Biomedical subjects

Naoki Tanimizu

Publications and source records attributed to Naoki Tanimizu.

6 recordsLinked to original sources

PubMed2005

Formation of multicellular epithelial structures.

The kidney is primarily comprised of highly polarized epithelial cells. Much has been learned recently about the mechanisms of epithelial polarization. However, in most experimental systems the orientation of this polarity is determined by external cues, such as growth of epithelial cells on a filter support. When Madin-Darby canine kidney (MDCK) cells are grown instead in a three-dimensional (3D) collagen gel, the cells form hollow cysts lined by a monolayer of epithelial cells, with their apical surfaces all facing the central lumen. We have found that expression of a dominant-negative (DN) form of the small GTPase Rac1 causes an inversion of epithelial polarity, such that the apical surface of the cells instead faces the periphery of the cyst. This indicates that the establishment of polarity and the orientation of polarity can be experimentally separated by growing cells in a 3D collagen gel, where there is no filter support to provide an external cue for orientation. DN Rac1 causes a defect in the assembly of laminin into its normal basement membrane network, and addition of a high concentration of exogenous laminin rescues the inversion of polarity caused by DN Rac1.

Animals↗

PubMed2004

Long-term culture of hepatic progenitors derived from mouse Dlk+ hepatoblasts.

We previously demonstrated that hepatoblasts can be isolated from mouse fetal liver based on the expression of delta-like [corrected] (Dlk), also known as Pref-1. Each Dlk+ hepatoblast forms a colony containing both albumin+ hepatocytes and cytokeratin 19+ (CK19) cholangiocytic cells on either type IV collagen or laminin. Here we show that extracellular matrices (ECMs) significantly affect the growth of Dlk+ cells. Dlk+ cells vigorously proliferated on type IV collagen-coated dishes in the presence of EGF and HGF during the first 5 days, but their proliferative capability declined thereafter. Dlk+ cells also proliferated on laminin-coated plates and some colonies continued to expand even beyond one month after plating. These hepatic progenitor cells proliferating on laminin (HPPL) efficiently proliferated even after replating. Moreover, they were induced to differentiate into hepatocytes and cholangiocytes by overlaying Engelbreth-Holm-Swarm sarcoma (EHS) gel and by embedding in type I collagen gel, respectively. HPPL acquired the metabolic functions of accumulating polysaccharides and detoxifying ammonium ions after hepatic differentiation. Surprisingly, HPPL expressed pancreatic genes such as Pdx1 when dexamethasone was depleted from the culture medium. Therefore, the long-term culture of hepatoblasts on laminin produces multi-potential hepatic progenitors, which possess a strong proliferative capability, differentiate into both hepatocytes and cholangiocytes, and potentially give rise to pancreatic cells.

Albumins↗

PubMed2004

Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors.

Hepatoblasts give rise to both mature hepatocytes and cholangiocytes. While Notch signaling has been implicated in the formation of bile ducts composed of cholangiocytes, little is known about the mechanism of lineage commitment of hepatoblasts. Here we describe the role of the Notch pathway in hepatoblast differentiation. Immunohistochemical analysis showed that Jagged1 was expressed in the cells surrounding the portal veins and Notch2 was expressed in most hepatic cells at mid gestation when ductal plates are formed surrounding the portal veins. Interestingly, the Jagged1+ cells were adjacent to ductal plates, suggesting that the Notch signaling is activated in hepatoblasts that undergo differentiation into cholangiocytes. In fact, expression of the Notch intracellular domain in Dlk+ hepatoblasts inhibited hepatic differentiation and significantly reduced the expression of albumin, a marker of both hepatoblasts and hepatocytes. Furthermore, the addition of Matrigel to the hepatoblast culture upregulated the expression of cytokeratin 7 and 19, integrin beta4, and HNF1beta, which are known to be expressed in cholangiocytes. By contrast, downregulation of the Notch signaling by siRNA specific for Notch2 mRNA as well as by the gamma-secretase inhibitor L-685,458 promoted the hepatic differentiation. Consistent with the previous finding that mature cholangiocytes strongly express HNF1beta, but barely express HNF1alpha, HNF4, and C/EBPalpha, activation of the Notch signaling upregulated HNF1beta expression, whereas it downregulated the expression of HNF1alpha, HNF4, and C/EBPalpha. These results suggest that the Notch signaling contributes to form a network of these transcription factors suitable for cholangiocyte differentiation.

Animals↗

PubMed2004

Expression of Dlk/Pref-1 defines a subpopulation in the oval cell compartment of rat liver.

