ETS Domain-containing Protein Research Articles

ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea

Intermittent hypoxia (IH) in patients with obstructive sleep apnea (OSA) syndrome elicited neuron injury (especially in the hippocampus and cortex), contributing to cognitive dysfunction. This study investigated the effects and clarified the mechanisms of ETS domain-containing protein Elk-4 (ELK4) on the cognitive function and neuroinflammation of mice with IH. Mouse microglia BV2 cells were induced with IH by exposure to fluctuating O2 concentrations (alternating from 5 % to 21 % every 30 min), and mice with OSA were developed and subjected to lentivirus-mediated gene intervention. ELK4 expression was significantly reduced in IH-induced microglia and brain tissues of mice with OSA. Overexpression of ELK4 attenuated oxidative stress, decreased the pro-inflammatory factors IL-1β, IL-6, and TNF-α, and increased the level of the anti-inflammatory factors IL-10 and TGF-β1, as well as the neuroprotective factor BDNF. ELK4 promoted the transcription of fibronectin type III domain-containing protein 5 (FNDC5) by binding to the promoter of FNDC5. Knockdown of FNDC5 in IH-induced microglia and animals reversed the protective effects of ELK4 on OSA-associated neuroinflammation and cognitive dysfunction. Overall, the results demonstrated that ELK4 overexpression repressed microglial activation by inducing the transcription of FNDC5, thus attenuating neuroinflammation and cognitive dysfunction induced by OSA.

The Elk-3 target Abhd10 ameliorates hepatotoxic injury and fibrosis in alcoholic liver disease

Alcoholic liver disease (ALD) and other forms of chronic hepatotoxic injury can lead to transforming growth factor β1 (TGFβ1)-induced hepatic fibrosis and compromised liver function, underscoring the need to develop novel treatments for these conditions. Herein, our analyses of liver tissue samples from severe alcoholic hepatitis (SAH) patients and two murine models of ALD reveals that the ALD phenotype was associated with upregulation of the transcription factor ETS domain-containing protein (ELK-3) and ELK-3 signaling activity coupled with downregulation of α/β hydrolase domain containing 10 (ABHD10) and upregulation of deactivating S-palmitoylation of the antioxidant protein Peroxiredoxin 5 (PRDX5). In vitro, we further demonstrate that ELK-3 can directly bind to the ABHD10 promoter to inhibit its transactivation. TGFβ1 and epidermal growth factor (EGF) signaling induce ABHD10 downregulation and PRDX5 S-palmitoylation via ELK-3. This ELK-3-mediated ABHD10 downregulation drives oxidative stress and disrupts mature hepatocyte function via enhancing S-palmitoylation of PRDX5’s Cys100 residue. In vivo, ectopic Abhd10 overexpression ameliorates liver damage in ALD model mice. Overall, these data suggest that the therapeutic targeting of the ABHD10-PRDX5 axis may represent a viable approach to treating ALD and other forms of hepatotoxicity.

Knockdown of lncRNA LINC00662 suppresses malignant behaviour of osteosarcoma cells via competition with miR-30b-3p to regulate ELK1 expression

PurposeOsteosarcoma is a type of bone malignancy that mainly occurred in teenagers. This investigation is aimed to clarify the effect of long non-coding RNA (lncRNA) LINC00662 on the proliferation, migration, and invasion in osteosarcoma and explore the underlying action mechanisms.MethodsThe mRNA expression of LINC00662 was determined by real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and transwell assays, respectively. A dual-luciferase reporter assay was used to validate the target relationships Between microRNA (miR)-30b-3p and LINC00662/ ETS domain-containing protein 1 (ELK1). Western blotting was performed to determine the protein expression of ELK1. Xenograft model was established to evaluate the effects of LINC00662 silencing on tumor growth in vivo.ResultsLncRNA LINC00662 and ELK1 were significantly increased, while miR-30b-3p was reduced in osteosarcoma tissues. The results of functional experiments indicated that transfection of small hairpin (sh)-LINC00662 and miR-30b-3p mimics repressed the migration, invasion, and proliferation of osteosarcoma cells. LncRNA LINC00662 also appeared to sponge miR-30b-3p in order to affect the expression of ELK1. Simultaneously, there were weak negative correlations between the expression of miR-30b-3p and LINC00662/ELK1 in osteosarcoma tissues. Rescue experiments suggested that ELK1 overexpression and downregulation of miR-30b-3p reversed the suppressive effects of sh-LINC00662 on the cell migration, invasion, and proliferation in osteosarcoma.ConclusionsThe current study indicated that knockdown of LINC00662 repressed cell migration, invasion, and proliferation through sponging miR-30b-3p to regulate the expression of ELK1 in osteosarcoma. These results may uncover a promising target for the treatment of osteosarcoma.

