Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations.

Wanson, Jean-Claude; Drochmans, Pierre; Mosselmans, Roger; Ronveaux, M. F.

doi:10.1083/jcb.74.3.858

Cited by 142 publications

(40 citation statements)

References 28 publications

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“…S5B) Because primary hepatocytes allow direct biochemical analyses, we isolated hepatocytes by a two-step collagenase perfusion followed by Percoll purification (25). Cells were harvested 4 d after CCl 4 exposure and cultured for 24 h to allow reconstitution of cell polarity and function before analyses (26). The yield of viable TOP1mt-deficient hepatocytes was decreased more than twofold compared with the matched WT cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Lack of mitochondrial topoisomerase I ( TOP1mt ) impairs liver regeneration

Khiati

Baechler

Factor

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl 4 ) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl 4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steadystate levels of ATP, O 2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl 4 -treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation.liver regeneration | mitochondrial topoisomerase I | cell proliferation | mitochondrial DNA replication | mitochondrial homeostasis

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Section: Resultsmentioning

confidence: 99%

Lack of mitochondrial topoisomerase I ( TOP1mt ) impairs liver regeneration

Khiati

Baechler

Factor

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Ruthenium red is a heavy metal conjugate that is too large to cross a functionally intact TJ and can be visualized with electron microscopy (35,36,83). Ruthenium red was added to the apical side of either untreated or HGF-treated Transwell filter cultures of MDCK cells during fixation and processing for electron microscopy.…”

Section: E and G)mentioning

confidence: 99%

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity

Pollack¹,

Apodaca²,

Mostov³

2004

American Journal of Physiology-Cell Physiology

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Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two-to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 g/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair. c-met protooncogene; transepithelial resistance; Madin-Darby canine kidney cell MORPHOGENETIC CELL REARRANGEMENTS are stimulated by hepatocyte growth factor (HGF), a fibroblast-derived growth factor (77) that has paracrine actions on a variety of cell types. In vivo, HGF is important for the development of the placenta, liver, limbs, and kidney (6,28,67,71,72,82,87) and induces morphogenesis of blood vessels (9,22,47,70). In addition, both HGF and the c-met protooncogene (c-met) (31, 86), a tyrosine kinase receptor (11,19,51,54) to which HGF binds with high affinity (25,32), are regulated in response to organ injury and stimulate epithelial morphogenesis that contributes to tissue repair and regeneration in liver, kidney, lung, gastric mucosa, and muscle (12,38,40,66,81,88). In vitro, many of the pleiotropic effects of HGF can be modeled and show a dependence on both the target cell type and environmental context: paracrine actions of HGF increase hepatocyte mitogenesis (20,41,49,50,90), inhibit growth of tumor cells (69, 78), activate motogenesis and invasiveness of epithelial and endothelial cells (9,18,63,64,70,75,84,85), stimulate wound repair (53), and induce morphogenesis of either epithelial tubes or pseudostrati...

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“…Primary hepatocyte culture provides a direct test ofthis question. Freshly isolated cells, plated in a complete culture medium, reconstitute many of the features of the liver in vivo, including intercellular structures that resemble canaliculi both morphologically (15) and functionally (16). The basolateral surface of individual cells attaches to the culture substratum, modeling the interaction of hepatocytes and basal lamina in the intact tissue.…”

Section: Introductionmentioning

confidence: 99%

Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver.

Bissell¹,

Arenson²,

Maher³

et al. 1987

J. Clin. Invest.

533

206

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The subendothelial space of normal rat liver contains the constituent proteins of a basal lamina, as judged by immunohistochemical study oftissue sections. However, it is unknown whether these proteins constitute a complex with effects on hepatocellular function. We have examined this question, using normal rat hepatocytes cultured on substrata of matrix proteins as a model of the interaction between cells and basal lamina in vivo. In cultures on a type I collagen substratum, albumin secretion decreased progressively after 2 d. By contrast, when cells were cultured on a laminin-rich gel matrix, albumin secretion was stable for at least 3 wk; other functions and ultrastructural morphology were similarly maintained. None of the individual matrix proteins effectively substituted for the gel matrix, suggesting that full support of hepatocellular function requires a complex of matrix proteins. We speculate that a cause of hepatocellular dysfunction in acute inflammation is disruption of this matrix and alteration of its interaction with the hepatocyte plasma membrane.

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Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations.

Cited by 142 publications

References 28 publications

Lack of mitochondrial topoisomerase I ( TOP1mt ) impairs liver regeneration

Lack of mitochondrial topoisomerase I ( TOP1mt ) impairs liver regeneration

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity

Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver.

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