title: 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.
authors.affiliation: Centre for Endocrine & Diabetes Sciences, Cardiff University, Cardiff CF14 4XN, United Kingdom.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Timothy
authors.initials: T
authors.lastname: Bowen
authors.forename: Fiona
authors.initials: F
authors.lastname: Grennan-Jones
authors.forename: Carol
authors.initials: C
authors.lastname: Paddon
authors.forename: Peter
authors.initials: P
authors.lastname: Giles
authors.forename: Jason
authors.initials: J
authors.lastname: Webber
authors.forename: Robert
authors.initials: R
authors.lastname: Steadman
authors.forename: Marian
authors.initials: M
authors.lastname: Ludgate
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000378:metabolism*; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013961:Thyroid Gland / Q000378:metabolism / Q000503:physiopathology
chemical_list: D011989:Receptors, Thyrotropin; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases