Poster presentationSession: Cell-Cell Junctions INectin-2 is a major component of the adherens junctions (AJs) between Sertoli cells and germ
cells in the testis. Recent studies have shown that male knockout mice of nectin-2 are sterile.
Cadmium (Cd), an environmental toxicant, is known to be also an endocrine disruptor that
affects spermatogenesis. In this study, we investigate whether cadmium chloride (CdCl2) plays a role in nectin-2 expression. CdCl2 negatively regulates mRNA and protein levels of nectin-2 in
mouse Sertoli cell line, TM4 cells. Luciferase reporter assays indicated that CdCl2 reduces
nectin-2 promoter activity within the region of nucleotides (nt) -246 and -211 (relative to the
translation start site) where putative transcription factors (TFs) binding motifs are identified.
However, site-directed mutational studies have shown that no specific motif is found to involve
in CdCl2-mediated nectin-2 gene repression. Hence, six consecutive cis-acting regions (each
contains 6 nucleotides) between nt -246 and -211 are mutated respectively to identify the cisacting
region involved in the CdCl2 effect. Results showed that the second 6-bp region (between
nt -240 and -235) is involved in CdCl2-mediated reduction of nectin-2 promoter activity. In
addition, putative TFs binding to this region are identified. By EMSAs, we found that DNA (nt -
240 to -235)-protein complexes are formed in a dose-dependent manner and CdCl2 treatment
could diminish the formation of the complexes. Antibody supershift assays have shown that
TFs, E2F1, Sp1 and KLF4, are present in the complexes. We also found that CdCl2 downregulates
the expression level of these TFs including E2F1, Sp1 and KLF4 in the nucleus. Apart
from transcriptional regulation, cycloheximide assay indicated that CdCl2 negatively modulates
nectin-2 protein level via post-translational modification and we are now investigating the
underlying mechanism. Taken together, CdCl2-mediated down-regulation of nectin-2 is
mediated through transcriptional modification by negatively affecting its basal gene transcription
and post-translational modification. [This work was supported by Hong Kong Research Grants
Council (HKU772009 and HKU773710) and CRCG Seed Funding for Basic Research.]published_or_final_versio
