Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size - implication for FasR-associated apoptosis

Abstract

Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases.

Keywords: ASMase; FasR; Hepatocyte; Keratin; Lipid raft; PKC.

Fig. 1.

K8/K18 IF modulation of three interrelated size parameters of CT-lipid rafts in hepatocytes. (A) Lateral (XY) and axial (XZ) fluorescence images of WT and K8-null hepatocyte lipid rafts labeled using Vybrant® Alexa Fluor® 488 (CTX-B labeling), showing that the lipid raft distribution is perturbed as result of the K8/K18 IF loss. (B) Percentage cell surface coverage by lipid rafts on WT and K8-null (K8n) hepatocytes, showing a decrease as result of the K8/K18 IF loss (n>74). (C) Surfaces of coalesced CT-lipid rafts on WT and K8-null (K8n) hepatocytes showing a reduction as result of the K8/K18 IF loss (n>74). (D) Volumes of coalesced CT-lipid rafts on WT and K8-null (K8n) hepatocytes showing a decrease as result of the K8/K18 IF loss (n>70). (E) Lateral (XY) and axial (XZ) fluorescence images of hK8 and K8G62C hepatocyte lipid rafts labeled using Vybrant^® Alexa Fluor^® 488 (CTX-B labeling), showing that the G62C mutation perturbs the lipid raft organization. (F) Percentage cell surface coverage by lipid rafts on hK8 and K8 G62C (G62C) hepatocytes, showing a decrease as result of the G62C mutation (n>40). (G) Surfaces of coalesced CT-lipid rafts on hK8 and K8 G62C (G62C) hepatocytes, showing a decrease as result of the G62C mutation (n>40). (H) Volumes of coalesced CT-lipid rafts on hK8 and K8 G62C (G62C) hepatocytes, showing a decrease as result of the G62C mutation (n>40). Quantitative results are mean±s.e.m. ***P<0.005 (t-test).

Fig. 2.

K8/K18 IF modulation of ASMase-dependent CT-lipid raft size. (A) Fluorescence images of the CT-lipid raft distributions at the surfaces of WT and K8-null hepatocytes, following a 2-h treatment with ASMase (50 µM DPM, 50 µM IPM) or lipid raft (2 mM MBC) inhibitors. The results show that the ASMase inhibition leads to a reduction in coalesced lipid raft size and distribution (CTRL, untreated control). (B) Percentage cell surface coverage by lipid rafts on WT and K8-null (K8n) hepatocytes following a 2-h treatment with ASMase or lipid raft inhibitors as in A, showing a strong reduction after these treatments (C, untreated control). n>25. (C) Surface of coalesced CT-lipid rafts on WT and K8-null (K8n) hepatocytes following a 2-h treatment with ASMase (or lipid raft inhibitors as in A, showing a surface reduction as result of the treatments (C, untreated control). n>25. (D) Volume of coalesced CT-lipid rafts on WT and K8-null hepatocyte surfaces following a 2-h treatment with ASMase or lipid raft inhibitors as in A, showing a reduction for both WT and K8-null hepatocytes (C, untreated control). n>25. Quantitative results are mean±s.e.m. **P<0.01; ***P<0.005 (t-test).

Fig. 3.

K8/K18 IF modulation of ASMase localization and activity. (A) Immunofluorescence confocal imaging of the ASMase localization in WT and K8-null hepatocytes, revealing no major differences. (B) XZ views of ASMase immunofluorescence images with or without a 2-h treatment with cisplatin (Cis; 5 µg/ml) in WT and K8-null hepatocytes. In the absence of cisplatin, a slight decrease in ASMase localization is seen at the surface of hepatocytes lacking K8/K18 IFs, whereas an increased localization is observed at the surface of both cell types, following the cisplatin treatment. (C) ASMase activity assessments in WT and K8-null hepatocytes with or without a 2-h treatment with cisplatin (Cis; 5 µg/ml) or PMA (100 nM), showing a decrease in ASMase activity in hepatocytes lacking K8/K18 IFs, under either inhibitory conditions (C: untreated control). Results are mean±s.e.m. **P<0.01; ***P<0.005 (t-test).

