miRNA-1 regulates endothelin-1 in diabetes

Aims

MicroRNAs (miRNAs) play important roles in several biological processes. In this study, we investigated the role of miR-1, an endothelin-1 (ET-1) targeting miRNA, in endothelial cells (ECs) and tissues of diabetic animals. ET-1 is known to be of pathogenetic significance in several chronic diabetic complications.

Main methods

PCR array was used to identify alterations of miRNA expression in ECs exposed to glucose. miR-1 expression was validated by TaqMan real-time PCR assay. Human retinal ECs (HRECs) and human umbilical vein ECs (HUVECs) exposed to various glucose levels with or without miR-1 mimic transfection, and tissues from streptozotocin-induced diabetic animals after two months of follow-up, were examined for miR-1 expression, as well as ET-1 and fibronectin (FN) mRNA and protein levels.

Key findings

Array analyses showed glucose-induced alterations of 125 miRNAs (out of 381) in ECs exposed to 25 mM glucose compared to 5 mM glucose. Fifty-one miRNAs were upregulated and 74 were downregulated. 25 mM glucose decreased miR-1 expression and increased ET-1 mRNA and protein levels. miR-1 mimic transfection prevented HG-induced ET-1 upregulation. Furthermore, glucose induced upregulation of FN, which is mediated partly by ET-1, was also prevented by such transfection.Diabetic animals showed decreased miR-1 expression in the retina, heart and kidneys. In parallel, ET-1 mRNA expressions were increased in these tissues of diabetic animals, in association with upregulation of FN.

Significance

These results indicate a novel glucose-induced mechanism of tissue damage, in which miR-1 regulates ET-1 expressions in diabetes. Identifying such mechanisms may lead to RNA based treatment for diabetic complications.     

AXR1-ECR1 and AXL1-ECR1 heterodimeric RUB-activating enzymes diverge in function in Arabidopsis thaliana

RELATED TO UBIQUITIN (RUB) modification of CULLIN (CUL) subunits of the CUL-RING ubiquitin E3 ligase (CRL) superfamily regulates CRL ubiquitylation activity. RUB modification requires E1 and E2 enzymes that are analogous to, but distinct from, those activities required for UBIQUITIN (UBQ) attachment. Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis. To further examine the extent of redundancy within the RUB pathway, we undertook biochemical and genetic characterizations of one such duplication event- the duplication of the genes encoding a subunit of the RUB E1 into AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1). In vitro, the two proteins have similar abilities to function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation and RUB1-ECR1 thioester formation. Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects in axr1-30, a severe loss-of-function AXR1 mutant, when the respective coding sequences are expressed from the same promoter, suggesting differential in vivo functions. These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.     

Group 1 CD1 genes in rabbit

CD1 is an Ag-presenting molecule that can present lipids and glycolipids to T cells. The CD1 genes were first identified in the human, and since then, homologs have been identified in every mammalian species examined to date. Over a decade ago, CD1B and CD1D homologs were identified in the rabbit. We have extended this earlier study by identifying additional CD1 genes with the goal of developing the rabbit as an animal model to study the function of CD1 proteins. We constructed a thymocyte cDNA library and screened the library with CD1-specific probes. Based on nucleotide sequence analyses of the CD1(+) cDNA clones obtained from the library, we have identified two CD1A genes and one CD1E gene as well as determined the complete sequence of the previously identified CD1B gene. The CD1E(+) cDNA clones lacked the transmembrane and cytoplasmic domains and, if translated, would encode for a soluble or secreted CD1E protein. In addition, expression studies demonstrated that the CD1 genes were expressed in peripheral lymphoid tissues as well as in skin, gut, and lung. Of interest is the finding that CD1A2, CD1B, and CD1E genes were found to be expressed by rabbit B cell populations. The rabbit, with a complex CD1 locus composed of at least two CD1A genes, one CD1B gene, one CD1D gene, and one CD1E gene, is an excellent candidate as an animal model to study CD1 proteins.     

Polymorphisms in detoxification genes CYP1A1, CYP2E1, GSTT1 and GSTM1 in gastric cancer susceptibility

Cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes are involved in activation and detoxification of many potential carcinogens. Genetic polymorphisms in those enzymes have been found to influence the interindividual susceptibility to cancer. Some polymorphisms of those enzymes have been associated specifically with susceptibility to gastric cancer. We conducted a study in a Costa Rican population, where gastric cancer incidence and mortality rates are among the highest in the world. We investigated whether such variations affected the risk of developing gastric cancer. Subjects included 31 with gastric cancer, 58 controls with gastric injures others than cancer and 51 normal controls confirmed by X-rays (double-contrast) or endoscopic diagnostic. DNA from peripheral white blood cell was obtained from all subjects. Deletion of GSTT1 and GSTM1 was assessed by multiplex PCR and genotyping of CYP2E1 was performed using a PCR-based restriction fragment length polymorphism assay with the restriction enzyme PstI and the gene CYP1A1 using the restriction enzyme MspI The prevalence of CYP1A1 Msp1 polymorphism, GSTT1 and GSTM1 null genotype was similar in the three groups of individuals (p = 0.73, p = 0.88 y p = 0.89 respectively). Our findings suggest that the polymorphism CYP2E1 PstI could be associated with a reduced risk of having gastric cancer (OR = 0.09, IC95%:0.01 - 0.83).     

Bmi1 cooperates with Dnmt1-associated protein 1 in gene silencing

Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.     

