Keratin 8/18 modulation of protein kinase C-mediated integrin-dependent adhesion and migration of liver epithelial cells

Abstract

Keratins are intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatocyte and hepatoma cell IFs are made solely of keratins 8/18 (K8/K18), the hallmark of all simple epithelia. Cell attachment/spreading (adhesion) and migration involve the formation of focal adhesions at sites of integrin interactions with extracellular matrix, actin adaptors such as talin and vinculin, and signaling molecules such as focal adhesion kinase (FAK) and member(s) of the protein kinase C (PKC) family. Here, we identify the novel PKCdelta as mediator of the K8/K18 modulation of hepatoma cell adhesion and migration. We also demonstrate a K8/K18-dependent relationship between PKCdelta and FAK activation through an integrin/FAK-positive feedback loop, in correlation with a reduced FAK time residency at focal adhesions. Notably, a K8/K18 loss results to a time course modulation of the receptor of activated C-kinase-1, beta1-integrin, plectin, PKC, and c-Src complex formation. Although the K8/K18 modulation of hepatocyte adhesion also occurs through a PKC mediation, these differentiated epithelial cells exhibit minimal migrating ability, in link with marked differences in protein partner content and distribution. Together, these results uncover a key regulatory function for K8/K18 IFs in the PKC-mediated integrin/FAK-dependent adhesion and migration of simple epithelial cells.

Figure 1.

PKC requirement in the K8/K18 modulation of hepatoma cell attachment and spreading. H4ev and shK8b cells were maintained in serum-free media for 24 h before cell dispersion and subsequent seeding in fibronectin-coated dishes. (A) Cell attachment was assayed in absence (C) or presence of BIM (B) or Gö (G). Cells were fixed at 10-min postseeding time, and attached cells were counted in 10 randomly chosen fields per dish. The number of attached cells at 1 h, in absence of inhibitor, was taken as 100%, based on our previous published data (Galarneau et al., 2007) (B) Cell spreading was assayed in absence (C) or presence of BIM (B), Gö (G), PMA (P), or in combination with BIM (P+B) or Gö (P+G). Cells were fixed at 60 min postseeding, and the area of at least 36 cells per time point was calculated. The graph shows the mean cell surface (%) values in reference to H4ev cell surface (100%). (C) DIC image of H4ev and shK8b cells fixed at 1 h postseeding and corresponding wide field fluorescence images of Alexa Fluor 488 anti-β1-integrin. The mean values ± SEM of three independent experiments, plus the p values <0.05, are provided.

Figure 2.

PKC requirement in the K8/K18 modulation of collective hepatoma cell migration. Scratch wounds were made in confluent cultures of H4ev and shK8b cells. (A) Phase-contrast images were taken immediately after scratching (0 h) and at 20 h postscratching. Cells were pretreated with vehicle (control) or PMA. (B) Quantification of the wound closure at 20 h postscratching of cells pretreated with vehicle (C), BIM (B), Gö (G), PMA (P), PMA + BIM (P+B), or PMA + Gö (P+G). The mean values ± SEM of three independent experiments, plus the p values <0.05, are provided.

Figure 3.

PKC requirement in the K8/K18 modulation of single-hepatoma cell locomotion. (A–D) Phase-contrast images of the cell locomotion were taken during 3 h after treatment with vehicle (A and B, respectively) and PMA (C and D). The dotted lines correspond to the locomotion paths during the 3-h period. Insets in C and D show migrating cells with lamellipodia. (E) Histograms showing the mean travel distance ± SEM (linear gap between white dot and star in A–D) calculated for 60 cells per experiment. Cells were treated with vehicle (Control, C), BIM (B), Gö (G), PMA (P), PMA + BIM (P+B), or PMA + Gö (P+G). (F) Western blotting on total cell extracts prepared from H4ev and shK8b showing the diminution in protein content of PKCδ (δ), PKCα (α), or PKCε (ε) at 48 h posttransfection of the respective siRNA. C, control siRNA. (G) Histogram showing the mean travel distance of H4ev and shK8b cells transfected with control siRNA (C) or with PKCα siRNA (α) and further treated with PMA (P). (H) Histograms showing the mean travel distance of H4ev and shK8b cells transfected with control siRNA (C) or with PKCδ siRNA (δ) and further treated with PMA (P). (I) Histogram showing the mean travel distance of H4ev and shK8b cells transfected with control siRNA (C) or with PKCε siRNA (ε) and further treated with PMA (P). The p values <0.05 are provided; *p < 0.01 relative to control.

Figure 4.

PKCδ and FAK activations in spreading H4ev and shK8b cells. Western blotting on total protein extracts from H4ev (H) and shK8b (S) cells at indicated postseeding times on fibronectin. (A) PKCδ phosphorylation on Thr-505 and (D) FAK phosphorylation on Tyr-397 (FAK; p-FAK) in presence of vehicle (control), PMA, or PMA + BIM. H4ev and shK8b densitometric quantifications of PKCδ (B and C) and FAK (E and F) phosphorylation levels normalized over the total respective PKCδ and FAK levels at 2 h postspreading, in absence (B and E) or presence of PMA and PMA + BIM (C and F). GAPDH was used as loading control. The mean densitometric values ± SEM of three independent experiments are provided. *p < 0.05 for H4ev versus shK8b, and H4ev + PMA versus shK8b + PMA conditions.

