Hiromu Kawahara

Interaction of intermediate filaments with nuclear lamina and cell periphery

Ultrastructural observations of the cytoskeleton suggest that the connection of the intermediate filaments (IFs) to actin microfilaments (MFs) at the plasma membrane and the nuclear lamina inside the nuclear membrane link signals received at the cell periphery to the nucleus. When these connections are viewed in three dimensions using detergent extracted cytoskeletal preparations from tissue cultures or slices made from tissue, the IFs are seen to run without interruption from the cell periphery to the nucleus and back. The IFs form side to side connections with the nuclear lamina and pore complexes. The nucleus and the centrioles are supported and held suspended in these extracted cells where all organelles and cytosol have been removed. The IFs are particularly dense in the ectoplasm where they form a sheet and provide the scaffolding which maintains the shape of the extracted cells. The IFs in the ectoplasm are attached to desmoplakin at cell-cell desmosome adhesions and to MFs where the cells are attached to the fibronectin substratum possibly through integrin linkages at adhesion plaques. This was graphically shown by immunogold labelling of IF cells treated with nickel. In this way, it was possible to visualize the loss of the cell-cell connections at desmosomes and the disruption of the IF-MF connections in the ectoplasm. The MFs after losing their connections with the IFs, redistribute to cover the entire cell periphery. The nickel treatment of primary liver cell cultures lead to the loss of several functions including formation of the bile canaliculus, the ability to secrete fluorescein diacetate and the ability to take up horseradish peroxidase (HRP) by endocytosis. These observations support the conclusion that the IF-MF connections at the cell periphery provide both structural and functional polarity of the liver cells including uptake and secretion and the formation of bile canaliculi.

Ethanol-induced phosphorylation of cytokeratin in cultured hepatocytes

We studied the effect of ethanol on the phosphorylation of cytokeratins (CKs) in cultured hepatocytes since CK filaments are regulated by phosphorylation and they are abnormal in alcoholic liver disease. Hepatocytes were obtained from 14-day-old rats and cultured for 48 hrs. The hepatocytes were exposed to ethanol (300 mM) for 30 min. The cells were extracted with the buffer containing Triton X-100. The residual insoluble cytoskeletons were analyzed by two dimensional (2D) gel electrophoresis and autoradiography. 2D gel electrophoresis showed CK 55 and CK 49 or 8 and 18 and actin. The CKs had several isoelectric variants. The most basic spot was the dominant protein which was not phosphorylated. The more acidic spots were phosphorylated. After ethanol treatment, the phosphorylation of CK 55 and CK 49 were markedly increased over controls. We compared these results, with the effect of vasopressin (10 nM), TPA (150 nM) and db-cAMP (1 mM) on the phosphorylation of CKs. Vasopressin and TPA caused the phosphorylation of CK 55 and 49 but db-cAMP did not. The results suggest that CKs are phosphorylated by protein kinase C through the phosphoinositide-linked transduction system activated by ethanol.

Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M(r) respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vivo substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.

Selective accumulation of IF proteins at a focal juxtanuclear site in COS-1 cells transfected with mouse keratin 18 cDNA.

COS-1 cells contain two keratins analogous to human keratins 8 (type II) and 18 (type I), and vimentin. Transfection of a plasmid, pSVK18, containing a mouse keratin 18 cDNA regulated by the SV40 early region promoter, was used to force the synthesis of exogenous (but homologous) type I keratin and to assess the effect of the oversynthesis of a keratin on endogenous keratins and vimentin intermediate filaments (IFs). Double immunofluorescence microscopy with monoclonal antimouse keratin 18 and monoclonal anti-human keratins 8 and 18 antibodies which cross-react with monkey keratins, showed that mouse keratin 18 formed typical IFs with the endogenous keratins but also accumulated in a focal area near the nucleus. Vimentin and its associated protein, p50, also colocalized at the juxtanuclear focal region, but the vimentin IFs of the outer cytoplasm vanished. Similar analyses with anti-tubulin and anti-actin antibodies indicated that the accumulated mouse keratin 18 colocalized with the centrosome but did not disturb the organization of microtubules or microfilaments. Anti-lamin and anti-SV40 large T antibodies showed that the oversynthesis of mouse keratin 18 had no effect on the distribution of these proteins. The accumulation was therefore selective for the cytoplasmic IF proteins. Electron microscopy and immunogold labeling of whole-mount detergent-extracted cells demonstrated that the accumulated IFs in the centrosomal region extended as a dense IF plexus-like network anchored to part of the nuclear surface.

Effects of chlorpromazine and low calcium on the cytoskeleton and the secretory function of hepatocytes in vitro

It has been established that the cytoskeleton plays an important role in the secretory function of hepatocytes. We studied the effect of chlorpromazine (CPZ) and low calcium (LC) on the cytoskeleton of primary cultured hepatocytes using double-labelling immunofluorescence and secretion of fluorescein diacetate (FD) into the bile canaliculi (BC). The hepatocytes were obtained from 14-day-old male rats. They were cultured for 24 h in serum-free Williams E medium with insulin and dexamethasone added to induce differentiation including bile canaliculus formation. After incubation with CPZ (200 microM) for 1 h, the BC became dilated and distorted and formed diverticula. Actin filaments around the BC became more prominent and the stress fibers decreased. CPZ did not affect the microtubules or cytokeratin filaments. Exposure to LC (20 microM) for 24 h caused a slight dilation of the BC. Actin spread out over the cell periphery and appeared non-filamentous. Actin filaments around the BC appeared unchanged and the stress fibers disappeared. Microtubules and cytokeratin filaments were unaffected. Secretion of FD into the BC occurred normally after treatment with CPZ or LC. These results support the idea that the integrity of actin is not necessary for secretory function and that microtubules and intermediate filaments play a role in this process. The dilatation and diverticular formation in the BC induced by CPZ treatment suggests that a cytochalasin-like loss of contraction of the BC may explain the CPZ-induced decrease in bile flow observed in vivo.

Ethanol-Induced Phosphorylation of Cytokeratins in Primary Cultured Hepatocytes

Phosphorylation of specific proteins by protein kinase A (PKA) or protein kinase C (PKC) may play a role in the regulation of assembly and disassembly and stability of intermediate filaments including intermediate filaments composed of cytokeratins (CKs). Initiation of the phosphorylation is through signal transduction by agonist receptor G protein activation of a catalyst such as cyclic AMP or phospholipase C which activate PKA or PKC, respectively.