Sp1 and CREB promote nectin-2 transcription to regulate adherens junctions assembly in the testis

Abstract

Nectin-2, a major protein component of the adherens junctions (AJs), is found between Sertoli cells and germ cells in the seminiferious epithelium. Recent studies have shown that the expression of nectin-2 gene in testis is crucial to maintain normal spermatogenesis since male knockout mice lacking nectin-2 gene are sterile and possess morphological abnormal spermatozoa. However, the molecular mechanisms governing its basal transcription remain poorly understood. By the use of Sertoli and germ cell-lines (TM4 and GC-2 Spd (ts) cells, respectively) in transient transfection studies, we showed that the minimal mouse nectin-2 promoter was located between nucleotides -316 and -211 relative to the translation start site. Two putative Specificity protein 1 (Sp1) motifs and one each of the cAMP response element (CRE), AP1 and AP2 motifs were identified within this region. Mutational studies showed that these two Sp1 motifs cooperated synergistically with the CRE motif, but not the AP1 and AP2 motifs, to regulate nectin-2 gene transcription in both TM4 and GC-2 Spd(ts) cells. By EMSAs, we found that an AP-1 consensus sequence was able to inhibit DNA-protein complex formation with the CRE/AP-1 motif, suggesting a cross-talk between the AP-1 transcription factor (c-Jun) and this CRE/AP-1 motif. Over-expressions of CRE-binding protein (CREB) and a serine-133-mutated CREB significantly increased the promoter activity, which suggests CREB to be one of crucial transcription factors involved in regulating nectin-2 gene transcription in a PKA-independent manner. It has been known that there is a cyclic expression of CREB throughout the spermatogenic cycle. Our present data provide new evidence that CREB regulates spermatogenesis by regulating nectin-2 gene transcription and controls the timely assembly and disassembly of AJs between Sertoli cells and spermatids.

This work was supported by grants from the University of Hong Kong (CRCG) and the Hong Kong Research Grant Council (HKU 7194/01M).