Final Report Summary - INVIVO_RA_NONCANON (Non-canonical signalling pathways relayed by retinoic acid affecting germ cell differentiation.)
Retinoic acid (RA) is a signalling molecule synthesized from vitamin A (retinol), which is essential for normal embryonic development, and after birth is indispensable for survival, growth, vision, reproduction and epithelial differentiation. Consequently vitamin A deficiency (VAD) represents a public health problem, particularly in developing countries where it represents an indisputable cause of morbidity and mortality for more than 250 million people worldwide each year. VAD causes growth retardation, night blindness, susceptibility to infections or anaemia, and afflicted children are at high risk for corneal ulcer and may become definitively blind; severe VAD is lethal. Even in industrialized countries a significant fraction of the population has insufficient retinol intake, the consequences of which have not been fully evaluated. Conversely, Vitamin A toxicity symptoms, such as vomiting, diarrhoea or headache, occur when an individual ingests vitamin A in large amounts (liver, food supplements). Together these findings emphasise that a vitamin A balanced diet is necessary for good health, and show the importance of retinoids for normal development and tissue homeostasis. Studies that further the understanding of the molecular mechanisms involved in RA signalling are therefore of paramount importance.
In vivo RA exerts its effects through binding to heterodimers of retinoic acid receptors (RARs) and retinoid X receptors, also called rexinoid receptors (RXRs). RARs and RXRs are present in three isotypes: RARalpha, RARbeta and RARgamma, and RXRalpha, RXRbeta and RXRgamma; these are designated hereafter as RARA, RARB and RARG, and RXRA, RXRB and RXRG, respectively. RARs act as RA-dependent transcriptional regulators, whereas there is now a consensus that RXRs do not bind RA, but instead bind unsaturated fatty acids or other ligands found in all cell types. RARs and RXRs belong to the non-steroid nuclear receptor (nsNR) family that includes thyroid hormone receptor (TR) and vitamin D3 receptor (VDR). The NRs act as transcription factors by binding to regulatory regions located in the DNA of their target genes, and they exhibit a conserved structure with a variable N-terminal activation function, a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD). Most NRs bind to DNA as protein dimers formed via the binding of two DBDs on the hormone response elements (HRE) and a strong interaction between the LBDs of the partners. According to the canonical RA signalling pathway, RAR/RXR heterodimers bind to RA response elements (RARE) found in the regulatory regions of the target genes. In the absence of the RAR ligand (that is RA), RAR/RXR binds co-repressors that associate with histone deacetylase (HDAC) and lead to transcriptional repression. Upon binding of RA to RAR, the RAR/RXR receptor complex changes conformation, releases the co-repressors, and binds to co-activators that recruit histone acetyl transferase (HAT) to bring about histone acetylation and transcriptional activation. The LBD is a pivotal element for NR function; not only does it permit ligand binding but it also enables interaction with other co-factors such as the dimerization partner, the co-activators, or the co-repressors.
In vitro crystal structure analysis of dimers of the LBD has shown that RARs can actually form functional heterodimers with RXRs. In vivo, pharmacological and functional genetic assays in mice have shown that RA regulates embryogenesis and organogenesis through activating exclusively RAR/RXR heterodimers. Heterodimerization with RXR is however not the only pathway that enables RAR function. According to in vitro data, RARs can act via several non-canonical signalling pathways independently of RXR: (i) homodimerization, (ii) heterodimerization with nsNRs other than RXR, (iii) interference with transcriptional activity of the Activator Protein 1 (AP-1)/DNA complex, (iv) interaction with the WNT/CTNNB1 signalling pathway or (v) non-genomic actions of RAR.
The objectives of the InVivo_RA_NonCanon project were to confirm the existence and characterize such pathways in vivo, using the seminiferous epithelium of the testis as a model system. To reach these objectives, working hypotheses have been constructed which integrate data aimed at uncovering genes differentially expressed in RA-deficient contexts and datasets identifying RAR binding sites genome wide. In accordance with our initial hypothesis, our data confirmed that in the Sertoli cells (SC), the supporting cells of the seminiferous epithelium, RARA acts independently of RXR to control RA-target gene expression. Although the specific non-canonical pathway has not yet been confirmed; the fellow identified a secreted glycoprotein as a downstream effector of RA that enables germ cell differentiation toward meiosis. Further studies are now needed to clarify the non-canonical RA signaling enabling gene expression and to deepen the role of RA target genes in cell differentiation.
