Retinoic acid-related mechanisms in oral keratinocytes
A scientific essay in Medical Sciences
DOCTORAL THESIS defended in public on 12th of June 2018
Retinoic acid (RA), the active derivative of vitamin A, is associated with orofacial clefting (OFC) through a mechanism which is not completely clear. Therefore, the major goal of this thesis was to investigate RA-related mechanisms in oral keratinocytes and thus to try elucidate the possible role of RA in the etiopathogenesis of OFC.
We start in chapter 1 with a the general introduction to the epidemiology, subtypes, and cellular and molecular mechanisms of OFC. We shortly discuss several causative genetic mutations underlying syndromic forms of OFC and then focus on the regulatory genes and signalling pathways mainly associated to non-syndromic OFC. We then discuss the impact of environmental risk factors combined with genetic background on the aetiology of OFC. Finally, we present the rationale and the aims of this thesis.
In chapter 2, we first present an overview of RA biosynthesis and signalling. Subsequently, we discuss the role of RA in the development of the epidermis, the limbs and the secondary palate which share common regulatory pathways. In conjunction with other regulatory molecules, RA promotes the development of the epithelium from the ectoderm. In the limbs, RA signalling initiates limb bud formation, digit separation and regulates chondrogenesis and osteogenesis. In the secondary palate, RA is a key factor for mesenchymal cell proliferation during palatal shelf outgrowth and elevation, and for differentiation and apoptosis of the palatal epithelium during palatal fusion. In addition, RA is also involved in bone formation in the hard palate. Finally, we discuss recent evidence on polymorphisms in the genes encoding proteins involved in RA biosynthesis and signalling which might affect RA levels and activity within embryonic tissues. We conclude that correct spatiotemporal RA biosynthesis and activity is crucial for embryonic development and that disturbances may lead to congenital diseases such as OFC.
In chapter 3, we investigated the effects of RA on the proliferation and gene expression in palatal keratinocytes obtained from OFC and non-OFC 127 patients. We observed that RA reduces palatal keratinocyte proliferation in a dose dependent-manner. We also observed a large inter-individual variation in response to RA, but no significant difference between OFC and non-OFC palatal keratinocytes. In addition, gene expression analyses show that RA downregulates the expression of early differentiation genes.
In chapter 4, we analyzed the effect of RA on the expression of OFC- related genes in oral keratinocytes derived from the gingiva and the palate. Gene ontology analysis of the differentially expressed genes demonstrates a significant enrichment for genes involved in angiogenesis, apoptosis and keratinocyte differentiation. Interestingly, the fraction of affected OFC-related genes was significantly higher than the fraction of total protein-coding genes. Deregulation of these genes by RA seems to induce apoptosis and inhibit differentiation, which might be relevant to the pathogenesis of OFC. In addition, RA seems to affect proliferation, adhesion and migration at the gene expression level which has to be further investigated using functional analyses.
In chapter 5, we hypothesized that the expression of genes related to key cellular processes for palate development such as proliferation, differentiation, adhesion, migration and apoptosis might be deregulated in keratinocytes derived from OFC patients. For this purpose, we performed genome-wide expression analysis on palatal keratinocytes derived from OFC patients and non-OFC controls. This analysis showed a clear difference in gene expression between the two cell types. Gene ontology analysis revealed a significant enrichment for the genes involved in adhesion and migration. Using bioinformatic techniques we identified 21 adhesion- and migration-related genes, all of which were downregulated in OFC keratinocytes. Finally, a scratch assay demonstrated significantly reduced migration of OFC keratinocytes as compared to non-OFC keratinocytes. This indicates that adhesion and migration programs are deregulated in OFC keratinocytes, which might be relevant to OFC pathogenesis.
Chapter 6 provides a general discussion of the main findings and implications, and future perspectives of the research conducted within this thesis.