Hsiang-Po Huang

Regulation of the Pancreatic Islet-Specific Gene BETA2 (neuroD) by Neurogenin 3

ABSTRACT The BETA2 (neuroD) gene is expressed in endocrine cells during pancreas development and is essential for proper islet morphogenesis. The objective of this study is to identify potential upstream regulators of the BETA2 gene during pancreas development. We demonstrated that the expression of neurogenin 3 (ngn3), an islet- and neuron-specific basic-helix-loop-helix transcription factor, partially overlaps that of BETA2 during early mouse development. More importantly, overexpression of ngn3 can induce the ectopic expression of BETA2 in Xenopus embryos and stimulate the endogenous RNA of BETA2 in endocrine cell lines. Furthermore, overexpression of ngn3 could cause a dose-dependent activation on the 1.0-kb BETA2 promoter in islet-derived cell lines. Deletion and mutation analyses revealed that two proximal E box sequences, E1 and E3, could bind to ngn3-E47 heterodimer and mediate ngn3 activation. Based on these results, we hypothesize that ngn3 is involved in activating the expression of BETA2 at an early stage of islet cell differentiation through the E boxes in theBETA2 promoter.

Sorafenib Overcomes Trail Resistance of Hepatocellular Carcinoma Cells through the Inhibition of Stat3

Purpose: Recombinant tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, many hepatocellular carcinoma (HCC) cells show resistance to TRAIL-induced apoptosis. Here, we report that sorafenib improves the antitumor effect of TRAIL-related agents in resistant HCC. Experimental Design: HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib and/or TRAIL-related agents (TRAIL or LBY135) and analyzed in terms of apoptosis and signal transduction. In vivo efficacy was determined in nude mice with PLC5 xenografts. Results: Sorafenib, the only approved drug for HCC, sensitizes resistant HCC cells to an agonistic DR5 antibody (LBY135) and TRAIL-induced apoptosis in TRAIL-resistant HCC cells. We found that STAT3 played a significant role in mediating TRAIL sensitization. Our data showed that sorafenib downregulated phospho-STAT3 (pSTAT3) and subsequently reduced the expression levels of STAT3-related proteins (Mcl-1, survivin, and cyclin D1) in a dose- and time-dependent manner in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the TRAIL-sensitizing effect of sorafenib. Moreover, SHP-1 inhibitor reversed downregulation of pSTAT3 and apoptosis induced by sorafenib, and silencing of SHP-1 by RNA interference abolished the effects of sorafenib on pSTAT3. Notably, sorafenib increased SHP-1 activity in PLC5 cells. Finally, sorafenib plus LBY135 significantly suppressed PLC5 xenograft tumor growth. Conclusions: Sorafenib sensitizes resistant HCC cells to TRAIL-induced apoptosis at clinical achievable concentrations, and this effect is mediated via the inhibition of STAT3. Clin Cancer Res; 16(21); 5189–99. ©2010 AACR.

Signal transducer and activator of transcription 3 is a major kinase-independent target of sorafenib in hepatocellular carcinoma

BACKGROUND & AIMS Recently, we reported that sorafenib sensitizes hepatocellular carcinoma (HCC) cells to TRAIL through the inhibition of signal transducer and activator of transcription 3 (STAT3). Here, we report that sorafenib inhibits HCC via a kinase-independent mechanism: SHP-1 dependent STAT3 inactivation. METHODS SC-1 is a sorafenib derivative that closely resembles sorafenib structurally but with no kinase inhibition activity. HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib or SC-1 and apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with Huh-7 xenografts. RESULTS SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis in all tested HCC cell lines. SC-1 down-regulated phosphorylation of phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested HCC cells. Expression of STAT3-driven genes, including Cyclin D1 and Survivin, was also repressed by SC-1. Luciferase reporter assay confirmed the inhibition of transcriptional activity of STAT3 in both sorafenib-treated and SC-1-treated cells. Ectopic expression of STAT3 in PLC5 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 up-regulated SHP-1 activity. Knockdown of SHP-1, but not SHP-2 or PTP-1B, by small interference RNA reduced apoptosis induced by SC-1. Finally, SC-1 reduced Huh-7 tumor growth significantly in vivo, which was associated with down-regulation of p-STAT3 and up-regulation of SHP-1 activity. CONCLUSIONS STAT3 is a major kinase-independent target of sorafenib in HCC.

Tbx19, a tissue-selective regulator of POMC gene expression.

Pituitary cell types arise in a temporally and spatially specific fashion, in response to combinatorial actions of transcription factors induced by transient signaling gradients. The critical transcriptional determinants of the two pituitary cell types that express the pro-opiomelanocortin (POMC) gene, the anterior lobe corticotropes, producing adrenocorticotropin, and the intermediate lobe melanotropes, producing melanocyte-stimulating hormone (MSHα), have remained unknown. Here, we report that a member of the T-box gene family, Tbx19, which is expressed only in the rostral ventral diencephalon and pituitary gland, commencing on e11.5, marks pituitary cells that will subsequently express the POMC gene and is capable of altering progression of ventral cell types and inducing adrenocorticotropin in rostral tip cells. It is suggested that Tbx19, depending on the presence of synergizing transcription factors, can activate POMC gene expression and repress the α glycoprotein subunit and thyroid-stimulating hormone β promoters.

