Normal Thyroid Structure and Function in Rhophilin 2-Deficient Mice

ABSTRACT Rhophilin 2 is a Rho GTPase binding protein initially isolated by differential screening of a chronically thyrotropin (TSH)-stimulated dog thyroid cDNA library. In thyroid cell culture, expression of rhophilin 2 mRNA and protein is enhanced following TSH stimulation of the cyclic AMP (cAMP) transduction cascade. Yeast two-hybrid screening and coimmunoprecipitation have revealed that the GTP-bound form of RhoB and components of the cytoskeleton are protein partners of rhophilin 2. These results led us to suggest that rhophilin 2 could play an important role downstream of RhoB in the control of endocytosis during the thyroid secretory process which follows stimulation of the TSH/cAMP pathway. To validate this hypothesis, we generated rhophilin 2-deficient mice and analyzed their thyroid structure and function. Mice lacking rhophilin 2 develop normally, have normal life spans, and are fertile. They have no visible goiter and no obvious clinical signs of hyper- or hypothyroidism. The morphology of thyroid cells and follicles in these mice were normal, as were the different biological tests performed to investigate thyroid function. Our results indicate that rhophilin 2 does not play an essential role in thyroid physiology.

Effect of glucocorticoids and oestrogen on interleukin-6 production by human thyrocytes from patients with Graves' disease and toxic multinodular goitre and from HTori3 cells

Interleukin-6 (IL-6) is a cytokine released by thyrocytes and is involved in disease processes such as autoimmune thyroid disease. The secretion of IL-6 can be stimulated by interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF), serum, TSH and agents which increase intracellular cyclic AMP levels. Antithyroid drugs such as methimazole inhibit IL-6 production by thyrocytes but the effects of glucocorticoids and oestrogen have not been investigated. The effects of dexamethasone and 17 beta-oestradiol on IL-1-, TNF-, TSH-, forskolin- and phorbol 12-myristate 13-acetate (PMA)-stimulated IL-6 release in serum-free conditions were studied in human thyrocytes derived from patients with Graves' disease and toxic multinodular goitres, and in the immortalised human thyrocyte cell line, HTori3. Dexamethasone inhibited IL-6 production under stimulated conditions. In serum-free conditions, no basal release of IL-6 was assayable. In all but one of the primary thyroid cultures, TSH did not stimulate IL-6 release above the lower detectable limit of the assay. In Graves' and multinodular goitre thyrocytes, inhibition of IL-1 (100 U/ml)-stimulated IL-6 release by dexamethasone (100 nmol/l) was 62.51% +/- 10.43 (S.E.M.), and in HTori3 cells it was 78.35% +/- 3.9. The degree of IL-1 stimulation of IL-6 release and inhibition by dexamethasone was not significantly different in thyrocytes derived from either Graves' or multinodular glands. 17 beta-Oestradiol had no effect on IL-1-stimulated IL-6 release in either primary thyroid cell culture or in HTori3 cells.

Human thyroid cells in monolayer retain the ability to secrete tri-iodothyronine in response to thyrotrophin

ABSTRACT Confluent monolayer cultures of human thyroid cells secreted low levels of immunoassayable triiodothyronine (T3) and this process could be stimulated by TSH in a concentration-dependent manner. The characteristics of the response to TSH were related to the age of the thyroid cell culture both in terms of the relative sensitivity to TSH and the quantity of T3 released. Cells which had been in culture for 2–3 days (primary cultures) secreted high levels of T3 under unstimulated and TSH-stimulated conditions with a median effective dose (ED50) for TSH of 0·030 mu. TSH/ml. However, cells which had been subcultured and consequently had been in culture for a longer period of 6–7 days secreted lower levels of T3 under basal and stimulated conditions. This was approximately 30% of that released from primary cultures with an ED50 for TSH of 0·1 mu. TSH/ml. Reorganization of human thyroid cells into follicular structures was seen during growth with TSH but these cultures showed little response to subsequent acute stimulation by TSH; the return of a diminished, less sensitive response to TSH was seen after a recovery period of 8 h. The time-course of T3 release was dependent on the TSH concentration with low TSH concentrations stimulating T3 secretion after increased incubation periods. Human thyroid cells had lost the ability to concentrate and organify free iodide after several days in culture but were still secreting T3. This indicates the presence of intracellular stores of T3 which are released on stimulation with TSH, rather than new synthesis of T3. J. Endocr. (1985) 104, 285–290