The Tissue Distribution Of The Novel Thyroid Hormone Receptor Isoform-TRβ△

Thyroid hormone has very important metabolic and developmental regulationeffects in almost all organs of the body.It exerts most of their functions at thegemomic level,through binding to thyroid hormone receptors (TRs),which arehighly conserved transcription factors of the nuclear receptor superfamily.Because ofdifferent transcript start position and alternative splicing,each subtype has severalisoforms,including TRα1,TRα2,TRα3,TRΔα1,TRΔα2,TRβ1,TRβ2,TRβ3,TRΔβ3.TRβΔis a novel TRβisoform which have isolated and identified in rat liver.It'smRNA/cDNA is only 108bps longer than that of TRβ1.The only difference betweenTRβΔand TRβ1 is an additional 36 amino-acid residues of TRβΔ.No specificantibody against the TRβΔisoform is available at present time,and the antibody toTRβ1 may recognised TRβΔas well.The reported tissue distribution results of theTRβ1 isoform do not account for this.In this paper,a study of the tissue distributionof the novel thyroid hormone receptor isoform-TRβΔis reported.Objectives:To detect the TRβΔmRNA in normal adult rat tissues.To investigate thetissue-specific and temporal-specific expression features of TRβΔand TRβ1 mRNAand to analyze the correlation between the two transcripts.To prepare TRβΔspecificpolyclonal antibody for investigating the tissue distribution of TRβΔproteins innormal adult rats.Methods:1.Bioinformatics method was used to determine the immunogenicity of the specificpeptide,encoding for amino acids 129～164 of the TRβΔprotein.Indirect ELISAwas established to determine the titer of the antiserum.The specificity of theantibody was determined by Western blot of the recombinant rat TRβΔand TRβ1protein with the prepared polyclonal antibody.2.RT-PCR was used to detect the TRβΔmRNA in the liver,brain,kidney,spleen,small intestine and bone of normal adult rats.The tissue distribution features ofthe TRβΔ,TRβ1and TRβΔ+TRβ1mRNΔin the liver,brain,kidney,spleen tissuesof adult rats were analyzed with RT-qPCR.The temporal-specific features werealso investigated in tissues of rats in different developmental stages.3.Nuclear extracts from the liver,brain,kidney,spleen tissues of adult rats were investigated by Western blot with the anti-TRβΔpolyclonal antibody andanti-TRβ1 monoclonal antibody to detect the protein expression.Results1.The TRβΔspecific peptide has one epitope.After purification,the anti-serum hasgained sufficient purity and sensitivity.The anti-TRβΔantibody could detectrecombinant TRβΔprotein but no cross-reactivity was seen with recombinantTRβ1 protein.2.The TRβΔmRNA could be detected not only in liver,but also in brain,kidney,spleen,small intestine and bone of normal adult rats.In adult tissues,TRβΔ,TRβ1and TRβΔ+TRβ1mRNA levels were much higher in liver,kidney,spleenthan that in brain.In all developmental periods,liver showed a higher expressionlevel than the other three tissues.For all kinds of mRNA,the expression peakswere in the developmental period and declined after sexual maturation.In alltissues and time periods,the mRNA level of TRβΔis much higher than that ofTRβ1 and is in dominant proportion of the TRβΔ+TRβ1 levels.3.TRβΔprotein was detected in the liver and kidney but not in the brain and spleentissues.Conclusions1.We have successfully prepared anti-TRβΔpolyclonal antibody with sufficientsensitivity and specificity to perform Western blot.2.The TRβΔis widely expressed at the transcriptional level in adult rat tissues.Thetissue-specific features of the TRβΔ,TRβ1 and so-called TRβ1 (actuallyTRβΔ+TRβ1)were quite similar.The temporal-specific expression features ofTRβΔis in accordance with other TRβisoforms in that the expression levels arerelatively high during developmental periods but tend to decline after sexualmaturation.The TRβΔsplicing pattern may be dominant in rat tissues.3.The TRβΔprotein exists in the liver and kidney tissues of normal adult rats.