Mcl-1 degradation during hepatocyte lipoapoptosis - PubMed (original) (raw)

Mcl-1 degradation during hepatocyte lipoapoptosis

Howard C Masuoka et al. J Biol Chem. 2009.

Abstract

The mechanisms of free fatty acid-induced lipoapoptosis are incompletely understood. Here we demonstrate that Mcl-1, an anti-apoptotic member of the Bcl-2 family, was rapidly degraded in hepatocytes in response to palmitate and stearate by a proteasome-dependent pathway. Overexpression of a ubiquitin-resistant Mcl-1 mutant in Huh-7 cells attenuated palmitate-mediated Mcl-1 loss and lipoapoptosis; conversely, short hairpin RNA-targeted knockdown of Mcl-1 sensitized these cells to lipoapoptosis. Palmitate-induced Mcl-1 degradation was attenuated by the novel protein kinase C (PKC) inhibitor rottlerin. Of the two human novel PKC isozymes, PKCdelta and PKC, only activation of PKC was observed by phospho-immunoblot analysis. As compared with Jurkat cells, a smaller PKC polypeptide and mRNA were expressed in hepatocytes consistent with an alternative splice variant. Short hairpin RNA-mediated knockdown of PKC reduced Mcl-1 degradation and lipoapoptosis. Likewise, genetic deletion of Pkc also attenuated Mcl-1 degradation and cytotoxicity by palmitate in primary hepatocytes. During treatment with palmitate, rottlerin inhibited phosphorylation of Mcl-1 at Ser(159), a phosphorylation site previously implicated in Mcl-1 turnover. Consistent with these results, an Mcl-1 S159A mutant was resistant to degradation and improved cell survival during palmitate treatment. Collectively, these results implicate PKC-dependent destabilization of Mcl-1 as a mechanism contributing to hepatocyte lipoapoptosis.

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Figures

FIGURE 1.

FIGURE 1.

Mcl-1 protein levels decrease in Huh-7 cells in response to treatment with saturated free fatty acids. Immunoblot of whole cell extracts was obtained from Huh-7 cells treated with 800 μ

m

palmitate (A), 600 μ

m

stearate (B), or 800 μ

m

oleate (C) for the indicated times. Endogenous human Mcl-1 in Huh-7 cells consistently migrated as a doublet of 42 kDa. Immunoblot analysis was performed for the proteins of interest, Mcl-1 (sc-819), Bcl-xL, and β-actin, a control for protein loading.

FIGURE 2.

FIGURE 2.

Palmitate destabilizes Mcl-1 protein and not mRNA. A, palmitate does not alter steady state Mcl-1 mRNA levels. Mcl-1 mRNA levels were measured by quantitative real time PCR performed on cDNA from Huh-7 cells treated with 800 μ

m

palmitate for the time intervals indicated. Ribosomal 18 S RNA was used as a copy control. Each point is the absolute number of copies of Mcl-1 mRNA divided by the number of copies of 18 S RNA determined by comparison with known standards. B, caspase inhibition does not prevent palmitate-induced loss of Mcl-1 protein level. Huh-7 cells were incubated in the presence of 800 μ

m

palmitate plus 50 μ

m

Z-VAD-fmk for the time intervals indicated. Whole cell extracts were obtained, and proteins were resolved by SDS-PAGE. Immunoblot analysis was performed for Mcl-1 and, as a control for protein loading, β-actin. C, MG-132, a proteasome inhibitor, blocks palmitate-induced loss of cellular Mcl-1 protein. Huh-7 cells were incubated in the presence of 800 μ

m

palmitate plus 10 μ

m

MG-132. At the time intervals indicated, total protein was isolated for immunoblot analysis. D, proteasome-resistant Mcl-1 mutant is resistant to cellular degradation. Huh-7 cells were transiently transfected with a construct expressing a human Mcl-1 with mutations (lysine to arginine) of five established ubiquitination sites (KR Mcl-1). Twenty four h after transfection, cells were treated with 800 μ

m

palmitate. Immunoblot analysis for Mcl-1 was performed on whole cell extracts obtained at the time intervals indicated. In contrast to endogenous Mcl-1, KR Mcl-1 migrated as a single 42-kDa band.

