Chris J. Vlahos

Increased Protein Kinase C Activity and Expression of Ca2+-Sensitive Isoforms in the Failing Human Heart

BACKGROUND Increased expression of Ca2+-sensitive protein kinase C (PKC) isoforms may be important markers of heart failure. Our aim was to determine the relative expression of PKC-beta1, -beta2, and -alpha in failed and nonfailed myocardium. METHODS AND RESULTS Explanted hearts of patients in whom dilated cardiomyopathy or ischemic cardiomyopathy was diagnosed were examined for PKC isoform content by Western blot, immunohistochemistry, enzymatic activity, and in situ hybridization and compared with nonfailed left ventricle. Quantitative immunoblotting revealed significant increases of >40% in PKC-beta1 (P<0.05) and -beta2 (P<0.04) membrane expression in failed hearts compared with nonfailed; PKC-alpha expression was significantly elevated by 70% in membrane fractions (P<0.03). PKC-epsilon expression was not significantly changed. In failed left ventricle, PKC-beta1 and -beta2 immunostaining was intense throughout myocytes, compared with slight, scattered staining in nonfailed myocytes. PKC-alpha immunostaining was also more evident in cardiomyocytes from failed hearts with staining primarily localized to intercalated disks. In situ hybridization revealed increased PKC-beta1 and -beta2 mRNA expression in cardiomyocytes of failed heart tissue. PKC activity was significantly increased in membrane fractions from failed hearts compared with nonfailed (1021+/-189 versus 261+/-89 pmol. mg-1. min-1, P<0.01). LY333531, a selective PKC-beta inhibitor, significantly decreased PKC activity in membrane fractions from failed hearts by 209 pmol. min-1. mg-1 (versus 42.5 pmol. min-1. mg-1 in nonfailed, P<0.04), indicating a greater contribution of PKC-beta to total PKC activity in failed hearts. CONCLUSIONS In failed human heart, PKC-beta1 and -beta2 expression and contribution to total PKC activity are significantly increased. This may signal a role for Ca2+-sensitive PKC isoforms in cardiac mechanisms involved in heart failure.

The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme involved in cellular responses to growth factors. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a naturally occuring bioflavinoid, was found to inhibit PtdIns 3-kinase with an IC50 of 1.3 micrograms/ml (3.8 microM); inhibition appears to be directed towards the ATP binding site of the kinase. Analogs of quercetin were also investigated as PtdIns 3-kinase inhibitors, with the most potent compounds exhibiting IC50s in the range of 1.7-8.4 micrograms/ml (5-19 microM). In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 greater than 30 micrograms/ml). These findings suggest that flavinoids may serve as potent inhibitors of PtdIns 3-kinase. Furthermore, the enzyme is much more sensitive to substituents at the 3-position of the flavinoid ring than are other protein and PtdIns kinases, suggesting that specific inhibitors of PtdIns 3-kinase can be developed to explore the biological role of the enzyme in cellular proliferation and growth factor response.

Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption.

Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+ reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.

Kinases as therapeutic targets for heart failure

Cardiac cells respond to external stimuli by activating signal-transduction cascades involving protein and lipid kinases. These enzymes are attractive therapeutic targets as they are responsible for the direct or indirect control of most signalling pathways in cells. Existing therapies for heart failure are directed against the renin-angiotensin system and the β-adrenoceptor, and prevent the initiation of signalling cascades. However, as molecular signalling events in the progression of heart failure are elucidated, new downstream signalling targets have emerged as candidates for therapeutic intervention.

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 ≈ 6 μM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

Inhibition of neutrophil oxidative burst and granule secretion by Wortmannin: Potential role of MAP kinase and renaturable kinases

Exposure of neutrophils to a variety of agonists including soluble chemoattractant peptides and cytokines results in degranulation and activation of the oxidative burst (effector functions) that are required for bacterial killing. At present, the signaling pathways regulating these important functions are incompletely characterized. Mitogen‐activated protein (MAP) kinases (MAPK) as well as members of a family of “renaturable kinases” are rapidly activated in neutrophils in response to diverse physiological agonists, suggesting that they may regulate cell activation. Antagonists of phosphatidyl inositol‐3‐(OH) kinase (PI3‐kinase) such as wortmannin (Wtmn) inhibit these effector responses as well as certain of the above‐mentioned kinases, leading to the suggestion that these enzymes lie downstream of PI3‐kinase in the pathway regulating the oxidative burst and granule secretion. However, an apparent discrepancy exists in that, while virtually obliterating activity of PI3‐kinase and the oxidase at low concentrations (ID50 < 20 nM), Wtmn has only variable inhibitory effects on MAPK even at substantially higher concentrations (75–100 nM). This raises the possibility that the inhibitory effects of Wtmn are mediated via other enzyme systems. The purpose of the current study was therefore to compare the effects of Wtmn on PI3‐kinase activity and on the chemoattractant‐activated kinases, and to determine the potential relationship of these pathways to microbicidal responses. In human neutrophils, both the oxidative burst and granule secretion induced by fMLP were inhibited by Wtmn but at markedly different concentrations: the oxidative burst was inhibited with an ID50 of <5 nM while granule secretion was only partially inhibited at concentrations exceeding 75 nM. Activation of both MEK‐1 and MAPK in response to fMLP was only partially inhibited by high doses of Wtmn (ID50 of > 100 nM and ≡75 nM, respectively). In contrast, Wtmn potently inhibited fMLP‐induced activation of the 63 and 69 kDa renaturable kinases (ID50 ≡ 5–10 nM). We speculate that the renaturable kinases may be involved in the regulation of the oxidative burst, whereas the MAPK pathway may play a role in other neutrophil functions such as granule secretion. J. Cell. Physiol. 172:94–108, 1997.

