PKC Activation in Niemann Pick C1 Cells Restores Subcellular Cholesterol Transport

Activation of protein kinase C (PKC) has previously been shown to ameliorate the cholesterol transport defect in Niemann Pick Type C1 (NPC1) cells, presumably by increasing the soluble levels of one of its substrates, vimentin. This activity would then restore the vimentin cycle in these cells and allow vimentin-dependent retrograde transport to proceed. Here, we further investigate the effects of PKC activation in NPC1 cells by evaluating different isoforms for their ability to solubilize vimentin and correct the NPC1 cholesterol storage phenotype. We also examine the effects of PKC activators, including free fatty acids and the PKC-specific activator diazoxide, on the NPC1 disease phenotype. Our results indicate that PKC isoforms α, βII, and ε have the greatest effects on vimentin solubilization. Furthermore, expression or activation of PKCε in NPC1 cells dramatically reduces the amount of stored cholesterol and restores cholesterol transport out of endocytic vesicles. These results provide further support for the contribution of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease

PKC activation in Niemann pick C1 cells restores subcellular cholesterol transport.

Activation of protein kinase C (PKC) has previously been shown to ameliorate the cholesterol transport defect in Niemann Pick Type C1 (NPC1) cells, presumably by increasing the soluble levels of one of its substrates, vimentin. This activity would then restore the vimentin cycle in these cells and allow vimentin-dependent retrograde transport to proceed. Here, we further investigate the effects of PKC activation in NPC1 cells by evaluating different isoforms for their ability to solubilize vimentin and correct the NPC1 cholesterol storage phenotype. We also examine the effects of PKC activators, including free fatty acids and the PKC-specific activator diazoxide, on the NPC1 disease phenotype. Our results indicate that PKC isoforms α, βII, and ε have the greatest effects on vimentin solubilization. Furthermore, expression or activation of PKCε in NPC1 cells dramatically reduces the amount of stored cholesterol and restores cholesterol transport out of endocytic vesicles. These results provide further support for the contribution of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease

Effects of PKC activation on sphingolipid transport.

<p>Human NPC1 3123 cells were treated with (B) 20µM DCP-LA, (C) 2µg/ml oleic acid, (D) 2µg/ml linoleic acid, or 100nM PMA for 48 hours before BODIPY-LacCer staining was performed. In untreated cells (A), transport of the lipid to the TGN is inhibited and staining is visible only in punctate endocytic vesicles. In contrast, in treated cells the lipid can be seen in the TGN (arrows) in treated cells, indicating release of the transport block that characterizes NPC1.</p

Rab9 release from insoluble vimentin fraction of NPC1 cell lysates.

<p>The insoluble vimentin fraction from NPC1 cell lysates was incubated with various PKC isoforms. All isoforms tested can effect Rab9 release to some degree from the insoluble vimentin fraction, with PKCα being the most effective and PKCγ being the least effective. The blots shown are representative of at least 3 independent experiments.</p

Effects of PKCs and fatty acids on cholesterol esterification in M12 NPC1 CHO cells.

<p>M12 cells were treated with 50µg/ml fatty acids for 2 days and then transfected with the indicated PKC isoforms. Following transfection, cholesterol transport was assessed by esterification assay. Both free fatty acids and PKCs alleviate the cholesterol transport defect of NPC1 cells and their effects appear to be additive.</p

Effects of transient PKC expression on vimentin solubilization in human NPC1 cells.

<p>Representative western blot analyses of soluble and insoluble vimentin levels in human NPC1 3123 (A) and human nullNPC1₀ (B) cells transfected with PKC ε, β, or α show that the three isoforms increase levels of soluble vimentin and Rab9 with a concurrent decrease of insoluble vimentin relative to untransfected cells (-). The levels of vimentin solubilized are similar to that seen in cells expressing Rab9 (Rab9). The blots shown are representative of at least 3 independent experiments.</p

Effects of fatty acids on vimentin solubilization and the NPC1 phenotype.

<p>(A) Human NPC1 3123 cells were treated with 50µg/ml linoleic or oleic acid for 24 hrs, after which the levels of soluble vimentin were analyzed by Western blotting. Cholesterol storage in NPC1 CHO (B) or human 3123 (C) cells was analyzed by filipin staining. Fluorescence intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values ±SEM from 3 independent experiments. * and *** denote statistically significant differences between treated and untreated cells with P<0.05 and P<0.0001, respectively, as determined by Student’s t-test.</p

Effects of transient PKC expression on the NPC1 phenotype.

<p>M12 cells were transfected with PKC isoforms or Rab9 for 48 hrs and then analyzed by filipin staining for cholesterol storage. Cells positive for transfection stain positive for GFP (left panel) and show decreased filipin staining (outlined cells, right panel) compared to surrounding untransfected cells, confirming the role of PKCs in mobilizing stored cholesterol from the NPC1 endosomes. Bar, 20μ.</p