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Kenneth R McLeish

Publications and source records attributed to Kenneth R McLeish.

At least 19 recordsLinked to original sources
PubMed2007

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets.

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED(50) of 15, 31, and 28 nM, respectively, with maximal response at 10(-7) M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC(50) for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.

Actins↗
PubMed2006

A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells.

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.

Animals↗
PubMed2006

Proteomic analysis of human neutrophils.

Proteomics is the study of the set of proteins, or proteome, expressed by a cell under specific conditions. Proteomics methodology consists of protein extraction, protein separation, and protein identification. Currently, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry are the most widespread methods for proteomic studies. The recent introduction of precast immobilized pH gradient gel strips, precast gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and well-designed electrophoresis equipment has made 2DE a highly reproducible and relatively simple method for protein separation. Inherent limitations of the procedure, however, require approaches in sample preparation that may be cell- or tissue-dependent. This chapter describes a methodology for proteomic analysis of human neutrophils and discusses its applications.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Proteomic analysis of human neutrophil granules.

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.

Actins↗
PubMed2005

Gamma-amino butyric acid type B receptors stimulate neutrophil chemotaxis during ischemia-reperfusion.

Serine/threonine kinase Akt, or protein kinase B, has been shown to regulate a number of neutrophil functions. We sought to identify Akt binding proteins in neutrophils to provide further insights into understanding the mechanism by which Akt regulates various neutrophil functions. Proteomic and immunoprecipitation studies identified gamma-amino butyric acid (GABA) type B receptor 2 (GABA(B)R2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABA(B)R2 demonstrated Akt association with the intact GABA(B)R. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABA(B)R2 Ab. Additionally, GABA(B)R2 and Akt colocalization was demonstrated by confocal microscopy. A GABA(B)R agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432, a GABA(B)R antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABA(B)R agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABA(B)R with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABA(B)R2 is present in human neutrophils and that it is functionally associated with Akt. Intraventricular baclofen pretreatment in rats subjected to a stroke model showed increased migration of neutrophils to the ischemic lesion. Thus, the GABA(B)R is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABA(B)R potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.

Animals↗
PubMed2005

Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils.

The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.

Amino Acid Sequence↗
PubMed2005

p38 MAPK/HSP25 signaling mediates cadmium-induced contraction of mesangial cells and renal glomeruli.

The environmental pollutant cadmium affects human health, with the kidney being a primary target. In addition to proximal tubules, glomeruli and their contractile mesangial cells have also been identified as targets of cadmium nephrotoxicity. Glomerular contraction is thought to contribute to reduced glomerular filtration, a characteristic of cadmium nephrotoxicity. Because p38 MAPK/HSP25 signaling has been implicated in smooth muscle contraction, we examined its role in cadmium-induced contraction of mesangial cells. We report that exposure of mesangial cells to cadmium resulted in 1) cell contraction, 2) activation of MAP kinases, 3) increased HSP25 phosphorylation coincident with p38 MAP kinase activation, 4) sequential phosphorylation of the two phosphorylation sites of mouse HSP25 with Ser15 being phosphorylated before Ser86, 5) reduction of oligomeric size of HSP25, and 6) association of HSP25 with microfilaments. Exposure of isolated rat glomeruli to cadmium also resulted in contraction and increased HSP25 phosphorylation. The cadmium-induced responses were inhibited by the specific p38 MAP kinase inhibitor SB-203580, and cadmium-induced phosphorylation of HSP25 was inhibited by expression of a dominant-negative p38 MAP kinase mutant. These findings tentatively suggest that cadmium-induced nephrotoxicity results, in part, from glomerular contraction due to p38 MAP kinase/HSP25 signaling-dependent contraction of mesangial cells. With regard to the cellular action of HSP25, these data support a change in paradigm: in addition to its well-established cytoprotective function, HSP25 may also be involved in processes that ultimately lead to adverse effects, as is observed in the response of mesangial cells to cadmium.

Actin Cytoskeleton↗
PubMed2005

Parathyroid hormone-mediated regulation of Na+-K+-ATPase requires ERK-dependent translocation of protein kinase Calpha.

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the alpha1 subunit through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. Based on previous studies we postulated that PTH regulates sodium pump activity through isoform-specific PKC-dependent activation of ERK. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH stimulated membrane translocation of PKCalpha by 102 +/- 16% and PKCbetaI by 41 +/- 7% but had no effect on PKCbetaII and PKCzeta. Both PKCalpha and PKCbetaI phosphorylated the Na+-K+-ATPase alpha1 subunit in vitro. PTH increased the activity of PKCalpha but not PKCbetaI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na+-K+-ATPase alpha1 subunit and PKCalpha, whereas the association between Na+-K+-ATPase alpha1 subunit and PKCbetaI remained unchanged. A PKCalpha inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na+-K+-ATPase alpha1 subunit and inhibition of Na+-K+-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKCalpha, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKCalpha and increased phosphorylation of the Na+-K+-ATPase alpha1 subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKCalpha inhibitory peptide. Inhibition of PKCalpha expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na+-K+-ATPase alpha1 subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKCalpha, and phosphorylation of the Na+-K+-ATPase alpha1 subunit. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by ERK-dependent activation of PKCalpha.