Signal transduction in neutrophil activation Phosphatidylinositol 3‐kinase is stimulated without tyrosine phosphorylation

Treatment of human neutrophils with the peptide f‐Met‐Leu‐Phe (FMLP) results in neutrophil activation concomitant with stimulation of phosphatidylinositol (PtdIns) 3‐kinase activity as measured by production of PtdIns‐3,4,5‐P3 in [32P]orthophosphate labeled cells. Antiphosphotyrosine immunoprecipitates were assayed for PtdIns 3‐kinase activity; essentially no activity was present in lysates from either stimulated or unstimulated cells. The 85 kDa regulatory subunit of PtdIns 3‐kinase, which normally serves as a substrate for tyrosine kinases, was not detected by SDS‐PAGE or Western blot analysis in antiphosphotyrosine immunoprecipitates. In addition, no radioactive band corresponding to PtdIns 3‐kinase was observed by SDS‐PAGE following antiPtdIns 3‐kinase immunoprecipitations. However, immunoprecipitates using polyclonal antibodies against PtdIns 3‐kinase showed high PtdIns 3‐kinase activity in neutrophil lysates and the 85kDa subunit of PtdIns 3‐kinase was detected in Western blots; no differences in activity were observed in FMLP‐stimulated and unstimulated cells. These results suggest that, in contrast to polypeptide growth factor signal transduction systems, the activation of PtdIns 3‐kinase by FMLP does not require tyrosine phosphorylation.

Complete Inhibition of Anisomycin and UV Radiation but Not Cytokine Induced JNK and p38 Activation by an Aryl-substituted Dihydropyrrolopyrazole Quinoline and Mixed Lineage Kinase 7 Small Interfering RNA

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nm for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nm or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1β or tumor necrosis factor-α-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase.

Studies on a platelet‐derived growth factor (PDGF) responsive osteosarcoma cell line, MG‐63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3‐kinase inhibitors on serum‐stimulated cell proliferation and PDGF‐stimulated DNA replication, actin rearrangements, or Ptdlns 3‐kinase activity. In a dose‐dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2‐(4‐morpholinyl)‐8‐phenyl‐4H‐1‐benzopyran‐4‐one), inhibited serum‐stimulated MG‐63 cell proliferation. The mitogenic effects of PDGF on MG‐63 cells, as determined by incorporation of [3H]‐thymidine, were also substantially inhibited in the presence of 0.10 μM wortmannin or 10 μM LY294002. Furthermore, MG‐63 cells stimulated by PDGF form distinct actin‐rich, finger‐like membrane projections which are completely inhibited by either 0.10 μM wortmannin or 10 μM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3‐phosphate in PDGF‐stimulated MG‐63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3‐kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 μM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 μM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3‐kinase inhibitors on serum‐ and PDGF‐stimulated MG‐63 cell proliferation and PDGF‐stimulated morphological changes and suggest a greater role for Ptdlns 3‐kinase in these processes. J. Cell. Biochem. 64:182–195.

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation.

Inflammation is critically involved in atherogenesis. Signaling from innate immunity receptors TLR2 and 4, IL-1 and IL-18 is mediated by MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1. We hypothesized that IRAK4 kinase activity is critical for development of atherosclerosis. IRAK4 kinase-inactive knock-in mouse was crossed with the ApoE-/- mouse. Lesion development was stimulated by carotid ligation. IRAK4 functional deficiency was associated with down-regulation of several pro-inflammatory genes, inhibition of macrophage infiltration, smooth muscle cell and lipid accumulation in vascular lesions. Reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE-/- mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist thereby validating the model in the relevant pathway context. Thus, IRAK4 functional deficiency inhibits vascular lesion formation in ApoE-/- mice, which further unravels mechanisms of vascular inflammation and identifies IRAK4 as a potential therapeutic target.