical Collecting Duct

Cilia are organelles that play diverse roles, from fluid movement to sensory reception. Polaris, a protein associated with cystic kidney disease in Tg737°rpkmice, functions in a ciliogenic pathway. Here, we explore the role of polaris in primary cilia on Madin-Darby canine kidney cells. The results indicate that polaris localization and solubility change dramatically during cilia formation. These changes correlate with the formation of basal bodies and large protein rafts at the apical surface of the epithelia. A cortical collecting duct cell line has been derived from mice with a mutation in the Tg737 gene. These cells do not develop normal cilia, which can be corrected by reexpression of the wild-type Tg737 gene. These data suggest that the primary cilia are important for normal renal function and/or development and that the ciliary defect may be a contributing factor to the cystic disease in Tg737°rpkmice. Further characterization of these cells will be important in elucidating the physiological role of renal cilia and in determining their relationship to cystic disease.

scholarly journals Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium

2021 ◽  
Author(s):  
Stephanie Probst ◽  
Johannes Fels ◽  
Bettina Scharner ◽  
Natascha A. Wolff ◽  
Eleni Roussa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Cell Fate ◽  
Catalase Activity ◽  
Metal Ion ◽  
Iron Homeostasis ◽  
Collecting Duct ◽  
Duct Cell ◽  
Plasmid Transfection

AbstractThe liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

scholarly journals Coupling of epithelial Na+ and Cl− channels by direct and indirect activation by serine proteases

2012 ◽  
Vol 303 (9) ◽  
pp. C936-C946 ◽  
Author(s):  
Veronika Gondzik ◽  
Wolf Michael Weber ◽  
Mouhamed S. Awayda
Keyword(s):  
Serine Proteases ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Epithelial Cell Line ◽  
Extracellular Proteases ◽  
Direct Role ◽  
Renal Epithelial Cell ◽  
Transport Pathways

The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na+ channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na+ transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na+ homeostasis. To explore this process, we examined the coordinated effect of this protease on Na+ and Cl− transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current ( Isc) was used to measure transport of these ions. The Isc exhibited an ENaC-dependent Na+ component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl− component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated IENaC but was without effects on ICFTR. Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na+ and Cl− transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na+ and Cl− transport in these epithelia.

scholarly journals The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice

Diabetologia ◽  
2012 ◽  
Vol 55 (12) ◽  
pp. 3331-3340 ◽  
Author(s):  
F. N. Pardo ◽  
J. Altirriba ◽  
M. Pradas-Juni ◽  
A. García ◽  
U. Ahlgren ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Beta Cell ◽  
Kinase Inhibitor ◽  
Pancreatic Beta Cell ◽  
Beta Cell Proliferation ◽  
Inhibitor Protein

scholarly journals Lipopolysaccharide directly inhibits bicarbonate absorption by the renal outer medullary collecting duct

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shuichi Tsuruoka ◽  
Jeffrey M. Purkerson ◽  
George J. Schwartz
Keyword(s):  
Lipid A ◽  
Kinase Inhibitor ◽  
Collecting Duct ◽  
Therapeutic Interventions ◽  
E Coli ◽  
Monophosphoryl Lipid A ◽  
Tlr4 Agonist ◽  
Medullary Collecting Duct ◽  
Phosphoinositide 3 Kinase

AbstractAcidosis is associated with E. coli induced pyelonephritis but whether bacterial cell wall constituents inhibit HCO3 transport in the outer medullary collecting duct from the inner stripe (OMCDi) is not known. We examined the effect of lipopolysaccharide (LPS), on HCO3 absorption in isolated perfused rabbit OMCDi. LPS caused a ~ 40% decrease in HCO3 absorption, providing a mechanism for E. coli pyelonephritis-induced acidosis. Monophosphoryl lipid A (MPLA), a detoxified TLR4 agonist, and Wortmannin, a phosphoinositide 3-kinase inhibitor, prevented the LPS-mediated decrease, demonstrating the role of TLR4-PI3-kinase signaling and providing proof-of-concept for therapeutic interventions aimed at ameliorating OMCDi dysfunction and pyelonephritis-induced acidosis.

Loss of RAF Kinase Inhibitor Protein Is a Frequent Event In Acute Myeloid Leukemia with a Monocytic Phenotype and Cooperates with Mutant RAS In Malignant Transformation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4185-4185
Author(s):  
Armin Zebisch ◽  
Albert Woelfler ◽  
Karin Hiden ◽  
Claudia Bodner ◽  
Franz Quehenberger ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Inhibitor Protein ◽  
Raf Kinase ◽  
Raf Kinase Inhibitor Protein ◽  
Frequent Event ◽  
Sirna Knockdown

