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LBP Proteins Modulate SF1-Independent Expression of P450scc in Human Placental JEG-3 Cells

Department of Pediatrics and The Metabolic Research Unit, University of California, San Francisco, San Francisco, California 94143-0978

Address all correspondence and requests for reprints to: Professor Walter L. Miller, M.D., Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-0978. E-mail: wlmlab{at}itsa.ucsf.edu.

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.

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Nuclear Receptors:   SF-1

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