Human liver cell trafficking mutants: characterization and whole exome sequencing.

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Gel-based proteomics of liver cancer progression in rat.

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.

Increased levels of a unique post-translationally modified betaIVb-tubulin isotype in liver cancer.

Identifying changes at the molecular level during the development of hepatocellular carcinoma is important for the detection and treatment of the disease. The characteristic structural reorganization of preneoplastic cells may involve changes in the microtubule cytoskeleton. Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, vesicle transport, and motility. They are comprised of multiple isotypes of alpha- and beta-tubulin. Changes in the levels of these isotypes may affect not only microtubule stability and sensitivity to drugs but also interactions with endogenous proteins. We employed a rat liver cancer model that progresses through stages similar to those of human liver cancer, including metastasis to the lung, to identify changes in the tubulin cytoskeleton during carcinogenesis. Tubulin isotypes in both liver and lung tissue were purified and subsequently separated by isoelectric focusing electrophoresis. The C-terminal isotype-defining region from each tubulin was obtained by cyanogen bromide cleavage and identified by mass spectrometry. A novel post-translational modification of betaIVb-tubulin in which two hydrophobic residues are proteolyzed from the C-terminus, thus exposing a charged glutamic acid residue, was identified. The unique form of betaIVb-tubulin was quantified in the liver tissue of all carcinoma stages and found to be approximately 3-fold more abundant in nodular and tumor tissue than in control tissue. The level of this form was also found to be increased in lung tissue with liver metastasis. This modification alters the C-terminal domain of one of the most abundant beta-tubulin isotypes in the liver and therefore may affect the interactions of microtubules with endogenous proteins.

Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice.

Transplantation of healthy cells to repair organ damage or replace deficient functions constitutes a major goal of cell therapy. However, the mechanisms by which transplanted cells engraft, proliferate, and function remain unknown. To investigate whether host liver sinusoidal endothelium could be replaced with transplanted liver sinusoidal endothelial cells, we developed an animal model of tissue replacement that utilized a genetic system to identify transplanted cells and induced host-cell perturbations to confer a proliferative advantage to transplanted cells. Under these experimental conditions, transplanted cells engrafted efficiently and proliferated to replace substantial portions of the liver endothelium. Tissue studies demonstrated that transplanted cells became integral to the liver structure and reacquired characteristic endothelial morphology. Characterization of transplanted endothelial cells by membrane markers and studies of cellular function, including synthesis and release of coagulation factor VIII, demonstrated that transplanted cells were functionally intact. Further analysis showed that repopulation of the livers of mice that model hemophilia A with healthy endothelial cells restored plasma factor VIII activity and corrected their bleeding phenotype. Our studies therefore suggest that transplantation of healthy endothelial cells should be considered for cell therapy of relevant disorders and that endothelial reconstitution with transplanted cells may offer an excellent paradigm for defining organ-specific pathophysiological mechanisms.

Altered protein expression at early-stage rat hepatic neoplasia.

Protein expression patterns were analyzed in a rat model of hepatic neoplasia to detect changes reflecting biological mechanism or potential therapeutic targets. The rat resistant hepatocyte model of carcinogenesis was studied, with a focus on the earliest preneoplastic lesion visible in the liver, the preneoplastic hyperplastic nodule. Expression differences were shown by two-dimensional polyacrylamide gel electrophoresis and image analysis. Polypeptide masses were measured by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and their sequences were obtained by tandem mass spectrometry. Alterations in expression of cytoskeletal and functional proteins were demonstrated, consistent with biological changes known to occur in the preneoplastic cells. Of particular interest was the differential expression of a serine protease inhibitor (serpin) with a role implicated in angiogenesis. Serpin, implicated in the inhibition of angiogenesis, is present in normal liver but has greatly reduced expression at the preneoplastic stage of liver cancer development. Immunofluorescence microscopy with antibodies to this serpin, kallistatin, supports the proteomic identification. Immunofluorescence microscopy with antibodies to the blood vessel marker von Willebrand factor provides evidence for neovascularization in the liver containing multiple preneoplastic nodules. These observations suggest that at an early stage of liver carcinogenesis reduction or loss of angiogenesis inhibitors may contribute to initiation of neoangiogenesis. A number of other identified proteins known to be associated with hepatomas are also present at early-stage neoplasia.

Cyclin D1 determines mitochondrial function in vivo.

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.

Cyclin D1 repression of nuclear respiratory factor 1 integrates nuclear DNA synthesis and mitochondrial function.

Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. Herein, cyclin D1 deficiency increased mitochondrial size and activity that was rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1), which induces nuclear-encoded mitochondrial genes, was repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis and mitochondrial function.

An optimal culture condition maintains human hepatocyte phenotype after long-term culture.

BACKGROUND: Long-term culture of primary hepatocytes from various species is impeded by a decrease of cell viability and a loss of hepatocyte-specific function. The aim of the present study was to investigate whether our optimal culture condition (OC) can maintain the phenotype of primary hepatocytes in long-term culture. METHODS: Primary human hepatocytes were cultured in either hepatocyte maintenance medium (HM) or OC for 2-4 weeks. Expression of hepatocyte-specific genes was determined by real-time quantitative RT-PCR. RESULTS: The level of albumin mRNA in human hepatocytes cultured in OC was 11-fold more than in HM and gene expression levels of alpha1-antitrypsin and transferrin were at approximately 40 and 11% of freshly isolated primary human hepatocytes. Electron microscopy revealed that cells in OC displayed hepatocyte properties (e.g. polarity, junctional complexes, bile canaliculi and glycogen particles). Cytochrome P4501A1/2 activity of hepatocytes cultured in OC was 15- and 17-fold higher than in HM at 2 and 4 weeks of culture, and DNA synthesis was higher. CONCLUSIONS: Using our optimal culture condition, we were able to maintain the phenotype of primary human hepatocytes in long-term culture. They not only maintain better liver-specific function, but also retain higher proliferative potential.

