Study of membrane-related effects of TSH in thyrocytes: TSH receptor localization and action, and Duox-TPO interaction

Thyroid hormone regulates growth and development throughout the animal kingdom. The thyroid which secretes it, is controlled by TSH and its receptor TSHR. TSH and its receptor TSHR act through TSHR-coupled G proteins to control thyroid functions, with a stronger coupling of the TSHR with Gs protein than with Gq protein in human thyrocytes. Gq is not activated by TSH/TSHR in dog, whereas dog TSHR activates it in CHO transfected cells. To better understand TSHR and its downstream effectors G proteins, we attempted to answer the questions by the role of “lipid rafts/caveolae” in TSH action.

Lipid rafts/caveolae are sphingolipids-cholesterol-enriched microdomains on plasma membrane that have been proposed to play a role in signal transduction. By concentrating the signal molecules, lipid rafts/caveolae increase the efficiency of the interactions between the molecules and sequestrate them from the bulk membranes. The compartmentation of signal proteins in lipid rafts/caveolae might provide a possible explanation for the relationship between TSHR and G proteins in human and dog thyroctyes.

To answer these questions, we first tested the existence of such lipid microdomains in human and dog thyrocytes. By northernblot and RT-PCR of caveolin-1 mRNA, we demonstrated its existence in thyrocytes. The immunohistochemistry of caveolin-1 showed that caveolin/caveolae are present on the apical membrane of thyrocytes, opposite to the TSHR localization on the basolateral membranes. The isolation of lipid rafts/caveolae by Triton X-100/OptiPrep density experiments showed that TSHR and Gq are not in the rafts, even though other proteins such as insulin receptor, flotillin-2 and partially Gs are present in these lipid domains, as expected. Testing the function of the TSH receptor on its main cascade (Gs-Adenylyl cyclase-cAMP) after treating the follicles with Methyl β-cyclodextrin (a cholesterol chelator), we observed no modification of the cAMP levels by this treatment. This is in agreement with our conclusion that the TSHR-Gs-cAMP pathway does not involve the lipid rafts/caveolae domain.

TSH-activated signalling does not take place in these membrane domains. Therefore, the differences between species, concerning the TSHR-G proteins coupling cannot be explained by the presence of these membrane domains.

2. Species specific thyroid signal transduction: conserved physiology, diverged mechanisms

As mentioned above, Gq proteins are activated in human but not in dog thyroid, in response to TSHR. However the dog TSH receptor is able to activate Gq, as demonstrated in transfected CHO cells. Thus, different thyroid signal transduction pathways exist in different species.

In this study, we investigated the effects of TSH on its two signal transduction cascades, the cAMP pathway and the phospholipase C – IP3 – DAG pathway, as measured by cAMP levels and inositol phosphate generation. We also measured the effects of TSH and of agents stimulating specifically one of these cascades, forskolin for the cAMP pathway and Ca++ ionophore (ionomycin) and phorbolmyristate ester (TPA) for the phospholipase C pathway, on markers of thyroid hormone synthesis (H2O2 generation and iodide binding to proteins) and on thyroid hormone secretion in vitro in the various thyroids.

We demonstrated that in all species investigated, the TSH receptor activates both hormone synthesis and secretion. While in some species, including humans, rats and mice, the TSH receptor activates both the cAMP and phospholipase C– IP3 – DAG cascades, in others (e.g. dog) it only stimulates the first. The cAMP pathway activates the limiting step in thyroid hormone synthesis, the generation of H2O2, in dog, rat and mice but not in human, pig, horse and beef. Thus physiology remains but the pathways to achieve it differ. On a practical point of view, these results allow to choose adequate animal models for investigating different aspects of human thyroid signalling.

3. Duoxes -TPO association and its regulation in human thyrocytes: the thyroxisome

Duox (Dual Oxidase) and TPO (thyroid peroxidase) are the crucial enzymes for the thyroid hormones biosynthesis (T3/T4). TPO uses the hydrogen peroxide (H2O2) produced by Duox1 and Duox2 isoenzymes to covalently link oxidized iodide to tyrosines of thyroglobulin and couple the iodinated tyrosines to form triiodothyronine (T3) and thyroxine (T4). An excess of H2O2 is considered to be toxic for cells although at appropriate concentrations H2O2 may carry out signalling functions. Even though thyrocytes show a better resistance to H2O2 than other cells, it would be beneficial for thyrocytes if Duox and TPO localize closely to increase the working efficiency and avoid an excessive H2O2 spillage. In this study, we explored the association of Duox with TPO, and the possible factors affecting their interaction in the human thyrocyte model. This association was established by co-immunoprecipitation approaches on purified plasma membranes from human thyrocytes and COS-7 transfected cells.

Our results show that 1) Duox and TPO localize closely at the plasma membranes of human thyrocytes, 2) this association is up-regulated through the Gq-PLC-Ca2+-PKC pathway and down-regulated through the Gs-cAMP-PKA pathway. 3) H2O2 directly increases the association of Duox and TPO. 4) Partial NH2- or COOH-terminal Duox1 and Duox2 proteins show different binding abilities with TPO in COS-7 transfected cells.

The association of the two proteins Duox and TPO thus supports our previous hypothesis of the thyroxisome, a pluriprotein plasma membrane complex in which elements of the iodination apparatus localize closely, thus optimizing working efficiency and minimizing H2O2 spillage. Defect in this association, independently of the catalytic efficiency of the enzyme, could therefore impair thyroid hormone synthesis and be harmful to thyroid cells, leading to thyroid insufficiency.