FIGURE 3.

FIGURE 3.

Overexpression of a ubiquitin-resistant Mcl-1 mutant (KR Mcl-1) protects Huh-7 cells from lipoapoptosis, and targeted Mcl-1 knockdown sensitizes cells to palmitate-induced lipoapoptosis. A and B, Huh-7 cells were transiently transfected with a plasmid expressing KR Mcl-1 or GFP as control. Twenty four h following transfection, the cells were treated with 800 μ

m

palmitate for 16 h at 37 °C. Apoptosis was quantified morphologically (A) as described under “Experimental Procedures” or biochemically (B) by measuring caspase-3/7 activity. *, statistically significant difference (p < 0.01) compared with GFP vehicle cells; **, statistically significant difference (p < 0.01) compared with GFP palmitate cells. C, Mcl-1 knockdown was performed by stable transfection of Huh-7 cells with a plasmid encoding shRNA directed to Mcl-1. Efficiency of the targeted knockdown of Mcl-1 was established by immunoblot analysis of whole cell extracts. D, knockdown of Mcl-1 increases sensitivity of Huh-7 cells to lipoapoptosis by palmitate. Huh-7 cells and Huh-7 cells stably transfected with shRNA targeted against Mcl-1 (shMcl-1) were incubated with 400 μ

m

palmitic acid for 16 h. Apoptosis was quantified morphologically (C) and biochemically (D) by measuring caspase-3/7 activity. **, statistically significant difference (p < 0.01) for palmitate-treated shMcl-1 cells versus the palmitate-treated parental cell line.

FIGURE 4.

FIGURE 4.

Primary hepatocytes isolated from mice overexpressing human Mcl-1 are resistant to palmitate-induced loss of Mcl-1 and lipoapoptosis. A, primary murine hepatocytes were isolated from wild type C57BL/6 mice and cultured overnight. Cells were treated with 400 μ

m

palmitate for the time intervals indicated, and whole cell extracts were procured. Immunoblot analysis was performed for murine Mcl-1 (Rockland) and β-actin. Murine Mcl-1 migrated as a doublet with a dominant band of ∼37 kDa. B, primary murine hepatocytes were obtained from C57BL/6 mice transgenic for human MCL-1. Studies were performed as described in A with Mcl-1 immunoblot using sc-819 anti-human Mcl-1. C, primary mouse hepatocytes from C57BL/6, wild type (WT), and transgenic mice for human MCL-1 (Mcl-1 Tg) were treated with 400 μ

m

palmitate for 8 h. Apoptosis was quantified based on nuclear morphology. *, statistically significant difference (p < 0.01) compared with vehicle-treated wild type cells; **, statistically significant difference (p < 0.001) compared with palmitate-treated wild type cells.

FIGURE 5.

FIGURE 5.

Inhibition of PKCθ, a novel PKC, blocks Mcl-1 degradation by palmitate. Huh-7 cells were incubated in the presence of 800 μ

m

palmitate plus 3 μ

m

GSK-3β inhibitor IX (A) or 10 μ

m

of the novel PKC inhibitor rottlerin (B). At the time intervals indicated, whole cell extracts were prepared for immunoblot analysis. C, ubiquitination of Mcl-1 is dependent on novel PKC activity. Whole cell extracts were prepared from Huh-7 cells stably expressing HA-tagged ubiquitin that were treated with 800 μ

m

palmitate and the proteasome inhibitor MG-132 for 8 h either with or without the PKCθ inhibitor rottlerin. Affinity pulldown (PD) was performed for S-tagged (S-tag) Mcl-1 using protein S-agarose followed by immunoblot (IB) analysis of ubiquitin with anti-HA peptide tag antibody.

FIGURE 6.

FIGURE 6.