Human Pompe disease induced pluripotent stem cells for pathogenesis modeling, drug testing and disease marker identification

Pompe disease is caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene, which encodes GAA. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease-induced pluripotent stem cells (PomD-iPSCs) from two patients with different GAA mutations and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to recombinant human GAA reversed the major pathologic phenotypes. Furthermore, l-carnitine treatment reduced defective cellular respiration in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria-related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for the development of novel therapeutic strategies for Pompe disease.

Mice Deficient in Collapsin Response Mediator Protein-1 Exhibit Impaired Long-Term Potentiation and Impaired Spatial Learning and Memory

Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. We generated viable and fertile CRMP-1 knock-out (CRMP-1−/−) mice with no evidence of gross abnormality in the major organs. CRMP-1−/− mice exhibited intense microtubule-associated protein 2 (MAP2) staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 (growth-associated protein-43) and PSD95 (postsynaptic density-95) (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knock-out mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1−/− mice showed a normal synapsin I labeling pattern in CA1 and normal paired-pulse facilitation. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory.

Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.

Background: EpCAM is highly expressed in ESCs. However, the role of EpCAM complex proteins in pluripotency reprogramming is still unknown. Results: Overexpression of EpCAM complex proteins significantly repressed the expression of p53 and enhanced reprogramming efficiency in MEFs. Conclusion: EpCAM signaling enhance reprogramming through suppression of the p53-p21 pathway. Significance: EpCAM signaling enhance reprogramming through suppression of the p53-p21 pathway. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.

Distinct Hepatitis B Virus Dynamics in the Immunotolerant and Early Immunoclearance Phases

ABSTRACT Little is known about hepatitis B virus (HBV) diversity changes within a host during the immunotolerant phase of chronic HBV infection. Such knowledge, nevertheless, may help in understanding how host immunity and HBV interact at the early stage of infection. In this study, serial serum samples were collected from a long-term (>17 years) follow-up cohort of seven patients, and multiple copies of the full-length viral genome from serially sampled sera were recovered and analyzed. Viral genetic diversity was positively correlated with host immunity, represented by levels of alanine aminotransferase (ALT), but was negatively correlated with the viral copy number. During the immunotolerant phase, when the host immunity was feeble (ALT < 20 U/liter), viral nucleotide diversity decreased while copy numbers increased. Rates of evolutionary change derived for different patients were in a very narrow range (1.6 × 10−5 to 5.4 × 10−5/site/year). As the disease progressed toward the immunoclearance phase (ALT > 20 U/liter), viral diversity increased but copy numbers decreased. Evolutionary rates varied among patients in accordance with their levels of ALT, ranging from 9.6 × 10−6 to 3.2 × 10−4/site/year. More than half (19/32 sites) of positively selected sites resided in immune epitopes, suggesting their possible role in host immunity. Our results demonstrate that host immunity is a dominant factor in HBV evolution. Different selective forces, including immune-mediated positive selection and virus-mediated negative selection, operate in tandem in shaping viral population dynamics within a host.

BETA3, a novel helix-loop-helix protein, can act as a negative regulator of BETA2 and MyoD-responsive genes.

Using degenerate PCR cloning we have identified a novel basic helix-loop-helix (bHLH) transcription factor, BETA3, from a hamster insulin tumor (HIT) cell cDNA library. Sequence analysis revealed that this factor belongs to the class B bHLH family and has the highest degree of homology with another bHLH transcription factor recently isolated in our laboratory, BETA2 (neuroD) (J. E. Lee, S. M. Hollenberg, L. Snider, D. L. Turner, N. Lipnick, and H. Weintraub, Science 268:836-844, 1995; F. J. Naya, C. M. M. Stellrecht, and M.-J. Tsai, Genes Dev. 8:1009-1019, 1995). BETA2 is a brain- and pancreatic-islet-specific bHLH transcription factor and is largely responsible for the tissue-specific expression of the insulin gene. BETA3 was found to be tissue restricted, with the highest levels of expression in HIT, lung, kidney, and brain cells. Surprisingly, despite the homology between BETA2 and BETA3 and its intact basic region, BETA3 is unable to bind the insulin E box in bandshift analysis as a homodimer or as a heterodimer with the class A bHLH factors E12, E47, or BETA1. Instead, BETA3 inhibited both the E47 homodimer and the E47/BETA2 heterodimer binding to the insulin E box. In addition, BETA3 greatly repressed the BETA2/E47 induction of the insulin enhancer in HIT cells as well as the MyoD/E47 induction of a muscle-specific E box in the myoblast cell line C2C12. In contrast, expression of BETA3 had no significant effect on the GAL4-VP16 transcriptional activity. Immunoprecipitation analysis demonstrates that the mechanism of repression is via direct protein-protein interaction, presumably by heterodimerization between BETA3 and class A bHLH factors.

Surface Marker Epithelial Cell Adhesion Molecule and E-cadherin Facilitate the Identification and Selection of Induced Pluripotent Stem Cells

The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1 (SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation.