Factors and Mechanisms Regulating the Expression of Human TLR2 Protein and mRNA

Persistent Link:
http://hdl.handle.net/10150/195594
Title:
Factors and Mechanisms Regulating the Expression of Human TLR2 Protein and mRNA
Author:
Dalvi, Minal
Issue Date:
2006
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Toll-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.
Type:
text; Electronic Dissertation
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Microbiology & Immunology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Halonen, Marilyn

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleFactors and Mechanisms Regulating the Expression of Human TLR2 Protein and mRNAen_US
dc.creatorDalvi, Minalen_US
dc.contributor.authorDalvi, Minalen_US
dc.date.issued2006en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.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.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology & Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairHalonen, Marilynen_US
dc.contributor.committeememberHalonen, Marilynen_US
dc.contributor.committeememberHarris, Daviden_US
dc.contributor.committeememberVercelli, Donataen_US
dc.contributor.committeememberBernstein, Harrisen_US
dc.contributor.committeememberBloom, Johnen_US
dc.identifier.proquest1898en_US
dc.identifier.oclc659746462en_US
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