A combined in vitro-in silico method for assessing the androgenic activities of bisphenol A and its analogues.

Interactions between endocrine-disruptor chemicals (EDCs) and androgen receptor (AR) have adverse effects on the endocrine system, leading to human reproductive dysfunction. Bisphenol A (BPA) is an EDC that can damage both the environment and human health. Although numerous BPA analogues have been produced as substitutes for BPA, few studies have evaluated their endocrine-disrupting abilities. We assessed the (anti)-androgenic activities of BPA and its analogues using a yeast-based reporter assay. The BPA analogues tested were bisphenol S (BPS), 4-phenylphenol (4PP), 4,4'-(9-fluorenyliden)-diphenol (BPFL), tetramethyl bisphenol F (TMBPF), and tetramethyl bisphenol A (TMBPA). We also conducted molecular docking and dynamics simulations to assess the interactions of BPA and its analogues with the ligand-binding domain of human AR (AR-LBD). Neither BPA nor its analogues had androgenic activity; however, all except BPFL exerted robust anti-androgenic effects. Consistent with the in vitro results, anti-androgenic analogues of BPA formed hydrogen bonding patterns with key residues that differed from the patterns of endogenous hormones, indicating that the analogues display in inappropriate orientations when interacting with the binding pocket of AR-LBD. Our findings indicate that BPA and its analogues disrupt androgen signaling by interacting with the AR-LBD. Overall, BPA and its analogues display endocrine-disrupting activity, which is mediated by AR.

Affinity prediction using deep learning based on SMILES input for D3R grand challenge 4.

Modern molecular docking comprises the prediction of pose and affinity. Prediction of docking poses is required for affinity prediction when three-dimensional coordinates of the ligand have not been provided. However, a large number of feature engineering is required for existing methods. In addition, there is a need for a robust model for the sequential combination of pose and affinity prediction due to the probabilistic deviation of the ligand position issue. We propose a pipeline using a bipartite graph neural network and transfer learning trained on a re-docking dataset. We evaluated our model on the released data from drug design data resource grand challenge 4 (D3R GC4). The two target protein data provided by the challenge have different patterns. The model outperformed the best participant by 9% on the BACE target protein from stage 2. Further, our model showed competitive performance on the CatS target protein.

Donor cell memory confers a metastable state of directly converted cells.

Generation of induced oligodendrocyte progenitor cells (iOPCs) from somatic fibroblasts is a strategy for cell-based therapy of myelin diseases. However, iOPC generation is inefficient, and the resulting iOPCs exhibit limited expansion and differentiation competence. Here we overcome these limitations by transducing an optimized transcription factor combination into a permissive donor phenotype, the pericyte. Pericyte-derived iOPCs (PC-iOPCs) are stably expandable and functionally myelinogenic with high differentiation competence. Unexpectedly, however, we found that PC-iOPCs are metastable so that they can produce myelination-competent oligodendrocytes or revert to their original identity in a context-dependent fashion. Phenotypic reversion of PC-iOPCs is tightly linked to memory of their original transcriptome and epigenome. Phenotypic reversion can be disconnected from this donor cell memory effect, and in vivo myelination can eventually be achieved by transplantation of O4+ pre-oligodendrocytes. Our data show that donor cell source and memory can contribute to the fate and stability of directly converted cells.

Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Reprogramming competence of OCT factors is determined by transactivation domains.

OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.

Generation of a human iPSC line (MPIi007-A) from a patient with Metachromatic leukodystrophy.

Here we have generated a human induced pluripotent stem cells (hiPSC) line (MPIi007-A) from skin fibroblasts of a 4-year-old male Metachromatic leukodystrophy (MLD) patient with a heterozygous 1178C > G (Thr393Ser) mutation in arylsulfatase A (ARSA) gene via retroviral expression of OCT4, SOX2, KLF4 and c-MYC. The MPIi007-A iPSC line displayed typical embryonic stem cell-like morphology, carried the ARSA gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and were capable of forming teratomas containing three germ layers. The MPIi007-A line can be used for the characterization of MLD-associated pathomechanisms and developing new therapeutic options.

Generation of a human iPSC line (MPIi006-A) from a patient with Pelizaeus-Merzbacher disease.

We established a human induced pluripotent stem cells (hiPSC) line (MPIi006-A) from fibroblasts of a 20-year-old male Pelizaeus-Merzbacher disease (PMD) patient with a hemizygous 643C>T mutation in proteolipid protein 1 (PLP1) gene using a retroviral delivery of OCT4, SOX2, KLF4 and c-MYC. The MPIi006-A iPSC line carried the mutation, displayed typical iPSC morphology, expressed pluripotent stem cell makers, exhibited normal karyotype and were capable of differentiating into cells representative of three germ layers.

Transcriptional regulation of endothelial cell behavior during sprouting angiogenesis.