Amino Acid Sequence↗
PubMed2004

Methylglyoxal: a stimulus to neutrophil oxygen radical production in chronic renal failure?

BACKGROUND: Chronic renal failure is characterized by oxidant stress, resulting in part from increased reactive oxygen species production by neutrophils. Plasma concentrations of methylglyoxal are increased in uraemia. Methylglyoxal activates p38 mitogen-activated protein kinase (MAPK) in endothelial cells. Activation of p38 MAPK in neutrophils enhances reactive oxygen species production through exocytosis of intracellular storage granules. We tested the hypothesis that methylglyoxal enhances reactive oxygen species production by activating p38 MAPK in neutrophils. METHODS: Neutrophils were exposed to methylglyoxal in vitro. Activation of p38 MAPK was determined by immunoblot analysis. Exocytosis was determined by measuring plasma membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules, respectively. Reactive oxygen species production was determined by measuring H(2)O(2) and O(-)(2) production. RESULTS: Methylglyoxal activated p38 MAPK and caused dose-dependent increases in CD35 and CD66b expression, which were blocked by the methylglyoxal scavenger, aminoguanidine, or the p38 MAPK inhibitor, SB203580. Methylglyoxal caused dose-dependent increases in basal and Staphylococcus aureus-stimulated H(2)O(2) production and basal and formyl-methionyl-leucyl-phenylalanine-stimulated O(-)(2) production. Enhancement of reactive oxygen species production was blocked by aminoguanidine and SB203580. CONCLUSIONS: Methylglyoxal enhances reactive oxygen species production in neutrophils through a process involving p38 MAPK-dependent exocytosis of intracellular storage granules. These findings, together with the observation that methylglyoxal concentrations are increased in renal failure, suggest a possible role for methylglyoxal as a uraemic toxin that contributes to the oxidant stress associated with renal failure.

Exocytosis↗
PubMed2004

Alterations in the renal elastin-elastase system in type 1 diabetic nephropathy identified by proteomic analysis.

Diabetes now accounts for >40% of patients with ESRD. Despite significant progress in understanding diabetic nephropathy, the cellular mechanisms that lead to diabetes-induced renal damage are incompletely defined. For defining changes in protein expression that accompany diabetic nephropathy, the renal proteome of 120-d-old OVE26 transgenic mice with hypoinsulinemia, hyperglycemia, hyperlipidemia, and proteinuria were compared with those of background FVB nondiabetic mice (n = 5). Proteins derived from whole-kidney lysate were separated by two-dimensional PAGE and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Forty-one proteins from 300 visualized protein spots were differentially expressed in diabetic kidneys. Among these altered proteins, expression of monocyte/neutrophil elastase inhibitor was increased, whereas elastase IIIB was decreased, leading to the hypothesis that elastin expression would be increased in diabetic kidneys. Renal immunohistochemistry for elastin of 325-d-old FVB and OVE26 mice demonstrated marked accumulation of elastin in the macula densa, collecting ducts, and pelvicalyceal epithelia of diabetic kidneys. Elastin immunohistochemistry of human renal biopsies from patients with type 1 diabetes (n = 3) showed increased elastin expression in renal tubular cells and the interstitium but not glomeruli. These results suggest that coordinated changes in elastase inhibitor and elastase expression result in increased tubulointerstitial deposition of elastin in diabetic nephropathy. The identification of these coordinated changes in protein expression in diabetic nephropathy indicates the potential value of proteomic analysis in defining pathophysiology.

Animals↗
PubMed2003

Identification of the p16-Arc subunit of the Arp 2/3 complex as a substrate of MAPK-activated protein kinase 2 by proteomic analysis.

The p38 MAPK pathway regulates multiple neutrophil functional responses via activation of the serine-threonine kinase MAPK-activated protein kinase 2 (MAPKAPK2). To identify substrates of MAPKAPK2 that mediate these responses, a proteomic approach was used in which in vitro phosphorylation of neutrophil lysates by exogenously added active recombinant MAPKAPK2 was followed by protein separation using two-dimensional electrophoresis. Peptide mass fingerprinting of peptides defined by MALDI-MS was then utilized to identify phosphorylated proteins detected by autoradiography. Six candidate substrates were identified, including the p16 subunit of the seven-member Arp2/3 complex (p16-Arc). In vitro studies confirmed that MAPKAPK2 interacts with and phosphorylates the A isoform, but not the B isoform, of p16-Arc with a stoichiometry of 0.6 to 0.7. MAPKAPK2 also phosphorylated p16-Arc in intact Arp2/3 complexes precipitated from neutrophil lysates. Mutation of serine-77 to alanine on the A isoform prevented phosphorylation by MAPKAPK2. The ability of MAPKAPK2 to phosphorylate one isoform of p16-Arc suggests a possible mechanism by which the p38 MAPK cascade regulates remodeling of the actin cytoskeleton.