We previously showed that Dlk, a transmembrane protein containing six epidermal growth factor like repeats in its extracellular domain, is strongly expressed in hepatoblasts in murine fetal liver. Here, we examined the expression of Dlk in oval cells, which are adult hepatic progenitors, in the rat 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) model. Reverse transcription polymerase chain reaction analysis showed that Dlk expression was significantly induced in the regenerating liver at day 12 and 14 after PH, when many oval cells were present in periportal areas. Immunofluoresence staining analysis revealed that Dlk(+) cells expressed oval cell markers, cytokeratin 19 (CK19) and alpha-fetoprotein, indicating that Dlk is expressed in oval cells. However, Dlk(+) cells accounted for only about 20% of total CK19(+) oval cells. Dlk(+) cells were localized more distantly from the portal vein than Dlk(-) cells, and were adjacent to mature hepatocytes, though Dlk(+) cells were surrounded by the basal membrane as other oval cells. Furthermore, at day 12 after PH, only 3% of Dlk(+) oval cells expressed Ki67, whereas about 13% of total oval cells expressed Ki67, indicating that Dlk(+) oval cells are less proliferative than Dlk(-) oval cells. Taken together, these results demonstrate that Dlk is expressed in a subpopulation of oval cells and that Dlk(+) cells represent intermediate cells between Dlk(-) oval cells and mature hepatocytes.

Animals↗

PubMed2003

Isolation of hepatoblasts based on the expression of Dlk/Pref-1.

Hepatoblasts are common progenitors for hepatocytes and biliary epithelial cells, although their nature remains largely unknown. In order to isolate and to characterize hepatoblasts, we searched for cell surface antigens expressed in mouse fetal hepatic cells by the signal sequence trap method and found that Dlk, also known as Pref-1, was strongly expressed in fetal liver. Immunohistochemical as well as northern analysis indicated that Dlk was highly expressed in the E10.5 liver bud. The strong expression continued until the E16.5 stage and was significantly downregulated thereafter. Using a monoclonal antibody against Dlk, we isolated Dlk+ cells either by a fluorescence-activated cell sorter or by an automatic magnetic cell sorter. Dlk+ cells isolated from fetal livers expressed albumin and formed colonies when cultured at low density with HGF and EGF for 5 days. Over 60% of colonies derived from E14.5 Dlk+ cells contained both albumin+ and cytokeratin 19+ cells, indicating that a majority of colony-forming Dlk+ cells are able to differentiate into both hepatocyte and biliary epithelial cell lineages. In addition, numerous microvilli were observed by electronmicroscopic analysis in most of those cultured cells, also indicating differentiation of Dlk+ cells under this condition. Furthermore, 7% of the colony-forming Dlk+ cells were not only bipotential but also highly proliferative, forming a large colony containing more than 100 cells during 5 days of culture. By transplantation of Dlk+ cells into the spleen, donor-derived hepatocytes were found in the recipient liver, indicating that Dlk+ cells differentiated into hepatocytes in vivo. These results indicate that Dlk+ cells are hepatoblasts and that Dlk is a useful marker to enrich highly proliferative hepatoblasts from fetal liver.

Animals↗

PubMed2002

Replacement of His12 or His119 of bovine pancreatic ribonuclease A with acidic amino acid residues for the modification of activity and stability.

In an attempt to produce a bovine pancreatic ribonuclease A (RNase A) with increased activity and stability, the catalytic pair of His12 and His119 was substituted with aspartic acid or glutamic acid, and aspartic acid, respectively, to evaluate the role of the two histidine residues in the activity and stability. Kinetic analysis revealed that k(cat)/K(m) values were significantly reduced for all mutant enzymes due to a decreased k(cat) rather than an increased K(m): the k(cat) values for both CpA and C>p of H12D and H12E decreased to about 1/1000; the k(cat) values of H119D decreased by 1/3300 for CpA and 1/80 for C>p. Thus, neither Asp nor Glu is able to act solely as an efficient catalytic residue of RNase A. Alkylation with iodoacetic acid (IAA) revealed that mutant enzymes had reduced reaction rates and that no modification was evident at Glu12 and Asp12 of H12E and H12D, respectively. This indicates that the low catalytic activity of mutant enzymes could be due to low basicity of Asp12 and Glu12. While the T(m) of H119D was almost the same as that of the wild-type enzyme, the T(m) of both H12D and H12E markedly decreased. It became apparent that His12 located at the bottom of the active site cleft contributes significantly to the structural stability of RNase A.

Journal Article↗