Petar N. Grozdanov

Identification of adult hepatic progenitor cells capable of repopulating injured rat liver

Oval cells appear and expand in the liver when hepatocyte proliferation is compromised. Many different markers have been attributed to these cells, but their nature still remains obscure. This study is a detailed gene expression analysis aimed at revealing their identity and repopulating in vivo capacity. Oval cells were activated in 2‐acetylaminofluorene–treated rats subjected to partial hepatectomy or in D‐galactosamine–treated rats. Two surface markers [epithelial cell adhesion molecule (EpCAM) and thymus cell antigen 1 (Thy‐1)] were used for purification of freshly isolated cells. Their gene expression analysis was studied with Affymetrix Rat Expression Array 230 2.0, reverse‐transcriptase polymerase chain reaction, and immunofluorescent microscopy. We found that EpCAM+ and Thy‐1+ cells represent two different populations of cells in the oval cell niche. EpCAM+ cells express the classical oval cell markers (alpha‐fetoprotein, cytokeratin‐19, OV‐1 antigen, a6 integrin, and connexin 43), cell surface markers recently identified by us (CD44, CD24, EpCAM, aquaporin 5, claudin‐4, secretin receptor, claudin‐7, V‐ros sarcoma virus oncogene homolog 1, cadherin 22, mucin‐1, and CD133), and liver‐enriched transcription factors (forkhead box q, forkhead box a2, onecut 1, and transcription factor 2). Oval cells do not express previously reported hematopoietic stem cell markers Thy‐1, c‐kit, and CD34 or the neuroepithelial marker neural cell adhesion molecule 1. However, oval cells express a number of mesenchymal markers including vimentin, mesothelin, bone morphogenetic protein 7, and Tweak receptor (tumor necrosis factor receptor superfamily, member 12A). A group of novel differentially expressed oval cell genes is also presented. It is shown that Thy‐1+ cells are mesenchymal cells with characteristics of myofibroblasts/activated stellate cells. Transplantation experiments reveal that EpCAM+ cells are true progenitors capable of repopulating injured rat liver. Conclusion: We have shown that EpCAM+ oval cells are bipotential adult hepatic epithelial progenitors. These cells display a mixed epithelial/mesenchymal phenotype that has not been recognized previously. They are valuable candidates for liver cell therapy. (HEPATOLOGY 2007.)

Novel hepatic progenitor cell surface markers in the adult rat liver

Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2‐acetylaminofluorene treatment followed by partial hepatectomy (2‐AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT‐PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO‐1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO‐1 cells and 6 are unique for the adult progenitors (CD133, claudin‐7, cadherin 22, mucin‐1, ros‐1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep‐CAM antibodies from D‐galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. Conclusion: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity. (HEPATOLOGY 2007;45:139–149.)

Bone marrow progenitors are not the source of expanding oval cells in injured liver

Liver progenitor/oval cells differentiate into hepatocytes and biliary epithelial cells, repopulating the liver when the regenerative capacity of hepatocytes is impaired. Recent studies have shown that hematopoietic bone marrow (BM) stem/progenitor cells can give rise to hepatocytes in diseased/damaged liver. One study has reported that BM cells can transdifferentiate into liver progenitor/oval cells, but it has not been proven that the latter can repopulate the liver. To answer this question, we have lethally irradiated female DPP4− mutant F344 rats and transplanted them with 50 million wild‐type male F344 BM cells. One month after transplantation, the recipient BM was reconstituted with male hematopoietic cells, determined by quantitative polymerase chain reaction using primers for Y chromosome–specific sry gene. In addition, DPP4+ cells, single or in clusters and predominantly in the periportal region, were detected in all liver sections of recipient rats. Animals were subjected to the following three different liver injury protocols for activation and expansion of oval cells: D‐galactosamine, retrorsine/partial hepatectomy (Rs/PH), and 2‐acetylaminofluorene/partial hepatectomy (2‐AAF/PH). In all three models, prominent expansion and accumulation of cytokeratin 19–positive (CK‐19+) oval cells was observed. However, most of the DPP4+ clusters dispersed over time, and their total number decreased. Very few oval cells (less than 1%) showed double DPP4/CK‐19 labeling. None of the small hepatocytic clusters in the Rs/PH or 2‐AAF/PH model were comprised of DPP4+ cells. These data demonstrate that the sources of oval cells and small hepatocytes in the injured liver are endogenous liver progenitors and that they do not arise through transdifferentiation from BM cells.

