The regulation of thyroglobulin (Tg) and its specific mRNA by interleukin-1 (IL-1) in cultured human thyrocytes was investigated. Specific binding of 125I-labelled IL-1 on thyrocytes was confirmed by solid-phase binding assay. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without recombinant human IL-1. TSH stimulated Tg release from cultured human thyrocytes in a dose- and time-dependent manner. Both IL-1 alpha and beta inhibited TSH-induced Tg release at concentrations ranging from 0.01 to 10 U/ml. The suppressive activities of IL-1 alpha and beta were similar. They did not alter the basal level of Tg release. Unstimulated human thyrocytes did not contain any detectable Tg mRNA, but TSH-stimulated thyrocytes expressed a single species of Tg mRNA (8.5 kb). Both IL-1 alpha and beta inhibited TSH-induced Tg mRNA in a dose-responsive manner. IL-1 (10 U/ml) caused maximal suppression of TSH-induced Tg mRNA to nearly basal levels. In contrast, the gamma-actin mRNA hybridization signal was not altered in control or treated cells. Furthermore, IL-1 stimulated [3H]thymidine uptake into thyrocyte DNA. These results demonstrate that IL-1 directly inhibits TSH-induced Tg gene expression and provide further support for a functional role of IL-1 as a local modulator of thyroid hormone synthesis.
The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of -107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gtl 1 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant fl-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA.The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.
The effect of TSH stimulation on cellular (c)-myc mRNA content in cultured thyroid cells was examined by Northern blot analysis. Polyadenylated mRNA was prepared from quiescent FRTL-5 rat thyroid cells and from cells stimulated by TSH for 8 h. A major 2.1 kilobase (kb) transcript was enhanced 5.5-fold by TSH. (Bu)2cAMP and forskolin each enhanced c-myc expression 2.7-fold. These data indicate that TSH, via cAMP as a second messenger, can regulate expression of a cellular oncogene in a non-neoplastic, specific target cell.
We have previously demonstrated that interferon-gamma (IFN-gamma) induced HLA-DR antigen and also inhibited thyrotropin (TSH)-induced triiodothyronine (T3) and thyroglobulin (Tg) secretion from cultured human thyrocytes. In order to further clarify the inhibitory effect of IFN-gamma on TSH-stimulated thyroid hormone secretion, we have examined human thyroid peroxidase (TPO) gene expression. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without IFN-gamma. Total RNA was extracted, separated and hybridized with 32P-labelled human TPO cDNA. Thyrocytes expressed four TPO mRNA transcripts (4.0, 3.2, 2.1 and 1.7kb, respectively), all of which were stimulated by TSH. IFN-gamma inhibited TSH-stimulated TPO mRNA in a dose dependent manner (0.01-10U/mL). 1 U/mL IFN-gamma caused maximal suppression of TSH-stimulated TPO mRNA levels to basal levels. IFN-gamma also inhibited 8-bromo-cyclic AMP-stimulated TPO mRNA levels. In contrast, the gamma-actin mRNA hybridization signal was not altered in control or treated cells. These results demonstrate that IFN-gamma directly inhibits TSH-stimulated TPO gene expression and provide further support for a role of IFN-gamma as a local modulator of thyroid hormone synthesis.
cDNAs for thyroid peroxidase (TPO) and thyroglobulin (Tg) have been cloned and sequenced. Using such cDNAs, we investigated the regulation of TPO and Tg gene expression by various agents in cultured human thyroid cells. Unstimulated human thyroid cells contained a major RNA species [3.2 kilobases (kb) in length] and several minor RNA species (less than 3.2 kb mRNA) of TPO, but no detectable Tg transcripts. However, TSH-stimulated thyroid cells contained four distinct TPO mRNA species (4.0, 3.2, 2.1, and 1.7 kb) and a single Tg mRNA species (8.5 kb). TSH stimulated the TPO and Tg mRNA levels in a dose- and time-dependent manner. The same results were obtained with 8-bromo-cAMP, a cAMP analog, but not with insulin or insulin-like growth factor I. Furthermore, inductions of these mRNAs by TSH and 8-bromo-cAMP were almost completely blocked by cycloheximide, a protein synthesis inhibitor. These results suggest that human thyroid cells contain four distinct TPO mRNAs and a single species of Tg mRNA, and the levels of all mRNAs are increased by TSH/cAMP stimulation. These increases are blocked by inhibiting protein synthesis, indicating that TSH stimulation of TPO and Tg mRNA levels may be mediated by newly synthesized protein(s).
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