Amelioration of Hyperglycemia-Induced Nephropathy by 3,3'-Diindolylmethane in Diabetic Mice.

Type 1 diabetes mellitus (insulin-dependent diabetes) is characterized by hyperglycemia caused by an insulin deficiency. Diabetic nephropathy is a major complication of hyperglycemia. 3,3'-diindolylmethane (DIM)-a natural compound produced from indole-3-carbinol, found in cruciferous vegetables-enhances glucose uptake by increasing the activation of the insulin signaling pathway in 3T3-L1 adipocytes. In this study, we investigated whether DIM could improve insulin-dependent diabetes and nephropathy in streptozotocin (STZ)-induced diabetic mice. In mice, STZ induced hyperglycemia, hunger, thirst, and abnormally increased kidney weight and serum creatinine, which is a renal functional parameter. DIM decreased STZ-increased high blood glucose levels and food and water intake in diabetic mice. DIM also improved diabetic nephropathy by inhibiting the expression of PKC-α, the marker of albuminuria, and TGF-ß1, an indicator of renal hypertrophy, in diabetic mice. Our findings suggest that DIM may ameliorate hyperglycemia and diabetic nephropathy through the inhibition of PKC-α and TGF-ß1 signaling.

Abnormal activation of calpain and protein kinase Cα promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients.

Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.

Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation.

Platelet activation and aggregation underlie acute thrombosis that leads to ST-elevation myocardial infarction (STEMI). L5-highly electronegative low-density lipoprotein (LDL)-is significantly elevated in patients with STEMI. Thus, we examined the role of L5 in thrombogenesis. Plasma LDL from patients with STEMI (n = 30) was chromatographically resolved into 5 subfractions (L1-L5) with increasing electronegativity. In vitro, L5 enhanced adenosine diphosphate-stimulated platelet aggregation twofold more than did L1 and induced platelet-endothelial cell (EC) adhesion. L5 also increased P-selectin expression and glycoprotein (GP)IIb/IIIa activation and decreased cyclic adenosine monophosphate levels (n = 6, P < .01) in platelets. In vivo, injection of L5 (5 mg/kg) into C57BL/6 mice twice weekly for 6 weeks shortened tail bleeding time by 43% (n = 3; P < .01 vs L1-injected mice) and increased P-selectin expression and GPIIb/IIIa activation in platelets. Pharmacologic blockade experiments revealed that L5 signals through platelet-activating factor receptor and lectin-like oxidized LDL receptor-1 to attenuate Akt activation and trigger granule release and GPIIb/IIIa activation via protein kinase C-α. L5 but not L1 induced tissue factor and P-selectin expression in human aortic ECs (P < .01), thereby triggering platelet activation and aggregation with activated ECs. These findings indicate that elevated plasma levels of L5 may promote thrombosis that leads to STEMI.

Serum protein kinase Cα as a diagnostic biomarker of cancers.

BACKGROUND: Several serum biomarkers such as antigens, soluble proteins, metabolites, and genes have been identified for the diagnosis of cancers and for monitoring the recurrence after cancer treatment. METHODS: In the present study, a protein kinase C (PKC) α-specific peptide substrate was phosphorylated with serum samples collected from cancer-bearing mice (U87, A431, HepG2, and A549) and the phosphorylation ratio was detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The phosphorylation ratio for peptide substrates significantly increased in the serum of cancer-bearing mice compared with the ratio found in control mice. The addition of a PKCα inhibitor induced a concentration-dependent decrease in phosphorylation ratios, but the non-PKCα inhibitors, rottlerin and H-89, did not significantly effect phosphorylation ratios. CONCLUSIONS: These results suggest that serum activated PKCα is a good biomarker applicable to cancer diagnosis.

The antiplatelet activity of magnolol is mediated by PPAR-ß/γ.

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, ß/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 µM) dose-dependently enhanced the activity and intracellular level of PPAR-ß/γ in platelets. In the presence of selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-ß/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-ß/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-ß/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-ß/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-ß/γ-dependent pathways.

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs.

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, ß/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid.

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

Plasma protein kinase C (PKC)alpha as a biomarker for the diagnosis of cancers.

Protein kinase C (PKC)alpha plays a key role in the differentiation, proliferation and apoptosis of cancer cells, and its activity is higher in cancer cells than in normal cells. In the present study, we investigated the existence of activated PKCalpha in plasma and its possibility for cancer diagnosis. Plasma samples were prepared from xenograft mouse models of cancer and from normal mice. Phosphorylation ratios for a PKCalpha-specific peptide substrate (Alphatomega) were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and activated PKCalpha was identified by western blot analysis. Increased levels of activated PKCalpha were found in the plasma of cancer-bearing mice (U87, A549, A431, HuH-7 and B16 melanoma) compared with the levels found in control mice. Phosphorylation ratios for peptide substrate increased with an increase in tumor size. Moreover, the addition of Ro-31-7549, a highly specific inhibitor of PKCalpha, produced a concentration-dependent reduction of phosphorylation ratios, whereas the non-PKCalpha inhibitors, rottlerin and H-89, did not significantly effect phosphorylation ratios. In addition, the level of activated PKCalpha decreased after cancer resection but increased if the cancer recurred. From these results, we suggest that (i) activated PKCalpha in plasma can be a useful biomarker for the diagnosis of cancers and (ii) the level of activated PKCalpha can be monitored to assess the recurrence of cancer after surgical removal. To our knowledge, this is the first report demonstrating the existence of activated PKCalpha in plasma and its possibility for cancer diagnosis.