Showing papers in "Journal of Molecular Cell Biology in 2015"

Ribosomal proteins: functions beyond the ribosome

Abstract: Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics.

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung

Abstract: The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells.

Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

Abstract: The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.

Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN

Abstract: Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN.

Caspase-1 activation by NLRP3 inflammasome dampens IL-33-dependent house dust mite-induced allergic lung inflammation

Abstract: The cysteine protease caspase-1 (Casp-1) contributes to innate immunity through the assembly of NLRP3, NLRC4, AIM2, and NLRP6 inflammasomes. Here we ask whether caspase-1 activation plays a regulatory role in house dust mite (HDM)-induced experimental allergic airway inflammation. We report enhanced airway inflammation in caspase-1-deficient mice exposed to HDM with a marked eosinophil recruitment, increased expression of IL-4, IL-5, IL-13, as well as full-length and bioactive IL-33. Furthermore, mice deficient for NLRP3 failed to control eosinophil influx in the airways and displayed augmented Th2 cytokine and chemokine levels, suggesting that the NLPR3 inflammasome complex controls HDM-induced inflammation. IL-33 neutralization by administration of soluble ST2 receptor inhibited the enhanced allergic inflammation, while administration of recombinant IL-33 during challenge phase enhanced allergic inflammation in caspase-1-deficient mice. Therefore, we show that caspase-1, NLRP3, and ASC, but not NLRC4, contribute to the upregulation of allergic lung inflammation. Moreover, we cannot exclude an effect of caspase-11, because caspase-1-deficient mice are deficient for both caspases. Mechanistically, absence of caspase-1 is associated with increased expression of IL-33, uric acid, and spleen tyrosine kinase (Syk) production. This study highlights a critical role of caspase-1 activation and NLPR3/ASC inflammasome complex in the down-modulation of IL-33 in vivo and in vitro, thereby regulating Th2 response in HDM-induced allergic lung inflammation.

The histone H3 lysine-27 demethylase Jmjd3 plays a critical role in specific regulation of Th17 cell differentiation

Abstract: Interleukin (IL) 17-producing T helper (Th17) cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases, such as multiple sclerosis, psoriasis, and ulcerative colitis. Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently, the participation of epigenetic modifications in the differentiation process has yet to be elucidated. We demonstrated here that histone H3 lysine-27 (H3K27) demethylation, predominantly mediated by the H3K27 demethylase Jmjd3, crucially regulated Th17 cell differentiation. Activation of naive CD4(+) T cells immediately induced high expression of Jmjd3. Genetic depletion of Jmjd3 in CD4(+) T cells specifically impaired Th17 cell differentiation both in vitro and in vivo. Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficient CD4(+) T cells. Importantly, Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis (EAE). Furthermore, inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro. At the molecular level, Jmjd3 directly bound to and reduced the level of H3K27 trimethylation (me3) at the genomic sites of Rorc, which encodes the master Th17 transcription factor Rorγt, and Th17 cytokine genes such as Il17, Il17f, and Il22. Therefore, our studies established a critical role of Jmjd3-mediated H3K27 demethylation in Th17 cell differentiation and suggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses.

Walking on multiple disease-gene networks to prioritize candidate genes.

Abstract: Uncovering causal genes for human inherited diseases, as the primary step toward understanding the pathogenesis of these diseases, requires a combined analysis of genetic and genomic data. Although bioinformatics methods have been designed to prioritize candidate genes resulting from genetic linkage analysis or association studies, the coverage of both diseases and genes in existing methods is quite limited, thereby preventing the scan of causal genes for a significant proportion of diseases at the whole-genome level. To overcome this limitation, we propose a method named pgWalk to prioritize candidate genes by integrating multiple phenomic and genomic data. We derive three types of phenotype similarities among 7719 diseases and nine types of functional similarities among 20327 genes. Based on a pair of phenotype and gene similarities, we construct a disease-gene network and then simulate the process that a random walker wanders on such a heterogeneous network to quantify the strength of association between a candidate gene and a query disease. A weighted version of the Fisher's method with dependent correction is adopted to integrate 27 scores obtained in this way, and a final q-value is calibrated for prioritizing candidate genes. A series of validation experiments are conducted to demonstrate the superior performance of this approach. We further show the effectiveness of this method in exome sequencing studies of autism and epileptic encephalopathies. An online service and the standalone software of pgWalk can be found at http://bioinfo.au.tsinghua.edu.cn/jianglab/pgwalk.

mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration

Abstract: Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration

The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation

Abstract: The transcription factor PU.1 is involved in regulation of macrophage differentiation and maturation. However, the role of PU.1 in alternatively activated macrophage (AAM) and asthmatic inflammation has yet been investigated. Here we report that PU.1 serves as a critical regulator of AAM polarization and promotes the pathological progress of asthmatic airway inflammation. In response to the challenge of DRA (dust mite, ragweed, and Aspergillus) allergens, conditional PU.1-deficient (PU/ER(T)(+/-)) mice displayed attenuated allergic airway inflammation, including decreased alveolar eosinophil infiltration and reduced production of IgE, which were associated with decreased mucous glands and goblet cell hyperplasia. The reduced asthmatic inflammation in PU/ER(T)(+/-) mice was restored by adoptive transfer of IL-4-induced wild-type (WT) macrophages. Moreover, after treating PU/ER(T)(+/-) mice with tamoxifen to rescue PU.1 function, the allergic asthmatic inflammation was significantly restored. In vitro studies demonstrate that treatment of PU.1-deficient macrophages with IL-4 attenuated the expression of chitinase 3-like 3 (Ym-1) and resistin-like molecule alpha 1 (Fizz-1), two specific markers of AAM polarization. In addition, PU.1 expression in macrophages was inducible in response to IL-4 challenge, which was associated with phosphorylation of signal transducer and activator of transcription 6 (STAT6). Furthermore, DRA challenge in sensitized mice almost abrogated gene expression of Ym-1 and Fizz-1 in lung tissues of PU/ER(T)(+/-) mice compared with WT mice. These data, all together, indicate that PU.1 plays a critical role in AAM polarization and asthmatic inflammation.

JMJD3 promotes chondrocyte proliferation and hypertrophy during endochondral bone formation in mice

Abstract: JMJD3 (KDM6B) is an H3K27me3 demethylase and counteracts polycomb-mediated transcription repression. However, the function of JMJD3 in vivo is not well understood. Here we show that JMJD3 is highly expressed in cells of the chondrocyte lineage, especially in prehypertrophic and hypertrophic chondrocytes, during endochondral ossification. Homozygous deletion of Jmjd3 results in severely decreased proliferation and delayed hypertrophy of chondrocytes, and thereby marked retardation of endochondral ossification in mice. Genetically, JMJD3 associates with RUNX2 to promote proliferation and hypertrophy of chondrocytes. Biochemically, JMJD3 associates with and enhances RUNX2 activity by derepression of Runx2 and Ihh transcription through its H3K27me3 demethylase activity. These results demonstrate that JMJD3 is a key epigenetic regulator in the process of cartilage maturation during endochondral bone formation.

Diagnosing phenotypes of single-sample individuals by edge biomarkers.

Abstract: Network or edge biomarkers are a reliable form to characterize phenotypes or diseases. However, obtaining edges or correlations between molecules for an individual requires measurement of multiple samples of that individual, which are generally unavailable in clinical practice. Thus, it is strongly demanded to diagnose a disease by edge or network biomarkers in one-sample-for-one-individual context. Here, we developed a new computational framework, EdgeBiomarker, to integrate edge and node biomarkers to diagnose phenotype of each single test sample. By applying the method to datasets of lung and breast cancer, it reveals new marker genes/gene-pairs and related sub-networks for distinguishing earlier and advanced cancer stages. Our method shows advantages over traditional methods: (i) edge biomarkers extracted from non-differentially expressed genes achieve better cross-validation accuracy of diagnosis than molecule or node biomarkers from differentially expressed genes, suggesting that certain pathogenic information is only present at the level of network and under-estimated by traditional methods; (ii) edge biomarkers categorize patients into low/high survival rate in a more reliable manner; (iii) edge biomarkers are significantly enriched in relevant biological functions or pathways, implying that the association changes in a network, rather than expression changes in individual molecules, tend to be causally related to cancer development. The new framework of edge biomarkers paves the way for diagnosing diseases and analyzing their molecular mechanisms by edges or networks in one-sample-for-one-individual basis. This also provides a powerful tool for precision medicine or big-data medicine.