Abstract Abstract 4185 RAF kinase inhibitor protein (RKIP) has been described as a negative regulator of the RAS-mitogen activated protein kinase (MAPK) signaling cascade and as a metastasis suppressor gene in solid tumors. As constitutive activation of this pathway occurs frequently in acute myeloid leukemia (AML), we investigated the role of RKIP in the pathogenesis of this disorder. Analysis of RKIP expression by Western blot revealed complete or partial loss in 21/96 (22%) AML patient samples and 5/19 (26%) AML cell lines, but in none of ten purified normal CD34+ hematopoietic stem and precursor cell specimens. To evaluate the biological consequences of RKIP loss in AML, we examined the effects of introducing an RKIP transgene in 32D murine hematopoietic cells and THP-1 AML cells, the latter characterized by low RKIP expression. Overexpression of RKIP induced a significant decrease in the number of viable cells in both cell lines which was due to a decrease in proliferation as measured by cell cycle analysis after bromodeoxyuridine (BrdU) labeling. However, no differences were shown in the percentage of apoptotic cells - subG1 peak – in these assays, a finding that was confirmed by analysis of PARP- and caspase-3 cleavage. Importantly, colony formation of leukemic THP-1 cells in soft agar was significantly decreased following RKIP reconstitution going along with a tumor suppressor function of RKIP in hematopoietic cells. In a next step, we analyzed RKIP expression in a previously described transcriptomic data set of 285 AML patients (Valk et al. NEJM 2004) and sought to identify cooperating alterations. 28/285 (10%) patients harbored mutations in either NRAS or KRAS, 84/285 (29%) in NPM1, 17/285 (6%) in CEBPA, 31/285 (11%) in the FLT3 tyrosine kinase domain and 68/285 (24%) a FLT3 internal tandem duplication. Out of these, only mutations in RAS were significantly associated with decreased expression of RKIP (P = 0.00005) suggesting co-existence of mutant RAS and RKIP loss in AML. To test for a functional synergism, we employed NIH3T3 cells, a system commonly used to study RAS biology. The oncogenic potential of mutant RAS – as assessed by its ability to transform cells and induce focus formation – could be significantly reduced by simultaneous overexpression of RKIP indicating cooperation between RAS mutations and RKIP loss in malignant transformation. We next analyzed if RKIP loss was associated with specific AML features and observed a highly significant correlation with monocytic phenotypes (FAB M4 and M5) in two independent patient cohorts (cohort one with 96 patients, P = 2.13 × 10-7; cohort two with 285 patients, P = 1.6 × 10-8). To test for a causative role of RKIP in monocytic differentiation, we analyzed immature HL-60 AML cells, a cell line which can be converted into a monocytic phenotype by incubation with 1,25-dihydroxyvitamin D3 (1,25 D3). Whereas siRNA knockdown of RKIP alone failed to induce the monocytic phenotype, 1,25 D3 driven effects could be significantly increased by siRNA knockdown of RKIP and inhibited by RKIP overexpression indicating that RKIP loss is involved in the development of a monocytic AML phenotype. In conclusion, our data demonstrate that loss of RKIP is a frequent event in AML, particularly in subgroups with a monocytic phenotype. RKIP acts as a tumor suppressor in hematopoietic cells and cooperates with mutant RAS in malignant transformation. Disclosures: No relevant conflicts of interest to declare.

ATM- and ATR-Induced Primary Ciliogenesis Promotes Cisplatin Resistance in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Author(s):  
Yu-Ying Chao ◽  
Bu-Miin Huang ◽  
I-Chen Peng ◽  
Yi-Shyun Lai ◽  
Wen-Tai Chiu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Molecular Mechanism ◽  
Primary Cilia ◽  
Kinase Inhibitor ◽  
Ductal Adenocarcinoma ◽  
Ataxia Telangiectasia Mutated ◽  
Development And Differentiation ◽  
Inhibitor Resistance

Abstract BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of its late diagnosis and chemoresistance. Primary cilia, the cellular antennae, are observed in most human cells to maintain development and differentiation. Primary cilia are gradually lost during the progression of pancreatic cancer and are eventually absent in PDAC. However, recent study showed that primary cilia regrowth contributes to the development of diverse kinase inhibitor resistance in lung cancer. We elucidated the role of regrowth primary ciliogenesis in PDAC chemoresistance and uncovered the underlying molecular mechanism.ResultsWe showed that cisplatin-resistant PDAC regrew primary cilia. Additionally, genetic or pharmacological disruption of primary cilia sensitized PDAC to cisplatin treatment. Mechanistically, ataxia telangiectasia mutated (ATM) and ATM and RAD3-related (ATR), tumor suppressors that initiate DNA damage responses, promoted the excessive formation of centriolar satellites (EFoCS) and autophagy activation. Disruption of EFoCS and autophagy inhibited primary ciliogenesis, sensitizing PDAC cells to cisplatin treatment. ConclusionsCollectively, our findings revealed an unexpected interplay among the DNA damage response, primary cilia, and chemoresistance in PDAC and deciphered the molecular mechanism by which ATM/ATR-mediated EFoCS and autophagy cooperatively regulate primary ciliogenesis.

Abstract P117: A Conserved Trans-membrane Domain Lysine in the Integrin Beta1 Subunit Regulates Renal Collecting Duct Cell Function

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Sijo Mathew ◽  
Jiang Chen ◽  
Zhenwei Lu ◽  
Charles Sanders ◽  
Roy Zent
Keyword(s):  
Cell Function ◽  
Collecting Duct ◽  
Duct Cell ◽  
Lysine Residue ◽  
Mutant Protein ◽  
Integrin Β1 ◽  
Binding Ability ◽  
Integrin Β3 ◽  
Collecting Duct Cells

Integrins are heterodimeric trans-membrane receptor proteins that mediate the interaction of cells with extracellular matrix proteins (ECM). They mediate various growth factor dependent cell-signaling pathways during the development of fibrosis that is characteristic of all forms of chronic kidney diseases. Although integrin β1 is the most abundant integrin subunit in kidney and can form complexes with 12 different α subunits, integrin β3 is the best studied integrin β subunit and serves as the canonical model for integrin function based on the high sequence homology between the trans-membrane and cytosolic domains of integrin β subunits. A conserved lysine residue towards the C-terminus of integrin β3 subunit is reported to be important for regulating the activation of integrin αIIbβ3 complexes; however the functional importance of this lysine is unknown in β1 integrins. We investigated the role of this lysine residue in integrin β1-dependent kidney collecting duct cell function. We expressed the mutant protein where the lysine is mutated to glutamic acid in collecting duct cells null for integrin β1. Collecting duct cells expressing mutant protein had decreased the adhesion of cells to collagen IV mediated by integrin α1β1 by 80% (0.95 vs 0.18). This mutation also decreased the ability of IMCD cells adhesion to collagen I mediated by integrin α2β1 by 82% (0.78 vs 0.15). In contrast to earlier reports in integrin β3, this mutation did not significantly alter the amount of active integrin β1 on the cell surface as estimated by FACS analysis; however we did observe a decrease in conformation specific antibody binding on cells adhered to collagen (0.70 vs 0.30). We also investigated the role of this lysine residue in complex formation of purified integrin β1 with integrin α1 and α2 TM/CT domains in phospholipid bicelles using fluorescence anisotropy. The dissociation constant for binding was estimated to be >3.2 mol and mutation of lysine residue did not significantly alter their binding ability. This contrasted with integrin αIIb β3 where we found fourfold decrease in binding ability (Kd 0.09 ± 0.03 mol and 0.33 ± 0.05 mol). Our data clearly suggest that conserved transmembrane lysine in both integrin β3 and integrin β1 regulate cell functions by distinct mechanisms.

Molecular Mechanism by Which Rituximab Sensitizes B-NHL Cells to TRAIL Apoptosis: Induction of RKIP and DR5 Expression Via Inhibition of NF-Kb, Snail, and YY1.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1596-1596
Author(s):  
Mario I. Vega ◽  
Melisa Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Yeung Kam ◽  
Stavroula Baritaki ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Proteasome Inhibitors ◽  
Underlying Mechanism ◽  
Inhibitor Protein ◽  
Rt Pcr ◽  
Yin Yang 1 ◽  
Induced Apoptosis

Abstract There have been significant advances in the treatment of patients with B-NHL using combination of rituximab and CHOP. However, a subset of patients does not initially respond or develop resistance to further treatments; hence, the need for alternative therapies to overcome resistance. TRAIL and agonist DR4/DR5 monoclonal antibodies have been examined clinically against a variety of tumors in Phase I/II. However, the majority of B-NHL derived from patients and cell lines are resistant to TRAIL-induced apoptosis. Recent findings demonstrated that treatment of TRAIL-resistant-B-NHL with rituximab sensitizes the tumor cells to TRAIL apoptosis. The underlying mechanism of rituximab-induced sensitization to TRAIL, however, is not clear. We have recently reported that treatment of tumor cells with sensitizing agents (example CDDP, proteasome inhibitors) resulted in the reversal of resistance to TRAIL via induction of Raf-1 kinase inhibitor protein (RKIP) and demonstrated the pivotal role of RKIP in the regulation of tumor cell sensitivity to TRAIL. Hence, since rituximab induces the expression of RKIP in B-NHL, we determined the role of RKIP induction by rituximab in the sensitization of B-NHL to TRAIL apoptosis. Various B-NHL cell lines were used as models for study. Treatment of B-NHL cells with rituximab (20 ng/ml) and TRAIL (5–10 ng/ml) resulted in significant potentiation of apoptosis and synergy was achieved. Rituximab induced the expression of RKIP as determined by RT-PCR and western concomitantly with inhibition of NF-kB. The inhibition of NF-kB resulted in upregulation of RKIP expression and was mediated, in large part, by inhibition of the transcription repressor Snail (downstream of NF-kB). Further, RKIP-induced inhibition of NF-kB by rituximab resulted in downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and concomitantly with the upregulation of DR5 expression. The role of RKIP induction by rituximab in the upregulation of DR5 and sensitization to TRAIL apoptosis was corroborated by the use of cells over expressing RKIP which were sensitive to TRAIL apoptosis in the absence of rituximab. Our findings reveal a novel mechanism of rituximab-induced sensitization of B-NHL to TRAIL apoptosis via inhibition of NF-kB and Snail and upregulation of RKIP and DR-5. The combination of rituximab and TRAIL may be effective in the treatment of B-NHL. Further, our studies suggest that agents other than rituximab that can induce RKIP can reverse resistance to TRAIL in B-NHL that are unresponsive to rituximab treatment.

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