Palmitate induces phosphorylation of PKCθ but not PKCδ. A, Huh-7 cells were incubated in the presence of 800 μ

m

palmitate over the time intervals indicated. Whole cell lysates were subjected to immunoblot analysis for phospho-Thr538 PKCθ and total PKCθ or (B) phospho-Thr505 PKCδ and total PKCδ. Immunoblots were also probed for α-tubulin or β-actin as loading controls. C, Northern blot analysis demonstrates expression of a _PKC_θ mRNA in Huh-7 cells that is smaller than the transcript in Jurkat cells.

FIGURE 7.

FIGURE 7.

shRNA-targeted knockdown of PKCθ prevents palmitate-induced loss of Mcl-1. A, immunoblot analysis for PKCθ using whole cell extracts from Huh-7 cells stably transfected with empty vector expression construct as a control and cells stably transfected with the PKCθ-targeted shRNA. B, stable cell lines (as described in A) were incubated with 800 μ

m

palmitate over 6 h. Immunoblot analysis was performed on whole cell extracts for Mcl-1 and β-actin as a control for protein loading. C, hepatocytes from _Pkc_θ−/− mice maintain Mcl-1 protein levels following palmitate treatment. Primary murine hepatocytes from _Pkc_θ knock-out mice were treated with palmitate 400 μ

m

. At desired time intervals, whole cell lysates were prepared and blotted for Mcl-1 and actin (cf. Fig. 4_A_). In primary hepatocytes from _Pkc_θ−/− mice, actin was detected as a doublet, possibly due to detection of both β and γ isoforms.

FIGURE 8.

FIGURE 8.

Inhibition, shRNA-mediated knockdown, and genetic deletion of _Pkc_θ reduce lipoapoptosis. A, Huh-7 cells were treated with the novel PKC inhibitor rottlerin (10 μ

m

) beginning 30 min prior to the addition of 800 μ

m

palmitate. After 16 h of incubation, apoptosis was quantified by characteristic nuclear morphologic changes. B, Huh-7 cells were stably transfected with an shRNA targeting PKCθ or the empty expression construct (Huh-7; see Fig. 7). After 16 h of incubation with 800 μ

m

palmitate, apoptosis was quantified by nuclear morphology. C, apoptosis was also quantified in primary murine hepatocytes from wild type and _Pkc_θ−/− mice at 8 h following addition of 400 μ

m

palmitate. *, statistically significant difference (p < 0.01) compared with vehicle-treated cells; **, statistically significant difference (p < 0.01) compared with palmitate-treated cells in the absence of rottlerin.

FIGURE 9.

FIGURE 9.

Mcl-1 Ser159 is phosphorylated in a PKC-dependent manner, and S159A mutation decreases palmitate-induced Mcl-1 degradation and apoptosis. A, affinity-purified S peptide-tagged Mcl-1 from Huh-7 cells treated with vehicle or 800 μ

m

palmitate was blotted using antiserum specific for phosphorylation at Ser159/Thr163. Mutation of either site to Ala prevents detection with this antibody. PD, pulldown; IB, immunoblot; WT, wild type. B, whole cell lysates of Huh-7 cells treated with MG-132 as well as rottlerin (Rott) (10 μ

m

) and palmitate (PA) (800 μ

m

) as indicated were blotted with anti-phospho-Mcl-1 or total Mcl-1. Veh, vehicle. The proteasome inhibitor MG-132 was included to prevent degradation and facilitate detection of phosphorylated Mcl-1. C, Huh-7 cells were stably transfected with a construct expressing either S peptide-tagged wild type Mcl-1 (left) or S159A Mcl-1 (right). The stable transfectants were treated with palmitate (800 μ

m

) for the indicated time and then subjected to immunoblot analysis with anti-S peptide antibody. The ratio of intensities of S peptide-tagged Mcl-1 to β-actin is shown. S peptide-tagged human Mcl-1 migrates as a single band of 42 kDa. D, stable transfectants were treated with 800 μ

m

palmitate for 16 h, and apoptosis was quantitated by characteristic morphologic features. *, statistically significant difference (p < 0.01) compared with vehicle-treated cells transfected with wild type Mcl-1;**, statistically significant difference (p < 0.001) compared with palmitate-treated cells transfected with wild type Mcl-1.

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