The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells.

Glypican-3 (Gpc3), a cell surface-linked heparan sulfate proteoglycan is highly expressed during embryogenesis and is involved in organogenesis. Its exact biological function remains unknown. We have studied the expression of Gpc3 in fetal and adult liver, in liver injury models of activation of liver progenitor cells: D-galactosamine and 2-acetylaminofluorene (2-AAF) administration followed by partial hepatectomy (PH) (2-AAF/PH); and in the Solt-Farber carcinogenic model: by initiation with a single dose of diethylnitrosamine and promotion with 2-AAF followed by PH treatment. Gpc3 expression was studied using complementary DNA microarrays, reverse transcriptase-polymerase chain reaction, in situ hybridization (ISH); ISH combined with immunohistochemistry (IHC) and immunofluorescent microscopy. We found that Gpc3 is highly expressed in fetal hepatoblasts from embryonic days 13 through 16 and its expression gradually decreases towards birth. Dual ISH with Gpc3 and α-fetoprotein (AFP) probes confirmed that only hepatoblasts and no other fetal liver cells express Gpc3. At 3 weeks after birth the expression of Gpc3 mRNA and protein was hardly detected in the liver. Gpc3 expression was highly induced in oval cell of D-gal and 2-AAF/PH treated animals. Dual ISH/IHC with Gpc3 riboprobe and cytokeratin-19 (CK-19) antibody revealed that Gpc3 is expressed in activated liver progenitor cells. ISH for Gpc3 and AFP performed on serial liver sections also showed coexpression of the two-oncofetal proteins. FACS isolated oval cells with anti-rat Thy1 revealed expression of Gpc3. Gpc3 expression persists in atypical duct-like structures and liver lesions of animals subjected to the Solt-Farber model of initiation and promotion of liver cancer expressing CK-19. In this work we report for the first time that the oncofetal protein Gpc3 is a marker of hepatic progenitor cells and of early liver lesions. Our findings show further that hepatic progenitor/oval cells are the target for malignant transformation in the Solt-Farber model of hepatic carcinogenesis.

SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs

Assembly of H/ACA RNPs in yeast is aided by at least two accessory factors, Naf1p and Shq1p. Although the function of Naf1p and its human ortholog NAF1 has been delineated in detail, that of Shq1p and its putative human ortholog SHQ1 remains obscure. We demonstrate that SHQ1 indeed functions in the biogenesis of human H/ACA RNPs and we dissect its mechanism of action. Like NAF1, SHQ1 binds the major H/ACA core protein and pseudouridine synthase NAP57 (aka dyskerin) but precedes the assembly role of NAF1 at nascent H/ACA RNAs because the interaction of SHQ1 with NAP57 in vivo and in vitro precludes that of NAF1 and of the other H/ACA core proteins that are present at the sites of H/ACA RNA transcription. The N-terminal heat shock protein 20-like CS domain of SHQ1 is dispensable for NAP57 binding. Consistent with its role as an assembly factor, SHQ1 localizes to the nucleoplasm and is excluded from nucleoli and Cajal bodies, the sites of mature H/ACA RNPs. In an in vitro assembly system of functional H/ACA RNPs that is dependent on NAF1, excess recombinant SHQ1 interferes with assembly. Importantly, knockdown of cellular SHQ1 prevents accumulation of a newly synthesized H/ACA reporter RNA and generally reduces the levels of endogenous H/ACA RNAs including telomerase RNA. In summary, the sequential action of SHQ1 and NAF1 is required for functional assembly of H/ACA RNPs in vivo and in vitro. This step-wise process could serve as an efficient means of quality control during H/ACA RNP assembly.

