RESUMO
Human prostatic acid phosphatase (hPAP) is intensely expressed in epithelial cells of the prostate gland following puberty. Its regulatory regions were analyzed in transgenic mice and cell line transfections, in order to clarify the mechanisms of tissue-specific gene expression. A construct containing the sequence of hPAP between the nucleotides -734 and +467 in front of the CAT reporter gene was significantly expressed in the prostate of transgenic mice, while the proximal promoter -734/+50 alone achieved low levels of CAT mRNA in all tissues analyzed. Five homologous sequences (A-E) for our previously identified prostatic GAAAATATGATA DNA-binding site were found in the area. The competitive reactions in electrophoretic mobility shift assays suggested that the same nuclear factor binds to the GAAAATATGATA and the sites C and E. The importance of the intronic area +57/+467 on the androgen-activated expression in prostatic cells was shown by the reporter construct containing heterologous promoter.
Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Próstata/enzimologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Ativação Transcricional/genética , Fosfatase Ácida , Animais , Sequência de Bases , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica/genéticaRESUMO
Overexpression of SSAT (polyamine catabolic enzyme) in female mice results in impaired ovarian folliculogenesis and uterine hypoplasia. To identify the molecular basis for this, the gene expression profiles in uterus and ovary and for comparison, liver and kidney, from non-transgenic (NT) and SSAT transgenic (ST) mice were compared. The mRNA abundance for lipoprotein lipase and glyceraldehyde-3-phosphate dehydrogenase was elevated in all four ST (>NT) tissues. The translation initiation factor-3 subunit 5 mRNA, and transcripts related to endogenous murine leukemia provirus (MLV-related) and murine retrovirus-related sequences (MuRRS) were decreased in ST tissues. A novel calmodulin-related mRNA was strongly induced in ST liver and kidney. SSAT overexpression was associated with increased levels of IGF-binding protein-2 (IGFBP-2) in the uterus and ovary, and a reduction in IGFBP-3 mRNA levels in the uterus. Exogenous spermidine and spermine elevated endogenous IGFBP-2 and SSAT mRNA abundance, whereas, putrescine stimulated IGFBP-2 mRNA abundance and transfected IGFBP-2 gene promoter activity in human (Hec-1-A) uterine cells. Sp1 and BTEB1 mRNAs that encode transcription factors for the IGFBP-2 gene also were induced in some ST tissues. The data suggest that SSAT and polyamines are important for the control of molecular pathways underlying reproductive tract tissue growth, phenotype, and function.
Assuntos
Acetiltransferases/genética , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cruzamentos Genéticos , Feminino , Regulação Enzimológica da Expressão Gênica , Rim/enzimologia , Fígado/enzimologia , Luciferases/genética , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , Ovário/enzimologia , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Útero/enzimologiaRESUMO
We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.