LBP proteins modulate SF1-independent expression of P450scc in human placental JEG-3 cells.
The cholesterol side-chain cleavage enzyme, P450scc, initiates biosynthesis of all steroid hormones. Adrenal and gonadal P450scc expression requires steroidogenic factor-1 (SF1), but P450scc expression in human placental JEG-3 cells utilizes an SF1-independent element at -155/-131 that is inactive in adrenals and gonads. We previously cloned two transcription factors, long terminal repeat binding protein (LBP)-1b and LBP-9, from JEG-3 cells. In transient transfection assays, LBP-1b activated the -155/-131 element whereas LBP-9 suppressed its LBP-1b-stimulated expression. To assess the roles of these factors on the intact P450scc gene, we stably expressed LBP-1b or LBP-9 in JEG-3 cells. All cell lines stably expressing a fusion protein of LBP-1b and enhanced green fluorescent protein increased P450scc expression, but cell lines stably expressing LBP-9 fused to enhanced green fluorescent protein either increased or decreased P450scc expression. 8-Br-cAMP induced endogenous LBP-9, but not LBP-1b expression. Glutathione-S-transferase pull-down assays showed that LBP-1b and LBP-9 can dimerize with themselves and with each other; LBP-1b residues 300-540 and LBP-9 residues 300-479 were required for dimer formation. Glutathione-S-transferase pull-down assays, bandshifts, and transient transfection assays showed that TReP-132 (another factor that can bind to -155/-131) does not interact with either LBP-1b or LBP-9, or influence their ability to induce or suppress transcription from the -155/-131 element. Gal4 transactivation assays showed that transcriptional repression activity by LBP-9 requires residues 100-200. RNAi interference of either LBP-1b or LBP-9 mRNAs decreased P450scc expression. LBP-1b is an important SF1-independent transcriptional activator stimulating P450scc expression in human placental JEG-3 cells, whereas LBP-9 modulates the action of LBP-1b, exerting both positive and negative effects.
Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-0978, USA.
Ningwu
N
Huang
Walter L
WL
Miller
D015152:Blotting, Northern; D045744:Cell Line, Tumor; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis*; D000242:Cyclic AMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D019281:Dimerization; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry; D010920:Placenta / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D012260:Ribonucleases / Q000378:metabolism; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; C515057:NR5A1 protein, human; D009841:Oligonucleotides; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C107467:SF1 protein, human; D054339:Steroidogenic Factor 1; C428128:TFCP2L1 protein, human; D014157:Transcription Factors; C085968:UBP1 protein, human; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005982:Glutathione Transferase; D012260:Ribonucleases