A Small Molecule, UAB126, Reverses Diet-Induced Obesity and its Associated Metabolic Disorders.

Targeting retinoid X receptor (RXR) has been proposed as one of the therapeutic strategies to treat individuals with metabolic syndrome, as RXR heterodimerizes with multiple nuclear receptors that regulate genes involved in metabolism. Despite numerous efforts, RXR ligands (rexinoids) have not been approved for clinical trials to treat metabolic syndrome due to the serious side effects such as hypertriglyceridemia and altered thyroid hormone axis. In this study, we demonstrate a novel rexinoid-like small molecule, UAB126, which has positive effects on metabolic syndrome without the known side effects of potent rexinoids. Oral administration of UAB126 ameliorated obesity, insulin resistance, hepatic steatosis, and hyperlipidemia without changes in food intake, physical activity, and thyroid hormone levels. RNA-sequencing analysis revealed that UAB126 regulates the expression of genes in the liver that are modulated by several nuclear receptors, including peroxisome proliferator-activated receptor α and/or liver X receptor in conjunction with RXR. Furthermore, UAB126 not only prevented but also reversed obesity-associated metabolic disorders. The results suggest that optimized modulation of RXR may be a promising strategy to treat metabolic disorders without side effects. Thus, the current study reveals that UAB126 could be an attractive therapy to treat individuals with obesity and its comorbidities.

CFTR dysfunction increases endoglin and TGF-ß signaling in airway epithelia.

Endoglin (ENG) regulates signaling by transforming growth factor-ß (TGF-ß), a genetic modifier of cystic fibrosis (CF) lung disease severity. We hypothesized that ENG mediates TGF-ß pathobiology in CF airway epithelia. Comparing CF and non-CF human lungs, we measured ENG by qPCR, immunoblotting and ELISA. In human bronchial epithelial cell lines (16HBE), we used CFTR siRNA knockdown and functional inhibition (CFTRINH -172) to connect loss of CFTR to ENG synthesis. Plasmid overexpression of ENG assessed the direct effect of ENG on TGF-ß transcription and signal amplification in 16HBE cells. We found ENG protein to be increased more than fivefold both in human CF bronchoalveolar fluid (BALF) and human CF lung homogenates. ENG transcripts were increased threefold in CF, with a twofold increase in TGF-ß signaling. CFTR knockdown in 16HBE cells tripled ENG transcription and doubled protein levels with corresponding increases in TGF-ß signaling. Plasmid overexpression of ENG alone nearly doubled TGF-ß1 mRNA and increased TGF-ß signaling in 16HBE cells. These experiments identify that loss of CFTR function increases ENG expression in CF epithelia and amplifies TGF-ß signaling. Targeting ENG may offer a novel therapeutic opportunity to address TGF-ß associated pathobiology in CF.

Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening.

The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte's Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research's HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens.

Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

4-Hydroxytamoxifen induces autophagic death through K-Ras degradation.

Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved. In this study, we gained insights into the estrogen receptor-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated autophagic degradation of K-Ras, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma, and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

High-throughput RNA interference screening: tricks of the trade.

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z' factor, which incorporates both standard deviation and signal window. A Z' factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z' factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z' factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.

A brain-specific SGK1 splice isoform regulates expression of ASIC1 in neurons.

Neurodegenerative diseases and noxious stimuli to the brain enhance transcription of serum- and glucocorticoid-induced kinase-1 (SGK1). Here, we report that the SGK1 gene encodes a brain-specific additional isoform, SGK1.1, which exhibits distinct regulation, properties, and functional effects. SGK1.1 decreases expression of the acid-sensing ion channel-1 (ASIC1); thereby, SGK1.1 may limit neuronal injury associated to activation of ASIC1 in ischemia. Given that neurons express at least two splice isoforms, SGK1 and SGK1.1, driven by distinct promoters, any changes in SGK1 transcript level must be examined to define the isoform induced by each stimulus or neurological disorder.

Simple chordates exhibit a proton-independent function of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) constitute a family of neuron-specific voltage-insensitive sodium channels gated by extracellular protons. Functions of ASICs in mammals include nociception, mechanosensation, and modulation of synaptic transmission. However, the role protons play in mediating the effects of ASICs remains elusive. We have examined ASICs from the ascidian Ciona intestinalis, a simple chordate organism whose nervous system in the larval stage exhibits high similarity to that of higher vertebrates. We found that the ascidian genome contains a single ASIC gene that gives rise to two splice forms analogous to the mammalian ASIC1 and ASIC2. CiASIC is expressed in most neurons of the larva but is absent in the adult. Despite high sequence similarity with mammalian counterparts, CiASIC is proton-insensitive when examined in heterologous systems or in larval neurons; the latter rules out the possibility that proton sensitivity is conferred by accessory proteins or particular factors present only in Ciona neurons. Down-regulation of the CiASIC transcript by double-stranded RNA disrupted the regular pattern of larval swimming, implying that proton-independent mechanisms mediate the effects of ASIC in vivo. Together the data identify ASIC as a highly conserved channel distinctive of chordate nervous systems and show that protons are not essential for ASIC function.

Effects of extracellular calcium on fASIC1 currents.

Fish ASIC1 (fASIC1) cloned from Opsanus tau, unlike the rat ASICs, requires Ca(2+) in the extracellular preconditioning solution (pH 7.4) to be activated. Here we show that fASIC1 is interacting with Ca(2+) in the same way as mammalian ASICs: extracellular Ca(2+) is increasing the proportion of channels available for activation by stabilizing the closed state of the channel; in the activation process Ca(2+) is released; H(+) compete for the binding site of Ca(2+) making the gating mechanism both Ca(2+) and H(+) dependent; H(+) stabilizes the desensitized state; Ca(2+) blocks the fASIC1 channel; and the affinity of the block is also modulated by H(+). The "Ca(2+) activation requirement" of fASIC1 reflects its greater affinity for steady-state desensitization by H(+) compared to mammalian ASIC1.

Proton sensitivity of ASIC1 appeared with the rise of fishes by changes of residues in the region that follows TM1 in the ectodomain of the channel.

The acid-sensitive ion channel 1 (ASIC1) is a neuronal Na+ channel insensitive to changes in membrane potential but is gated by external protons. Proton sensitivity is believed to be essential for the role of ASIC1 in modulating synaptic transmission and nociception in the mammalian nervous system. To examine the structural determinants that confer proton sensitivity, we cloned and functionally characterized ASIC1 from different species of the chordate lineage: lamprey, shark, toadfish and chicken. We observed that ASIC1s from early vertebrates (lamprey and shark) were proton insensitive in spite of a high degree of amino acid conservation (66-67%) with their mammalian counterparts. Sequence analysis showed that proton-sensitive ASIC1s could not be distinguished from proton-insensitive channels by any signature in the protein sequence. Chimeras made with rat ASIC1 (rASIC1) and lamprey or shark indicated that most of the ectodomain of rASIC1 was required to confer proton sensitivity and the distinct kinetics of activation and desensitization of the rat channel. Proton-sensitive chimeras contained the segment D78-E136, together with residues D351, Q358 and E359 of the rat sequence. However, none of the functional chimeras containing only part of the rat extracellular domain retained the kinetics of activation and desensitization of rASIC1, suggesting that residues distributed in several regions of the ectodomain contribute to allosteric changes underlying activation and desensitization. The results also demonstrate that gating by protons is not a feature common to all ASIC1 channels. Proton sensitivity arose recently in evolution, implying that agonists different from protons activate ASIC1 in lower vertebrates.

Expression of ENaC and serum- and glucocorticoid-induced kinase 1 in the rat intestinal epithelium.

Increase in epithelium sodium channel (ENaC) activity induced by aldosterone in the distal tubule of the kidney has been attributed to serum- and glucocorticoid-induced kinase 1 (sgk1). The distal colon constitutes another classical aldosterone-responsive epithelium that expresses both ENaC and sgk1 in an aldosterone-dependent manner. However, the site of expression and the temporal relationship of the aldosterone induction of these two proteins have not been investigated. Here, we examined the distribution and abundance of sgk1 in the rat intestine under basal conditions and after changes in the concentration of aldosterone and glucocorticoids. Results indicate that sgk1 is expressed in the distal colon and also in the ileum and jejunum. Abundance of sgk1 was high in control animals, and it did not change significantly after sodium depletion or after a single dose of aldosterone; however, it decreased after adrenalectomy. In contrast, the three subunits of ENaC were markedly induced in the distal colon by acute and chronic increases in aldosterone levels. Results indicate differential regulation of sgk and ENaC subunits by aldosterone in the distal colon. Distribution of sgk1 in the intestine beyond the aldosterone-responsive segments suggests that sgk1 may additionally regulate other sodium transporters in the intestinal epithelium.

The extracellular domain determines the kinetics of desensitization in acid-sensitive ion channel 1.

The acid-sensitive ion channel 1 (ASIC1alpha or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that has the broadest distribution in the nervous system. Hallmarks of ASIC1alpha are gating by external protons and rapid desensitization. In sensory neurons ASIC1 may constitute a nociceptor for pain induced by local acidification, whereas in central neurons it may modulate synaptic activity. To gain insight into the functional roles of ASIC1, we cloned and examined the properties of the evolutionarily distant species toadfish (Opsanus tau), approximately 420-million year divergent from mammals. Analysis of the protein sequence from fish ASIC1 revealed 76% amino acid identity with the rat orthologue. The regions of highest conservation are the second transmembrane domain and the ectodomain, whereas the amino and carboxyl termini and first transmembrane domain are poorly conserved. At the functional level, fish ASIC1 is gated by external protons with a half-maximal activation at pHo 5.6 and a half-maximal inactivation at pHo 7.30. The fish differs from the rat channel on having a 25-fold faster rate of desensitization. Functional studies of chimeras made from rat and fish ASIC1 indicate that the extracellular domain specifically, a cluster of three residues, confers the faster desensitization rate to the fish ASIC1.