Overexpression of MRP3 in HeLa-UGT1A9 Cells Enhances Glucuronidation Capability of the Cells

Background: The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low systemic bioavailability of flavonoids. Objective: The study aims to investigate the dynamic interplay between multiple UGTs and multiple efflux transporters inside the cells. Methods: A new HeLa-UGT1A9-MRP3 cell was established to overexpress two dominant efflux transporters MRP3 and BCRP, and two UGT isoforms UGT1A9 and UGT1A3. The metabolism and glucuronides excretion for a model flavonoid genistein were determined in HeLa-UGT1A9-MRP3 cells and HeLa-UGT1A9-Con cells that overexpressed one UGT (1A9) and one efflux transporter (BCRP). Results: The excretion rate grew nearly 6-fold, cellular clearance of glucuronides increased about 3-fold, and a fraction of genistein metabolized (fmet) increased (14%, p<0.01) in the new cells. Small interfering (siRNA)-mediated MRP3 functional knockdown resulted in markedly decreases in the excretion rates (26%-78%), intracellular amounts (56%-93%), cellular clearance (54%-96%) in both cells, but the magnitude of the differences in HeLa-UGT1A9-Con cells were relatively small. Reductions in fmet values were similarly moderate (11%-14%). In contrast, UGT1A9 knockdown with siRNA caused large decreases in the excretion rates (46%-88%), intracellular amounts (80%-97%), cellular clearance (80%-98%) as well as fmet value (33%-43%, p<0.01) in both UGT1A9 cells. Comparisons of the kinetic parameters and profiles of genistein glucuronidation and UGT mRNA expression suggest that HeLa-UGT1A9-MRP3 has increased expression of both MRP3 and UGT1A3. Conclusion: The newly engineered HeLa-UGT1A9-MRP3 cells are an appropriate model to study the kinetic interplay between multiple UGTs and efflux transporters. It's a promising biosynthetic tool to obtain flavonoids glucuronides of high purity.

In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.

LINGO1 is a regulatory subunit of large conductance, Ca2+-activated potassium channels

LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson’s disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca2+-activated K+ (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a “functional knockdown” of these currents and may contribute to the tremor associated with increased LINGO1 levels.

Pectoral Fin Anomalies in tbx5a Knockdown Zebrafish Embryos Related to the Cascade Effect of N-Cadherin and Extracellular Matrix Formation

Functional knockdown of zebrafish tbx5a causes hypoplasia or aplasia of pectoral fins. This study aimed to assess developmental pectoral fin anomalies in tbx5a morpholino knockdown zebrafish embryos. The expression of cartilage-related genes in the tbx5a morphant was analyzed by DNA microarray, immunostaining, and thin-section histology to examine the detailed distribution of the extracellular matrix (ECM) during different pectoral fin developmental stages. Chondrogenic condensation (CC) in the tbx5a morpholino knockdown group was barely recognizable at 37 h postfertilization (hpf); the process from CC to endoskeleton formation was disrupted at 48 hpf, and the endoskeleton was only loosely formed at 72 hpf. Microarrays identified 18 downregulated genes in tbx5a-deficient embryos, including 2 fin morphogenesis-related (cx43, bbs7), 4 fin development-related (hoxc8a, hhip, axin1, msxb), and 12 cartilage development-related (mmp14a, sec23b, tfap2a, slc35b2, dlx5a, dlx1a, tfap2b, fmr1, runx3, cdh2, lect1, acvr2a, mmp14b) genes, at 24 and 30 hpf. The increase in apoptosis-related proteins (BAD and BCL2) in the tbx5a morphant influenced the cellular component of pectoral fins and resulted in chondrocyte reduction throughout the different CC phases. Furthermore, tbx5a knockdown interfered with ECM formation in pectoral fins, affecting glycosaminoglycans, fibronectin, hyaluronic acid (HA), and N-cadherin. Our results provide evidence that the pectoral fin phenotypic anomaly induced by tbx5a knockdown is related to disruption of the mesoderm and ECM, consequently interfering with mesoderm migration, CC, and subsequent endoskeleton formation.

iPSC-Based Phenomic Screen Revealed an Impact of Uncontrolled NFkB Activity at the Initiating Stages of AML1-ETO Related Leukemia

Onset of acute myeloid leukemia (AML) has been accounted for by cooperation between multiple genetic alterations which induce abnormal control of various cellular pathways. Among the previously listed leukemogenic lesions, AML1-ETO fusion (AE) generated by translocation (8;21) (q22;q22) is one of the common mutations observed in 20-40% of patients. AE affects transcriptional regulation associated with hematopoietic differentiation, while 60% of AE-positive AML cases are shown to have together other types of mutation of genes involved in cell proliferation, such as receptor tyrosine kinase (RTK) c-kit and FLT3. However, the detailed mechanisms of how they work in the very early stages of leukemogenesis and what unknown "cooperative " cues function in those periods. From this viewpoint, in order to identify novel cellular molecules involved in the acquisition of leukemic phenotypes, we have conducted the gene-trap strategy-based phenomic screen in the use of pluripotent stem cell (PSC)-derived hematopoietic culture. Through our screening, we found that the functional knockdown of a gene NSFL1c, also known as p47, enhanced the activities of hematopoietic progenitor cells harboring AE to show leukemic phenotype both in vivo and in vitro: Cells differentiated from AE-PSCs which have additionally the poly A trapping sequence inserted in NSFL1c locus showed doubled efficiency in engraftment into immunodeficient NOG mice than cells without trapping (3.1% v.s. 1.3%, 16 weeks after intra bone marrow transplantation), and also showed the significantly higher colony replating efficacy in methylcellulose colony forming assay. In order to clear what lineages of cells were most responsible for those phenomena, we next performed those colony forming experiments after sorting of CD34+CD43+CD13- immature cells, CD34+/-CD43+CD13+ myeloid cells and CD71+CD41+ erythro-megakaryocytic subpopulations of cells. As a result, CD34+/-CD43+CD13+ myeloid precursors showed the strongest tendencies to emerge highly proliferative clones followed by CD34+CD43+CD13- immature progenitors in the presence of NSF1c trapping. Interestingly, those activities were cancelled in the absence of AE expression. NSFL1c is one of the component of NEMO complex which binds to ubiquitinated NEMO and induces its degradation, resulting in reduced IKK and elevated NFkB activities. In AML, elevated NF-κB pathways have been detected in more than 30% of patients. Although NF-κB signaling networks have proved induced by inflammatory and immune signals, and previous studies in vivo and in vitro showed their abnormal activities make leukemia cells escape from cell death and go into abnormal proliferation, the detailed mechanisms of how they are involved in the pathogenesis, in particular at the very early stages of the leukemogenesis, are well not defined. Our data therefore reflect the novel mechanisms behind the deviation of progenitor cell fate from normal to abnormal pathway leading to the emergence of leukemic initiating cells, and suggested the myeloid-biased leukemogenic potentials. Disclosures No relevant conflicts of interest to declare.

Sculpting ion channel functional expression with engineered ubiquitin ligases

The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels.

PXD101 potentiates hormonal therapy and prevents the onset of castration-resistant phenotype modulating androgen receptor, HSP90, and CRM1 in preclinical models of prostate cancer

Aberrant activation or ‘reactivation’ of androgen receptor (AR) during androgen ablation therapy shows a potential cause for the development of castration-resistant prostate cancer. This study tested the hypothesis that PXD101, a potent pan histone deacetylase (HDAC) inhibitor, may prevent onset of castration-resistant phenotype and potentiate hormonal therapy. A panel of human prostate cancer cells with graded castration-resistant phenotype and in vivo models were used to verify this hypothesis. In this report, we demonstrated that hormonal manipulation favors the onset of castration-resistant phenotype increasing HDAC expression and activity as well as modulating expression and activity of AR, EGFR, HER2, and Akt. Consistent with these observations, the functional knockdown of HDACs by PXD101 prevented the onset of castration-resistant phenotype with a significant downregulation of AR, EGFR, HER2, and Akt expression/activity. The dysregulation of functional cooperation between HDAC6 with hsp90, on the one hand, and between GSK-3β with CRM1, on the other hand, may explain the biological effects of PXD101. In this regard, the HDAC6 silencing or the functional knockdown of hsp90 by 17AAG resulted in the selective downregulation of AR, EGFR, HER2, and Akt expression/activity, while the decreased phosphorylation of GSK-3β mediated by PXD101 increased the nuclear expression of CRM1, which in turn modified the AR and survivin recycling with increased caspase 3 activity. HDAC inhibitors retain the ability to prevent the onset of castration-resistant phenotype and, therefore, merit clinical investigation in this setting. However, additional data are needed to develop clinical treatment strategies for this disease stage.