Factors and Mechanisms Regulating the Expression of Human TLR2 Protein and mRNA
Committee ChairHalonen, Marilyn
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PublisherThe University of Arizona.
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AbstractToll-like receptors (TLRs) are innate immune receptors that recognize pathogen associated molecular patterns like lipopolysaccharide and peptidoglycan. TLR2 plays an important and possibly critical role in interacting with the most prominent portion of the microbial load encountered by humans and many animal species on a continual basis: i.e., gram-positive bacteria and fungi. TLR2 has been reported to be one of the candidate genes to influence protection from asthma and allergies through complex gene environment interactions.TLR2 expression on monocytes from adult humans can be modulated dose dependently by interaction with the TLR2 synthetic ligand Pam3Cys. Stimulation with concanavalin-A and phorbol 12-myristate 13-acetate resulted in a significant decrease of surface TLR2 in 30 minutes and altered expression continued for at least 24 hours, suggesting that shedding and/or internalization could be potential mechanisms for immediate TLR2 down-regulation and the prolonged effect suggested alterations in transcription or translation. Mitogen stimulated purified monocytes showed that TLR2 expression can be influenced by lymphocyte dependent and independent pathways.TLR2 protein expression was found to be significantly lower in T homozygous individuals as compared to heterozygous individuals with respect to a SNP in TLR2 intron, further implicating active regulatory mechanisms. Our studies also showed that monocytes obtained from umbilical cord express significantly lower TLR2 than adult monocytes. Thus, it appeared that both genetic factors and environmental factors modulate the TLR2 expression.A report that TLR2 mRNA existed in several alternatively spliced forms involving the 5'untranslated region (5'UTR) region suggested to us that this might be one possible mechanism for regulating receptor expression. By cloning and sequencing the TLR2 mRNA from monocytes, Hela cells, Monomac-6 and adult mononuclear cells we identified 5 new variants in addition to the previously identified variants and showed that TLR2 gene is composed of 4 non-coding exons and one coding exon. The 5'-UTR of variant A (exon1+exon2+coding exon) and variant E (exon1+coding exon), were cloned and placed into a luciferase-gene-containing expression vector. The relative luciferase activity of construct with variant E 5'UTR was significantly higher than variant A 5'UTR construct. These results thus demonstrate that alternative splicing may be a mechanism to modulate protein expression.
Degree ProgramMicrobiology & Immunology