Site-Specific Anti-C-ERB A Antibodies Recognizing Native Thyroid Hormone Receptors: Their use to Detect the Expression and Localization of α and β C-ERB A Proteins in Rat Liver

Macchia, Enrico; Falcone, M; Giorgilli, Gianfranco; Bogazzi, Fausto; Antonangeli, L; Baccarini, S; Fontanini, Gabriella; Torresani, Janine; DeGroot, Leslie J.; Pinchera, Aldo

doi:10.3109/10799899209074792

Cited by 12 publications

(13 citation statements)

References 29 publications

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“…Several immunohistochemical studies have reported the presence of TR proteins in the liver of rodents and humans (Tagami et al 1990, Strait et al 1991, Macchia et al 1992, Rodd et al 1992, Falcone et al 1994. Although not all hepatocytes were stained in these studies, none of these studies mention a topographical distribution of TR isoforms even though most known T 3 -dependent genes are expressed in the liver in different zones.…”

Section: Discussionmentioning

confidence: 92%

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver

Zandieh-Doulabi¹,

Dop²,

Schneiders³

et al. 2003

Journal of Endocrinology

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Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-encoding TR 1 and TR 2 and their respective isoforms, and c-erbA-which encodes TR 1, TR 2 and TR 3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TR 1 isoform in rat liver. We were therefore also interested to see whether TR 1 and TR 2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TR 1 and TR 2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TR 1 and TR 2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TR 1 is broader than that of TR 1. Moreover, the expression of the TR 2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TR 1 protein.From our data it appears that both the TR 1 and TR 2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.

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Section: Discussionmentioning

confidence: 92%

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver

Zandieh-Doulabi¹,

Dop²,

Schneiders³

et al. 2003

Journal of Endocrinology

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“…The characterization of these antipeptide polyclonal antibodies, competition studies (preadsorbing each antibody with the specific peptide or with the whole protein expressed in Escherichia coli) and immunoprecipitation analysis (immunoprecipitation of the protein translated in vitro and of the thyroid hormone receptor expressed in E. coli) were reported previously (26,27). Alpha-2-431 antibody activity could be blocked by an excess amount of immunizing peptide and by TR alpha-2 expressed in B. coli in both ELISA and western blotting analyses, but only a partial competition was obtained in our immuno¬ histochemical studies.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the ontogeny of thyroid hormone receptor isotypes in rat brain and liver using an immunohistochemical technique

Falcone

Miyamoto²,

Fierro-Renoy³

et al. 1994

European Journal of Endocrinology

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We performed an immunohistochemical study on rat brain and liver during fetal and neonatal life using rabbit antipeptide polyclonal antibodies able to recognize each thyroid hormone receptor (TR) isoform. The expression of TR alpha-1, alpha-2 and beta-1 proteins from 14 days of gestation to 21 days after birth was evaluated. Frozen tissues from 14 (F14), 17 (F17) and 21 (F21)-day-old fetuses and from 5 (N5), 16 (N16) and 21 (N21)-day old newborn rats were stained with anti-TR antibodies using an avidin-biotin-peroxidase system. The antipeptide antibodies utilized in the present study were characterized previously: alpha-144 antibody recognizes both TR alpha-1 and alpha-2; alpha-2-431 antibody is specific for TR variant alpha-2, and beta-62 antibody specifically reacts with the TR beta-1 isoform. The expression of TR alpha-1 was deduced by comparing the staining obtained with alpha-144 and alpha-2-431 antibodies. We demonstrated that each TR isoform is expressed in rat brain from 14 days of gestation and that the alpha isoform was predominant in the early stage. The three TR isoforms were expressed in both neural cell nuclei and in glial cell nuclei. As far as the liver is concerned, at F14 the expression of TR isoforms was weaker in hepatocytes when, on the contrary, TR alpha was clearly detected in hematopoietic cells. The expression of TRs in hepatocytes becomes evident later. The data that we obtained, although not quantitative, emphasize the presence of each TR isoform in brain and liver from 14 days of fetal rat life.

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“…In this perspective, several attempts were conducted by different groups, the main strategy being based on the production of polyclonal antipeptide antibodies after selection of restricted sequences in regions that diverge between the known c-ErbA isoforms. In most studies, these antibodies were characterized mainly with recombinant c-ErbA iso¬ forms, and then used for immunohistochemical detec¬ tion (16)(17)(18)(19)(20)(21). Only a few reports provided data that indicate a discrimination between T3 receptor isoforms at the level of nuclear extracts (22,23).…”

mentioning

confidence: 98%

Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian α- or β-type triiodothyronine receptors and interfere differently with hormone binding

Bismuth¹,

Lenoir²,

Teboul³

et al. 1995

European Journal of Endocrinology

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In our first report, rabbit antibodies directed to recombinant polypeptides of human alpha-type c-ErbA sequences recognized natural triiodothyronine (T3) receptors (TR) in adipocytes (mouse Ob 17 cell line) but not in liver (mouse, rat). Moreover, some of them, directed to the sequence 150-228, markedly interfered with hormone binding to adipocyte T3 receptors. We now raised antibodies against shorter synthetic peptides within this alpha-type 150-228 c-ErbA sequence, which encompasses part of the hinge (D) domain and N-terminus of the E domain (alpha-150-166 and alpha 172-191) and against a beta-type c-ErbA sequence (beta 204-220 aligned on alpha 150-166, and differing by eight amino acids). Our present antibodies, which bear the expected c-ErbA alpha- or beta-type specificity, immunoprecipitated the TR in nuclear extracts, with a different pattern between tissues: exclusive precipitation by anti-c-ErbA alpha antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA beta antibodies in rat or mouse liver, which also expresses some alpha-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our previously described antibodies to other c-ErbA alpha or beta sequences (anti-alpha 144-162, anti-alpha 1 403-410 and anti-beta 62-82), roughly verifies results of c-erbA mRNA expression in these tissues. Slight differences appeared in the extent of alpha-type TR recognition by antibodies directed to alpha 172-191, whether TR were liganded or not to T3 before antibody addition. This evokes a different conformation of this region after hormone binding. Most interestingly, these anti-alpha 172-191 antibodies lowered the Ka for T3 and extensively dissociated the adipocyte T3-TR complexes; they interfered poorly with the binding of T3 in liver nuclear extracts. This strongly supports the concept that internal sequences in c-ErbA alpha, more precisely in a restricted C-terminal part of the D domain, are necessary for efficient T3 binding, which also need the C-terminal part of domain E.

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Site-Specific Anti-C-ERB A Antibodies Recognizing Native Thyroid Hormone Receptors: Their use to Detect the Expression and Localization of α and β C-ERB A Proteins in Rat Liver

Cited by 12 publications

References 29 publications

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver

Evaluation of the ontogeny of thyroid hormone receptor isotypes in rat brain and liver using an immunohistochemical technique

Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian α- or β-type triiodothyronine receptors and interfere differently with hormone binding

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