Aki Matsunobu

Osteoblast migration into type I collagen gel and differentiation to osteocyte-like cells within a self-produced mineralized matrix: a novel system for analyzing differentiation from osteoblast to osteocyte.

Osteoblasts are believed to differentiate into osteocytes, becoming embedded in bone, or to undergo apoptosis after the bone formation phase. The regulation of this terminal differentiation seems to be critical for bone homeostasis. However the mechanism remains unclear and there is no assay system currently available to analyze this process. To address this issue, we developed a new model in which osteoblasts are cultured on a type I collagen gel layer with osteogenic supplements β-glycerophosphate and ascorbic acid. Cellular behavior was analyzed by electron microscopy, immunohistochemistry and real-time RT-PCR. Osteoblasts gradually migrated into the gel, produced collagen fibrils, and differentiated to osteocytic cells with bone lacunae- and canaliculi-like mineralization. Osteocalcin, DMP-1 and SOST protein expression was mainly expressed in the migrated cells within the mid-layer of the gel. Osteoblastic (ALP and osteocalcin) and osteocytic (PHEX, DMP-1 and SOST) mRNA expression was significantly increased compared with those of the cells cultured on plastic dishes alone after 21 days. The number of TUNEL-positive apoptotic cells gradually increased, reaching a maximum at 28 days. The cells were distributed at the surface and in the mid-layer of the gel at 7 days and after 14 days of culture, respectively. These data indicate that our model reproduces transition from osteoblasts to osteocytes, suggesting the following: 1) migration of osteoblasts into collagen gel may play a critical role in osteocytic differentiation; and 2) spatiotemporal gene expression and apoptosis may be involved in the terminal differentiation of osteoblasts. Our model will make it possible to study the mechanism of transition from osteoblast to osteocyte, and both cell type-related diseases including osteoporosis and osteonecrosis.

Interaction between adipose tissue stromal cells and gastric cancer cells in vitro.

Adipose tissue exists in the gastric submucosa and subserosa. Thus, adipose tissue stromal cells (ATSCs), which include mesenchymal stem cells (MSCs), seem critical for the progression of gastric cancer but their interaction with the cancer cells is unknown. We demonstrated an interaction between these cells, using immunohistochemistry, Western blot and the collagen gel invasion assay system, in which the adenocarcinoma cells (well and poorly differentiated types, MKN28 and MKN45, respectively) were cultured on a ATSC-embedded or ATSC-non-embedded gel. ATSCs promoted the expression of the growth marker, proliferation cell nuclear antigen but inhibited that of the apoptosis marker, single-stranded DNA, in the cancer cell types. ATSCs accelerated the invasion of only MKN28 into the gel and promoted the expression of mitogen-activated protein kinase (MAPK, pERK-1/2) but decreased that of the molecularly targeted protein, HER2, in the cancer cells. ATSCs did not affect the expression of the prostaglandin biosynthetic enzyme cyclooxgenase-2 (COX-2) in the cancer cells. The COX-2 inhibitor celecoxib did not affect the morphology or invasion of the cancer cells. The cancer cell types in turn promoted the display of the myofibroblast marker, α-smooth muscle actin, whereas they decreased that of some MSC markers, e.g., CD44 and CD105, in ATSCs. The data suggest that (1) ATSCs influence the progression of gastric cancer by increasing their growth/invasion and decreasing their apoptosis through MAPK activation in a COX-2-independent way; (2) ATSCs adversely affect HER2-targeted therapy; (3) the cancer cells induce the cancer-associated myofibroblast phenotype in ATSCs.

Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture

Adipokine-producing fatty tissues, composed of preadipocytes, adipocytes, and mesenchymal stem cells, surround the kidney. To study the interaction between renal tubular cells and adipose tissue, we cocultured adipose tissue fragments and MDCK cells. MDCK cells in the coculture showed a taller columnar shape with improved organization of their microvilli and basal lamina than that seen in MDCK cell monoculture. The adipose tissue-induced change in morphology was replicated when we added leptin to MDCK cells cultured alone. Adiponectin abolished the leptin effect. Adipose tissue fragments inhibited MDCK cell division and also the formation of single-stranded DNA, an indicator of apoptosis. The fragments promoted the expression of polarity-associated proteins, including the tight junction molecules, ZO-1, atypical protein kinase C, and Cdc42. Further, the fragments also accelerated the expression of pendrin, the chloride/iodide transporter in the MDCK cells. In turn, MDCK cells decreased the number of preadipocytes and CD44+/CD105+ mesenchymal stem cells in the fragments, and promoted adiponectin production from the fragments. Thus, our study shows that adipose tissue fragments promote the hypertrophy, polarization, and differentiation of MDCK cells by attenuating their growth and apoptosis through opposing endocrine or paracrine effects of leptin and adiponectin. Further, MDCK cells inhibit the regeneration of preadipocytes and mesenchymal stem cells in adipose tissue.

