| Mouse renin gene (mREN) transcription is controlled by a transcriptional enhancer (mE) with unknown mechanisms. My research goal was to uncover molecular mechanisms for the enhancer-mediated regulation of REN transcription.Within mE, three cis-acting elements, Ea, Eb, and Ec, were identified using mutagenesis, EMSA, and transient transfection analysis in mouse kidney renin-expressing As4.1 cells. Ea is negative, contains a CCAAT box, and binds transcription factor NF-Y. The inhibitory function of Ea is achieved through antagonism of Eb. Eb and Ec are positive, form a direct repeat of TGACCT, and bind nuclear receptors, such as RARalpha and RXRalpha.EcEb, as a RA responsive element (RARE), mediates the retinoic-acid (RA) induced activation of mREN promoter activity, suggesting that the RA signaling pathway is one of the upstream signaling pathways controlling mE. The role of RARalpha in this signaling was confirmed with overexpression of RARalpha403, a dominant negative mutant. RNase protection analysis revealed that RA increases the endogenous REN mRNA in mouse kidneys and As4.1 cells, and the mRNA from a hybrid SV40TAG transgene in As4.1 cells, demonstrating the important role of RA in activation of REN expression.Interestingly, the RA-induced response is antagonized by interleukin-1beta. Consistently, EcEb is the specific target of this negative interleukin-1beta signaling. This finding has revealed the underlying mechanism for the interleukin-1beta mediated inhibition of mREN transcription.The aforementioned discoveries facilitated the investigation of the human REN enhancer (hE). hE lacks Ea, but possesses EcEb with a single-base mismatch in Eb. Although the mismatch decreases the basal hE activity, hE still mediates the RA-induced response through its imperfect RARE, implying that hE is functional. The imperfect RARE of hE existed in all samples of human genomic DNA examined by polymorphism study. Therefore, the reduced activity of hE is probably conserved in humans for appropriate stimulation of hREN transcription through species-specific mechanisms.Taken together, my study has demonstrated that communication between the upstream signaling molecules and the specific enhancer elements is the molecular basis of the enhancer-mediated regulation of REN transcription. Therefore, my findings will benefit future studies of the related physiological significance. |
