RT-PCR Analysis of TSH-dependent transcripts in primary cultures of human thyrocytes
TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate more ..
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TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate (cAMP). The TSH receptor (THSR) is mainly expressed in thyroid follicular cells and regulates their growth and function. This cell based assay was conducted on a TSHR transfected HEK 293 cell line, measuring cAMP stimulation using antibody-based Homogeneous Time Resolved Fluorescence (HTRF).
Our goal is to identify small molecules that will inhibit (antagonize) the thyroid-stimulating hormone receptor (TSHR), a member of the 7 transmembrane-spanning receptor (7TMR) family of cell surface receptors. TSHR-mediated hyperthyroidism is important in thyroid pathology. The development of small molecule antagonists of TSHR may lead to therapeutic approaches for TSHR-mediated hyperthyroidism caused by constitutively activating mutations or stimulating auto-antibodies associated with Graves' disease. Graves' disease is the leading cause of hyperthyroidism and is caused by autoantibodies that stimulate TSHR (TsAbs) causing thyroid growth and unregulated overproduction of thyroid hormones and existing treatments have liabilities (agranulocytosis). In addition, TSHRs are known to be expressed in multiple extrathyroidal tissues including bone, brain, kidney, testis, fat and cells of the immune system but the role of the TSHR in these tissues is not clear. Therefore, TSHR antagonists/inverse agonists could be used as probes of extrathyroidal TSHR function. Finally, TSHR inverse agonists, which are a subclass of antagonists that inhibit agonist-independent (or basal or constitutive) TSHR signaling, could be used to inhibit TSH-independent signaling in patients with recurrent or metastatic thyroid cancer who are receiving thyroid hormone to suppress TSH.
This assays looks for effects on TSH-dependent functions in primary human thyrocytes. Total RNA is purified using RNeasy Micro kits. RT-PCR is performed in 25-ul reactions using cDNA prepared from 100 ng or less of total RNA and Universal PCR Master Mix (Applied Biosystems). mRNA levels are measured using primers and probes from Applied Biosystems. Quantitative RT-PCR results are normalized to GAPDH to correct for differences in RNA input.
Thyroid tissue samples were collected from normal thyroid tissue from patients undergoing total thyroidectomy for thyroid cancer at the National Institutes of Health Clinical Center. Surgical specimens were maintained in Hanks' balanced salt solution (HBSS) on ice, minced into small pieces, and digested with 3 mg/ml collagenase type IV (Life Technologies). Monodispersed cells were plated in 10 ml DMEM with 10% FBS in 10-cm tissue culture dishes and incubated at 37C in a humidified5%CO2 incubator, and after 24 h, the primary cultures of adherent thyroid cells were obtained. For determination of thyroglobulin (TG), thyroid peroxidase (TPO), TSHR, sodium iodide symporter (NIS), or deiodinase type 2 (DIO2) mRNAexpression, thyrocytes were seeded into 12-well plates at a density of 1 x 105 cells per well. The cells were incubated in DMEM with 2% FBS and 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 48 h in the presence or absence of compound.
Active compounds which modulated TSHR pathway transcripts were given a score of 100, inactive compounds were given a score of 0.
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Data Table (Concise)