Nuclear factor kappa B and tumor necrosis factor‐alpha modulation of transcription of the mouse testis‐ and pre‐implantation development‐specific Rnf33/Trim60 gene

We have previously reported a mouse Rnf33/Trim60 gene that is temporally expressed in the pre-implantation embryo. The Rnf33 structural gene is composed of a short noncoding exon 1 and an intronless coding exon 2. In the present work, Rnf33 was shown to be expressed in the mouse testis and in the testicular cell lines TM3 and TM4. To elucidate Rnf33 transcriptional modulation, a 2.5-kb Rnf33 sequence, inclusive of the upstream regulatory region, exon 1 and the associated intronic sequence, was dissected in transient transfection and luciferase assays. An initiator and an atypical TATA-box were shown to act as the core promoter elements of the gene. Deletion and mutagenesis of the 2.5-kb sequence in luciferase constructs further demonstrated that an intronic and palindromic kappa B (κB) sequence was an important cis element targeted by the nuclear factor-κB (NF-κB) subunits p65/RELA and p50/NFκB1, and also through modulation by tumor necrosis factor α. Transcriptional up-regulation of Rnf33 by NF-κB and tumor necrosis factor-α was directly demonstrated in TM3 and TM4 cells by real-time PCR quantification of the Rnf33 mRNA levels. Small interfering RNA knockdown of p65 and p50 confirmed Rnf33 down-regulation by p65/p50. Spermatogenesis is regulated by a wide range of stimuli, including NF-κB, which, in turn, is regulated by other signals. Hence, demonstration of NF-κB-regulated Rnf33 expression in testicular cells, particularly in Sertoli cells, implicates functional involvement of the putative RNF33 protein in spermatogenesis through association of the RNF33 protein with the microtubule via interaction with kinesin motor proteins, as previously demonstrated [Huang et al., submitted].