Hot spots of DNA double-strand breaks and genomic contacts of human rDNA units are involved in epigenetic regulation

Abstract: DNA double-strand breaks (DSBs) are involved in many cellular mechanisms, including replication, transcription, and genome rearrangements. The recent observation that hot spots of DSBs in human chromosomes delimit DNA domains that possess coordinately expressed genes suggests a strong relationship between the organization of transcription patterns and hot spots of DSBs. In this study, we performed mapping of hot spots of DSBs in a human 43-kb ribosomal DNA (rDNA) repeated unit. We observed that rDNA units corresponded to the most fragile sites in human chromosomes and that these units possessed at least nine specific regions containing clusters of extremely frequently occurring DSBs, which were located exclusively in non-coding intergenic spacer (IGS) regions. The hot spots of DSBs corresponded to only a specific subset of DNase-hypersensitive sites, and coincided with CTCF, PARP1, and HNRNPA2B1 binding sites, and H3K4me3 marks. Our rDNA-4C data indicate that the regions of IGS containing the hot spots of DSBs often form contacts with specific regions in different chromosomes, including the pericentromeric regions, as well as regions that are characterized by H3K27ac and H3K4me3 marks, CTCF binding sites, ChIA-PET and RIP signals, and high levels of DSBs. The data suggest a strong link between chromosome breakage and several different mechanisms of epigenetic regulation of gene expression.

X chromosome inactivation and active X upregulation in therian mammals: facts, questions, and hypotheses

Abstract: X chromosome inactivation is a mechanism that modulates the expression of X-linked genes in eutherian females (XX). Ohno proposed that to achieve a proper balance between X-linked and autosomal genes, those on the active X should also undergo a 2-fold upregulation. Although some support for Ohno's hypothesis has been provided through the years, recent genomic studies testing this hypothesis have brought contradictory results and fueled debate. Thus far, there are as many results in favor as against Ohno's hypothesis, depending on the nature of the datasets and the various assumptions and thresholds involved in the analyses. However, they have confirmed the importance of dosage balance between X-linked and autosomal genes involved in stoichiometric relationships. These facts as well as questions and hypotheses are discussed below.

Non-homologous functions of the AlkB homologs

Abstract: The DNA repair enzyme AlkB was identified in E. coli more than three decades ago. Since then, nine mammalian homologs, all members of the superfamily of alpha-ketoglutarate and Fe(II)-dependent dioxygenases, have been identified (designated ALKBH1-8 and FTO). While E. coli AlkB serves as a DNA repair enzyme, only two mammalian homologs have been confirmed to repair DNA in vivo. The other mammalian homologs have remarkably diverse substrate specificities and biological functions. Substrates recognized by the different AlkB homologs comprise erroneous methyl- and etheno adducts in DNA, unique wobble uridine modifications in certain tRNAs, methylated adenines in mRNA, and methylated lysines on proteins. The phenotypes of organisms lacking or overexpressing individual AlkB homologs include obesity, severe sensitivity to inflammation, infertility, growth retardation, and multiple malformations. Here we review the present knowledge of the mammalian AlkB homologs and their implications for human disease and development.

Deregulation of splicing factors and breast cancer development

Abstract: It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single-nucleotide polymorphisms (SNPs) within the DNA sequences of cancer-related genes, which can strongly affect the activity of splicing factors and influence the splice site choice. However, it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection. There is now increasing evidence to demonstrate that the expression profile of ten splicing factors (including SRs and hnRNPs) and eight RNA-binding proteins changes in breast cancer cells compared with normal cells. These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences. Thus, a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis. Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes, this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events.

miRNA-181 regulates embryo implantation in mice through targeting leukemia inhibitory factor

