Tatyana Tchaikovskaya

Elevated Expression of the miR-17–92 Polycistron and miR-21 in Hepadnavirus-Associated Hepatocellular Carcinoma Contributes to the Malignant Phenotype

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.

MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads

Transforming growth factor‐beta / bone morphogenetic protein (TGFβ/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFβ ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR‐23b cluster miRNAs comprising miR‐23b, miR‐27b, and miR‐24‐1 and miR‐10a, miR‐26a, and miR‐30a as up‐regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR‐23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR‐23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFβ signaling molecules, as putative miR‐23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3′ UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down‐regulation of constructs containing Smad 3, 4, or 5, 3′ UTRs by a mixture of miR‐23b cluster mimics. Knockdown of miR‐23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC‐3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR‐23b mimics during bile duct differentiation of HBC‐3 cells blocked the process. Conclusion: Our data provide a model in which miR‐23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down‐regulating Smads and consequently TGFβ signaling. Concomitantly, low levels of the miR‐23b miRNAs are needed in cholangiocytes to allow TGFβ signaling and bile duct formation. (HEPATOLOGY 2009.)

Glutathione S-transferase hGSTM3 and ageing-associated neurodegeneration: relationship to Alzheimer's disease

Glutathione S-transferases (GSTs) are detoxification enzymes that can counter ageing-associated oxidative and chemical stresses. The transcript of a distinct subclass of human GSTs (hGSTM3) was shown by RNA blot analysis to be widely distributed in different regions of adult brain. HPLC profiles indicated that the hGSTM3 subunit was the second most abundant GST subunit in brain. Immunocytochemistry performed with hGSTM3-specific antisera, showed prominent staining of neuritic plaques, neurofibrillary tangles and microglia in sections of hippocampus obtained from patients with Alzheimers disease. The staining pattern was distinct from that obtained with normal brains. Because hGSTM3 is rich in cysteine residues and readily undergoes S-glutathiolation reactions, deposition of this protein could originate from cross-links produced by oxidative stress.

Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

Summary Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.

HUMAN TESTICULAR GLUTATHIONE S-TRANSFERASES : INSIGHTS INTO TISSUE-SPECIFIC EXPRESSION OF THE DIVERSE SUBUNIT CLASSES

Cytosolic glutathione S-transferase (GST) subunits from human testis were resolved by HPLC and unambiguously identified by combined use of peptide sequence-specific antisera and electrospray ionization mass spectrometry (ESI MS). Allelic variants of hGSTP1, hGSTM1 and hGSTA2 were distinguished on the basis of observed differences in their molecular masses. Relative amounts of the multiple different subunit types in various human tissues were determined from HPLC profiles. From this type of analysis, tissues from hGSTM1 null allele individuals were readily discerned at the protein level; liver was the only tissue in which the hGSTM1 subunit was the major mu-class GST. hGSTM4 and hGSTM5 subunits were found at very low levels in all tissues examined. By far the tissue richest in the unique hGSTM3 subunit was testis, although brain also has significant levels.

Rat glutathione S-transferase M4-4: An isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

The Proximal Promoter Governs Germ Cell-Specific Expression of the Mouse Glutathione Transferase mGstm5 Gene

To explain the tissue‐selective expression patterns of a distinct subclass of glutathione S‐transferase (GST), transgenic mice expressing EGFP under control of a 2 kb promoter sequence in the 5′‐flanking region of the mGstm5 gene were produced. The intent of the study was to establish whether the promoter itself or whether posttranscriptional mechanisms, particularly at the levels of mRNA translation and stability or protein targeting, based on unique properties of mGSTM5, determine the restricted expression pattern. Indeed, the transgene expression was limited to testis as the reporter was not detected in somatic tissues such as brain, kidney or liver, indicating that the mGstm5 proximal promoter is sufficient to target testis‐specific expression of the gene. EGFP expression was also more restricted vis‐a‐vis the natural mGstm5 gene and exclusively found in germ but not in somatic cells. Real‐time quantitative PCR (qPCR) data were consistent with alternate transcription start sites in which the promoter region of the natural mGstm5 gene in somatic cells is part of exon 1 of the germ cell transcript. Thus, the primary transcription start site for mGstm5 is upstream of a TATA box in testis and downstream of this motif in somatic cells. The 5′ flanking sequence of the mGstm5 gene imparts germ cell‐specific transcription. Mol. Reprod. Dev. 76: 379–388, 2009.

Differentiation in stem/progenitor cells along fetal or adult hepatic stages requires transcriptional regulators independently of oscillations in microRNA expression

ABSTRACT Understanding mechanisms in lineage differentiation is critical for organ development, pathophysiology and oncogenesis. To determine whether microRNAs (miRNA) may serve as drivers or adjuncts in hepatic differentiation, we studied human embryonic stem cell‐derived hepatocytes and primary hepatocytes representing fetal or adult stages. Model systems were used for hepatic lineage advancement or regression under culture conditions with molecular assays. Profiles of miRNA in primary fetal and adult hepatocytes shared similarities and distinctions from pluripotent stem cells or stem cell‐derived early fetal‐like hepatocytes. During phenotypic regression in fetal or adult hepatocytes, miRNA profiles oscillated to regain stemness‐associated features that had not been extinguished in stem cell‐derived fetal‐like hepatocytes. These oscillations in stemness‐associated features were not altered in fetal‐like hepatocytes by inhibitory mimics for dominantly‐expressed miRNA, such as hsa‐miR‐99b, ‐100, ‐214 and ‐221/222. The stem cell‐derived fetal‐like hepatocytes were permissive for miRNA characterizing mature hepatocytes, including mimics for hsa‐miR‐122, ‐126, ‐192, ‐194 and ‐26b, although transfections of the latter did not advance hepatic differentiation. Examination of genome‐wide mRNA expression profiles in stem cell‐derived or primary fetal hepatocytes indicated targets of highly abundant miRNA regulated general processes, e.g., cell survival, growth and proliferation, functional maintenance, etc., without directing cell differentiation. Among upstream regulators of gene networks in stem cell‐derived hepatocytes included HNF4A, SNAI1, and others, which affect transcriptional circuits directing lineage development or maintenance. Therefore, miRNA expression oscillated in response to microenvironmental conditions, whereas lineage‐specific transcriptional regulators, such as HNF4A, were necessary for directing hepatic differentiation. This knowledge will be helpful for understanding the contribution of stem cells in pathophysiological states and oncogenesis, as well as for applications of stem cell‐derived hepatocytes. HIGHLIGHTSThe role of microRNAs in differentiation of stem cells is not well understood.This study found microRNA levels oscillated during differentiation of hepatocytes.Many functions but not differentiation were regulated by microRNAs in hepatocytes.Regulators of gene networks independently maintained hepatic differentiation.These insights will advance how stem cells may contribute in disease mechanisms and therapies.