Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

Abstract Background: Disrupted iron homeostasis in the substantia nigra (SN) is an important mechanism in Parkinson’s disease (PD). In our previous studies using cultured neurons, we showed that 6-hydroxydopamine (6-OHDA) treatment increased the expression of divalent metal transporter-1 (DMT1) gating iron influx and decreased the expression of ferroportin 1 (FPN1) gating iron efflux, leading to increased iron deposition. In astrocytes, 6-OHDA increased both DMT1 and FPN1 expressions and iron traffic. However, the underlying mechanisms mediating these processes remain elusive. Hypoxia-inducible factors (HIFs) are important regulators of iron homeostasis.Methods: Using western blots, we observed HIFs, DMT1 and FPN1 expressions in primary cultured astrocytes and ventral mesencephalic (VM) treated with 6-OHDA, inhibitors of HIF-1α and HIF-2α, protein kinase C (PKC) inhibitor and PKC activator, radical scavenger and inducible NO synthase (iNOS) inhibitor . The ferrous iron traffic of astrocytes was determined by measuring the quenching or reversing of calcein fluorescence.Results: Using primary rat cell cultures, we observed that 6-OHDA treatment significantly increased the expressions of HIF-1α and HIF-2α in cultured astrocytes but not in VM neurons. Moreover, we observed that HIF-2α inhibitor, but not HIF-1α inhibitor reversed the 6-OHDA-induced upregulation of DMT1 and FPN1 and ferrous iron traffic. Blocking protein kinase C (PKC) pathway by bisindolylmaleimide I hydrochloride (Bisl) or N-acetyl-l-cysteine (NAC) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibits Phorbol 12-myristate 13-acetate (PMA) or 6-OHDA induced activation of HIF-2α, whereas activating by PMA or 6-OHDA was sufficient to activate HIF-2α. Notably, blocking of PKC delta (PKC δ) phosphorylation by NAC, or L-NAME inhibits 6-OHDA induced activation of HIF-2α.Conclusion: Our data indicate that astrocytic HIF-2α, but not HIF-1α, may be an important regulator of DMT1 and FPN1 expressions by activating the PKC -dependent pathway.

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Hypoxia-inducible factor 2α but not hypoxia-inducible factor-1α increases iron traffic in astrocytes

10.21203/rs.3.rs-24083/v1 ◽

2020 ◽

Author(s):

Manman Xu ◽

Zhiqiang Chen ◽

Xizhen Ma ◽

Hangrui Xu ◽

Changsheng Fan ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Ferrous Iron ◽

Iron Homeostasis ◽

Hypoxia Inducible Factor ◽

Western Blots ◽

Divalent Metal Transporter 1 ◽

Divalent Metal Transporter ◽

Kinase C ◽

Cultured Astrocytes

Abstract Background Disrupted iron homeostasis in the substantia nigra (SN) is an important mechanism in Parkinson’s disease (PD). In our previous studies using cultured neurons, we showed that 6-hydroxydopamine (6-OHDA) treatment increased the expression of divalent metal transporter-1 (DMT1) gating iron influx and decreased the expression of ferroportin 1 (FPN1) gating iron efflux, leading to increased iron deposition. In astrocytes, 6-OHDA increased both DMT1 and FPN1 expressions and iron traffic. However, the underlying mechanisms mediating these processes remain elusive. Hypoxia-inducible factors (HIFs) are important regulators of iron homeostasis. Methods Using western blots, we observed HIFs, DMT1 and FPN1 expressions in primary cultured astrocytes and ventral mesencephalic (VM) treated with 6-OHDA, inhibitors of HIF-1α and HIF-2α, protein kinase C (PKC) inhibitor and PKC activator, radical scavenger and inducible NO synthase (iNOS) inhibitor. The ferrous iron traffic of astrocytes was determined by measuring the quenching or reversing of calcein fluorescence. Results Using primary rat cell cultures, we observed that 6-OHDA treatment significantly increased the expressions of HIF-1α and HIF-2α in cultured astrocytes but not in VM neurons. Moreover, we observed that HIF-2α inhibitor, but not HIF-1α inhibitor reversed the 6-OHDA-induced upregulation of DMT1 and FPN1 and ferrous iron traffic. Blocking protein kinase C (PKC) pathway by bisindolylmaleimide I hydrochloride (Bisl) or N-acetyl-l-cysteine (NAC) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibits Phorbol 12-myristate 13-acetate (PMA) or 6-OHDA induced activation of HIF-2α, whereas activating by PMA or 6-OHDA was sufficient to activate HIF-2α. Notably, blocking of PKC delta (PKC δ) phosphorylation by NAC, or L-NAME inhibits 6-OHDA induced activation of HIF-2α. Conclusion Our data indicate that astrocytic HIF-2α, but not HIF-1α, may be an important regulator of DMT1 and FPN1 expressions by activating the PKC -dependent pathway.

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Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets

Biochemical Journal ◽

10.1042/bj2430667 ◽

1987 ◽

Vol 243 (3) ◽

pp. 667-678 ◽

Cited By ~ 29

Author(s):

K A Williams ◽

W Murphy ◽

R J Haslam

Keyword(s):

Creatine Kinase ◽

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Platelet Activating Factor ◽

Human Platelets ◽

Prostaglandin D2 ◽

Guanine Nucleotide Binding Protein ◽

Kinase C

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.

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