Cellular and molecular characterization of the sterol-regulatory element-binding protein-1

Human cells maintain lipid homeostasis by regulated cleavage of membranebound transcription factors, so-called sterol-regulatory element-binding proteins (SREBPs). The mature forms of SREBP-1 and -2 are transcriptional activators of lipogenic genes controlling cholesterol, fatty acids, and triglyceride biosynthesis and uptake. As the SREBPs play a central role in the regulation of the lipoprotein metabolism, we supposed that specific sequence variations, which correspond to single-nucleotide polymorphisms in these genes, and certain drugs, that influence the expression of SREBP, may result in alterations in plasma lipoprotein concentrations. A syndrome characterized by hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and lipodystrophy has been found to be associated with antiretroviral treatment (ART) including protease inhibitors. A marker predicting this syndrome has been identified in the gene encoding the sterolregulatory element-binding protein-1c (SREBP-1c), a regulator of triglycerides, cholesterol, insulin and adipocytes. A possible inhibition of SREBP-1c-dependent genes by the protease inhibitor indinavir and its possible reversal by the lipid-lowering drug simvastatin were studied in cell culture. The effects of indinavir and simvastatin on SREBP-1c-dependent genes were compared with the effects of indinavir and simvastatin on SREBP1c-independent genes. In fact, indinavir inhibited the SREBP-1c-dependent genes encoding the lipoprotein lipase and the fatty acid synthase in a dosedependent manner but not the SREBP-1c-independent gene encoding the low-density lipoprotein receptor. Furthermore, simvastatin antagonized the indinavir-induced SREBP-1c-inhibition. Thus, indinavir inhibits important effector genes of the SREBP-1c pathway, which may explain major antiretroviral treatment-related adverse effects. A single-nucleotide polymorphism (3' 322C/G SNP) identified in the sterolregulatory element-binding protein-1c (SREBP-1c) gene was predictive of highly active antiretroviral therapy-related hyperlipoproteinemia. Increases in cholesterol, triglyceride and insulin were less frequently associated with homozygous SREBP-1c-3' 322G (genotype 22) than with heterozygous/homozygous SREBP-1c-3'322C (genotypes 11/12). The
differences in messenger RNA conformation can explain the pharmacogenetic
basis of these findings. The mRNA stability of both homozygous genotypes of
SREBP-1c-3’322C/G was compared in the stably transfected T-REx cell lines
using a real-time quantitative polymerase chain reaction method. The mRNA
of the SREBP-1c-3’322C isoform (genotype 11) was shown to have a more
abundance decay rate than 3’322G isoform (genotype 22). Thus, the
sequence variation (3’322C/G SNP) in the coding 3’ end of the gene affects
the secondary structure of the SREBP-1c mRNA, influences its degradation
rate and, therefore, causes differences in the regulation of SREBP-1c
expression.
In the process of this thesis, three new splice variants of the human SREBP-1
gene that shared different combinations of the SREBP-1a and -1c exons at
the 3’ end of mRNA were identified. The splice variant containing exons 17,
18a and 18c was designated as SREBP-1d, the splice variant containing
exons 17, 18a, 18c and 19c was termed as SREBP-1e, and the splice variant
containing exons 17, 18c and 20f was named as SREBP-1f. Analysis of tissue
distribution showed that the new splice variants SREBP-1e and -1d were
ubiquitously found in various human tissues and tumor-derived cells, whereas
wild-type SREBP-1c and SREBP-1f transcripts were relatively tissue-specific.
This high abundance led us to the hypothesis that splice variants SREBP-1e
and SREBP-1d play a more general role in regulating cellular lipid levels as
compared to other isoforms.
This thesis concludes that the sterol-regulatory element-binding protein
(SREBP)-1c is crucial in the metabolic side-effects associated with highly
active antiretroviral therapy using protease inhibitors. Moreover, regulation
mechanism mediated by the transcription factor SREBP-1 is a model of a
complex gene regulation system composed of different related levels:
promoter regulation of effector genes, differences in mRNA stability and tissue
specific splice variants in different quantities.