Lipopolysaccharide (LPS), a glycolipid found in the cell wall of Gram-negative bacteria, exerts pleiotropic biological effects in different cell types. LPS is mainly recognized by the Toll-like receptor (TLR) 4/MD2/Cluster of differentiation 14 complex (CD14). We previously demonstrated that LPS produced a direct action on thyroid cells, including up-regulation of thyroglobulin gene expression. This work aimed to study further the effect of LPS on thyroid function and to elucidate the mechanism by which LPS is recognized by the thyroid cell. We could detect the transcript and protein expression of TLR4, MD2, and CD14 in thyroid cells, and that these proteins are localized at the plasma membrane. The sodium iodide symporter (NIS) is the transporter involved in the iodide uptake, the first step in thyroid hormonogenesis. We demonstrated that LPS increases the TSH-induced iodide uptake and NIS protein expression. The LPS agonist lipid A reproduced LPS effect, whereas the LPS antagonist, polymyxin B, abrogated it. By the use of anti-TLR4 blocking antibodies and the transient expression of TLR4 dominant-negative forms, we evidenced the involvement of TLR4 in the LPS action. The enrichment of TLR4 expressing Fisher rat thyroid cell line-5 (FRTL-5) cells confirmed that TLR4 confers LPS responsiveness to thyroid cells. In conclusion, we revealed for the first time that all the components of the LPS receptor complex are expressed in thyroid cells. Evidence that the effects of LPS on rodent thyroid function involve TLR4-induced signaling was obtained. The fact that thyroid cells are able to recognize and respond to LPS supports a role of the endotoxin as a potential modifier of thyroid function.
Nitric oxide (NO) is a free radical that mediates a wide array of cell functions. It is generated from L-arginine by NO-synthase (NOS). Expression of NOS isoforms has been demonstrated in thyroid cells. Previous reports indicated that NO donors induce dedifferentiation in thyrocytes. However, the functional significance of endogenous thyrocyte-produced NO has not been explored. This work aimed to study the influence of endogenous NO on parameters of thyroid cell function and differentiation in FRTL-5 cells. We observed that treatment with the NOS inhibitor, Nu-nitro-L-arginine methyl ester (L-NAME), increased the TSHstimulated iodide uptake. The TSH-induced sodium iodide symporter (NIS) and thyroglobulin (TG) mRNA expressions were increased after incubation with L-NAME. In transient transfection assays, TSH-stimulated transcriptional activities of NIS and TG promoters were increased by L-NAME. An increment of the TSH-stimulated cell proliferation was observed after NOS inhibition. Similar results were obtained when the action of another NOS inhibitor, N g -monomethyl-L-arginine, was analysed for most of these studies. The production of NO, which was not detectable in basal conditions, was increased by TSH. Our data provide strong evidence that endogenous NO could act as a negative signal for TSH-stimulated iodide uptake and thyroid-specific gene expression as well as proliferation in thyrocytes. These findings reveal a possible new inhibitory pathway in the regulation of thyroid cell function.
The bacterial lipopolysaccharide (LPS) is a biological activator that induces expression of multiple genes in several cell types. LPS has been proposed as an etiopathogenic agent in autoimmune diseases. However, whether LPS affects the expression of autoantigens has not been explored. Thyroglobulin (TG) is a key protein in thyroid hormonogenesis and one of the major thyroid autoantigens. This study aimed to analyze the action of LPS on TG gene expression in Fisher rat thyroid cell line FRTL-5 thyroid cells. We demonstrate that LPS increases the TSH-induced TG protein and mRNA level. Evidence that the effect of LPS is exerted at the transcriptional level was obtained by transfecting the minimal TG promoter. The C element of the TG promoter, which contains sequences for paired box domain transcription factor 8 (Pax8) and thyroid transcription factor (TTF)-1 binding, is essential for full TG promoter expression under TSH stimulation. The transcriptional activity of a construct containing five tandem repeats of the C site is increased by LPS, indicating a possible involvement of the C site in the LPS-induced TG gene transcription. We demonstrate that the TG promoter mutated at the Pax8 or TTF-1 binding element in the C site does not respond to LPS. In band shift assays, binding of Pax8 and TTF-1 to the C site is increased by LPS. The Pax8 and TTF-1 mRNA and protein levels are augmented by LPS. The half-lives of TG, Pax8, and TTF-1 are increased in endotoxin-treated cells. Our results reveal the ability of LPS to stimulate the expression of TG, a finding of potential pathophysiological implication.
These findings favor a long-term inhibitory role of the NO/cGMP pathway on parameters of thyroid hormone biosynthesis. A novel property of NO to inhibit TPO and TG mRNA expression is supported. The NO action on iodide uptake could involve cGK mediation. The long-term inhibition of steps of thyroid hormonogenesis by NO could be of interest in thyroid pathophysiology.
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