The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic

SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) required for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. SHQ1 binds dyskerin/NAP57, the catalytic subunit of human H/ACA RNPs, and this interaction is modulated by mutations causing X-linked dyskeratosis congenita. We report the crystal structure of the C-terminal domain of yeast SHQ1, Shq1p, and its complex with yeast dyskerin/NAP57, Cbf5p, lacking its catalytic domain. The C-terminal domain of Shq1p interacts with the RNA-binding domain of Cbf5p and, through structural mimicry, uses the RNA-protein-binding sites to achieve a specific protein-protein interface. We propose that Shq1p operates as a Cbf5p chaperone during RNP assembly by acting as an RNA placeholder, thereby preventing Cbf5p from nonspecific RNA binding before association with an H/ACA RNA and the other core RNP proteins.

Pathogenic NAP57 mutations decrease ribonucleoprotein assembly in dyskeratosis congenita

X-linked dyskeratosis congenita (DC) is a rare bone marrow failure syndrome caused by mostly missense mutations in the pseudouridine synthase NAP57 (dyskerin/Cbf5). As part of H/ACA ribonucleoproteins (RNPs), NAP57 is important for the biogenesis of ribosomes, spliceosomal small nuclear RNPs, microRNAs and the telomerase RNP. DC mutations concentrate in the N- and C-termini of NAP57 but not in its central catalytic domain raising questions as to their impact. We demonstrate that the N- and C-termini together form the binding surface for the H/ACA RNP assembly factor SHQ1 and that DC mutations modulate the interaction between the two proteins. Pinpointing impaired interaction between NAP57 and SHQ1 as a potential molecular basis for X-linked DC has implications for therapeutic approaches, e.g. by targeting the NAP57-SHQ1 interface with small molecules.

Thymus cell antigen-1-expressing cells in the oval cell compartment†

Thymus cell antigen‐1 (Thy‐1)‐expressing cells proliferate in the liver during oval cell (OC)‐mediated liver regeneration. We characterized these cells in normal liver, in carbon tetrachloride‐injured liver, and in several models of OC activation. The gene expression analyses were performed using reverse‐transcriptase polymerase chain reaction (RT‐PCR), quantitative RT‐PCR (Q‐RT‐PCR) of cells isolated by fluorescence‐activated cell sorting (FACS), and by immunofluorescent microscopy of tissue sections and isolated cells. In normal liver, Thy‐1+ cells are a heterogeneous population: those located in the periportal region do not coexpress desmin or alpha smooth muscle actin (α‐SMA). The majority of Thy‐1+ cells located at the lobular interface and in the parenchyma coexpress desmin but not α‐SMA, i.e., they are not resident myofibroblasts. Although Thy‐1+ cells proliferate moderately after carbon tetrachloride injury, in all models of OC‐mediated liver regeneration they proliferate quickly and expand significantly and disappear from the liver when the OC response subsides. Activated Thy‐1+ cells do not express OC genes but they express genes known to be expressed in mesenchymal stem cells (CD105, CD73, CD29), genes considered specific for activated stellate cells (desmin, collagen I‐a2, Mmp2, Mmp14) and myofibroblasts (α‐SMA, fibulin‐2), as well as growth factors and cytokines (Hgf, Tweak, IL‐1b, IL‐6, IL‐15) that can affect OC growth. Activated in vitro stellate cells do not express Thy‐1. Subcloning of Thy‐1+ cells from OC‐activated livers yield Thy‐1+ fibroblastic cells and a population of E‐cadherin+ mesenchymal cells that gradually discontinue expression of Thy‐1 and begin to express cytokeratins. However, upon transplantation these cells do not differentiate into hepatocytes or cholangiocytes. Activated Thy‐1+ cells produce predominantly latent transforming growth factor beta. Conclusion: Thy‐1+ cells in the OC niche are activated mesenchymal‐epithelial cells that are distinct from resident stellate cells, myofibroblasts, and oval cells. (HEPATOLOGY 2009.)

Distant positioning of proteasomal proteolysis relative to actively transcribed genes

While it is widely acknowledged that the ubiquitin–proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.