Figure 5.

Residency of FAK, paxillin, and vinculin at FAs in spreading H4ev and shK8b cells. (A) Confocal live cell images of H4ev (left) and shK8b (right) cells expressing GFP-FAK were taken before photobleaching the regions of interest (ROIs) (white squares) containing FAs. Time-lapse sequence (below, in seconds) of the selected ROIs show recovery after photobleaching for H4ev cells (ev, top row) and shK8b cells (K8, bottom row). PB, prebleach; AB, immediately after photobleaching. Kinetics of recovery of GFP-FAK (B), GFP-paxillin (C), and GFP-vinculin (D) after photobleaching of FAs from H4ev (top) or shK8b (below) cells is illustrated. The measured fluorescence intensity in eight independent ROIs per cell type was normalized and the mean values ± SEM are shown. The histogram in E depicts the recovery half-time ± SEM obtained from FRAP analyses of GFP-FAK (FAK), GFP-paxillin (Pax), and GFP-vinculin (Vinc) in H4ev and shK8b cells. The histogram in (E) depicts the recovery half-time ± SEM obtained from FRAP analyses of GFP-FAK in cells pretreated with vehicle (C) or BIM (B).

Figure 6.

Complex formation between RACK1, β1-integrin, plectin, PKCs, and Src in spreading H4ev and shk8b cells. Cells were maintained in serum-free media for 24 h before their seeding on fibronectin, proteins were solubilized with Empigen, RACK1 was immunoprecipitated, and the flowthrough was kept. (A) Western blotting with antibodies against β1-integrin, plectin, PKCα, PKCδ, PKCε, Src, and K8 in the flow through from the RACK1 immunoprecipitates at the indicated postseeding times. (B) Western blotting with antibodies against the corresponding proteins, after RACK1 immunoprecipitation (IP: αRACK1).

Figure 7.

Colocalization of β1-integrin with talin, RACK1, plectin, PKCs, Src, and K8 in spreading H4ev and shk8b cells. (A) Cell were allowed to spread for 1 h and then fixed and colabeled with β1-integrin antibody and the corresponding antibody for the second protein. Images were captured with a FV1000 confocal system. (B) The Pearson's colocalization r of each protein versus β1-integrin was computed for a region rich in β1-integrin (boxed area in A).

Figure 8.

(A) PKC requirement in the K8/K18 modulation of WT and K8-null hepatocyte attachment. The attachment assay was performed in absence (C) or presence of BIM (B) or Gö (G). The cells were fixed at 30 min postseeding, and attached cells were counted in 10 randomly chosen fields per dish. The number of attached cells at 3 h, in absence of inhibitor, was taken as 100%. The mean values ± SEM of two independent experiments are indicated. (B) PKC requirement in the K8/K18 modulation of hepatocyte spreading. WT and K8-null hepatocytes were seeded in absence (C) or presence of BIM (B), Gö (G), PMA (P) or with PMA + BIM (P+B) or PMA + Gö (P+G) and allowed to spread. Images from fixed cells were captured at 60 min postseeding, and the area of 30 cells per time point was calculated. The graph shows the mean cell surface (%) values ± SEM, in reference to WT cell surface (100%). (C) Scratch wound assay on WT and K8-null hepatocyte monolayers, revealing the absence of hepatocyte collective cell migration. Phase contrast images were taken immediately after scratching (0 h) and at 20 h postscratching. Cells were pretreated with vehicle (Control) or PMA. (D) Hepatocyte migration was quantified during the 20-h period, after pretreatment with vehicle (C), BIM (B), Gö (G), or PMA (P). (E) Western blotting on total protein extracts from WT (W) and K8-null (K) hepatocytes at the indicated postseeding times, showing the kinetics of FAK phosphorylation on Tyr-397 (p-FAK) in absence (Control) or presence of PMA or PMA + BIM. GAPDH was used as loading control.

Figure 9.

Intracellular distributions of RACK1, β1-integrin, plectin, PKCs, Src, and K8 in hepatoma cells and hepatocytes. Suspensions or monolayers of H4ev (H) and shK8b (S) cells (A) or WT (W) and K8-null hepatocytes (K) (B) separated into fractions enriched in cytosolic (F1), membrane/organelle (F2), nuclear (F3), and cytoskeletal (F4) proteins were analyzed by Western blotting for changes in the relative distributions of the individual proteins. The relative contents the same proteins in the total protein extracts also were determined.

Figure 10.

Schematic representations of the integrin/FAK-dependent signaling in adhering and migrating H4ev and shK8b cells. (A) Kinetics of H4ev and shK8b cell attachment and spreading, along with changes in PKC versus FAK phosphorylation and RACK1/β1-integrin/plectin/PKC/c-Src complex assembly. (B) In H4ev cells, the K8/K18 modulation of the integrin/FAK-dependent signaling occurs through a plectin/RACK1/PKC mediation. It involves FAK phosphorylation at Tyr-397 (P-FAK) through a feedback loop, and subsequent activation of downstream c-Src-dependent signaling cascades regulating cell adhesion and migration; note that part of the PKC-mediated migration remains c-Src independent. (C) In light of the immunocomplex and proteome data, a coherent conclusion would be that the K8/K18 loss in shK8b cells leads to a mislocalization of plectin and RACK1, which in turn perturbs their interplay with PKC and integrin related-signaling proteins at FAs.