Fig. 4.

K8/K18 IF modulation of CT-lipid raft size through PKC activation. (A) Immunofluorescence confocal imaging of the CT-lipid raft distributions at the surfaces of WT and K8-null hepatocytes, following a 2-h treatment with two PKC activators [5 µg/ml cisplatin (Cis) or 100 nM PMA] or a PKC inhibitor (1 µM BIM). (B) Volume assessments of coalesced CT-lipid rafts on WT and K8-null hepatocytes following 2-h treatments with PKC activators or PKC inhibitor as in A, revealing that a PKC activation increases the lipid raft size, whereas a PKC inhibition reduces it in both cell types (n>25). Results are mean±s.e.m. *P<0.05; ***P<0.005 (t-test).

Fig. 5.

K8/K18 IF modulation of PKCδ translocation to the surface membrane versus internalization. (A) Immunofluorescence confocal imaging of PKCδ–EGFP in transfected WT and K8-null hepatocytes treated with PMA (100 nM) and fixed at 1, 5 and 15 min post treatment. (B) Percentage assessments of WT or K8-null hepatocytes exhibiting a PKCδ–EGFP translocation at the surface membrane as a function of time after treatment with PMA (100 nM), showing a rapid translocation to the surface in both cell types, with even faster kinetics in hepatocytes lacking K8/K18 IFs (n>70). (C) Percentage assessments of WT or K8-null hepatocytes exhibiting a PKCα–EGFP translocation at the surface membrane as a function of time after a PMA treatment (100 nM), showing no difference in both cell types (n>60). (D) Lateral (XY) and axial (XZ) immunofluorescence confocal imaging of PKCδ–EGFP in transfected WT and K8-null hepatocytes treated with PMA (100 nM) and fixed at different time points, revealing less PKCδ–EGFP internalization in hepatocytes lacking K8/K18 IFs (CTRL: untreated control). Quantitative results are mean±s.e.m.

Fig. 6.

K8/K18 IF modulation of PKCδ localization in lysosomes. Live-cell confocal imaging of PKCδ–EGFP in transfected WT and K8-null hepatocytes along with Lysotracker-labeled lysosomes, following treatment with PMA (100 nM), showing less PKCδ–EGFP and lysosome colocalization in hepatocytes lacking K8/K18 IFs. Yellow arrows point to colocalizations.

Fig. 7.

K8/K18 IF-dependent lipid raft size as a modulatory parameter of FasR-mediated apoptosis. (A) Nuclear fragmentation assessments in response to a Jo2 (0.5 µg/ml)+Protein A (PA, 0.1 µg/ml) treatment of WT and K8-null hepatocyte for 7 h with or without a 1 h pre-treatment with PKC activators [5 µg/ml cisplatin (Cis) or 100 nM PMA] or PKC inhibitor (1 µM BIM), showing that PKC modulations affect FasR-dependent nuclear fragmentation (C, untreated control). (B) Nuclear fragmentation analysis following a Jo2 (0.5 µg/ml)+Protein A (PA, 0.1 µg/ml) treatment of WT and K8-null hepatocytes for 7 h without or with a 1 h pre-treatment with ASMase inhibitors (50 µM DPM or 50 µM IPM,), indicating that ASMase inhibition affects FasR-dependent nuclear fragmentation (C: untreated control). (C,D) Nuclear fragmentation assessments in response to a Jo2 (0.5 µg/ml)+Protein A (PA, 0.1 µg/ml) treatment for 7 h with or without a pre-treatment with the ASMase inhibitor DPM at different concentrations in WT (C) or K8-null (D) hepatocytes. (E) Scale representation of the optimal range of CT-lipid raft volume for seeing apoptosis, showing where WT and K8-null hepatocytes are positioned on the scale versus different treatment conditions. Quantitative results are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.005 (t-test).