Endocrine Abstracts

The thyrotropin receptor (TSHR) is expressed in the thyroid and in precursors undergoing lineage-specific differentiation, e.g adipogenesis. The TSHR is activated by TSH, thyroid stimulating antibodies (TSAB) and gain-of-function mutations (TSHR*). To investigate the role of extra-thyroidal TSHR activation we compared the gene-expression profiles of non-modified human subcutaneous preadipocytes with the parallel TSHR* population. Twenty-seven genes were significantly upregulated including hyaluronan synthases 1 & 2. (HAS1 & HAS2). HA accumulates in adipose/connective tissues of patients with thyroid dysfunction. TSHR activation occurs in hyperthyroidism (TSAB) and hypothyroidism (elevated TSH) and may participate in controlling HA production; we have investigated further.

TSHR activation/cross-linking were achieved by TSHR*, TSH or TSHR monoclonals in preadipocyte-fibroblasts from orbital (n=12) and non-orbital (n=11) tissues. HA production was evaluated by QPCR for HAS 1 & 2, ELISA or metabolic labelling plus chromatography. cAMP mimetics were used to investigate signalling effects; in reporter assays (A293 cells transfected with proximal HAS1 promoter driven plasmids, containing/not a functional cAMP-response element, CRE) and in chromatin-immunoprecipitation (ChIP) of preadipocyte-fibroblasts (using a CREB antibody).

HA expression was higher in TSHR* expressing preadipocytes than the control populations (non-orbital reached significance). cAMP mimetics induced higher and more rapid HA expression in non-orbital than orbital preadipocyte-fibroblasts. Incubation with TSH or monoclonal TSAB induced modest but significant increases in HA production in orbital and non-orbital cells; a TSHR monoclonal without bioactivity behaved similarly. Reporter assays revealed that the HAS1 proximal promoter CRE is functional, with significant increases in light output only in the presence of CRE and cAMP mimetic; ChIP assays produced a HAS1 product with a ΔΔC_t of 3.3 cycles indicating a 4–6 fold increase in amplification.

We conclude that TSHR activation is implicated in HA production in preadipocyte-fibroblasts which, along with thyroid hormone level variation, explains the HA over-production in patients with thyroid dysfunction.