Toshihiro Mitaka

CELL ADHESION KINASE BETA FORMS A COMPLEX WITH A NEW MEMBER, HIC-5, OF PROTEINS LOCALIZED AT FOCAL ADHESIONS

Cell adhesion kinase β (CAKβ/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKβ-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor β1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB. This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5. The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitrowith the extreme C-terminal region (residue 801 to the end) of CAKβ. CAKβ was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKβ with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKβ. Coimmunoprecipitation of Hic-5 with CAKβ, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKβ and Myc-tagged Hic-5, was lost when the CAKβ amino acid residues 741–903 were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAKβ was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress. These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAKβ, implying possible involvement of Hic-5 in the downstream signaling of CAKβ.

The current status of primary hepatocyte culture

Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient‐rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient‐rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth.

Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells.

Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood‐forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow‐derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co‐cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell–cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcirption factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3‐dioxygenase, are improved in hepatocyte/BMSC co‐cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro.

Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation of primary cultured rat hepatocytes

In the present study, we showed the role of the liver‐enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We used primary rat hepatocytes cultured in modified L‐15 medium. The cells proliferated and, after the addition of 2% dimethyl sulfoxide (DMSO) from day 4, they stopped growing and gradually differentiated. During hepatic proliferation, expression of hepatocyte nuclear factors (HNF)1α, HNF4, C/EBPα, and C/EBPβ mRNAs was depressed, whereas that of HNF3α and HNF3β transcripts was enhanced. After the addition of DMSO, the expression of HNF1α, HNF3γ, and HNF4 returned to the level in isolated cells and HNF1β mRNA expression gradually increased. However, expression of C/EBPα and C/EBPβ mRNAs was partially recovered. The mitoinhibitory agents, IL‐1β, IL‐6, TGF‐β, and activin A, were examined to determine whether they could induce differentiation of proliferating hepatocytes as shown in cells treated with DMSO. Although these factors inhibited cell growth, the cells did not differentiate. The expression pattern of HNF3γ mRNA was quite different in the cells cultured with DMSO and those cultured with cytokines. Therefore, hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription factors. Thus, expression of HNF3γ, C/EBPα, and C/EBPβ may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1α, HNF1β, HNF3α, HNF3β, and HNF4 as well as suppression of C/EBPδ. J. Cell. Physiol. 174:273–284, 1998.

Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates the size of the luminal space surrounded by polarized epithelial cells. Grhl2 promotes epithelial barrier function and the formation of large lumen by up-regulating Cldn3, Cldn4, and Rab25. The results reveal a molecular network regulating epithelial lumen formation.

BAG-1 accelerates cell motility of human gastric cancer cells

BAG-1 is a Hsp70/Hsc70-binding protein that interacts with Bcl-2, Raf-1, steroid hormone receptors, Siah-1, and hepatocyte growth factor (HGF) receptors, implying multiple functions for the BAG-1 protein. Here, we provide evidence that gene transfer-mediated overexpression of BAG-1 markedly enhances the motility of human gastric cancer cells. Two independent in vitro migration assays showed that the BAG-1-expressing MKN74 cells exhibited more active migration compared with control transfectants or parent MKN74 cells. In MKN74 cells, the overexpression of BAG-1 affected neither cell adhesion capability nor migration responses to HGF. The promotive effect of BAG-1 on cell migration was similarly observed in transfectants of another human gastric cancer MKN45 cell line. In BAG-1 transfected gastric cancer MKN74 cells, BAG-1 colocalized with cytokeratin as well as actin filaments, and was concentrated at membrane ruffles induced by lysophosphatidic acid (LPA). Taken together, these studies demonstrate that BAG-1 has a novel function as promoter of cell migration in human gastric cancer cells, possibly through cooperation with cytoskeletal proteins.

Reconstruction of 3D stacked-up structures by rat small hepatocytes on microporous membranes

The three‐dimensional (3D) culture of hepatocytes is essential for the reconstruction of functional hepatic tissues in vitro. In the present experiment, we developed a 3D‐culture method in order to reconstruct hepatic cordlike structures by stacking up two‐dimensional (2D) tissues composed of rat small hepatocytes (SHs), which are hepatic progenitor cells. Pairs of membranes were prepared and the cells were separately cultured on each membrane. After the SH colonies had developed, one membrane was inverted on top of the other to form an SH bilayer. Thereafter, we investigated whether the stacked cells were organized into differentiated tissues. In the 3D stacked‐up structures, bile canaliculi (BC) started to form and gradually developed into anastomosing networks. Transmission electron microscopy revealed that the SHs of the upper and lower layers adhered to one another, and that BC formed between them. Bile canalicular proteins localized on the lumina of the tubular structures. Furthermore, the cells within the structures exhibited mRNA transcription of the hepatic‐differentiation markers and maintained a relatively high level of albumin secretion. We conclude that highly differentiated 3D tissues, including functional BC, can be reconstructed by stacking up layers of SHs. This 3D stacked‐up culture is useful for the reconstruction of tissue‐engineered livers.

Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled‐up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver‐enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4α, HNF6, CCAAT/enhancer binding proteins (C/EBP) α, and C/EBPβ, tryptophan 2,3‐dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel™ or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4α, HNF6, C/EBPα, C/EBPβ, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)‐like structure may induce the morphological changes of SHs. J. Cell. Biochem. 87: 16–28, 2002.

The LG1-3 tandem of laminin alpha 5 harbors the binding sites of lutheran/basal cell adhesion molecule and alpha 3 beta 1/alpha 6 beta 1 integrins

The laminin-type globular (LG) domains of laminin α chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, α-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin α5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin α5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified α chains, such that all or part of α5LG was replaced with analogous segments of human laminin α1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing α5 domains LN through LG3 fused to human laminin α1LG4–5. However, Lu/B-CAM did not bind other recombinant laminins containing α5LG3 unless α5LG1–2 was also present. A recombinant α5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the α5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin α5. Our results also suggest that the binding site for Lu/B-CAM on laminin α5 may overlap with that of integrins α3β1 and α6β1.

The LG1-3 tandem of laminin α5 harbors the binding sites of Lutheran/B-CAM and α3β1/α6β1 integrins

The laminin-type globular (LG) domains of laminin α chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, α-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin α5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin α5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified α chains, such that all or part of α5LG was replaced with analogous segments of human laminin α1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing α5 domains LN through LG3 fused to human laminin α1LG4–5. However, Lu/B-CAM did not bind other recombinant laminins containing α5LG3 unless α5LG1–2 was also present. A recombinant α5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the α5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin α5. Our results also suggest that the binding site for Lu/B-CAM on laminin α5 may overlap with that of integrins α3β1 and α6β1.