Honggang Ye

Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues.

The hepatocyte nuclear factor 3alpha (HNF-3alpha) and 3beta proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription factor family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-29 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-29, Caco-2, HepG2, HeLa, A549, and H441). We demonstrate that differentiation of Caco-2 cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3alpha and HNF-3beta mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühns crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, 24, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of keratinocyte growth factor in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription factor is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.

Increased levels of forkhead box M1B transcription factor in transgenic mouse hepatocytes prevent age-related proliferation defects in regenerating liver

The forkhead box (Fox) family of transcription factors share homology in the winged helix/forkhead DNA-binding domain and play important roles in regulating cellular proliferation, differentiation, longevity, and cellular transformation. Forkhead box M1B (FoxM1B) is a ubiquitously expressed member of the Fox transcription factor family whose expression is restricted to proliferating cells and that mediates hepatocyte entry into DNA synthesis and mitosis during liver regeneration. Recent cDNA microarray studies indicated that age-related defects in cellular proliferation are associated with diminished expression of the FoxM1B transcription factor. Here, we show that increased levels of FoxM1B in regenerating liver of old transgenic mice restore the sharp peaks in hepatocyte DNA replication and mitosis that are the hallmarks of young regenerating mouse liver. Restoration of the young regenerating liver phenotype is associated with increased expression of numerous cell cycle regulatory genes that include cyclin D1, cyclin A2, cyclin F, cyclin B1, cyclin B2, Cdc25B, and p55cdc. Cotransfection assays in the human hepatoma HepG2 cell line demonstrated that FoxM1B protein stimulated expression of both the cyclin B1 and cyclin D1 promoters, suggesting that these cyclin genes are a direct FoxM1B transcriptional target. These results suggest that FoxM1B controls the transcriptional network of genes that are essential for cell division and exit from mitosis. Our results indicate that reduced expression of the FoxM1B transcription factor contributes to the decline in cellular proliferation observed in the aging process.

Premature Expression of the Winged Helix Transcription Factor HFH-11B in Regenerating Mouse Liver Accelerates Hepatocyte Entry into S Phase

ABSTRACT Two-thirds partial hepatectomy (PH) induces differentiated cells in the liver remnant to proliferate and regenerate to its original size. The proliferation-specific HNF-3/fork head homolog-11B protein (HFH-11B; also known as Trident and Win) is a family member of the winged helix/fork head transcription factors and in regenerating liver its expression is reactivated prior to hepatocyte entry into DNA replication (S phase). To examine whether HFH-11B regulates hepatocyte proliferation during liver regeneration, we used the −3-kb transthyretin (TTR) promoter to create transgenic mice that displayed ectopic hepatocyte expression of HFH-11B. Liver regeneration studies with the TTR–HFH-11B mice demonstrate that its premature expression resulted in an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This liver regeneration phenotype is associated with protracted expression of cyclin D1 and C/EBPβ, which are involved in stimulating DNA replication and premature expression of M phase promoting cyclin B1 and cdc2. Consistent with the early hepatocyte entry into S phase, regenerating transgenic livers exhibited earlier expression of DNA repair genes (XRCC1, mHR21spA, and mHR23B). Furthermore, in nonregenerating transgenic livers, ectopic HFH-11B expression did not elicit abnormal hepatocyte proliferation, a finding consistent with the retention of the HFH-11B transgene protein in the cytoplasm. We found that nuclear translocation of the HFH-11B transgene protein requires mitogenic signalling induced by PH and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than in wild type.

The winged helix transcriptional activator HFH-8 is expressed in the mesoderm of the primitive streak stage of mouse embryos and its cellular derivatives.

The hepatocyte nuclear factor 3/fork head homolog (HFH) proteins are an extensive family of transcription factors which share homology in the winged helix DNA binding domain. Members of the winged helix family have been implicated in cell fate determination during pattern formation, in organogenesis and in cell type-specific gene expression. In this study, we used in situ hybridization to identify the cellular expression pattern of the winged helix transcription factor, HFH-8, during mouse embryonic development. We showed that HFH-8 expression initiates during the primitive streak stage of mouse embryogenesis in the extraembryonic mesoderm and in the lateral mesoderm which gives rise to the somatopleuric and splanchnopleuric mesoderm. During organogenesis, HFH-8 expression is found in the splanchnic mesoderm in close apposition of the gut endoderm, suggesting a role in mesenchymal-epithelial induction of lung and gut morphogenesis. HFH-8 expression continues in lateral mesoderm-derived tissue throughout mouse development. HFH-8 expression is observed in the mesenchymal cells of the oral cavity, esophagus, trachea, lung, intestine, dorsal aorta and intersomitic arteries, but not in the vasculature of the head, liver, kidney or heart. Consistent with these embryonic expression studies, adult HFH-8 expression is restricted to the endothelium and connective fibroblasts of the alveolar sac and in the lamina propria and smooth muscle of the intestine. We also show that several adult endothelial cell lines maintain abundant HFH-8 expression. Furthermore, we used our determined HFH-8 consensus sequence to identify putative target genes expressed in pulmonary and intestinal mesenchymal cells. Cotransfection assays with one of these target promoters, P-selectin, demonstrated that HFH-8 expression was required for IL-6 stimulation of P-selectin promoter activity and suggest that HFH-8 is involved in mediating its cell-specific transcriptional activation in response to cytokines.

Genesis, a Winged Helix transcriptional repressor, has embryonic expression limited to the neural crest, and stimulates proliferation in vitro in a neural development model

Abstract A novel repressor of the Winged Helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis (also called HFH2), was previously found to be exclusively expressed in primitive embryonic cell lines. In this study in situ cRNA hybridization experiments revealed that Genesis was expressed during embryogenesis only in developing neural crest cells. Its expression diminished upon their terminal differentiation into sympathetic and parasympathetic neurons. Based on that finding, Genesis was retrovirally transduced into pluripotent N-Tera-2 clone D1 (NT2/D1) teratocarcinoma cells, which are a well-described in vitro model of neural development. Retinoic acid (RA) treatment will drive these cells to differentiation toward the neuronal lineage and cause an increase in expression of the cyclin-dependent kinase inhibitor p21 protein, which leads to an inhibition in cellular proliferation. Although RA-induced expression of neuronal differentiation markers was not influenced by forced overexpression of Genesis in NT2-D1 cells, proliferation of Genesis-transduced cells continued following RA treatment. RA was unable to induce the expression of the cyclin-dependent kinase inhibitor p21 in the Genesis-transduced cells, but Go/G1 tumor suppressor p53 expression was induced normally. Therefore, Genesis may play a role in the regulation of primitive neural crest development by preventing terminal quiescence through inhibition of p21 protein expression. These data also lend evidence for the hypothesis that proliferation and differentiation pathways are not irrevocably linked, but can function independently.