Statin-dependent activation of protein kinase Cδ in acute promyelocytic leukemia cells and induction of leukemic cell differentiation

Abstract

Statins are HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase inhibitors, which block the conversion of HMG-CoA to mevalonate and have potent cholesterol lowering properties. Beyond their importance in the generation of lipid lowering effects, the regulatory effects of statins on the mevalonate pathway have a significant impact on multiple other cellular functions. There is now extensive evidence that statins have anti-inflammatory and anti-neoplastic properties, but the precise mechanisms by which such responses are generated are not well understood. In the present study we demonstrate that statins engage a member of the protein kinase C (PKC) family of proteins, PKCδ, in acute promyelocytic leukemia (APL) cells. Our study shows that atorvastatin and fluvastatin induce proteolytic activation of PKCδ in the APL NB4 cell line, which expresses the t(15;17) translocation. Such engagement of PKCδ results in induction of its kinase domain and downstream regulation of pathways important for statin-dependent leukemia cell differentiation. Our research shows that the function of PKCδ is essential for statin-induced leukemic cell differentiation, as demonstrated by studies involving selective targeting of PKCδ using siRNAs. We also demonstrate that the potent enhancing effects of statins on all-trans retinoic acid (ATRA)-induced gene expression for CCL3 and CCL4 requires the function of PKCδ, suggesting a mechanism by which statins may promote ATRA-induced antileukemic responses. Altogether, our data establish a novel function for PKCδ as a mediator of statin-induced differentiation of APL cells and antileukemic effects.

Figure 1

Effects of statin-treatment of APL cells on the proteolytic cleavage/activation of PKCδ. A, Top, NB4 cells were incubated in the presence or absence of atorvastatin or fluvastatin (10 μmol/L), for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKCδ. Middle, the same blot was then stripped and re-probed with an anti-PKCδ antibody to confirm the specificity of the detected active fragment. Bottom, the same blot was re-probed with an anti-GAPDH antibody to control for protein loading. B, NB4 cells were treated in the presence or absence of atorvastatin (10 μmol/L) for the indicated times. Cell lysates were immunoprecipitated (IP) with an antibody against PKCδ or control non-immune rabbit immunoglobulin (RIgG). The immunoprecipitates were then subjected to in vitro kinase assays, using histone H1 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of histone H1 was detected by autoradiography. Bottom, the blot was subsequently immunoblotted with an anti-PKCδ antibody

Figure 2

Targeting of PKCδ in APL cells. A, NB4 cells were nucleofected with either control siRNA or siRNA targeting PKCδ. Expression of PKCδ mRNA was then evaluated after 72 hrs from nucleofection by quantitative real time RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as fold increase over control samples and represent means ± S.E. of seven independent experiments. B, NB4 cells were nucleofected with either control siRNA or siRNA targeting PKCδ and treated with atorvastatin (2 μmol/L), for 48 hrs. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against PKCδ. The blot was subsequently immunoblotted with an anti-GAPDH antibody to control for protein loading.

Figure 3

Reversal of statin-induced differentiation of APL cells by PKCδ - knockdown. A, NB4 cells were treated with atorvastatin (2μmol/L) for 48hrs as indicated, in the presence or absence of FPP (10 μM) or GGPP (10 μM), and induction of CD11b expression was assessed by flow cytometry. Means + SE of three experiments are shown. B, NB4 cells were incubated for 48 hours in the presence or absence of DMSO (solvent control) or fluvastatin (3μmol/L). The cells were subsequently stained with a phycoerythrin-conjugated anti-CD11b monoclonal antibody and analyzed by flow cytometry. Data are expressed as cells positive for CD11B and represent means + SE of 3 independent experiments.

Figure 4

Effects of PKCδ knockdown on CCL3 and CCL4 gene expression. NB4 cells nucleofected as described in material and methods with control siRNA or PKCδ – siRNA were treated for 48 hours with DMSO (solvent control), ATRA (0.5 μM), atorvastatin (2 μM), or the combination of ATRA and atorvastatin. Expression of mRNAs for CCL3 (A) or CCL4 (B) was evaluated by quantitative real time RT-PCR (TaqMan), using GAPDH for normalization. Data are expressed as fold increase over control samples and represent means ± S.E. of 3 independent experiments for panel A and 2 independent experiments for panel B.