Actin-Related Protein 2↗
PubMed2003

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils.

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.

Androstadienes↗
PubMed2003

Heat shock protein 27 controls apoptosis by regulating Akt activation.

Activation of the serine-threonine kinase Akt by cytokines, chemokines, and bacterial products delays constitutive neutrophil apoptosis, resulting in a prolonged inflammatory response. We showed previously that Akt exists in a signaling complex with p38 MAPK, MAPK-activated protein kinase-2 (MAPKAPK-2), and heat shock protein-27 (Hsp27); and Hsp27 dissociates from the complex upon neutrophil activation. To better understand the regulation of this signaling module, the hypothesis that Akt phosphorylation of Hsp27 regulates its interaction with Akt was tested. The present study shows that Akt phosphorylated Hsp27 on Ser-82 in vitro and in intact cells, and phosphorylation of Hsp27 resulted in its dissociation from Akt. Additionally, the interaction between Hsp27 and Akt was necessary for activation of Akt in intact neutrophils. Constitutive neutrophil apoptosis was accelerated by sequestration of Hsp27 from Akt, and this enhanced rate of apoptosis was reversed by introduction of constitutively active recombinant Akt. Our results define a new mechanism by which Hsp27 regulates apoptosis, through control of Akt activity.

Apoptosis↗
PubMed2003

MAPK-activated protein kinase-2 participates in p38 MAPK-dependent and ERK-dependent functions in human neutrophils.

Many neutrophil responses, including chemotaxis, exocytosis, respiratory burst activity and chemokine synthesis, are mediated by p38 MAPK. MAPK-activated protein kinase-2 (MK2) is activated by p38 MAPK in human neutrophils. The present study tested the hypothesis that MK2 mediates multiple p38 MAPK-dependent responses in human neutrophils by comparing the effect of the p38 MAPK inhibitor, SB203580, with an MK2 inhibitory peptide. Both SB203580 and MK2 inhibitory peptide attenuated respiratory burst activity, exocytosis, and chemotaxis. Lipopolysaccharide (LPS)-induced IL-8 production was inhibited by SB203580, but not by the MK2 inhibitory peptide. Inhibition of chemotaxis and respiratory burst activity by SB203580 was less than that of MK2 inhibitory peptide. Inhibition of extracellular signal-regulated kinase (ERK) activity by PD98059 attenuated superoxide release and chemotaxis, and simultaneous treatment with SB203580 and PD98059 demonstrated additive inhibition. ERK phosphorylated MK2 in vitro and activated MK2 in f-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These data suggest that MK2 mediates both ERK- and p38 MAPK-dependent neutrophil responses.

Chemotaxis, Leukocyte↗
PubMed2003

Effects of high-flux hemodialysis on oxidant stress.

BACKGROUND: Neutrophil oxygen radical production is increased in end-stage renal disease (ESRD) patients and it is further enhanced during dialysis with low-flux cellulosic membranes. This increased oxygen radical production may contribute to the protein and lipid oxidation observed in ESRD patients. We tested the hypothesis that high-flux hemodialysis does not increase oxygen radical production and that it is not associated with protein oxidation. METHODS: Neutrophil oxygen radical production was measured during dialysis with high-flux dialyzers containing polysulfone and cellulose triacetate membranes. Free sulfhydryl and carbonyl groups and advanced oxidation protein products were measured to assess plasma protein oxidation. RESULTS: Pre-dialysis, neutrophil oxygen radical production was significantly greater than normal and increased significantly as blood passed through the dialyzer in the first 30 minutes of dialysis. Post-dialysis, however, neutrophil oxygen radical production had decreased and was not different from normal. Pre-dialysis, significant plasma protein oxidation was evident from reduced free sulfhydryl groups, increased carbonyl groups, and increased advanced oxidation protein products. Post-dialysis, plasma protein free sulfhydryl groups had increased to normal levels, while plasma protein carbonyl groups increased slightly, and advanced oxidation protein products remained unchanged. CONCLUSIONS: The results of this study show that neutrophil oxygen radical production normalizes during high-flux dialysis, despite a transient increase early in dialysis. This decrease in oxygen radical production is associated with an improvement in some, but not all, measures of protein oxidation.

Blood Proteins↗