A Promising Culture Model for Analyzing the Interaction between Adipose Tissue and Cardiomyocytes

The heart has epicardial adipose tissue that produces adipokines and mesenchymal stem cells. Systemic adipose tissue is involved in the pathophysiology of obesity-related heart diseases. However, the method for analyzing the direct interaction between adipose tissue and cardiomyocytes has not been established. Here we show the novel model, using collagen gel coculture of adipose tissue fragments (ATFs) and HL-1 cardiomyocytes, and electron microscopy, immunohistochemistry, real-time RT-PCR, and ELISA. HL-1 cells formed a stratified layer on ATF-nonembedded gel, whereas they formed almost a monolayer on ATF-embedded gel. ATFs promoted the apoptosis, lipid accumulation, and fatty acid transport protein (FATP) expression of FATP4 and CD36 in HL-1 cells, whereas ATFs inhibited the growth and mRNA expression of myosin, troponin T, and atrial natriuretic peptide. Treatment of leptin (100 ng/ml) and adiponectin (10 μg/ml) neither replicated nor abolished the ATF-induced morphology of HL-1 cells, whereas that of FATP4 and CD36 antibodies (25 μg/ml) never abolished it. HL-1 cells prohibited the development of CD44+/CD105+ mesenchymal stem cell-like cells and lipid-laden preadipocytes from ATFs. HL-1 cells increased the production of adiponectin in ATFs, whereas they decreased that of leptin. The data indicate that our model actively creates adipose tissue-HL-1 cardiomyocyte interaction, suggesting first that ATFs may be related to the lipotoxiciy of HL-1 cells via unknown factors plus FATP4 and CD36 and second that HL-1 cells may help to retain the static state of ATFs, affecting adipokine secretion. Our model will serve to study adipose tissue-cardiomyocyte interaction and mechanisms of obesity-related lipotoxicity and heart diseases.

FLUID FLOW STRESS AFFECTS PERITONEAL CELL KINETICS: POSSIBLE PATHOGENESIS OF PERITONEAL FIBROSIS

♦ Background: Peritoneal fibrosis is an essential precursor condition to the development of encapsulating peritoneal sclerosis (EPS). This serious complication leads to a high mortality rate in peritoneal dialysis (PD) patients. Although several factors, including highly concentrated glucose in the dialysis solution, are believed to be potent agents for peritoneal fibrosis, the underlying mechanism remains unclear. During PD, the dialysis solution continuously generates fluid flow stress to the peritoneum under peristalsis and body motion. Fluid flow stress has been implicated as playing a critical role in the physiologic responses of many cell types. We therefore hypothesized that fluid flow stress may be involved in the pathogenesis of peritoneal fibrosis leading to EPS. ♦ Methods: To generate fluid flow stress, culture containers were placed on a rotatory shaker in a thermostatic chamber. In this system, the shaker rotated at a speed of 25 rpm with a radius of 1.5 cm. Mesothelial cells were cultured in low-glucose (1000 mg/L) or high-glucose (4500 mg/L) complete medium with and without flow stress. ♦ Results: Fluid flow stress promoted hyperplasia and epithelial-mesenchymal transition (EMT) of mesothelial cells independent of glucose concentration. Fluid flow stress inhibited expression of ERK (extracellular signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) in mesothelial cells. Administration of ERK and p38 MAPK inhibitors replicated the stress-induced morphology of mesothelial cells. ♦ Conclusions: The present data indicate that fluid flow stress promotes hyperplasia and EMT of mesothelial cells via the MAPK axis, suggesting that fluid flow stress may be involved in the pathogenesis of peritoneal fibrosis.

Culture Models for Studying Thyroid Biology and Disorders

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types.

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming.

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.

The Air Liquid-interface, a Skin Microenvironment, Promotes Growth of Melanoma Cells, but not Their Apoptosis and Invasion, through Activation of Mitogen-activated Protein Kinase

The air-liquid interface (ALI) is a common microenvironment of the skin, but it is unknown whether the ALI affects melanoma cell behaviors. Using a collagen gel invasion assay, immunohistochemistry, and Western blots, here we show that melanoma cell proliferation in cultures with an ALI is higher than melanoma cell proliferation in submerged cultures. Bromodeoxyuridine (BrdU) uptake, an indicator of cell proliferation, of melanoma cells at the ALI was about 3 times that of submerged cells, while ALI and submerged melanoma cells had similar levels of single-stranded DNA (a marker of apoptosis). The ALI enhanced the expression of Raf-1, MEK-1 and pERK-1/2 components of the mitogen-activated protein kinase (MAPK) cascade, in cells more than the submerged condition did. The increases in BrdU uptake and pERK-1/2 expression promoted by ALI was abolished by the MEK inhibitor, PD-98059. ALI-treated and submerged melanoma cells did not infiltrate into the collagen gel, and they showed no significant difference in the expression of the invasion- and motility-related molecules, matrix metalloproteinase-1 and -9, laminin 5, and filamin A. Our data indicate that the ALI, a skin microenvironment, accelerates the growth, but not the apoptosis or invasion, of melanoma cells through MAPK activation.

The Influence of the Skin Microenvironment Air-Liquid Interface on Melanoma

Shuji Toda1, Shigehisa Aoki1, Kazuyoshi Uchihashi1, Aki Matsunobu1, Mai Yakushiji1, Akifumi Ootani2, Eisuke Koike3 and Nobuhisa Yonemitsu4 1Department of Pathology & Microbiology, Faculty of Medicine & Graduate School of Medicine, Saga University, Saga 849-8501, 2Department of Internal Medicine Faculty of Medicine & Graduate School of Medicine, Saga University, Saga 849-8501, 3Department of Surgery, Koike Hospital, Saga 840-0861, 4Department of Pathology, Sasebo Chuo Hospital, Nagasaki 857-1195 Japan