Thyrotropin receptor activation increases hyaluronan production in preadipocyte fibroblasts: contributory role in hyaluronan accumulation in thyroid dysfunction.
ABSTRACT The thyrotropin receptor (TSHR) is expressed during lineage-specific differentiation (e.g. adipogenesis) and is activated by TSH, thyroid-stimulating antibodies, and gain-of-function mutations (TSHR*). Comparison of gene expression profiles of nonmodified human preadipocytes (n = 4) with the parallel TSHR* population revealed significant up-regulation of 27 genes including hyaluronan (HA) synthases (HAS) 1 and 2. The array data were confirmed by quantitative PCR of HAS1 and HAS2 and enzyme-linked immunosorbent assay measurement of HA; all values were significantly increased (p < 0.03) in TSHR*-expressing preadipocytes (n = 10). Preadipocytes (n = 8) treated with dibutyryl (db)-cAMP display significantly increased HAS1 and HAS2 transcripts, HAS2 protein, and HA production (p < 0.02). HAS1 or HAS2 small interfering RNA treatment of db-cAMP-stimulated preadipocytes (n = 4) produced 80% knockdown in HAS1 or 61% knockdown in HAS2 transcripts (compared with scrambled), respectively; the corresponding HA production was reduced by 49 or 38%. Reporter assays using A293 cells transfected with HAS1 promoter-driven plasmids containing or not containing the proximal CRE and treated with db-cAMP revealed that it is functional. Chromatin immunoprecipitation, using a cAMP-responsive element-binding protein antibody, of db-cAMP-treated preadipocytes (n = 4) yielded products for HAS1 and HAS2 with relative fold increases of 3.3 +/- 0.8 and 2.6 +/- 0.9, respectively. HA accumulates in adipose/connective tissues of patients with thyroid dysfunction. We investigated the contributions of TSH and thyroid-stimulating antibodies and obtained small (9-24%) but significant (p < 0.02) increases in preadipocyte HA production with both ligands. Similar results were obtained with a TSHR monoclonal antibody lacking biological activity (p < 0.05). We conclude that TSHR activation is implicated in HA production in preadipocytes, which, along with thyroid hormone level variation, explains the HA overproduction in thyroid dysfunction.
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ABSTRACT: Hyaluronan synthase 1 (HAS1) is one of three isoenzymes responsible for cellular hyaluronan synthesis. Interest in HAS1 has been limited because its role in hyaluronan production seems to be insignificant compared to the two other isoenzymes, HAS2 and HAS3, which have higher enzymatic activity. Furthermore, in most cell types studied so far, the expression of its gene is low and the enzyme requires high concentrations of sugar precursors for hyaluronan synthesis, even when overexpressed in cell cultures. Both expression and activity of HAS1 are induced by pro-inflammatory factors like interleukins and cytokines, suggesting its involvement in inflammatory conditions. Has1 is upregulated in states associated with inflammation, like atherosclerosis, osteoarthritis, and infectious lung disease. In addition, both full length and splice variants of HAS1 are expressed in malignancies like bladder and prostate cancers, multiple myeloma, and malignant mesothelioma. Interestingly, immunostainings of tissue sections have demonstrated the role of HAS1 as a poor predictor in breast cancer, and is correlated with high relapse rate and short overall survival. Utilization of fluorescently tagged proteins has revealed the intracellular distribution pattern of HAS1, distinct from other isoenzymes. In all cell types studied so far, a high proportion of HAS1 is accumulated intracellularly, with a faint signal detected on the plasma membrane and its protrusions. Furthermore, the pericellular hyaluronan coat produced by HAS1 is usually thin without induction by inflammatory agents or glycemic stress and depends on CD44-HA interactions. These specific interactions regulate the organization of hyaluronan into a leukocyte recruiting matrix during inflammatory responses. Despite the apparently minor enzymatic activity of HAS1 under normal conditions, it may be an important factor under conditions associated with glycemic stress like metabolic syndrome, inflammation, and cancer.Frontiers in Immunology 01/2015; 6:43. DOI:10.3389/fimmu.2015.00043
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ABSTRACT: : The pathophysiology of thyroid eye disease (TED) is complex and incompletely understood. Orbital fibroblasts (OFs) seem to be the key effector cells that are responsible for the characteristic soft tissue enlargement seen in TED. They express potentially pathogenic autoantigens, such as thyrotropin receptor and insulin-like growth factor-1 receptor. An intricate interplay between these autoantigens and the autoantibodies found in Graves disease may lead to the activation of OFs, which then leads to increased hyaluronan production, proinflammatory cytokine synthesis, and enhanced differentiation into either myofibroblasts or adipocytes. Some of the OFs in TED patients seem to be derived from infiltrating fibrocytes. These cells originate from the bone marrow and exhibit both fibroblast and myeloid phenotype. In the TED orbit, they may mediate the orbital expansion and inflammatory infiltration. Last, lymphocytes and cytokines are intimately involved in the initiation, amplification, and maintenance of the autoimmune process in TED.Journal of neuro-ophthalmology: the official journal of the North American Neuro-Ophthalmology Society 06/2014; 34(2):177-85. DOI:10.1097/WNO.0000000000000132 · 1.09 Impact Factor
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ABSTRACT: Context: Graves' orbitopathy (GO) is caused by expansion of the orbital contents by excess adipogenesis and over-production of hyaluronan (HA). Immunosuppressive and anti-inflammatory treatments of GO are not always effective and can have side-effects, while targeting GO-associated tissue remodelling might be a more logical therapeutic strategy. Previously, we reported that signalling cascades through IGF1R and TSHR within orbital preadipocyte/fibroblasts drove adipogenesis and HA production. Our current study, combined stimulation of IGF1R and TSHR increase HA accumulation, which we hypothesize is by activation of PI3K1A/PI3K1B respectively. The central aim of this study was to investigate whether PI3K/mTORC1 inhibitors affected adipogenesis and/or HA production within orbital preadipocyte/fibroblasts. Methods: Human orbital preadipocytes were treated with/without inhibitors, LY294002 (PI3K-1A/mTORC1), AS-605240 (PI3K-1B) or PI103 (PI3K-1A/mTORC1) in serum-free medium for 24 h or cultured in adipogenic medium for 15 days. QPCR was used to measure HAS2 transcripts and the terminal adipogenesis differentiation marker LPL. HA accumulation in the medium was measured by ELISA. Results: Unlike AS-605240, both LY294002 (10 μM) and PI-103 (5 μM) significantly decreased HAS2 transcripts/HA-accumulation and adipogenesis. Since PI-103 and LY294002 are dual PI3K/mTOR inhibitors, we investigated inhibition of mTORC1 (rapamycin 100 nM), which significantly decreased adipogenesis but had no effect on HAS2 transcripts/HA, implicating PI3K-1A in the latter. Conclusions: Combined inhibition of PI3K-1A and mTORC1 signalling in vitro decreased both HA accumulation and adipogenesis. Since PI3K and mTOR inhibitors are clinically used to treat other conditions, they have potential to be repositioned to be used as an alternative non-immunosuppressive therapy of GO.The Journal of Clinical Endocrinology and Metabolism 04/2014; 99(7):jc20134182. DOI:10.1210/jc.2013-4182 · 6.31 Impact Factor