Thyrotropin Receptor Activation Increases Hyaluronan Production in Preadipocyte Fibroblasts

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. 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. The thyrotropin receptor (TSHR) 2The abbreviations used are: TSHRthyrotropin receptorTSABthyroid-stimulating antibodiesmAbmonoclonal antibodyHAhyaluronanHASHA synthase(s)QPCRquantitative PCRELISAenzyme-linked immunosorbent assaysiRNAsmall interfering RNAdbdibutyryl cAMPCREcAMP-responsive elementCREBCRE-binding proteinGAGglycosaminoglycan(s)GDGraves diseaseGOGraves ophthalmopathyCMcomplete mediumChIPchromatin immunoprecipitationTCNtranscript copy numbersAPRTadenosine phosphoribosyl transferase. is a G-protein-coupled receptor, which, in addition to its well characterized role in controlling thyrocyte function and growth (1Vassart G. Dumont J.E. Endocr. Rev. 1992; 13: 596-611PubMed Google Scholar), has been shown to be up-regulated during lineage-specific differentiation of adult precursors found in bone marrow and adipose tissue, e.g. preadipocyte adipogenesis to mature fat cells (2Crisp M.S. Lane C. Halliwell M. Wynford-Thomas D. Ludgate M. J. Clin. Endocrinol. Metab. 1997; 82: 2003-2005Crossref PubMed Scopus (122) Google Scholar, 3Abe E. Marians R.C. Yu W. Wu X.B. Ando T. Li Y. Iqbal J. Eldeiry L. Rajendren G. Blair H.C. Davies T.F. Zaidi M. Cell. 2003; 115: 151-162Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). To investigate a potential role in these tissues, we performed microarray analyses of human preadipocytes transduced with a gain-of-function mutant TSHR and the equivalent nonmodified populations. Hyaluronan synthases 1 and 2 (HAS1 and HAS2) are two of the three synthases that produce hyaluronan (HA) and were among a small number of genes whose expression was significantly increased in the mutant TSHR population. thyrotropin receptor thyroid-stimulating antibodies monoclonal antibody hyaluronan HA synthase(s) quantitative PCR enzyme-linked immunosorbent assay small interfering RNA dibutyryl cAMP cAMP-responsive element CRE-binding protein glycosaminoglycan(s) Graves disease Graves ophthalmopathy complete medium chromatin immunoprecipitation transcript copy numbers adenosine phosphoribosyl transferase. HA is a ubiquitous linear polysaccharide component of the extracellular matrix, which influences cellular proliferation and migration following injury and plays an important biological role in tissue remodeling, wound healing, and the phenotypic transformation of cells (4Turley E.A. Noble P.W. Bourguignon L.Y. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar). HA occupies a large hydrodynamic volume acting as a lubricant, support, and cushion in different tissues. It is synthesized on the inner surface of the plasma membrane and extruded to the extracellular matrix by three differentially regulated HAS enzymes about the control of which very little is known (5Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar). HAS1 has a tissue-specific expression, being present, for example, in dermal fibroblasts but absent in oral mucosal fibroblasts (6Meran S. Thomas D. Stephens P. Martin J. Bowen T. Phillips A. Steadman R. J. Biol. Chem. 2007; 282: 25687-25697Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar); HAS2 is inducible, and HAS3 is constitutively expressed in most cell types. The skin and adipose/connective tissue of individuals with thyroid dysfunction accumulate glycosaminoglycans (GAG), predominantly HA (7Smith T.J. Bahn R.S. Gorman C.A. Endocr. Rev. 1989; 10: 366-391Crossref PubMed Scopus (253) Google Scholar). HA is hydrophilic and thus generates the widespread build-up of mucopolysaccharide that produces edema in hypothyroidism. In contrast, the deposition of HA is assumed to be more localized in hyperthyroid conditions such as Graves disease (GD) in which the orbital and pretibial regions are the most affected and can result in Graves ophthalmopathy (GO) and pretibial myxoedema, respectively (8Weetman A.P. Q. J. Med. 1993; 86: 473-477Crossref PubMed Scopus (13) Google Scholar). The major cause of thyroid dysfunction is autoimmunity, and several immunomodulators, e.g. interleukin-1 and transforming growth factor β (both macrophage products), can induce/enhance HA production in vitro (9Kaback L.A. Smith T.J. J. Clin. Endocrinol. Metab. 1999; 84: 4079-4084Crossref PubMed Google Scholar, 10Wang H.S. Tung W.H. Tang K.T. Wong Y.K. Huang G.J. Wu J.C. Guo Y.J. Chen C.C. J. Cell. Biochem. 2005; 95: 256-267Crossref PubMed Scopus (40) Google Scholar). Furthermore, serum IgG from patients with GD can induce hyaluronan production in cultured GD (but not normal) fibroblasts. The effect appears to be mediated by the receptor for IGF-1 and related activating antibodies (11Smith T.J. Hoa N. J. Clin. Endocrinol. Metab. 