RESUMO
The peroxisome proliferator activated receptor alpha (PPARalpha) plays a key role in regulating fatty acid metabolism by regulating expression of genes involved in fatty acid oxidation. To identify endogenous transcripts that could be used as surrogate markers for on-target activity of PPARalpha agonists, we employed a global profiling approach using DNA microarrays. The HK-2 cell line derived from proximal tubules of the human kidney, showed induction of several genes, including pyruvate dehydrogenase kinase 4 (PDK-4) and adipocyte differentiation related protein (ADRP) by PPARalpha ligands. HK-2 cells express detectable levels of PPARalpha and its dimerization partner the retinoid X receptor (RXRalpha) proteins. Induction of PDK-4 in these cells correlates with induction of PDK-4 in the liver of fat-fed hamsters. The magnitude of fibrate induction of PDK-4 in the liver also mirrors the decrease in serum triglyceride levels. Thus, induction of PDK-4 by PPARalpha agonists in the HK-2 cell model closely correlates with its induction in vivo and may represent an early marker for PPARalpha agonist action.
Assuntos
Ácidos Graxos/metabolismo , Isoenzimas/biossíntese , Túbulos Renais Proximais/fisiologia , Proteínas de Membrana/biossíntese , Proteínas Quinases/biossíntese , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Animais , Butiratos/farmacologia , Células Cultivadas , Cricetinae , Ativação Enzimática , Fenofibrato/farmacologia , Regulação da Expressão Gênica/fisiologia , Humanos , Hipolipemiantes/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , Ligantes , Fígado/enzimologia , Masculino , Proteínas de Membrana/genética , Mesocricetus , Análise de Sequência com Séries de Oligonucleotídeos , Perilipina-2 , Compostos de Fenilureia/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Triglicerídeos/sangueRESUMO
Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.