Peroxisome proliferator‑activated receptor β/δ regulates cerebral vasospasm after subarachnoid hemorrhage via modulating vascular smooth muscle cells phenotypic conversion.

Cerebral vasospasm (CVS) is a common complication of subarachnoid hemorrhage (SAH) with high deformity rates and cerebral vascular smooth muscle cells (VSMCs) phenotypic switch is considered to be involved in the regulation of CVS. However, to the best of the authors' knowledge, its underlying molecular mechanism remains to be elucidated. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been demonstrated to be involved in the modulation of vascular cells proliferation and maintains the autoregulation function of blood vessels. The present study investigated the potential effect of PPARβ/δ on CVS following SAH. A model of SAH was established by endovascular perforation on male adult Sprague-Dawley rats, and the adenovirus PPARβ/δ (Ad-PPARβ/δ) was injected via intracerebroventricular administration prior to SAH. The expression levels of phenotypic markers α-smooth muscle actin and embryonic smooth muscle myosin heavy chain were measured via western blotting or immunofluorescence staining. The basilar artery diameter and vessel wall thickness were evaluated under fluorescence microscopy. SAH grade, neurological scores, brain water content and brain swelling were measured to study the mechanisms of PPARβ/δ on vascular smooth muscle phenotypic transformation. It was revealed that the expression levels of synthetic proteins were upregulated in rats with SAH and this was accompanied by CVS. Activation of PPARβ/δ using Ad-PPARβ/δ markedly upregulated the contractile proteins elevation, restrained the synthetic proteins expression and attenuated SAH-induced CVS by regulating the phenotypic switch in VSMCs at 72 h following SAH. Furthermore, the preliminary study demonstrated that PPARβ/δ downregulated ERK activity and decreased the expression of phosphorylated (p-)ETS domain-containing protein Elk-1 and p-p90 ribosomal S6 kinase, which have been demonstrated to serve an important role in VSMC phenotypic change. Additionally, it was revealed that Ad-PPARβ/δ could positively improve CVS by ameliorating the diameter of the basilar artery and mitigating the thickness of the vascular wall. Furthermore, subsequent experiments demonstrated that Ad-PPARβ/δ markedly reduced the brain water content and brain swelling and improved the neurological outcome. Taken together, the present study identified PPARβ/δ as a useful regulator for the VSMCs phenotypic switch and attenuating CVS following SAH, thereby providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

Synergistic anti-inflammatory effects of quercetin and catechin via inhibiting activation of TLR4-MyD88-mediated NF-κB and MAPK signaling pathways.

The synergistic anti-inflammatory effect of quercetin and catechin was investigated using lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. Results showed that the combined treatment of quercetin with catechin synergistically attenuated LPS-stimulated increase of some proinflammatory molecules, including nitric oxide, tumor necrosis factor α, interleukin-1β, nitric oxide synthase, and cyclooxygenase-2. Moreover, it exhibited significantly (p<0.05) stronger inhibitory effect on nuclear translocation of nuclear factor-κB (NF-κB) by suppressing the phosphorylation of NF-κB p65 and p50 submits and on the phosphorylation of ETS domain-containing protein and c-Jun N-terminal kinase than any of quercetin or catechin alone. Besides, the cotreatment of quercetin with catechin significantly (p<0.05) restored the impaired expression of toll-like receptor 4, myeloid differentiation primary response gene 88, and some downstream effectors (IRAK1, TRAF6, and TAK1). These results suggest that quercetin and catechin possessed synergistic anti-inflammatory effects, which may be attributed to their roles in suppressing the activation of TLR4-MyD88-mediated NF-κB and mitogen-activated protein kinases signaling pathways.

Ablation of low‐molecular‐weight FGF2 isoform accelerates murine osteoarthritis while loss of high‐molecular‐weight FGF2 isoforms offers protection

FGF2 is an essential growth factor implicated in osteoarthritis (OA), and deletion of full-length FGF2 (Fgf2ALLKO ) leads to murine OA. However, the FGF2 gene encodes both high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms, and the effects of selectively ablating individual isoforms, as opposed to total FGF2, has not been investigated in the context of OA. We undertook this study to examine whether mice lacking HMW FGF2 (Fgf2HMWKO ) or LMW FGF2 (Fgf2LMWKO ) develop OA and to further characterize the observed OA phenotype in Fgf2ALLKO mice. Fgf2HMWKO mice never developed OA, but 6- and 9-month-old Fgf2LMWKO and Fgf2ALLKO mice displayed signs of OA, including eroded articular cartilage, altered subchondral bone and trabecular architecture, and increased OA marker enzyme levels. Even with mechanical induction of OA, Fgf2HMWKO mice were protected against OA, whereas Fgf2LMWKO and Fgf2ALLKO displayed OA-like changes of the subchondral bone. Before exhibiting OA symptoms, Fgf2LMWKO or Fgf2ALLKO joints displayed differential expression of genes encoding key regulatory proteins, including interleukin-1β, insulin-like growth factor 1, bone morphogenetic protein 4, hypoxia-inducible factor 1, B-cell lymphoma 2, Bcl2-associated X protein, a disintegrin and metalloproteinase with thrombospondin motifs 5, ETS domain-containing protein, and sex-determining region Y box 9. Moreover, Fgf2LMWKO OA cartilage exhibited increased FGF2, FGF23, and FGFR1 expression, whereas Fgf2HMWKO cartilage had increased levels of FGFR3, which promotes anabolism in cartilage. These results demonstrate that loss of LMW FGF2 results in catabolic activity in joint cartilage, whereas absence of HMW FGF2 with only the presence of LMW FGF2 offers protection from OA.

NDUFA4L2 is associated with clear cell renal cell carcinoma malignancy and is regulated by ELK1.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common and lethal cancer of the adult kidney. However, its pathogenesis has not been fully understood till now, which hinders the therapeutic development of ccRCC. NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) was found to be upregulated and play an important role in ccRCC. We aimed to further investigate the underlying mechanisms by which NDUFA4L2 exerted function and its expression level was upregulated.MethodsThe Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data were mined to verify the change of NDUFA4L2 expression level in ccRCC tissues. The correlation between expression level of NDUFA4L2 and cell proliferation/apoptosis was explored by Gene Set Enrichment Analysis (GSEA). Protein-protein interaction (PPI) network of NDUFA4L2 was constructed. Biological process and involved pathways of NDUFA4L2 were analyzed by gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The transcription factors (TFs) which can induce the expression of NDUFA4L2 were explored in clinical samples by correlation analysis and its regulation on the expression of NDUFA4L2 was verified by knockdown experiment.ResultsNDUFA4L2 was verified to be overexpressed in ccRCC tissues and its expression level was increased accordingly as the American Joint Committee on Cancer (AJCC) stage progressed. A high NDUFA4L2 level predicted the poor prognosis of ccRCC patients and correlated with enhanced cell proliferation and anti-apoptosis. NDUFA4L2 may interact with 14 tumor-related proteins, participate in growth and death processes and be involved in ccRCC-related pathways, such as insulin-like growth factor 1 (IGF-1), mammalian target of Rapamycin (mTOR) and phosphoinositide 3 kinase serine/threonine protein kinase (PI3K/AKT). ETS domain-containing protein ELK1 level positively correlated with the level of NDUFA4L2 in ccRCC tissues and ELK1 could regulate the expression of NDUFA4L2 in ccRCC cells.DiscussionNDUFA4L2 upregulation was associated with ccRCC malignancy. NDUFA4L2 expression was regulated by ELK1 in ccRCC cells. Our study provided potential mechanisms by which NDUFA4L2 affected ccRCC occurrence and progression.

