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KCTD13 modulates SOX9 through KEAP1-mediated ubiquitination in Mouse Testicular Development
AHMED CHAHDI, PhD.
Nemours Children's Hospital, Orlando, FL, USA.
Background: Cryptorchidism or undescended Testis (UDT) is the most common genitourinary birth defect that occurs in 1-3% of full-term and 15-30% of premature males, with 80% of patients having unilateral UDT. Surgery is used to correct UDT between 6-12 months after birth. Despite orchidopexy in infancy, 13% of men with unilateral UDT after orchidopexy are infertile and have a 10X higher chance of developing testicular cancer. We identified
KCTD13 copy number variants in boys UDT.
Kctd13 encodes a substrate-specific adaptor of the BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex.
Kctd13 knockout (KO) mice exhibit UDT with reduced androgen receptor (AR) and SOX9 expression, suggesting that
KCTD13 modulates testicular development via AR-SOX9 signaling. We aim to elucidate the molecular mechanisms by which
Kctd13 regulates testicular development.
Methods: To validate SOX9 as a downstream target of KCTD13, we performed a genetic rescue experiment by crossing
Kctd13-KO mice with CAG-
Sox9 overexpressing mice, using the
Amh-Cre transgene to drive
Sox9 expression specifically in Sertoli cells. Cell culture, cell transfection, immunoblotting, co-immunoprecipitation, immunofluorescence and cell fractionation were used to study the effect of KCTD13 on SOX9 signaling.
Results:
In vivo,
KCTD13 loss results in SOX9 downregulation in testicular tissues and decreased fertility. Reintroduction of SOX9 (
Sox9+ mice) increased SOX9 protein and restored fertility. Furthermore, in
Sox9+ mice testicular tissues the increased SOX9 ubiquitination was accompanied with enhanced binding of SOX9 to KEAP1.
In vitro, ectopic expression of KCTD13 decreased KEAP1-mediated SOX9 ubiquitination and blocked SOX9 degradation and disrupted the physical association between KEAP1 and SOX9. In a series of domain function analysis of KCTD13 and SOX9, we found that both the BTB and CTD domains of KCTD13 are critical for binding the C-terminal trans-activator domain of SOX9. KCTD13 BTB and CTD domains were also crucial for binding to KEAP1. Ectopic expression of KCTD13 decreased nuclear association between SOX9, TCF4 and βcatenin. Furthermore, KCTD13 dose-dependently blocked binding of SOX9 and TCF4 to βcatenin and correlated with the
in vitro SOX9-luciferase assay. KCTD13 mutants lacking the BTB and CTD domains failed to inhibit SOX9-luciferase activity. Together, herein the findings uncover a novel molecular mechanism by which KCTD13 promotes SOX9 stability through disruption of KEAP1/SOX9 complex and inhibits the expression of SOX9 target genes by blocking the nuclear association between SOX9, TCF4 and βcatenin.
Conclusion: in the cytosol, KCTD13 prevents SOX9 degradation by blocking KEAP1 binding to SOX9. In the nucleus, KCTD13 blocks binding of TCF4 and SOX9 to catenin resulting in inhibition of SOX9 transcriptional activity and expression of SOX9 target genes.
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