2004; 89: 5076-5080Crossref PubMed Scopus (192) Google Scholar). Activation of the TSHR occurs in most patients with thyroid dysfunction through thyroid-stimulating antibodies (TSAB) in hyperthyroid GD or elevated TSH in hypothyroidism. In light of our array data, we hypothesize that TSAB or supraphysiological TSH target and activate the TSHR and stimulate the overproduction of HA. We report our findings on HA production in response to activation and/or cross-linking of the TSHR achieved using ligands and gain-of-function TSHR mutations naturally occurring in toxic adenoma and familial hyperthyroidism (reviewed in Ref. 12Paschke R. Ludgate M. N. Engl. J. Med. 1997; 337: 1675-1681Crossref PubMed Scopus (215) Google Scholar). All of the reagents were obtained from Sigma-Aldrich, and tissue culture were from tissue was with and from several (n = patients with all and disease with a activity and (n = predominantly from patients for preadipocytes were obtained from adipose tissue and preadipocytes were obtained by both as L. G. D. C. D. Ludgate M. PubMed Scopus Google Scholar). Preadipocytes were cultured in serum medium and were used number not all were in all mutant TSHR was the preadipocyte using produced in our D. K. R. Wynford-Thomas D. M. Ludgate M. Scopus Google Scholar). in the which increased of cAMP with the equivalent nonmodified cell all as L. G. D. C. D. Ludgate M. PubMed Scopus Google Scholar). The nonmodified and mutant TSHR preadipocyte from different individuals were cultured in in RNA was from the cells using to the RNA was using an to and in vitro The was to and the data were using the data were to differentially expressed genes using an P. PubMed Scopus Google with a Y. Y. J. R. for the data were and and The expression of gain-of-function TSHR nonmodified preadipocytes were in or in and were for and treated for or with dibutyryl cAMP preadipocytes in were treated for with 1 monoclonal TSAB 1 TSHR monoclonal antibodies lacking and and 1 monoclonal antibody to human thyroid The cell were in in RNA was following the and 1 was using for for the three HAS were using to are in To PCR were to the and the was confirmed by was using on a 1 of containing to in a volume of the to a in the of was for used and of in a measurement of transcripts in the used 1 of a in the Comparison with for in of values for In transcripts for a were that values be expressed relative to In a all of the were in the was in in all to preadipocytes were in in but in the medium but serum and in for the to from nonmodified preadipocytes or not with db-cAMP or TSHR and preadipocytes in were to and HA was in by using and using a enzyme-linked immunosorbent assay to the The results are expressed as the ± preadipocytes from individuals were cultured in and treated for with The were in containing 1 and 1 The of were by and the was membrane as M. D. G. Smith Ludgate M. J. Endocrinol. 2005; PubMed Scopus Google Scholar). The were with antibodies to HAS1 and HAS2 from and antibodies were used and the were The were using an for 1 and by Preadipocytes (n = 4) were in and were to culture medium The cells were in of culture medium containing of and 1 of or to produce a of the of the the cells were in and treated for with RNA and culture were for and HAS1 and HAS2 were for the of using the from the K. K. K. M. A. M. M. T. 2005; PubMed Scopus Google Scholar). The assays three plasmids to of The of HAS1 proximal and the cAMP-responsive element the is but the CRE has been to in not a all and the the the proximal and thus the In all the regions were the of the as Thomas G.J. Steadman R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In the was to control for response to cAMP was in several different cell including and all 1 in in medium containing All of the cell were transfected with of and of using to the in the cells were treated for or with ± or dibutyryl the cells were in To of cell was to a and with an volume of assay 2 for and the was using an the results are expressed in The activity in a of cell was using assay to the and was on an The results are expressed in relative light The results are expressed as relative light of values to activity light were all performed in Preadipocytes (n = were cultured in in 80% were for and treated or not for with dibutyryl Chromatin was and assays were performed using the to the the cells were with and with for The were to small from to antibody to was used for the The human HAS1 CRE was by PCR the following were to and The human HAS2 CRE was using the following and The data were using the with or In all < was The is in the To investigate a role for TSHR activation in the lineage-specific differentiation of we the gene expression profiles of control preadipocytes from individuals with the with to for the of variation, genes whose expression was significantly different the HAS1 and HAS2 were among the 27 genes whose expression was significantly up-regulated and in the populations. To the array data, a preadipocyte were using for HAS transcript measurement and to HA The results are in 2. In all of the the the values were in the of HAS1 values were S. R. Biochem. 