Mediating the expansion of vascular beds in many physiological and pathological settings, angiogenesis requires dynamic changes in endothelial cell behavior. However, the molecular mechanisms governing endothelial cell activity during different phases of vascular growth, remodeling, maturation, and quiescence remain elusive. Here, we characterize dynamic gene expression changes during postnatal development and identify critical angiogenic factors in mouse retinal endothelial cells. Using actively translating transcriptome analysis and in silico computational analyses, we determine candidate regulators controlling endothelial cell behavior at different developmental stages. We further show that one of the identified candidates, the transcription factor MafB, controls endothelial sprouting in vitro and in vivo, and perform an integrative analysis of RNA-Seq and ChIP-Seq data to define putative direct MafB targets, which are activated or repressed by the transcriptional regulator. Together, our results identify novel cell-autonomous regulatory mechanisms controlling sprouting angiogenesis.Angiogenesis is a complex process that requires coordinated changes in endothelial cell behavior. Here the authors use Ribo-tag and RNA-Seq to determine temporal profiles of transcriptional activity during postnatal retinal angiogenesis, identifying transcriptional regulators of the process.

Small-molecule binding of the axin RGS domain promotes ß-catenin and Ras degradation.

Both the Wnt/ß-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both ß-catenin and Ras, via targeting the Wnt/ß-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating ß-catenin and Ras degradation through enhancement of the ß-catenin destruction complex activating GSK3ß. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both ß-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/ß-catenin and Ras pathways.

Distinct Signaling Requirements for the Establishment of ESC Pluripotency in Late-Stage EpiSCs.

It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.

Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.

The ability to generate integration-free induced hepatocyte-like cells (iHeps) from somatic fibroblasts has the potential to advance their clinical application. Here, we have generated integration-free, functional, and expandable iHeps from mouse somatic fibroblasts. To elicit this direct conversion, we took advantage of an oriP/EBNA1-based episomal system to deliver a set of transcription factors, Gata4, Hnf1a, and Foxa3, to the fibroblasts. The established iHeps exhibit similar morphology, marker expression, and functional properties to primary hepatocytes. Furthermore, integration-free iHeps prolong the survival of fumarylacetoacetate-hydrolase-deficient (Fah(-/-)) mice after cell transplantation. Our study provides a novel concept for generating functional and expandable iHeps using a non-viral, non-integrating, plasmid-based system that could facilitate their pharmaceutical and biomedical application.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile.

Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3-6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.

Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells.

BACKGROUND: Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3ß inhibitor that activates the Wnt/ß-catenin pathway, to promote hair re-growth in vitro and in vivo. METHODOLOGY/ PRINCIPAL FINDINGS: Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/ß-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/ß-catenin pathway. VPA analogs and other GSK3ß inhibitors that activate the Wnt/ß-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2) also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. CONCLUSIONS/ SIGNIFICANCE: Our findings indicate that small molecules that activate the Wnt/ß-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.

Ras stabilization through aberrant activation of Wnt/ß-catenin signaling promotes intestinal tumorigenesis.

Although the guanosine triphosphate/guanosine diphosphate loading switch is a major regulatory mechanism that controls the activity of the guanosine triphosphatase Ras, we report a distinct mechanism for regulating Ras activity through phosphorylation-mediated degradation and describe the role of this second regulatory mechanism in the suppression of cellular transformation and tumors induced by Ras mutations. We found that negative regulators of Wnt/ß-catenin signaling contributed to the polyubiquitin-dependent degradation of Ras after its phosphorylation by glycogen synthase kinase 3ß (GSK3ß) and the subsequent recruitment of ß-TrCP-E3 ligase. We found a positive association between tumorigenesis and Ras stabilization resulting from the aberrant activation of Wnt/ß-catenin signaling in adenomas from two mouse models of colon cancer, human colonic tumors from various stages, and colon polyps of patients with familial adenomatous polyposis. Our results indicated that GSK3ß plays an essential role in Ras degradation and that inhibition of this degradation pathway by aberrant Wnt/ß-catenin signaling may contribute to Ras-induced transformation in colorectal tumorigenesis.

KR-POK interacts with p53 and represses its ability to activate transcription of p21WAF1/CDKN1A.

Transcriptional regulation by p53 is thought to play a role in its ability to suppress tumorigenesis. However, there remain gaps in understanding about how p53 regulates transcription and how disrupting this function may promote cancer. Here we report a role in these processes for the kidney cancer-related gene KR-POK (ZBTB7C), a POZ domain and Krüppel-like zinc finger transcription factor that we found to physically interact with p53. Murine embryonic fibroblasts isolated from genetically deficient mice (Kr-pok(-/-) MEFs) exhibited a proliferative defect relative to wild-type mouse embryonic fibroblasts (MEF). The zinc finger domain of Kr-pok interacted directly with the DNA binding and oligomerization domains of p53. This interaction was essential for Kr-pok to bind the distal promoter region of the CDKN1A gene, an important p53 target gene encoding the cell-cycle regulator p21WAF1, and to inhibit p53-mediated transcriptional activation of CDKN1A. Kr-pok also interacted with the transcriptional corepressors NCoR and BCoR, acting to repress histone H3 and H4 deacetylation at the proximal promoter region of the CDKN1A gene. Importantly, Kr-pok(-/-) MEFs displayed an enhancement in CDKN1A transactivation by p53 during the DNA damage response, without any parallel changes in transcription of either the p53 or Kr-pok genes themselves. Furthermore, Kr-pok promoted cell proliferation in vitro and in vivo, and its expression was increased in more than 50% of the malignant human kidney cancer cases analyzed. Together, our findings define KR-POK as a transcriptional repressor with a pro-oncogenic role that relies upon binding to p53 and inhibition of its transactivation function.

