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Murine Relaxin-Like Factor Promoter: Functional Characterization and Regulation by Transcription Factors Steroidogenic Factor 1 and DAX-1

Department of Physiology (P.K., J.L., P.S., I.H., M.P.) and Turku Graduate School of Biomedical Sciences (P.K., P.S.), University of Turku, 20520 Turku, Finland

Address all correspondence and requests for reprints to: Matti Poutanen, Ph.D., Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: . matti.poutanen{at}utu.fi

The gene for mouse relaxin-like factor (RLF), a member of the insulin/IGF/relaxin family of hormones, appears to be predominantly expressed in testicular Leydig cells. Mice deficient in RLF have revealed a role for this peptide in testicular descent, but the regulatory mechanisms of its function are still insufficiently characterized. In the present study we showed that the RLF promoter was active in both mLTC-1 Leydig cells and luteinized KK-1 granulosa tumor cells. Interestingly, the activity of the RLF promoter as well as the expression of endogenous RLF correlated with the amount of steroidogenic factor 1 (SF-1) expression in the four cell lines tested. The highest transcriptional activity (29-fold over promoterless plasmid) was detected in mLTC-1 using the 188-bp promoter fragment immediately 5' of the CAP site, containing three consensus sequences for SF-1 binding. However, the promoter fragments including the 188-bp promoter also showed significant SF-1-independent promoter activity in both mLTC-1 and KK-1 cells, 8-fold induced over the promoterless construct. Mutagenesis studies showed that all three SF-1-binding sites were needed to obtain maximal SF-1-dependent trans-activation. The most distal SF-1-binding site at position -144 to -136 showed the highest affinity toward SF-1, but the promoter fragments, including the SF-1-binding site at position -115 to -107, showed the strongest response to SF-1 in terms of transcriptional activation. Moreover, DAX-1 inhibited RLF promoter activity in mLTC-1 Leydig tumor cells and totally abolished SF-1-dependent RLF expression in nonsteroidogenic HEK-293 cells. DAX-1 especially inhibited binding of SF-1 to the binding motifs locating at positions -64 to -56 and -115 to -107, whereas no decrease was seen in the expression of SF-1. Taken together, these observations suggest that the 188-bp RLF promoter includes elements for both SF-1-dependent and -independent gene expression in steroidogenic cells. The data, furthermore, indicate differential binding affinities for the three SF-1 binding motifs toward SF-1, of which the motif locating at position -115 to -107 was the most critical for the promoter activity.

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