Protein Kinase C – Possible Therapeutic Target to Treat Cardiovascular Diseases

Bynagari-Settipalli, Yamini S.; Chari, Ramya; Kilpatrick, Laurie E.; Kunapuli, Satya P.

doi:10.2174/187152910793743869

Cited by 20 publications

(9 citation statements)

References 213 publications

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“…PKC is a multi‐gene family with at least 12 members. There are three families of PKC: conventional (α, βI, βII, γ), which are Ca 2+ and lipid activated, the novel (δ, ε, η, θ) and atypical (ζ, ν, µ, ι), which are Ca 2+ independent and activated by distinct lipid moieties (Bynagari‐Settipalli et al ., 2010). The role of each PKC isoform in cell signalling has not been elucidated.…”

Section: Discussionmentioning

confidence: 99%

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low‐density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

Xie

Liu

Lü

et al. 2012

British J Pharmacology

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BACKGROUND AND PURPOSEOne key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O2•-) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism. EXPERIMENTAL APPROACHHUVECs were incubated with ox-LDL with/without pyridoxine, N G -nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O2•-was measured using lucigenin chemiluminescence, and O2•--sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCa and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCa/bII, total PKC, phospho-PKCa, total PKCa and p47phox were measured by Western blot. KEY RESULTS Ox-LDL significantly increased O2•-production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O2•-generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCa. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments. CONCLUSIONS AND IMPLICATIONSPyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCa, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction. AbbreviationsCHCI, chelerythrine chloride; DHE, dihydroethidium; eNOS, endothelial NOS; HUVEC, human umbilical vein endothelial cell; L-NAME, N G -nitro-L-arginine methylester; O2•-, superoxide; ox-LDL, oxidized low-density lipoprotein

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Section: Discussionmentioning

confidence: 99%

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low‐density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

Xie

Liu

Lü

et al. 2012

British J Pharmacology

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“…The phospho-PKC ζ/λ (Thr410/403) (Cell Signaling Technology, Beverly, MA, USA) [24–26] and phospho-PKCζ (pT560) (Epitomics, Burlingame, CA, USA) [27, 28] antibodies have been validated by other investigators, as well as by the company (product datasheets). Furthermore, platelets do not express PKC λ isoform [29, 30], so the phospho-PKC ζ/λ (Thr410/403) antibody will detect PKCζ in platelets only when it is phosphorylated at Thr410. Both antibodies have also been shown to be reactive in both human and mouse tissues (company product datasheets).…”

Section: Methodsmentioning

confidence: 99%

Differential dephosphorylation of the Protein Kinase C-zeta (PKCζ) in an integrin αIIbβ3-dependent manner in platelets

Mayanglambam

Bhavanasi

Vijayan

et al. 2011

Biochemical Pharmacology

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Protein kinase C-zeta (PKCζ), an atypical isoform of the PKC family of protein serine/threonine kinases, is expressed in human platelets. However, the mechanisms of its activation and the regulation of its activity in platelets are not known. We have found that under basal resting conditions, PKCζ has a high phosphorylation status at the activation loop threonine 410 (T410) and the turn motif (autophosphorylation site) threonine 560 (T560), both of which have been shown to be important for its catalytic activity. After stimulation with agonist under stirring conditions, the T410 residue was dephosphorylated in a time- and concentration-dependent manner, while the T560 phosphorylation remained unaffected. The T410 dephosphorylation could be significantly prevented by blocking the binding of fibrinogen to integrin αIIbβ3 with an antagonist, SC-57101; or by okadaic acid used at concentrations that inhibits protein serine/threonine phosphatases PP1 and PP2A in vitro. The dephosphorylation of T410 residue on PKCζ was also observed in PP1cγ null murine platelets after agonist stimulation, suggesting that other isoforms of PP1c or another phosphatase could be responsible for this dephosphorylation event. We conclude that human platelets express PKCζ, and it may be constitutively phosphorylated at the activation loop threonine 410 and the turn motif threonine 560 under basal resting conditions, which are differentially dephosphorylated by outside-in signaling. This differential dephosphorylation of PKCζ might be an important regulatory mechanism for platelet functional responses.

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“…PKCs are classified in to three groups based on their structure and cofactor requirements 12 1) Classical isoforms (cPKCs: α, βI, βII, γ) contain both C1 and C2 domains, and thus require both DAG and Ca 2+ for activation. 2) Novel isoforms (nPKCs: δ, θ, η, ε) contain C1 domain and lack calcium binding C2 domain.…”

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confidence: 99%

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Bynagari-Settipalli

Lakhani

Jin

et al. 2012

ATVB

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Objective— Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results— A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA 2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca 2+ i ) mobilization, 2 signaling molecules, upstream of cPLA 2 activation. Thus, PKCs inhibit cPLA 2 activation by inhibiting ERK and Ca 2+ i mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl 3 -induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion— We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

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Protein Kinase C – Possible Therapeutic Target to Treat Cardiovascular Diseases

Cited by 20 publications

References 213 publications

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low‐density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low‐density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

Differential dephosphorylation of the Protein Kinase C-zeta (PKCζ) in an integrin αIIbβ3-dependent manner in platelets

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

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