MicroRNA-326 suppresses the proliferation, migration and invasion of cervical cancer cells by targeting ELK1.

Although microRNAs (miRNAs or miRs) are able to function as oncogenes or tumor suppressors, the role of miR-326 in regulating human cervical cancer cells remains unclear. In the present study, the expression of miR-326 was identified to be downregulated in cervical cancer cell lines and primary tumor samples, and the overexpression of miR-326 decreased cell proliferation, migration and invasion in cervical cell lines. Bioinformatics prediction and experimental validation results revealed that the function of miR-326 was achieved by targeting and repressing ETS domain-containing protein Elk-1 (ELK1) expression. ELK1 was targeted directly by miR-326, which was downregulated in human cervical cancer tissues compared with that in adjacent normal tissues. The results of the present study suggest that miR-326, a potential tumor suppressor, may be used in the treatment of cervical cancer.

Pravastatin attenuates the action of the ETS domain-containing protein ELK1 to prevent atherosclerosis in apolipoprotein E-knockout mice via modulation of extracellular signal-regulated kinase 1/2 signal pathway.

Oxidative stress plays an important role in atherosclerosis, a vascular disease with high morbidity and mortality. The ETS domain-containing protein ELK1 is an oxidative stress-sensitive factor modulated by the extracellular signal-regulated kinase (ERK) 1/2 pathway. However, the role of ELK1 in the prevention of atherosclerosis by pravastatin remains unclear. In the present study, male apolipoprotein E-knockout (apoE-/- ) mice fed a diet containing 1.25% cholesterol (w/w) were divided into two groups, one treated with pravastatin (80mg/kg, 2-2.4mg/mouse per day) for 8weeks and the other not. Male C57BL/6J mice fed with a normal diet were used as a control group. Human umbilical vein endothelial cells (HUVEC) were cultured and treated with pravastatin (10μmol/L) for 18hours before testing for the presence or absence of 100μmol/L H2 O2 (24hours). Examination of pathological sections from mice aortas revealed that pravastatin treatment almost prevented atherosclerotic plaque formation. Pravastatin also inhibited increases in serum and aortic levels of oxidized low-density lipoprotein and aortic malondialdehyde levels and decreases in aortic reduced glutathione, and the activities of superoxide dismutase, catalase and glutathione peroxidase. H2 O2 -induced increases in reactive oxygen species in HUVECs were reversed by pravastatin by 48%. Pravastatin blocked the phosphorylation of ELK1 and ERK1/2 proteins and reduced mRNA levels of early growth response 1, a known atherogenic transcription factor upregulated by the ROS/ERK/ELK1 pathway, in mice. In conclusion, pravastatin attenuates the action of ELK1 induced by oxidative stress to prevent atherosclerosis, which is dependent partly on modulation of ERK1/2 signalling.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFβ1 is considered central to the pathogenesis of IPF. A major mechanism of TGFβ1 activation in the lung involves the epithelially restricted αvβ6 integrin. Expression of the αvβ6 integrin is dramatically increased in IPF. How αvβ6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the β6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvβ6 integrin expression in vitro. We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvβ6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition.

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.

Gene expression changes in human mesenchymal stem cells from patients with osteoporosis.

The aim of the present study was to investigate the underlying molecular mechanisms of osteoporosis and to identify novel candidate genes involved in this disease. The gene expression profile of GSE35958 was downloaded from Gene Expression Omnibus, including five samples of human mesenchymal stem cells from patients with osteoporosis and four control samples. Differentially expressed genes (DEGs) were initially identified following an analysis using Student’s t-test. Subsequently, a protein-protein interaction (PPI) network of the significant pathways was constructed, based on the Human Protein Reference Database. In the significant pathways, DEGs were screened using cut-off criteria of FDR<0.1 and |log2FC|>1.5. A co-change network for pathways was also constructed using the method of cumulative hypergeometric probability distribution. Finally, the transcriptional regulatory network for DEGs was constructed based on the TRANSFAC database. In total, 1,127 DEGs, including 554 upregulated and 573 downregulated DEGs, were screened. The constructed PPI network for the DEGs involved in the two significant pathways, including focal adhesion and lysosome, demonstrated that the five DEGs with a high degree (>60) were β-catenin, SHC-transforming protein 1, RAC-α serine/threonine-protein kinase, caveolin 1 and filamin A, with degrees of 135, 117, 117, 73 and 63, respectively. The pathway with the degree of 22 in the constructed co-change network was neuroactive ligand receptor interaction. The nine genes with a high (≥9) degree in the constructed transcriptional regulatory network were REL-associated protein, upstream stimulatory factor 1, specificity protein 1, Fos-related antigen 1, cyclin-dependent kinase inhibitor 1A, upstream stimulatory factor 2, ETS domain-containing protein Elk-1, JUND and retinoic acid receptor α, with degrees of 29, 27, 19, 18, 17, 13, 11, 11 and 9, respectively. The DEGs with high degree in the PPI and transcriptional regulatory networks may be candidate target molecules, which may be used to monitor, diagnose and treat osteoporosis.

Active ERK2 is sufficient to mediate growth arrest and differentiation signaling.

Although extracellular signal-regulated kinases (ERK1/2) have been shown to be required in Raf/MEK/ERK pathway signaling, its sufficiency for mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously described ERK2 mutants that exhibit enhanced autophosphorylation of TEY sites in the activation loop in terms of their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. We demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp substitutions that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation, whereas expression of ERK2-I84A and ERK2-R65S/D319N is not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D only mildly increased ERK kinase activity in cells, as assessed using the ERK substrates p90(RSK) and ETS domain-containing protein (ELK1). However, ERK2-L73P/S151D expression effectively induced down-regulation of androgen receptors, Retinoblastoma (Rb) protein and E2F1 transcription factor, and up-regulation of p16(INK4A) and p21(CIP1), accompanied by cell-cycle arrest and morphological differentiation in LNCaP cells and neurite-like processes in PC12 cells. These effects and the TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introduction of the active site-disabling Lys52Arg mutation, suggesting that its autoactivation drives this signaling. Moreover, introduction of mutations Asp316/319Ala or Asp319Asn, which impair the common docking site/D-domain-based physical interaction of ERK, did not significantly affect ERK2-L73P/S151D signaling, suggesting that ERK2 mediates growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Thus, our study presents convincing evidence of ERK sufficiency for Raf/MEK/ERK signaling.

PRK1/PKN1 controls migration and metastasis of androgen-independent prostate cancer cells.

The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer.

Transcriptional and epigenetic substrates of methamphetamine addiction and withdrawal: evidence from a long-access self-administration model in the rat.

Methamphetamine use disorder is a chronic neuropsychiatric disorder characterized by recurrent binge episodes, intervals of abstinence, and relapses to drug use. Humans addicted to methamphetamine experience various degrees of cognitive deficits and other neurological abnormalities that complicate their activities of daily living and their participation in treatment programs. Importantly, models of methamphetamine addiction in rodents have shown that animals will readily learn to give themselves methamphetamine. Rats also accelerate their intake over time. Microarray studies have also shown that methamphetamine taking is associated with major transcriptional changes in the striatum measured within a short or longer time after cessation of drug taking. After a 2-h withdrawal time, there was increased expression of genes that participate in transcription regulation. These included cyclic AMP response element binding (CREB), ETS domain-containing protein (ELK1), and members of the FOS family of transcription factors. Other genes of interest include brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor, type 2 (TrkB), and synaptophysin. Methamphetamine-induced transcription was found to be regulated via phosphorylated CREB-dependent events. After a 30-day withdrawal from methamphetamine self-administration, however, there was mostly decreased expression of transcription factors including junD. There was also downregulation of genes whose protein products are constituents of chromatin-remodeling complexes. Altogether, these genome-wide results show that methamphetamine abuse might be associated with altered regulation of a diversity of gene networks that impact cellular and synaptic functions. These transcriptional changes might serve as triggers for the neuropsychiatric presentations of humans who abuse this drug. Better understanding of the way that gene products interact to cause methamphetamine addiction will help to develop better pharmacological treatment of methamphetamine addicts.

Distinction of Cell Types in Dicyema japonicum (Phylum Dicyemida) by Expression Patterns of 16 Genes

Dicyemids (phylum Dicyemida) are endoparasites, or endosymbionts, typically found in the renal sac of benthic cephalopod molluscs. The body organization of dicyemids is very simple, consisting of only 9 to 41 somatic cells. Dicyemids appear to have no differentiated tissues. Although categorization of somatic cells, to some types, is based on differences in the pattern of cilia and their position in the body, whether or not these cells are functionally different remains to be revealed. To provide insight into the functional differentiation, we performed whole mount in situ hybridization (WISH) to detect expression patterns of 16 genes, i.e., aquaglyceroporin, F-actin capping protein, aspartate aminotransferase, cathepsin-L-like cysteine peptidase, Ets domain-containing protein, glucose transporter, glucose-6-phosphate 1-dehydrogenase, glycine transporter, Hsp 70, Hsp 90, isocitrate dehydrogenase subunit alpha, Rad18, serine hydroxymethyltransferase, succinate-CoA ligase, valosin-containing protein, and 14-3-3 protein. In certain genes, regional specific expression patterns were observed among somatic cells of vermiform stages and infusoriform larvae of dicyemids. The WISH analyses also revealed that the Ets domain-containing protein and Rad18 are molecular markers for agametes.

Detection of Treatment-Induced Changes in Signaling Pathways in Gastrointestinal Stromal Tumors Using Transcriptomic Data

Cell signaling plays a central role in the etiology of cancer. Numerous therapeutics in use or under development target signaling proteins; however, off-target effects often limit assignment of positive clinical response to the intended target. As direct measurements of signaling protein activity are not generally feasible during treatment, there is a need for more powerful methods to determine if therapeutics inhibit their targets and when off-target effects occur. We have used the Bayesian Decomposition algorithm and data on transcriptional regulation to create a novel methodology, Differential Expression for Signaling Determination (DESIDE), for inferring signaling activity from microarray measurements. We applied DESIDE to deduce signaling activity in gastrointestinal stromal tumor cell lines treated with the targeted therapeutic imatinib mesylate (Gleevec). We detected the expected reduced activity in the KIT pathway, as well as unexpected changes in the p53 pathway. Pursuing these findings, we have determined that imatinib-induced DNA damage is responsible for the increased activity of p53, identifying a novel off-target activity for this drug. We then used DESIDE on data from resected, post-imatinib treatment tumor samples and identified a pattern in these tumors similar to that at late time points in the cell lines, and this pattern correlated with initial clinical response. The pattern showed increased activity of ETS domain-containing protein Elk-1 and signal transducers and activators of transcription 3 transcription factors, which are associated with the growth of side population cells. DESIDE infers the global reprogramming of signaling networks during treatment, permitting treatment modification that leverages ongoing drug development efforts, which is crucial for personalized medicine.

Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.

Serum response element associated transcription factors in mouse embryos: Serum response factor, YY1, and PEA3 factor

Many mammalian transcription factors, including human and mouse serum response factors (SRFs), are post-translationally modified with O-linked N-acetylglucosamine monosaccharides on multiple serine and/or threonine residues. Nuclear extracts were prepared from 9.5 to 19 days postcoitum mouse embryos and subsequently were fractionated by wheat germ agglutinin (WGA)-agarose affinity chromatography. SRF binds WGA-agarose and apparently is O-glycosylated. On the other hand, the low molecular weight serum response element (SRE)-binding proteins, including the previously named band I and band II factors, did not bind WGA-agarose. Furthermore, we showed that the fastest migrating complex contains the Yin-Yang 1 (YY1) factor. YY1 binds to the c-fos SRE and skeletal alpha-actin muscle regulatory element (MRE), but not the cardiac alpha-actin MRE. Nuclear extracts from NIH/3T3 fibroblasts contain similar, if not identical, SRE-binding complexes. Besides these SRE-binding factors, mouse PEA3-binding factor, presumably an ETS domain-containing protein, was found to bind SRF protein. This physical interaction, between SRF and ETS domain proteins, was shown to involve the DNA-binding domain-containing region of SRF and not the carboxyl-terminal transactivation domain.