Regulation of Fanconi anaemia complementation group a (Fanca) by gonadotropin releasing hormone : a role for Fanca in reproduction

Abstract

Binding of gonadotropin releasing hormone (GnRH) to its receptor on the surface of gonadotroph cells within the anterior pituitary triggers the pulsatile release of the gonadotropin hormones, luteinising hormone and follicle stimulating (LH and FSH) into the blood stream. These hormones then act on their target organs, the gonads, to stimulate and regulate spermatogenesis in males and folliculogenesis in females. LH and FSH are heterodimeric glycoproteins comprised of a common a subunit (aGSU) and a unique beta subunit (LH(3 or FSH(3), each encoded by a single gene. The processes by which GnRH regulates gonadotropin gene transcription are not fully characterised but variation in GnRH pulsatility, differential activation of GnRH receptor and coupling to second messenger signalling pathways, all contribute to give distinct, subunit-specific effects on gene transcription. The identification of transcripts and proteins regulated by GnRH could further characterise the biomolecular processes by which GnRH controls LH and FSH production.Previous work performed within the laboratory, using the technique of differential display RT-PCR (DD-RT-PCR), identified that a DNA damage repair gene, Fanconi Anaemia complementation group a (Fanca), is differentially expressed in response to hormone. FANCA is mutated in > 60% of cases of Fanconi anaemia (FA), a genetically heterogeneous autosomal recessive disorder characterised by bone marrow failure, endocrine tissue cancer susceptibility and infertility.Detailed analysis of Fanca mRNA expression using northern blotting, semi¬ quantitative RT-PCR and quantitative RT-PCR analysis reveals that GnRH induces a rapid and transient increase in Fanca mRNA within L|3T2 gonadotroph cells. Indirect immunofluorescence and western blotting analysis show that Fanca protein is expressed in the cytoplasm and nucleus of L|3T2 cells and GnRH induces a transient 2-fold increase in Fanca protein levels within both cellular compartments. Furthermore, treatment with inhibitors of nuclear import and export demonstrated that Fanca protein actively shuttles between the nucleus and cytoplasm of gonadotroph cells, via a CRM-1 (yeast chromosome region maintenance protein 1) dependent mechanism.Transient transfection assays using wildtype and dominant negative, point mutated forms of FANCA reveal that wildtype FANCA protein is required for GnRH induced activity of the aGSU promoter, but not LH|3 or FSH[3 promoter activity. The construction of adenoviral vectors expressing wildtype and mutant FANCA proteins and the characterisation of siRNAs to knockdown expression of Fanca in L|3T2 cells will allow the confirmation of a novel role for Fanca in gonadotropin gene transcription.The discovery that GnRH regulates Fanca expression, which in turn regulates GnRH induced aGsu transcription, provides the first molecular evidence of a role for Fanca in the control of fertility.