Interaction with PDZK1 is required for expression of organic anion transporting protein 1A1 on the hepatocyte surface.

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

Vascular binding, blood flow, transporter, and enzyme interactions on the processing of digoxin in rat liver.

The roles of vascular binding, flow, transporters, and enzymes as determinants of the clearance of digoxin were examined in the rat liver. Digoxin is metabolized by Cyp3a and utilizes the organic anion transporting polypeptide 2 (Oatp2) and P-glycoprotein (Pgp) for influx and excretion, respectively. Uptake of digoxin was found to be similar among rat periportal (PP) and perivenous (PV) hepatocytes isolated by the digitonin-collagenase method. The Km values for uptake were 180 +/- 112 and 390 +/- 406 nM, Vmax values were 13 +/- 8 and 18 +/- 4.9 pmol/min/mg protein, and nonsaturable components were 9.2 +/- 1.3 and 10.7 +/- 2.5 microl/min/mg for PP and PV, respectively. The evenness of distribution of Oatp2 and Pgp was confirmed by Western blotting and confocal immunofluorescent microscopy. When digoxin was recirculated to the rat liver preparation in Krebs-Henseleit bicarbonate (KHB) for 3 h in absence or presence of 1% bovine serum albumin (BSA) and 20% red blood cell (rbc) at flow rates of 40 and 10 ml/min, respectively, biexponential decays were observed. Fitted results based on compartmental analyses revealed a higher clearance (0.244 +/- 0.082 ml/min/g) for KHB-perfused livers over the rbc-albumin-perfused livers (0.114 +/- 0.057 ml/min/g) (P < 0.05). We further found that binding of digoxin to 1% BSA was modest (unbound fraction = 0.64), whereas binding to rbc was associated with slow on (0.468 +/- 0.021 min(-1)) and off (1.81 +/- 0.12 min(-1)) rate constants. We then used a zonal, physiologically based pharmacokinetic model to show that the difference in digoxin clearance was attributed to binding to BSA and rbc and not to the difference in flow rate and that clearance was unaffected by transporter or enzyme heterogeneity.

Impaired response of biliary lipid secretion to a lithogenic diet in phosphatidylcholine transfer protein-deficient mice.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that is highly expressed in liver and catalyzes intermembrane transfer of phosphatidylcholines in vitro. To explore a role for PC-TP in the hepatocellular trafficking of biliary phosphatidylcholines, we characterized biliary lipid secretion using Pctp(-/-) and wild-type littermate control mice with C57BL/6J and FVB/NJ genetic backgrounds, which express PC-TP at relatively high and low levels in liver, respectively. Eight-week-old male Pctp(-/-) and wild-type mice were fed a chow diet or a lithogenic diet, which served to upregulate biliary lipid secretion. In chow-fed mice, the absence of PC-TP did not reduce biliary phospholipid secretion or alter the phospholipid composition of biles. However, the responses in secretion of biliary phospholipids, cholesterol, and bile salts to the lithogenic diet were impaired in Pctp(-/-) mice from both genetic backgrounds. Alterations in biliary lipid secretion could not be attributed to transcriptional regulation of the expression of canalicular membrane lipid transporters, but possibly to a defect in their trafficking to the canalicular membrane. These findings support a role for PC-TP in the response of biliary lipid secretion to a lithogenic diet, but not specifically in the hepatocellular transport and secretion of phosphatidylcholines.

The role of extrahepatic retinol binding protein in the mobilization of retinoid stores.

Although the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP(-/-)) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP(-/-)). By comparing hRBP(-/-), RBP(-/-), and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical. Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed.

Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation.

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver.

To determine whether disruption of the hepatic sinusoidal endothelium will facilitate engraftment of transplanted cells, we treated Fischer 344 (F344) rats lacking dipeptidyl peptidase IV (DPPIV) activity with cyclophosphamide (CP). Electron microscopy showed endothelial injury within 6 hours following CP, and, after 24 and 48 hours, the endothelium was disrupted in most hepatic sinusoids. CP did not affect Kupffer cell function. Similarly, CP had no obvious effects on hepatocytes. Intrasplenic transplantation of F344 rat hepatocytes followed by their localization with DPPIV histochemistry showed 3- to 5-fold increases in the number of transplanted cells in CP-treated animals. Transplanted cells integrated in the liver parenchyma more rapidly in CP-treated animals, and hybrid bile canaliculi developed even 1 day after cell transplantation, which was not observed in control animals. To demonstrate whether improved cell engraftment translated into superior liver repopulation, recipient animals were conditioned with retrorsine and two-thirds partial hepatectomy (PH), which induces transplanted cell proliferation. CP treatment of these animals before cell transplantation significantly increased the number and size of transplanted cell foci. In conclusion, disruption of the hepatic sinusoidal endothelium was associated with accelerated entry and integration of transplanted cells in the liver parenchyma. These results provide insights into hepatocyte engraftment in the liver and will help in optimizing liver-directed cell therapy.