Persicaria hydropiper (L.) spach and its flavonoid components, isoquercitrin and isorhamnetin, activate the Wnt/ß-catenin pathway and inhibit adipocyte differentiation of 3T3-L1 cells.

Obesity, which is related to metabolic syndrome and is associated with liver disease, represents an epidemic problem demanding effective therapeutic strategies. Evidence shows that the Wnt/ß-catenin pathway is closely associated with obesity and that small molecules regulating the Wnt/ß-catenin pathway can potentially control adipogenesis related to obesity. Eleven plant extracts activating the Wnt/ß-catenin pathway were screened by using HEK 293-TOP cells retaining the Wnt/ß-catenin signaling reporter gene. An extract of Persicaria hydropiper (L.) Spach was found to activate Wnt/ß-catenin signaling. P. hydropiper is grown worldwide in temperate climates and is found widely in Southeast Asia. The P. hydropiper extract inhibited the differentiation of adipocyte 3T3-L1 cells. Isoquercitrin and isorhamnetin, constituents of P. hydropiper, also activated Wnt/ß-catenin signaling and suppressed the differentiation of 3T3-L1 cells. These results indicate that isoquercitrin in P. hydropiper suppresses the adipogenesis of 3T3-L1 cells via the inhibition of Wnt/ß-catenin signaling. P. hydropiper and isoquercitrin may therefore be potential therapeutic agents for obesity and its associated disorders.

MEK1/2 inhibitors AS703026 and AZD6244 may be potential therapies for KRAS mutated colorectal cancer that is resistant to EGFR monoclonal antibody therapy.

Epidermal growth factor receptor (EGFR) monoclonal antibodies (mAb) are used widely to treat metastatic colorectal cancer (mCRC) patients, but it is now clear that patients harboring K-ras mutation are resistant to EGFR mAbs such as cetuximab (Erbitux) and panitumumab (Vectibix). For this reason, current recommendations for patient care involve diagnosing the K-ras mutational status of patients prior to EGFR mAb therapy. In this study, we investigated the ability of two MEK inhibitors currently in clinical trials, AS703026 and AZD6244, to address the challenge posed by the resistance of K-ras mutated colorectal cancers to EGFR mAb. AS703026 and AZD6244 were tested in various cell-based assays and tumor xenograft studies, focusing on isogenic human colorectal tumor cell lines that expressed only WT or mutant K-Ras (D-WT or D-MUT). The EGFR mAb cetuximab inhibited the Ras-ERK pathway and proliferation of D-WT cells in vitro and in vivo, but it did not inhibit proliferation of D-MUT cells in either setting. In contrast, AS703026 and AZD6244 effectively inhibited the growth of D-MUT cells in vitro and in vivo by specific inhibition of the key MEK downstream target kinase ERK. Inhibition of MEK by AS703026 or AZD6244 also suppressed cetuximab-resistant colorectal cancer cells attributed to K-ras mutation both in vitro and in vivo. Our findings offer proof-of-concept for the use of MEK inhibitors as an effective therapy in K-ras mutated CRC.

Bone morphogenetic protein 4 stimulates neuronal differentiation of neuronal stem cells through the ERK pathway.

Bone morphogenic protein 4 (BMP4), a member of the TGF-beta superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker beta-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/beta-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3beta (GSK3beta) activating the Wnt/beta-catenin pathway. The BMP4- mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.

H-Ras is degraded by Wnt/beta-catenin signaling via beta-TrCP-mediated polyubiquitylation.

Ras is an important proto-protein that is regulated primarily by GDP/GTP exchange. Here, we report a novel regulatory mechanism whereby turnover of both endogenous and overexpressed H-Ras protein is controlled by beta-TrCP-mediated ubiquitylation, proteasomal degradation and the Wnt/beta-catenin signaling pathway. The interaction of H-Ras with the WD40 domain of beta-TrCP targeted H-Ras for polyubiquitylation and degradation. This process was stimulated by Axin or adenomatous polyposis coli (Apc), and was inhibited by Wnt3a. Ras-mediated cellular transformation was also inhibited by the expression of beta-TrCP and/or Axin. In vivo regulation of Ras stability by Wnt/beta-catenin signaling was determined via measurements of the status of Ras in the intestines of mice stimulated with recombinant Wnt3a by intravenous tail vein injection. The regulation of Ras stability by Wnt/beta-catenin signaling provides a mechanical basis for crosstalk between the Wnt/beta-catenin and the Ras-ERK pathways involved in transformation.

Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway.

BACKGROUND: Valproic acid (VPA), a commonly used mood stabilizer that promotes neuronal differentiation, regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3beta (GSK3beta). However, the mechanism by which VPA promotes differentiation is not understood. RESULTS: We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects, however, are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through beta-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos. CONCLUSION: We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos.