Biomedical subjects

Joan Papillon

Publications and source records attributed to Joan Papillon.

12 recordsLinked to original sources

PubMed2006

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha.

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.

Animals↗

PubMed2006

The 3'-untranslated region of the Ste20-like kinase SLK regulates SLK expression.

Ste20-like kinase, SLK, a germinal center kinase found in kidney epithelial cells, signals to promote apoptosis. Expression of SLK mRNA and protein and kinase activity are increased during kidney development and recovery from ischemic acute renal failure. The 3'-untranslated region (3'-UTR) of SLK mRNA contains multiple adenine and uridine-rich elements, suggesting that 3'-UTR may regulate mRNA stability. This was confirmed in COS cell transient transfection studies, which showed that expression of the SLK open-reading frame plus 3'-UTR mRNA was reduced by 35% relative to the open-reading frame alone. To further characterize the SLK-3'-UTR, this nucleotide sequence was subcloned downstream of enhanced green fluorescent protein (EGFP) cDNA. In COS, 293T, and glomerular epithelial cells, expression of EGFP mRNA and protein was markedly reduced in the presence of the SLK-3'-UTR. After transfection and subsequent addition of actinomycin D, EGFP mRNA remained stable in cells for at least 6 h, whereas EGFP-SLK-3'-UTR mRNA decayed with a half-life of approximately 4 h. A region containing five AUUUA motifs within the SLK-3'-UTR destabilized EGFP mRNA. Deletion of this region from the SLK-3'-UTR, in part, restored mRNA stability. By UV cross-linking and SDS-PAGE, the SLK-3'-UTR bound to protein(s) of approximately 30 kDa in extracts of COS cells, glomerular epithelial cells, and kidney. Cotransfection of HuR (a RNA binding protein of approximately 30 kDa) increased the steady-state mRNA level of EGFP-SLK-3'-UTR but not EGFP. Thus the SLK-3'-UTR may interact with kidney RNA-binding proteins to regulate expression of SLK mRNA during kidney development and after ischemic injury.

3' Untranslated Regions↗

PubMed2005

Induction of apoptosis by the Ste20-like kinase SLK, a germinal center kinase that activates apoptosis signal-regulating kinase and p38.

Expression and activity of the germinal center kinase, Ste20-like kinase (SLK), are increased during kidney development and recovery from ischemic acute renal failure. In this study, we characterize the activation and functional role of SLK. SLK underwent dimerization via the C-terminal domain, and dimerization enhanced SLK activity. In contrast, the C-terminal domain of SLK did not dimerize with a related kinase, Mst1, and did not affect Mst1 activity. Phosphorylation/dephosphorylation of SLK were not associated with changes in kinase activity. SLK induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1) and increased ASK1 activity, indicating that ASK1 is a substrate of SLK. Moreover, SLK stimulated phosphorylation of p38 mitogen-activated protein kinase via ASK1, but not c-Jun N-terminal kinase nor extracellular signal-regulated kinase. Chemical anoxia and recovery during re-exposure to glucose (ischemia-reperfusion injury in cell culture) stimulated SLK activity. Overexpression of SLK enhanced anoxia/recovery-induced apoptosis, release of cytochrome c, and activities of caspase-8 and -9, and apoptosis was reduced significantly with p38 and caspase-9 inhibitors. Induction of the endoplasmic reticulum stress response by anoxia/recovery or tunicamycin (monitored by induction of Bip or Grp94 expression, phosphorylation of eukaryotic translation initiation factor 2alpha subunit, expression of CHOP, and activation of caspase-12) was attenuated in cells that overexpress SLK. Thus, SLK is an anoxia/recovery-dependent kinase that is activated via homodimerization and that signals via ASK1 and p38 to promote apoptosis. Attenuation of the protective aspects of the endoplasmic reticulum stress response by SLK may contribute to its proapoptotic effect.

Animals↗

PubMed2005

Actin cytoskeleton regulates extracellular matrix-dependent survival signals in glomerular epithelial cells.

Adhesion of rat glomerular epithelial cells (GEC) to collagen activates focal adhesion kinase (FAK) and the Ras-extracellular signal-regulated kinase (ERK) pathway and supports survival (prevents apoptosis). The present study addresses the relationship between actin organization and the survival phenotype. Parental GEC (adherent to collagen) and GEC stably transfected with constitutively active mutants of mitogen-activated protein kinase kinase (R4F-MEK) or FAK (CD2-FAK) (on plastic) showed ERK activation, low levels of apoptosis, and a cortical distribution of F-actin. Parental GEC adherent to plastic showed increased apoptosis, disorganization of cortical F-actin, and formation of prominent stress fibers. Assembly of cortical F-actin was, at least in part, mediated via ERK. However, disruption of the actin cytoskeleton with cytochalasin D or latrunculin B in parental GEC (on collagen) and in GEC that express R4F-MEK or CD2-FAK (on plastic) decreased ERK activation and increased apoptosis. Expression of a constitutively active RhoA (L(63)RhoA) induced assembly of cortical F-actin, promoted ERK activation, and supplanted the requirement of collagen for survival. Adhesion of GEC to collagen increased phosphatidylinositol-4,5-bisphosphate (PIP(2)). Downregulation or sequestration of PIP(2) by transfection with an inositol 5'-phosphatase or the plextrin-homology domain of phospholipase C-delta1 decreased F-actin content and survival. Moreover, overexpression of wild-type or K256E mutant alpha-actinin-4 with increased affinity for F-actin increased apoptosis. These results demonstrate a reciprocal relationship between collagen-induced cortical F-actin assembly and collagen-dependent survival signaling, including ERK activation. Appropriate remodeling of the actin cytoskeleton may be necessary for facilitating survival, as both disassembly and excessive crosslinking affect survival adversely.

Actinin↗

PubMed2005

Role of the endoplasmic reticulum unfolded protein response in glomerular epithelial cell injury.

C5b-9-induced glomerular epithelial cell (GEC) injury in vivo (in passive Heymann nephritis) and in culture is associated with damage to the endoplasmic reticulum (ER) and increased expression of ER stress proteins. Induction of ER stress proteins is enhanced via cytosolic phospholipase A(2) (cPLA(2)) and limits complement-dependent cytotoxicity. The present study addresses another aspect of the ER unfolded protein response, i.e. activation of protein kinase R-like ER kinase (PERK or pancreatic ER kinase), which phosphorylates eukaryotic translation initiation factor 2-alpha (eIF2alpha), thereby generally suppressing translation and decreasing the protein load on a damaged ER. Phosphorylation of eIF2alpha was enhanced significantly in glomeruli of proteinuric rats with passive Heymann nephritis, compared with control. In cultured GECs, complement induced phosphorylation of eIF2alpha and reduced protein synthesis, and complement-stimulated phosphorylation of eIF2alpha was enhanced by overexpression of cPLA(2). Ischemia-reperfusion in vitro (deoxyglucose plus antimycin A followed by glucose re-exposure) also stimulated eIF2alpha phosphorylation and reduced protein synthesis. Complement and ischemia-reperfusion induced phosphorylation of PERK (which correlates with activation), and fibroblasts from PERK knock-out mice were more susceptible to complement- and ischemia-reperfusion-mediated cytotoxicity, as compared with wild type fibroblasts. The GEC protein, nephrin, plays a key role in maintaining glomerular permselectivity. In contrast to a general reduction in protein synthesis, translation regulated by the 5'-end of mouse nephrin mRNA during ER stress was paradoxically maintained, probably due to the presence of short open reading frames in this mRNA segment. Thus, phosphorylation of eIF2alpha and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury.

Animals↗

PubMed2005

Activation of the extracellular signal-regulated kinase by complement C5b-9.

Extracellular signals may be transmitted to nuclear or cytoplasmic effectors via the mitogen-activated protein kinases. In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of phospholipases and protein kinases. This study addresses the complement-mediated activation of the extracellular signal-regulated kinase (ERK). C5b-9 induced ERK threonine202/tyrosine204 phosphorylation (which correlates with activation) in GEC in culture and PHN in vivo. Expression of a dominant-inhibitory mutant of Ras reduced complement-mediated activation of ERK, but activation was not affected significantly by downregulation of protein kinase C. Complement-induced ERK activation resulted in phosphorylation of cytosolic phospholipase A2 and was, in part, responsible for phosphorylation of mitogen-activated protein kinase-associated protein kinase-2, but did not induce phosphorylation of the transcription factor, Elk-1. Activation of ERK was attenuated by drugs that disassemble the actin cytoskeleton (cytochalasin D, latrunculin B), and these compounds interfered with the activation of ERK by mitogen-activated protein kinase kinase (MEK). Overexpression of a constitutively active RhoA as well as inhibition of Rho-associated kinase blocked complement-mediated ERK activation. Complement cytotoxicity was enhanced after disassembly of the actin cytoskeleton but was unaffected after inhibition of complement-induced ERK activation. However, complement cytotoxicity was enhanced in GEC that stably express constitutively active MEK. Thus complement-induced ERK activation depends on cytoskeletal remodelling and affects the regulation of distinct downstream substrates, while chronic, constitutive ERK activation exacerbates complement-mediated GEC injury.

Actins↗

PubMed2003

The actin cytoskeleton facilitates complement-mediated activation of cytosolic phospholipase A2.

Cytosolic PLA(2)-alpha (cPLA(2)) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic Ca(2+) concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-gamma 1 and the 1,2-diacylglycerol (DAG)-PKC pathway. Ca(2+) and PKC are essential for membrane association and increased catalytic activity of cPLA(2). This study addresses the role of the actin cytoskeleton in cPLA(2) activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [(3)H]AA release, as well as the complement-induced increase in cPLA(2) activity. These effects were due to inhibition of [(3)H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [(3)H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [(3)H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytochalasin D, latrunculin B, nor calyculin A affected association of cPLA(2) with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [(3)H]AA. Thus in GEC, cPLA(2) activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPLA(2), the actin cytoskeleton may contribute to the pathophysiology of GEC injury.

Actin Cytoskeleton↗

PubMed2003

Extracellular matrix regulates glomerular epithelial cell survival and proliferation.

Glomerular epithelial cell (GEC) injury and apoptosis may contribute to sclerosis in glomerulonephritis. The present study addresses signals that regulate survival of GEC in culture and in the acute puromycin aminonucleoside nephrosis (PAN) model of GEC injury in vivo. Compared with GEC on plastic substratum, adhesion to collagen increased activation of focal adhesion kinase (FAK), c-Src, and ERK and facilitated survival (prevented apoptosis). GEC on plastic exhibited increased caspase-8 and -9 activities, increased expression of the proapoptotic protein, Bax, and decreased the antiapoptotic protein, Bcl-XL, compared with collagen. Stable expression of constitutively active mutants of FAK (CD2-FAK) or MEK (R4F-MEK) activated the ERK pathway and supplanted the requirement of collagen for survival. In contrast, expression of a Ras mutant that activates phosphatidylinositol 3-kinase but blocks ERK activation or pharmacological inhibition of the ERK pathway decreased survival on collagen. Glomeruli isolated from rats with PAN revealed increased beta1-integrin expression, along with increased activation of FAK, c-Src, and ERK, compared with controls. EGF receptor activation was undetectable in PAN. Therefore, adhesion to collagen, resulting in activation of FAK and the Ras-ERK pathway, supports GEC survival. Analogous signals for GEC survival are activated in PAN.

Animals↗

PubMed2003

Renal expression and activity of the germinal center kinase SK2.

Rat fetal kidney mRNA was analyzed by RT-PCR to identify protein kinases. This screening demonstrated expression of a protein kinase consistent with SK2, a group II germinal center kinase and homolog of human Ste20-like kinase (SLK). SK2 mRNA, protein expression, and kinase activity were increased in rat fetal kidney homogenates (embryonic days 17-21) compared with adult controls. In adult kidneys subjected to cross-clamping of the renal artery, followed by reperfusion, SK2 mRNA, protein expression, and kinase activity were increased compared with untreated contralateral controls. By immunohistochemistry, SK2 expression was evident mainly in the cytoplasm of tubular epithelial cells in fetal and adult kidneys. There was also some expression in developing and mature podocytes, but staining of the interstitium was negative. In cultured renal tubular epithelial cells, SK2 kinase activity was increased after incubation with serum, or after exposure to chemical anoxia plus reexposure to glucose. Stable overexpression of SLK reduced cell proliferation and increased apoptosis and exacerbated apoptosis and necrosis after chemical anoxia plus reexposure to glucose. Thus SK2 is a renal epithelial protein kinase whose expression and activity are increased during development and recovery from acute renal failure, where tubular epithelial regeneration may recapitulate developmental processes. The actions of SK2 appear to be antiproliferative and may facilitate cell injury.

Acute Kidney Injury↗

PubMed2003

Inhibition of cyclooxygenases reduces complement-induced glomerular epithelial cell injury and proteinuria in passive Heymann nephritis.

In the passive Heymann nephritis (PHN) model of rat membranous nephropathy, complement induces glomerular epithelial cell injury and proteinuria, which is partially mediated by eicosanoids. Glomerular cyclooxygenase (COX)-1 and -2 are up-regulated in PHN and contribute to prostanoid generation. In the current study, we address the role of COX isoforms in proteinuria, using the nonselective COX inhibitor indomethacin and the COX-2-selective inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU). Four groups of rats with PHN were treated twice daily, from day 7 through 14 with vehicle, 1 mg/kg DFU, 10 mg/kg DFU, or 2 mg/kg indomethacin. Vehicle-treated rats with PHN showed significant proteinuria on day 14 (163 +/- 15 mg/d, n = 19), compared with normal rats (10 +/- 4 mg/d, n = 3, p < 0.001). Treatment with DFU (1 or 10 mg/kg) reduced proteinuria significantly (by ~33%), compared with vehicle, but to a lesser extent than indomethacin (56% reduction). Glomerular eicosanoid generation was reduced significantly in the DFU and indomethacin groups, compared with vehicle. There were no significant differences among vehicle- or DFU-treated groups in [(3)H]inulin clearance, or in glomerular expression of COX-1 and -2. DFU did not affect the autologous immune response. In cultured rat glomerular epithelial cells, COX inhibition reduced complement-induced cytotoxicity, and this reduction was reversed by the thromboxane A(2) analog 9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F(2alpha) (U46619). Thus, in experimental membranous nephropathy, selective inhibition of COX-2 reduces proteinuria, without adversely affecting renal function. However, inhibition of both COX-1 and -2 is required to achieve a maximum cytoprotective and antiproteinuric effect.

Actins↗

PubMed2002

Complement activates the c-Jun N-terminal kinase/stress-activated protein kinase in glomerular epithelial cells.

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A(2) (cPLA(2)), and products of cPLA(2)-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA(2) in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and alpha-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H(2)O(2) activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA(2) and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.

Animals↗

PubMed2002

Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells.

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of cytosolic phospholipase A(2) (cPLA(2)). This study addresses the role of endoplasmic reticulum (ER) stress proteins (bip, grp94) in GEC injury. GEC that overexpress cPLA(2) (produced by transfection) and "neo" GEC (which expresses cPLA(2) at a lower level) were incubated with complement (40 min), and leakage of constitutively expressed bip and grp94 from ER into cytosol was measured to monitor ER injury. Greater leakage of bip and grp94 occurred in complement-treated GEC that overexpress cPLA(2), as compared with neo, implying that cPLA(2) activation perturbed ER membrane integrity. After chronic incubation (4-24 h), C5b-9 increased bip and grp94 mRNAs and proteins, and the increases were dependent on cPLA(2). Expression of bip-antisense mRNA reduced stimulated bip protein expression and enhanced complement-dependent GEC injury. Glomerular bip and grp94 proteins were up-regulated in proteinuric rats with PHN, as compared with normal control. Pretreatment of rats with tunicamycin or adriamycin, which increase ER stress protein expression, reduced proteinuria in PHN. Thus, C5b-9 injures the ER and enhances ER stress protein expression, in part, via activation of cPLA(2). ER stress protein induction is a novel mechanism of protection from complement attack.

Animals↗