1999; PubMed Scopus Google Scholar, M. Lane C. J. Ludgate M. Google Scholar, C. Ludgate M. S. J. Endocrinol. 2002; PubMed Scopus Google Scholar, J. S. Phillips A. Steadman R. J. Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. L. G. A. Ludgate M. J. Clin. Endocrinol. Metab. PubMed Scopus Google Scholar), the transcript and HAS2 transcripts were more HAS1 values in the of HAS transcript and HA in the culture medium in preadipocytes (n = from fat control or activating mutant TSHR ± ± < ± ± < ± ± < in a HAS1 and HAS2 and HA in the culture medium were significantly increased in preadipocytes TSHR with significant were nonmodified preadipocytes or the cells transduced with the not both Activation of the as in the the cAMP we used db-cAMP to investigate the increases were by elevated the results are shown in 2. of nonmodified preadipocytes with db-cAMP (n = 8) for significantly increased HAS1 (p < 0.02) and HAS2 (p < treatment was for were significant increases in HAS1 (p < HAS2 (p < and HA by (p < HAS3 were not significantly by the treatment not To the db-cAMP treatment an equivalent in HAS1 and HAS2 in the preadipocytes, was with an antibody to the protein corresponding to the were the of and to The HAS2 antibody produced a with a protein of and used to produce a of HAS2 (n = revealed that its expression was increased from ± to ± (p < by treatment with the cAMP protein was increased by db-cAMP but was the of is shown in In of the of protein expression data for we HAS1 to preadipocytes from In HAS1 produced a in transcripts for HAS1 (p < a in HAS2 (p < and in HA the cells were with HAS1 produced an 80% in transcripts for HAS1 (p < in and a in HA (p < in the culture medium using HAS2 produced a in transcripts for a in HAS1 transcripts, and a in HA < in conditions In db-cAMP cells HAS2 in a 61% in HAS2 transcripts by a in HA (both < but not HAS1 transcript The proximal of HAS1 has a CRE relative to the are two The HAS2 a CRE and relative to the but the CRE in the HAS1 is not to be E. L. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus (215) Google Scholar). used a in which is the control of the proximal of HAS1 containing the which was transfected different cell and The cell to produce in light treated with db-cAMP or was the were for the in addition to several these were the the proximal HAS1 from the and the proximal HAS1 from in which the CRE been by shown in two that significant increases in light were in the of an CRE and a cAMP and that the containing an CRE for of the HAS1 in light was obtained using but in A293 cells transfected with the in which the CRE was not To the CRE is in we chromatin immunoprecipitation on preadipocytes treated for with using an antibody to CRE protein, of the HAS1 CRE using to the CRE produced a relative fold of 3.3 ± 0.8 (n = In contrast, PCR products were using the to produce a the a of the we the HAS2 was a relative fold of 2.6 ± (n = using a that the The produced an of as in PCR products were obtained using of the or in the of the CRE or and in the HAS2 The performed thus constitutively TSHR or reagents to both of which target the preadipocyte population. To the in in patients with thyroid in which a small of cells the we investigated the of and (TSAB) TSHR ligands in preadipocytes from The results are in small but significant increases in HA were by TSH < and a monoclonal TSAB < level < was obtained with a TSHR antibody of biological a TSHR antibody that produced significant in HA The that the TSHR antibodies be an effect by was by the of response to an monoclonal antibody to thyroid not the cells ± ± ± < ± ± < ± ± < ± ± ± in a adipose of in hyperthyroid patients is the in which overproduction of HA is a major to the tissue remodeling, which occurs in using PCR by and an of and HA the in orbital preadipocytes a constitutively TSHR with or not we performed a of on orbital preadipocytes, from of individuals and normal) revealed that HA in the culture medium was significantly increased in the cells with as shown in the and = ± ± < = ± ± < and = ± ± < = ± ± < = ± ± of preadipocytes to both ligands significantly that of preadipocytes (p < = ± ± of preadipocytes to both ligands significantly that of preadipocytes (p < of preadipocytes to both ligands significantly that of preadipocytes (p < 0.05). in a treated with db-cAMP and normal) HAS1 and HAS2 transcripts by a of HAS2 with HAS1 was were significantly increased and (p < and HA in the culture