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

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