Cloning and functional characterization of the 5′ regulatory region of ovine Hormone Sensitive Lipase (HSL) gene

dc.contributor.author	Lampidonis, AD	en
dc.contributor.author	Stravopodis, DJ	en
dc.contributor.author	Voutsinas, GE	en
dc.contributor.author	Messini-Nikolaki, N	en
dc.contributor.author	Stefos, GC	en
dc.contributor.author	Margaritis, LH	en
dc.contributor.author	Argyrokastritis, A	en
dc.contributor.author	Bizelis, I	en
dc.contributor.author	Rogdakis, E	en
dc.date.accessioned	2014-06-06T06:48:23Z
dc.date.available	2014-06-06T06:48:23Z
dc.date.issued	2008	en
dc.identifier.issn	03781119	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.gene.2008.09.001	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/4119
dc.subject	Glucose response	en
dc.subject	Hormone Sensitive Lipase (HSL)	en
dc.subject	Ovis aries	en
dc.subject	Promoter	en
dc.subject	Transcriptional regulation	en
dc.subject.other	glucocorticoid receptor	en
dc.subject.other	hormone sensitive lipase	en
dc.subject.other	luciferase	en
dc.subject.other	transcription factor Sp1	en
dc.subject.other	animal cell	en
dc.subject.other	article	en
dc.subject.other	gene control	en
dc.subject.other	gene deletion	en
dc.subject.other	gene function	en
dc.subject.other	gene insertion	en
dc.subject.other	gene isolation	en
dc.subject.other	gene sequence	en
dc.subject.other	genetic transcription	en
dc.subject.other	human	en
dc.subject.other	human cell	en
dc.subject.other	molecular cloning	en
dc.subject.other	mouse	en
dc.subject.other	nonhuman	en
dc.subject.other	polymerase chain reaction	en
dc.subject.other	priority journal	en
dc.subject.other	promoter region	en
dc.subject.other	sequence analysis	en
dc.subject.other	3T3-L1 Cells	en
dc.subject.other	5' Untranslated Regions	en
dc.subject.other	Animals	en
dc.subject.other	Base Sequence	en
dc.subject.other	Cloning, Molecular	en
dc.subject.other	Humans	en
dc.subject.other	Mice	en
dc.subject.other	Models, Biological	en
dc.subject.other	Models, Genetic	en
dc.subject.other	Molecular Sequence Data	en
dc.subject.other	Mutagenesis, Site-Directed	en
dc.subject.other	Sequence Homology, Nucleic Acid	en
dc.subject.other	Sheep	en
dc.subject.other	Sterol Esterase	en
dc.subject.other	Transcription, Genetic	en
dc.subject.other	Ovis	en
dc.subject.other	Ovis aries	en
dc.title	Cloning and functional characterization of the 5′ regulatory region of ovine Hormone Sensitive Lipase (HSL) gene	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.gene.2008.09.001	en
heal.publicationDate	2008	en
heal.abstract	Hormone Sensitive Lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL enzymatic activity is increased by adrenergic agonists, such as catecholamines and glucagons, which induce cyclic AMP (cAMP) intracellular production, subsequently followed by the activation of Protein Kinase A (PKA) and its downstream signaling cascade reactions. HSL constitutes the critical enzyme in the modulation of lipid stores and the only component being subjected to hormonal control in terms of the recently identified Adipose Triglyceride Lipase (ATGL). In order to acquire detailed knowledge with regard to the mechanisms regulating ovine HSL (ovHSL) gene transcription activity, we initially isolated and cloned the 5′ proximal and distal promoter regions through a genome walking approach, with the utilization of the already characterized ovHSL cDNAs. As evinced by BLAST analysis and a multiple alignment procedure, the isolated genomic fragment of 2.744 kb appeared to contain the already specified 5′-untranslated region (5′-UTR), which was interrupted by a relatively large intron of 1.448 kb. Regarding the upstream remaining part of 1.224 kb, it was demonstrated to represent a TATA-less promoter area, harboring several cis-regulatory elements that could be putatively recognized by relatively more general transcription factors, mainly including Stimulating protein 1 (Sp1), CCAAT-box Binding Factors (CBFs), Activator Protein 2 (AP2) and Glucocorticoid Receptor (GR), as well as other cis-acting regions denominated as Insulin Response Element (IRE), Glucose Response Element (GRE), Fat Specific Element (FSE) and cAMP Response Element (CRE), which could likely function in a nourishment (i.e. glucose)-/hormone-dependent fashion. When different genomic fragments were directionally (5′ to 3′) cloned into a suitable reporter vector upstream of a promoter-less luciferase gene and transiently transfected into 3T3-L1 (mouse fibroblasts) as well as T24 (human bladder cancer) cell lines, strong promoter activities were unambiguously detected, with the - 140/+ 18 nucleotide sequence bearing the highest transcriptional response, thus indicating that the 1.224 kb 5′ flanking region, isolated by genome walking, veritably contains the ovHSL gene promoter. Of particular significance are the observations that the functional promoter fragments could trigger the transcriptional activity of luciferase gene only under high concentration of glucose conditions in both cell lines. © 2008 Elsevier B.V. All rights reserved.	en
heal.journalName	Gene	en
dc.identifier.issue	1-2	en
dc.identifier.volume	427	en
dc.identifier.doi	10.1016/j.gene.2008.09.001	en
dc.identifier.spage	65	en
dc.identifier.epage	79	en