Contact details of the researcher funded by the project InVivo_RA_NonCanon:
Dr Nadege Vernet - Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)
CNRS UMR 7104 - Inserm U 964
Dept. of Functional genomics & cancer
1, rue Laurent Fries – BP 10142
67404 Illkirch CEDEX (France)
Tel: +33 (0) 388 65 5706
E. mail : vernet@igbmc.fr
Contact details of the scientist in charge of the project InVivo_RA_NonCanon:
Dr Norbert Ghyselinck - Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)
CNRS UMR 7104 - Inserm U 964
Dept. of Functional genomics & cancer
Group Leader of the “Retinoic acid signaling pathways driving stem spermatogonia ontogenesis and differentiation” Team
1, rue Laurent Fries – BP 10142
67404 Illkirch CEDEX (France)
Tel: +33 (0) 388 65 5674
E. mail : norbert.ghyselinck@igbmc.fr
In vivo RA exerts its effects through binding to heterodimers of retinoic acid receptors (RARs) and retinoid X receptors, also called rexinoid receptors (RXRs). RARs and RXRs are present in three isotypes: RARalpha, RARbeta and RARgamma, and RXRalpha, RXRbeta and RXRgamma; these are designated hereafter as RARA, RARB and RARG, and RXRA, RXRB and RXRG, respectively. RARs act as RA-dependent transcriptional regulators, whereas there is now a consensus that RXRs do not bind RA, but instead bind unsaturated fatty acids or other ligands found in all cell types. RARs and RXRs belong to the non-steroid nuclear receptor (nsNR) family that includes thyroid hormone receptor (TR) and vitamin D3 receptor (VDR). The NRs act as transcription factors by binding to regulatory regions located in the DNA of their target genes, and they exhibit a conserved structure with a variable N-terminal activation function, a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD). Most NRs bind to DNA as protein dimers formed via the binding of two DBDs on the hormone response elements (HRE) and a strong interaction between the LBDs of the partners. According to the canonical RA signalling pathway, RAR/RXR heterodimers bind to RA response elements (RARE) found in the regulatory regions of the target genes. In the absence of the RAR ligand (that is RA), RAR/RXR binds co-repressors that associate with histone deacetylase (HDAC) and lead to transcriptional repression. Upon binding of RA to RAR, the RAR/RXR receptor complex changes conformation, releases the co-repressors, and binds to co-activators that recruit histone acetyl transferase (HAT) to bring about histone acetylation and transcriptional activation. The LBD is a pivotal element for NR function; not only does it permit ligand binding but it also enables interaction with other co-factors such as the dimerization partner, the co-activators, or the co-repressors.
In vitro crystal structure analysis of dimers of the LBD has shown that RARs can actually form functional heterodimers with RXRs. In vivo, pharmacological and functional genetic assays in mice have shown that RA regulates embryogenesis and organogenesis through activating exclusively RAR/RXR heterodimers. Heterodimerization with RXR is however not the only pathway that enables RAR function. According to in vitro data, RARs can act via several non-canonical signalling pathways independently of RXR: (i) homodimerization, (ii) heterodimerization with nsNRs other than RXR, (iii) interference with transcriptional activity of the Activator Protein 1 (AP-1)/DNA complex, (iv) interaction with the WNT/CTNNB1 signalling pathway or (v) non-genomic actions of RAR.
The objectives of the InVivo_RA_NonCanon project were to confirm the existence and characterize such pathways in vivo, using the seminiferous epithelium of the testis as a model system. To reach these objectives, working hypotheses have been constructed which integrate data aimed at uncovering genes differentially expressed in RA-deficient contexts and datasets identifying RAR binding sites genome wide. In accordance with our initial hypothesis, our data confirmed that in the Sertoli cells (SC), the supporting cells of the seminiferous epithelium, RARA acts independently of RXR to control RA-target gene expression. Although the specific non-canonical pathway has not yet been confirmed; the fellow identified a secreted glycoprotein as a downstream effector of RA that enables germ cell differentiation toward meiosis. Further studies are now needed to clarify the non-canonical RA signaling enabling gene expression and to deepen the role of RA target genes in cell differentiation.
Contact details of the researcher funded by the project InVivo_RA_NonCanon:
Dr Nadege Vernet - Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)
CNRS UMR 7104 - Inserm U 964
Dept. of Functional genomics & cancer
1, rue Laurent Fries – BP 10142
67404 Illkirch CEDEX (France)
Tel: +33 (0) 388 65 5706
E. mail : vernet@igbmc.fr
Contact details of the scientist in charge of the project InVivo_RA_NonCanon:
Dr Norbert Ghyselinck - Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)
CNRS UMR 7104 - Inserm U 964
Dept. of Functional genomics & cancer
Group Leader of the “Retinoic acid signaling pathways driving stem spermatogonia ontogenesis and differentiation” Team
1, rue Laurent Fries – BP 10142
67404 Illkirch CEDEX (France)
Tel: +33 (0) 388 65 5674
E. mail : norbert.ghyselinck@igbmc.fr