A szarvasmarha neonatális Fc receptor transzkripciós szabályozása

The neonatal Fc receptor (FcRn), like other MHC class I molecules, is composed of an α-chain and a β2-microglobulin (β2m). This receptor has been detected in the bovine mammary gland, small intestine, lower respiratory system, and endothelial cells. While epithelial FcRn is involved in IgG transport through these barriers, the FcRn expressed in capillary pendothelial cells is responsible for regulating the IgG catabolism. Due to these crucial immunological functions, the gene regualtion of the bovine FcRn (bFcRn) may contribute to the immune homeostasis. Although gene expression is controlled at multiple levels, one of the most important is transcriptional regulation. Accordingly the sequences of the human and mouse FcRn α-chain cis-regulatory region have been published and their preliminary examination has been achieved, but their transcriptional regulation has not been adequately unravelled. In order to reveal the regulation of the bFcRn transcription, the 5’-flanking sequence of the bFcRn α-chain gene was isolated, cloned and functionally examined. The bFcRn α-chain cis-regulatory region was induced by NFκB in the luciferase reporter gene assays of human and bovine cell models. Three functional κB binding sites were identified in the cis-regulatory region using site-directed mutagenesis accompanied by luciferase reporter gene assays, and it was verified that these κB sites were responsible for the complete NFκB responsiveness of the bFcRn cis-regulatory region. The κB binding sites were also tested in gel retardation assay verifying their binding ability to NFκB complex with p65 content. These in vitro findings indicated, in accordance with the present in vivo data, that the bFcRn was under the control of an important transcriptional pathway activated during infection and inflammation. The β2m is a chaperone of FcRn and other MHC class I (-like) proteins ensuring the appropriate function of these molecules. To fulfil this function, it is expressed ubiquitously under constitutive and cytokine-induced transcriptional controls. Transcriptional elements of the β2m cis-regulatory region have been experimentally well characterized in human, and it has been found that a κB and an ISRE sites were responsible for the cytokine-induced regulation. The 5’-flanking sequence of the bovine β2m (bβ2m) has been isolated and cloned in order to assess its cytokine-induced gene expression in relation to FcRn. Although the ISRE site was conserved in the cattle, there was a deletion in the bβ2m κB site compared to the human orthologue, and there was no NFκB responsiveness of the bβ2m cis-regulatory region in the luciferase reporter gene assays of human and bovine cell models. To the contrary, the bβ2m κB site did bind the NFκB complex with p65 content in gel retardation assay rendering these 6 in vitro results controversial. In vivo data about the mRNA level of the bβ2m upon LPS induction are also contradictory, therefore the NFκB inducibility of the bβ2m cis-regulatory region cannot be deduced from the present data. The functionality of the bβ2m ISRE site was confirmed in vitro by gel retardation and luciferase reporter gene assays, thus, the bβ2m ISRE site mediated the IFN-γ induction similarly to its human orthologue, and there were no differences in the ISRE-mediated transcriptional regulation of this gene in cattle. In order to establish a species-specific system that can be used to analyze gene regulation in bovine, the full length coding sequence of the bovine p65 (bp65) subunit of NFκB was isolated and cloned. The cloned bp65 was expressed in mammalian cells, and it induced the NFκB-specific luciferase reporter gene expression. Using gel retardation assay, it was demonstrated that the cloned bp65 bound to the consensus κB sequence. The comparison of the bp65 with its human and mouse orthologues at amino acid level showed high homology in both the DNA-binding domain, known as Rel homology domain (RHD) and the transactivation domain (TAD). The phylogenetic analysis at DNA level provided a new insight into the evolution of the NFκB family, and it was able to resolve the topology of the mammalian p65 molecules. Although, the RHD was conserved in vertebrates, the TAD sequences deviated from each other, and showed faster molecular evolution than RHD sequences