Abstract: Embryo implantation is a crucial step in mammalian reproduction. However, little is known regarding the physiological roles of microRNAs in the regulation of embryo implantation. Here we show that a minimum uterine expression of miR-181 is essential for the onset of embryo implantation. Both transient and prolonged transgenic expression of miR-181 led to impaired implantation, which can be rescued by exogenous administration of leukemia inhibitory factor (LIF). Mechanistically, miR-181 is able to directly target LIF and downregulate LIF expression, thereby inhibiting embryo implantation. We also show that miR-181 expression is regulated by the transcriptional factor Emx2, and the Emx2 – miR-181 axis plays an important role in regulating embryo implantation. Taken together, these results reveal a novel function of miR-181 in embryo implantation through the regulation of LIF, and also indicate a potential link between miR-181 dysregulation and human embryo implantation defects.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth

Abstract: The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

PI3K/mTOR mediate mitogen-dependent HDAC1 phosphorylation in breast cancer: a novel regulation of estrogen receptor expression

Abstract: Histone deacetylase 1 (HDAC1) is an important epigenetic controller involved in transcriptional regulation through modification of chromatin structure. Genetic and epigenetic changes and deregulation of signal transduction pathways have been implicated in the development of breast cancer. Downregulation of estrogen receptor α (ERα) expression is one of the mechanisms behind the acquisition of endocrine resistance. Sustained and increased hormone and growth factor receptor signaling in breast cancer cells contribute to resistance to endocrine therapy. Both HDACs and the PI3K/mTOR signaling pathway are becoming promising targets in breast cancer, reversing also acquired hormone resistance. Here we show how mitogens, activating the PI3K/mTOR pathway, trigger the phosphorylation of HDAC1 in breast cancer cells, which is completely dependent on the activity of the p70 S6 kinase (S6K1). Our findings show that S6K1, overexpressed in many breast cancers, controls HDAC1-dependent transcriptional regulation of ERα levels upon mitogenic stimuli, controlling HDAC1 recruitment to the ERα promoter. Furthermore, cell treatment with both mTOR and HDACs inhibitors shows an additive effect in inhibiting breast cancer proliferation. This confirms the novel cross-talk between the HDAC1 and PI3K pathways with clinical implications towards the treatment of this malignant disease.

RBM20 is an essential factor for thyroid hormone-regulated titin isoform transition