Roles for N-glycosylation in the dynamics of Edg-1/S1P1 in sphingosine 1-phosphate-stimulated cells

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid mediator released from activated platelets. To date, 5 seven-transmembrane-spanning receptors, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4 and Edg-8/S1P5, have been identified as specific Sph-1-P receptors. Our recent novel studies established that Edg-1/S1P1 is glycosylated in its N-terminal extracellular portion and further identified the specific glycosylation site as asparagine 30. We also demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. These studies revealed a possible regulatory role for the N-glycan on Edg-1/S1P1 in the dynamics of the receptor, such as its lateral and internal movements within the membrane, in ligand-stimulated mammalian cells. Published in 2004.     

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

Rap1 and SPA-1 in hematologic malignancy

Rap1 is a member of the Ras family of GTPases and, depending on the cellular context, has an important role in the regulation of proliferation or cell adhesion. In lymphohematopoietic tissues, SPA-1 is a principal Rap1 GTPase-activating protein. Mice that are deficient for the SPA-1 gene develop age-dependent progression of T-cell immunodeficiency followed by a spectrum of late onset myeloproliferative disorders, mimicking human chronic myeloid leukemia. Recent studies reveal that deregulated Rap1 activation in SPA-1-deficient mice causes enhanced expansion of the bone marrow hematopoietic progenitors, but induces progressive unresponsiveness or anergy in T cells. Rap1 and its regulator, SPA-1, could, therefore, provide unique molecular targets for the control of human hematologic malignancy.     

DEC1 expression in 1p-aberrant oligodendroglial neoplasms

BACKGROUND: Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). MATERIALS AND METHODS: 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. RESULTS: DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. CONCLUSION: DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice

The formation of beta-D-glucopyranosides (glucuronides) by the UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that facilitates the elimination of small hydrophobic molecules such as drugs, dietary constituents, steroids, and bile acids. We elucidate here that an anti-oxidative response leads to induction of UGT1A1 through the Nrf2-Keap1 pathway. When human HepG2 cells were treated with the prooxidants tert-butylhydroquinone and beta-naphthoflavone, cellular UGT1A1 glucuronidation activities were increased. The induction of UGT1A1 proceeded following the overexpression of Nrf2 and was blocked following overexpression of Keap1, demonstrating that Keap1 suppresses Nrf2 activation of the UGT1A1 gene. Loss of function analysis for Nrf2 conducted by small interfering RNA revealed that induction of UGT1A1 was not seen in Nrf2 knock-out cells. To examine the contribution of oxidants toward the regulation of human UGT1A1 in vivo, transgenic mice bearing the human UGT1 locus (Tg-UGT1) were treated with tert-butylhydroquinone. Human UGT1A1 was markedly increased in small and large intestines as well as in liver. Gene mapping experiments including transfections of UGT1A1 reporter gene constructs into HepG2 cells coupled with functional analysis of Nrf2 expression and binding to anti-oxidant-response elements (ARE) resulted in identification of an ARE in the phenobarbital-response enhancer module region of the UGT1A1 gene. The ARE flanks the recently identified Ah receptor xenobiotic-responsive element. The results suggest that Nrf2-Keap1-dependent UGT1A1 induction by prooxidants might represent a key adaptive response to cellular oxidative stress that defends against a variety of environmental insults, including electrophile attacks and chemical carcinogenesis.     

Absent or delayed adrenarche in Pit-1/POU1F1 deficiency

Mutations of the PIT1/POU1F1 gene are responsible for a rare variant of anterior hypopituitarism, including deficiency of growth hormone, prolactin and thyrotropin. In 8 ethnically diverse POU1F1-deficient patients (4 different mutations) with normal circulating levels of cortisol and adrenocorticotropic hormone, and with spontaneous onset and progression of puberty, we observed an absence or delay of adrenarche (median circulating dehydroepiandrosterone-sulfate -6.2 SD); in each of the 4 postmenarcheal females, pubarche (i.e. appearance of pubic hair) was also absent or delayed. The absence/delay of adrenarche in POU1F1-deficient patients and the absence/delay of pubarche in POU1F1-deficient females suggest that a POU1F1-dependent factor contributes to the normal development of adrenarche and female pubarche.     

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.     

Polymorphic Variation of CYP1A1 and CYP1B1 Genes in a Haryana Population

Cytochrome P450 (CYP) 1A1 and CYP1B1 are important phase I xenobiotic metabolizing enzymes involved in the metabolism of numbers of toxins, endogenous hormones, and pharmaceutical drugs. Polymorphisms in these phase I genes can alter enzyme activity and are known to be associated with cancer susceptibility related to environmental toxins and hormone exposure. Their genotypes may also display ethnicity-dependent population frequencies. The present study was aimed to determine the frequencies of commonly known functional polymorphisms of CYP1A1 and CYP1B1 genes in a Haryana state population of North India. The allelic frequency of CYP1A1 polymorphism m1 (MspI) was 29.65% and m2 (Ile⁴⁶²Val) was 24.85%. The frequency of CYP1B1 polymorphism m1 (Val⁴³²Leu) was 45.85% and m2 (Asn⁴⁵³Ser) was 16.2%. We observed inter- and intra-ethnic variation in the frequency distribution of these polymorphisms. Analysis of polymorphisms in these genes might help in predicting the risk of cancer. Our results emphasize the need for more such studies in high-risk populations.