was but the not The response of orbital preadipocytes to TSH and TSAB was more and is in significant response was obtained with in preadipocytes from and three not the results of the and preadipocytes we and increases in HA in response to TSAB (p < and TSH (p < in the In contrast, was significant in HA in the culture medium of preadipocytes from (n = with Furthermore, was a significant (p < in the HA response to TSH and TSAB of and orbital microarray data, a significant up-regulation in HAS1 and HAS2 in preadipocytes a gain-of-function a role for TSHR activation in HA is patients the of thyroid dysfunction in the and of in In both and the is to of in the and tissues J. Clin. Endocrinol. Metab. PubMed Scopus Google Scholar). The is in but to the pretibial and in hyperthyroid The are in the two conditions and in to treatment J. J. Clin. Endocrinol. Metab. PubMed Scopus Google Scholar), to the that reduced thyroid hormone TSH is Smith T.J. Y. L. S. J. Clin. PubMed Scopus Google that thyroid a effect on in human skin fibroblasts in vitro and in in S. A. PubMed Scopus (40) Google and P. K. P. Endocrinol. PubMed Google Scholar). role for thyroid hormone in HA has been J. Clin. Endocrinol. Metab. PubMed Scopus Google Scholar), are A. Biochem. PubMed Scopus Google Scholar, S. R. Biochem. 1999; PubMed Scopus Google Scholar). thyroid to gene expression, the a we an from the and an from the CRE in the HAS1 and the HAS2 three the of thyroid hormone of HAS expression the skin in and in addition to Furthermore, of has the of TSHR transcripts and in adipose tissues and pretibial skin M. Lane C. J. Ludgate M. Google Scholar, C. Ludgate M. S. J. Endocrinol. 2002; PubMed Scopus Google Scholar). we confirmed our microarray data and the using preadipocyte fibroblasts from orbital and Preadipocytes from all to constitutively TSHR of HAS1 and HAS2 to increased HA in the culture with nonmodified We are that the of constitutively TSHR the preadipocytes has the that all cells the receptor and increased cAMP it can the of the TSHR on such for example, adipogenesis or production of extracellular matrix, it not the in in which preadipocyte fibroblasts the In M. Lane C. J. Ludgate M. Google we that of preadipocytes and the TSHR using All of our were using and we confirmed the of TSHR transcripts in all of using not The in HA by TSHR ligands was more that obtained in the activating cells the in the of cells the TSHR in the two a In the transforming growth factor differentiation of fibroblasts to we a of increases in extracellular and results from increased HA (6Meran S. Thomas D. Stephens P. Martin J. Bowen T. Phillips A. Steadman R. J. Biol. Chem. 2007; 282: 25687-25697Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, J. S. Phillips A. Steadman R. J. Full Text Full Text PDF PubMed Scopus Google or HA Thomas G.J. Steadman R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). HA is but not on its for differentiation to It is not the HA be a of phenotypic by the and be the of The level of HA we obtained with TSH is to that by of for dermal in the human TSH to a response R. A. L. G. A. Ludgate M. J. Clin. Endocrinol. Metab. PubMed Scopus Google Scholar). Smith and Hoa (11Smith T.J. Hoa N. J. Clin. Endocrinol. Metab. 2004; 89: 5076-5080Crossref PubMed Scopus (192) Google orbital fibroblasts from a TSH not HA from the in HA production by human in response to TSH Hoa N. P. Smith T.J. 2007; PubMed Scopus Google Scholar), findings are with of several increased of HA in or thyroid cells in response to the hormone J. G. Biol. 1989; 13: PubMed Scopus Google Scholar, Y. M. Y. N. J. 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Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The of was that the the appears to a it produced an in light with the in all cell types. In contrast, it was in the A293 cells that we were to a in light in response to db-cAMP and a in light and was to the containing the The HAS1 gene a HAS1 as a potential target for by of several target genes as and found that all to cAMP with a in activity E. L. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The results we obtained are in with and that HAS1 is regulated by the protein and the results of our assays that it has in The that the HAS2 CRE is functional. In in addition to the well effect of thyroid our results that TSHR activation plays a role in production of the extracellular matrix component HA by preadipocyte fibroblasts from fat we to the receptor activation occurring in GD patients in response to TSAB or in patients with elevated The is to be of in The an for the production of HA occurring in patients with thyroid dysfunction that to widespread or localized HA of and hyperthyroid respectively. We the of monoclonal antibodies and from and of from The of and Phillips is