Abstract: Dear Editor, Diastolic heart failure (DHF) and systolic heart failure (SHF) are two main subsets of chronic heart failure that are commonly encountered in clinical practice (Chatterjee and Massie, 2007). Although there have been considerable advances in the treatment of SHF, the molecular and biochemical mechanisms mediating the structural remodeling and principal functional derangement in both SHF and DHF remain unclear. In particular, effective therapeutic regimens are still lacking for DHF; thus, it is imperative to better understand the molecular and biochemical mechanisms of DHF in an effort to seek novel therapeutic avenues for patients suffering from DHF. DHF is manifested by stiffer ventricular walls, while SHF has overly compliant ventricular walls. Therefore, altering ventricular wall stiffness will be a potential therapeutic strategy for both DHF and SHF. Recently, increasing attention has been paid by researchers and cardiologists to titin in regard to developing an alternative therapy for both DHF and SHF. Titin, a giant sarcomeric protein, is a key determinant of myocardial passive tension and is largely responsible for the diastolic properties of the heart (LeWinter and Granzier, 2014). Two major classes of titin are co-expressed in mammalian cardiac muscles, namely the smaller N2B isoform (3.0 MDa) and the larger N2BA isoform (3.2–3.7 MDa). The larger N2BA isoform is more compliant and develops lower passive tension, while the N2B isoform is stiffer and develops higher passive tension. At varying ratios of N2BA to N2B isoforms, the sarcomeres develop an intermediate level of passive tension affecting myofibrillar extensibility and passive force generation and altering the stiffness of the cardiac walls (Cazorla et al., 2000; Freiburg et al., 2000). Therefore, the manipulation of their ratios has been considered a therapy for reducing pathological diastolic stiffness or increasing pathological systolic stiffness. However, the mechanisms regulating titin isoform transition remain elusive. Recently, RNA binding motif 20 (RBM20), a splicing factor, has been identified as a major regulator of titin isoform transition (Guo et al., 2012), while the thyroid hormone-triiodothyronine (T3) has also been reported to regulate titin isoform transition (Wu et al., 2007; Kruger et al., 2008). Nevertheless, prior to this report, the relationship between RBM20 and T3 in the regulation of titin isoform transition remains unknown. The present study was designed to evaluate the role of RBM20 in T3-regulated titin isoform transition, and the molecular signaling mechanisms linking T3 and RBM20. There are indications that thyroid gland maturation and titin isoform transition occur in the fetus at approximately the same time (Polk, 1995; Kruger et al., 2006). Indeed, in the presence of RBM20, T3 treatment in primary cultures of neonatal rat cardiomyocytes (CMs) increased the smaller N2B isoform expression (Kruger et al., 2008). However, it is unknown whether T3 can still regulate titin isoform transition in the absence of RBM20. Hence, we examined the influence of T3 on titin isoform transition in the absence of RBM20. In homozygous RBM20 knockout rats (Rbm20−/−), only the largest N2BA isoform (∼3.9 MDa) is present (Guo et al., 2010), and in heterozygous RBM20 knockout rats (Rbm20+/−), the intermediate sized titin N2BA isoforms (3.2–3.7 MDa) are co-expressed with the smaller N2B isoform, while, in adult wild type rats (Rbm20+/+), the small, stiffer N2B isoform predominates (Guo et al., 2012). This makes the RBM20 deficient rat model a unique tool for this study. We isolated and cultured neonatal ventricular cardiomyocytes (NVCMs) of Rbm20+/+, Rbm20+/−, and Rbm20−/− rats. With the addition of T3 in a serum-starved medium, the ratios of N2B to N2BA isoforms increased significantly, from ∼23% to ∼59% of total titin isoforms (N2B+N2BA) in Rbm20+/+ rats (Figure 1A) and from ∼6% to ∼23% of total titin isoforms in Rbm20+/− group (Figure 1B). However, increased N2B expression did not occur in Rbm20−/− NVCMs with T3 supplementation. Virtually no N2B was expressed in either treated or untreated Rbm20−/− NVCMs (Figure 1C). These results suggest that RBM20 plays an indispensable role in the regulation of titin isoform transition triggered by T3. Figure 1 Effect of T3 on titin isoform transition in RBM20 deficient NVCMs and rats. (A, B, D, and E) N2B-titin isoform increased with T3 treatment in primary cultures of Rbm20+/+ and Rbm20+/− NVCMs and rats when compared with control without T3 treatment. ... In order to further confirm whether T3-regulated titin isoform transition is RBM20-dependent, we performed in vivo assays. We treated the Rbm20+/+, Rbm20+/−, and Rbm20−/− rats independently with T3 and propylthiouracil (PTU, a drug that inhibits the secretion of thyroid hormone). After a 90-day treatment with subcutaneously implanted T3 pellets in Rbm20+/+, Rbm20+/−, and Rbm20−/− rats, we harvested the hearts, and observed titin isoform transitions by resolving titin bands with a 1% SDS-agarose gel. The N2B isoform was significantly increased in Rbm20+/+ and Rbm20+/− rat hearts (Figure 1D), with increases of N2B isoform from ∼84% to 92% of total titin in Rbm20+/+ rats, and from ∼16% to 36% of total titin in Rbm20+/− rats (Figure 1E). No N2B isoform was expressed in Rbm20−/− rat hearts under T3 treatment when compared with control groups that were implanted with placebo (Figure 1F). An equal number of age-matched rats were used for the PTU treatment by feeding a diet containing 0.15% PTU. After 3 months of treatment, with low thyroid status, the N2BA isoform was increased and the N2B isoform was decreased from ∼87% to 80% of total titin in Rbm20+/+ rats (Figure 1G). The N2B isoform was nearly undetectable with PTU treatment in Rbm20+/− rats (Figure 1H). However, no changes were observed in Rbm20−/− rats treated with PTU when compared with control groups without PTU treatment (Figure 1I). These data further confirm that RBM20 is an essential factor for thyroid hormone-regulated titin isoform transition. Next, we examined the mechanisms linking RBM20 and T3 in the regulation of titin isoform transition. Western blot analysis using anti-RBM20 antibody indicated two bands (∼135 and 145 kDa) in control primary cultures of Rbm20+/+ and Rbm20+/− NVCMs (Figure 1J and K). With supplementation of T3, the intensity of the 145 kDa band increased in primary cultures of Rbm20+/+ and Rbm20+/− NVCMs when compared with the control without T3, while supplementation of LY 294002 (LY, a PI3K inhibitor) with or without T3 produced slightly decreased intensity of the 145 kDa band, and a significant reduction in the intensity of the 135 kDa band (Figure 1J and K). These results suggest that RBM20 is likely posttranslationally modified. If this is the case, the posttranslational modification of RBM20 could be phosphorylation, given that the phosphorylation of serine–arginine (SR) proteins (Guo et al., 2012) is essential for pre-mRNA splicing (Prasad et al., 1999). A previous study reported that T3 regulates titin isoform transition via the PI3K/Akt pathway (Kruger et al., 2008). Our results also demonstrated that T3 supplementation increased AKT activation and the N2B isoform, while LY supplementation decreased AKT activation and the N2B isoform (Supplementary Figures S1A–C and S2A–C) in primary cultures. In in vivo assays with rats treated with T3 and PTU, we found that the activation and phosphorylation of Akt were increased with T3 treatment, and deceased with PTU treatment (Supplementary Figures S3A–C and S4A–C). These results suggest that the phosphorylation of RBM20 is crucial for titin isoform transition triggered by T3. The mechanism could be that first T3 activates Akt and then Akt phosphorylates RBM20, and finally the phosphorylated RBM20 regulates titin isoform transition. Band density analysis also demonstrated that total RBM20 expression was increased with T3 supplementation when compared with control without T3 supplementation in the medium. Additionally, LY supplementation reduced the total amount of RBM20 when compared with control without T3 or LY supplementation (Figure 1J and K). These results imply that T3-regulated titin isoform transition could also activate the mTOR pathway and increase gene expression of RBM20. The relative expression of Rbm20 mRNA was also validated by real-time PCR, and the results revealed that the mRNA level increased with T3 supplementation while it decreased with LY supplementation (Supplementary Figure S5A and B). Therefore, we conclude that titin isoform transition triggered by T3 is linked to RBM20 via the PI3K/Akt/mTOR signaling pathway, phosphorylating RBM20 and/or increasing gene expression of RBM20 (Supplementary Figure S6). Taken together, these findings present significant clinical implications for patients with SHF or DHF. Our data suggest that T3 treatment in SHF may increase the stiffness of ventricular walls through upregulating the stiffer N2B isoform. In particular, patients with DHF can be benefited from the treatment to decrease the stiffer N2B isoform. Overall, our data should strengthen the understanding of the regulatory mechanism of titin isoform transition, which might help to decipher the mechanisms of developmental or diseased titin isoform transition, and further, they provide new insights for improving diastolic and systolic dysfunction. [Supplementary material is available at Journal of Molecular Cell Biology online. We thank Mark D. Hanna for expert technical assistance and laboratory management. We are grateful to the support of Agricultural Experiment Station at University of Wyoming and the Regional Hatch Project (NC1184). This work was supported by a pilot grant from NIH-P20-GM-103432 and Startup Funds from the University of Wyoming.]

Ndfip1 represses cell proliferation by controlling Pten localization and signaling specificity

Abstract: Pten controls a signaling axis that is implicated to regulate cell proliferation, growth, survival, migration, and metabolism. The molecular mechanisms underlying the specificity of Pten responses to such diverse cellular functions are currently poorly understood. Here we report the control of Pten activity and signaling specificity during the cell cycle by Ndfip1 regulation of Pten spatial distribution. Genetic deletion of Ndfip1 resulted in a loss of Pten nuclear compartmentalization and increased cell proliferation, despite cytoplasmic Pten remaining active in regulating PI3K/Akt signaling. Cells lacking nuclear Pten were found to have dysregulated levels of Plk1 and cyclin D1 that could drive cell proliferation. In vivo, transgene expression of Ndfip1 in the developing brain increased nuclear Pten and lengthened the cell cycle of neuronal progenitors, resulting in microencephaly. Our results show that local partitioning of Pten from the cytoplasm to the nucleus represents a key mechanism contributing to the specificity of Pten signaling during cell proliferation.

FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro

Abstract: The cranial neural crest (CNC) cells play a vital role in craniofacial development and regeneration. They are multi-potent progenitors, being able to differentiate into various types of tissues. Both pre-migratory and post-migratory CNC cells are plastic, taking on diverse fates by responding to different inductive signals. However, what sustains the multipotency of CNC cells and derivatives remains largely unknown. In this study, we present evidence that FGF8 signaling is able to sustain progenitor status and multipotency of CNC-derived mesenchymal cells both in vivo and in vitro. We show that augmented FGF8 signaling in pre-migratory CNC cells prevents cell differentiation and organogenesis in the craniofacial region by maintaining their progenitor status. CNC-derived mesenchymal cells with Fgf8 overexpression or control cells in the presence of exogenous FGF8 exhibit prolonged survival, proliferation, and multi-potent differentiation capability in cell cultures. Remarkably, exogenous FGF8 also sustains the capability of CNC-derived mesenchymal cells to participate in organogenesis such as odontogenesis. Furthermore, FGF8-mediated signaling strongly promotes adipogenesis but inhibits osteogenesis of CNC-derived mesenchymal cells in vitro. Our results reveal a specific role for FGF8 in the maintenance of progenitor status and in fate determination of CNC cells, implicating a potential application in expansion and fate manipulation of CNC-derived cells in stem cell-based craniofacial regeneration.

Synergistic control of sex hormones by 17β-HSD type 7: a novel target for estrogen-dependent breast cancer

Abstract: 17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17β-HSD type 7 (17β-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17β-HSD7 in breast carcinoma. Inhibition of 17β-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17β-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17β-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.

Ectodermal progenitors derived from epiblast stem cells by inhibition of Nodal signaling

Abstract: The ectoderm has the capability to generate epidermis and neuroectoderm and plays imperative roles during the early embryonic development. Our recent study uncovered a region with ectodermal progenitor potential in mouse embryo at embryonic day 7.0 and revealed that Nodal inhibition is essential for its formation. Here, we demonstrate that through brief inhibition of Nodal signaling in vitro, mouse embryonic stem cell (ESC)-derived epiblast stem cells (ESD-EpiSCs) could be committed to transient ectodermal progenitor populations, which possess the ability to give rise to neural or epidermal ectoderm in the absence or presence of BMP4, respectively. Mechanistic studies reveal that BMP4 recruits distinct transcriptional targets in ESD-EpiSCs and ectoderm-like cells. Furthermore, FGF-Erk signaling may also be alleviated during the generation of ectoderm-like cells. Thus, our data suggest that instructive interactions among several extracellular signals participate in the commitment of ectoderm from ESD-EpiSCs, which shed new light on the understanding of the formation of ectoderm during the gastrulation in early mouse embryo development.

Network-based identification of feedback modules that control RhoA activity and cell migration

Abstract: Cancer cell migration enables metastatic spread causing most cancer deaths. Rho-family GTPases control cell migration, but being embedded in a highly interconnected feedback network, the control of their dynamical behavior during cell migration remains elusive. To address this question, we reconstructed the Rho-family GTPases signaling network involved in cell migration, and developed a Boolean network model to analyze the different states and emergent rewiring of the Rho-family GTPases signaling network at protrusions and during extracellular matrix-dependent cell migration. Extensive simulations and experimental validations revealed that the bursts of RhoA activity induced at protrusions by EGF are regulated by a negative-feedback module composed of Src, FAK, and CSK. Interestingly, perturbing this module interfered with cyclic Rho activation and extracellular matrix-dependent migration, suggesting that CSK inhibition can be a novel and effective intervention strategy for blocking extracellular matrix-dependent cancer cell migration, while Src inhibition might fail, depending on the genetic background of cells. Thus, this study provides new insights into the mechanisms that regulate the intricate activation states of Rho-family GTPases during extracellular matrix-dependent migration, revealing potential new targets for interfering with extracellular matrix-dependent cancer cell migration.

A key role for PTP1B in dendritic cell maturation, migration and T cell activation

Abstract: Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naive T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.

Hepatitis B virus hijacks CTHRC1 to evade host immunity and maintain replication

Abstract: Hepatitis B virus (HBV) infection causes acute and chronic liver diseases, but is not directly cytopathic. Liver injury results from repeated attempts of the cellular immune response system to control the viral infection. Here, we investigate the roles of cellular factors and signaling pathways involved in the regulation of HBV replication to reveal the mechanism underlying HBV infection and pathogenesis. We show that collagen triple helix repeat containing 1 (CTHRC1) expression is elevated in HBV-infected patients and in HBV-transfected cells through epigenetic modification and transcriptional regulation. CTHRC1 facilitates HBV replication in cultured cells and BALB/c mice by activating the PKCα/ERK/JNK/c-Jun cascade to repress the IFN/JAK/STAT pathway. HBV-activated CTHRC1 downregulates the activity of type I interferon (IFN), the production of IFN-stimulated genes (ISGs), and the phosphorylation of signal transducer and activator of transcription 1/2 (STAT1/2), whereas it upregulates the phosphorylation and ubiquitination of type I IFN receptors (IFNARα/β). Thus, our results show that HBV uses a novel mechanism to hijack cellular factors and signal cascades in order to evade host antiviral immunity and maintain persistent infection. We also demonstrate that CTHRC1 has a novel role in viral infection.

Durable pluripotency and haploidy in epiblast stem cells derived from haploid embryonic stem cells in vitro

Abstract: Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells. When injected into the cytoplasm of an oocyte, androgenetic haEpiSC (ahaEpiSCs) can support embryonic development until midgestation (E12.5). Together, these results demonstrate durable pluripotency in mouse haESCs and haEpiSCs, as well as the valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

Cigarette smoke-induced cell cycle arrest in spermatocytes [GC-2spd(ts)] is mediated through crosstalk between Ahr–Nrf2 pathway and MAPK signaling

Abstract: Our earlier studies have demonstrated that the cigarette smoke in the form of cigarette smoke condensate (CSC) causes growth arrest of a mouse spermatocyte cell line [GC-2spd(ts)] through activation of the AHR-NRF2 pathway. The present study demonstrates the CSC-activated p38 and ERK MAPK signaling in GC-2spd(ts) via arylhydrocarbon receptor (AHR). Pharmacological inhibition by using AHR-antagonist, or p38 MAPK and ERK (MEK1) inhibitors significantly abrogates CSC-induced growth arrest by AHR and MAPK inactivation. QRT-PCR, western blot, and immunofluorescence of Ahr-target of Nrf2, and stress-inducible growth suppressive Atf3 and E2f4 following treatments indicate a crosstalk among these pathways. Regulation of Atf3 by Nrf2 and Ahr through RNA interference suggests the existence of a cross-regulatory loop between the targets. CSC induction of E2f4 via Atf3 and its regulation by pharmacological inhibitors reveal a possible regulatory mechanism of growth inhibitory CSC. SiRNA silencing of Ahr, Nrf2, Atf3, and E2f4 genes and downregulation of cyclins by CSC corroborate the growth inhibitory effect of cigarette smoke. Thus, the data obtained suggest that the CSC-mediated MAPKs and AHR-NRF2 crosstalks lay the molecular basis for the growth arrest and cell death of spermatocytes.

An information theoretic method to identify combinations of genomic alterations that promote glioblastoma

Abstract: Tumors are the result of accumulated genomic alterations that cooperate synergistically to produce uncontrollable cell growth. Although identifying recurrent alterations among large collections of tumors provides a way to pinpoint genes that endow a selective advantage in oncogenesis and progression, it fails to address the genetic interactions behind this selection process. A non-random pattern of co-mutated genes is evidence for selective forces acting on tumor cells that harbor combinations of these genetic alterations. Although existing methods have successfully identified mutually exclusive gene sets, no current method can systematically discover more general genetic relationships. We develop Genomic Alteration Modules using Total Correlation (GAMToC), an information theoretic framework that integrates copy number and mutation data to identify gene modules with any non-random pattern of joint alteration. Additionally, we present the Seed-GAMToC procedure, which uncovers the mutational context of any putative cancer gene. The software is publicly available. Applied to glioblastoma multiforme samples, GAMToC results show distinct subsets of co-occurring mutations, suggesting distinct mutational routes to cancer and providing new insight into mutations associated with proneural, proneural/G-CIMP, and classical types of the disease. The results recapitulate known relationships such as mutual exclusive mutations, place these alterations in the context of other mutations, and find more complex relationships such as conditional mutual exclusivity.