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3-AD Prohormone and its Uses

3-AD Prohormone3-AD- prohormone, 1 μM T3, 10 nM T4) with a range of 100 to 1,000 nM, based on the TSH receptor number, were tested. We have observed that the dose response of TSH treatment on cAMP level in cells expressing a lower number of receptors is lower.

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What is 3-AD Prohormone?

This suggests that the level of the receptor number and/or the intracellular cAMP level is directly related, but not necessarily linearly proportional. Since the lower receptor number did not affect significantly the expression level of L-T4 or its conversion to the active thyroid hormones, the lower response to TSH treatment could be due to lower functional TSHR numbers in these cells. Moreover, a high TSH dose (1 µM) that should saturate the TSHR at higher TSH doses led to low cAMP level in these cells ([Supplementary Fig. 3A,B](#S1){ref-type=”supplementary-material”}). Nevertheless, the expression level of TSHR mRNA remained unchanged in any of these treatments ([Supplementary Fig. 3C](#S1){ref-type=”supplementary-material”}).

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Furthermore, we performed western blotting analyses using rabbit polyclonal antibodies against human TSHR extracellular domain, intracellular domain and TSH-induced signaling[@b44]. These results demonstrate that these antibodies recognize specifically TSHR protein, but do not detect changes in TSHR protein level after these treatments ([Supplementary Fig. 3D](#S1){ref-type=”supplementary-material”}). The results also showed that none of these treatments affected the level of TSHR protein expression ([Supplementary Fig. 3E](#S1){ref-type=”supplementary-material”}). cAMP signaling pathway activation by L-T4 or TSH treatment ———————

Pro Hormones – What You Need To Know

Previously, we have reported that the thyroid hormones L-T4 and T3 are potent stimulators of adenylate cyclase (AC) in human TSHR-CHO cells. Moreover, thyroid hormone-dependent AC stimulation was associated with a significant increase in cAMP accumulation[@b31]. Therefore, we examined whether L-T4 or T3 induce a significant activation of AC in these cells. To assess the cAMP synthesis in these cells, we used a FRET-based cAMP biosensor, that was developed by the laboratory of Dr. Thomas E. Mayer[@b32]. This biosensor was based on the Gαs-βγ heterotrimer that was reconstituted with a membrane-targeted circularly permuted yellow fluorescent protein (cpYFP) and a cyan fluorescent protein (CFP). A single-chain fragment of the α subunit of the heterotrimeric G protein, fused with a modified C-terminal of the Yellow Fluorescent Protein (sensor YC-GECO1.1), was used as the bioluminescent reporter of cAMP level.

What is TSH Treatment?

As shown in [Fig. 1A](#f1){ref-type=”fig”}, L-T4 or TSH treatment induced a significant increase in cAMP biosensor activity, respectively. Activation of AC was also associated with an increase in cAMP level in TSHR-CHO cells ([Fig. 1B](#f1){ref-type=”fig”}). Our results revealed that L-T4 or T3 activated AC in a concentration-dependent manner and that this activation was associated with a significant increase in cAMP level in TSHR-CHO cells. L-T4-dependent AC activation was similar in WT and mutant receptors ([Supplementary Fig. 4A,B](#S1){ref-type=”supplementary-material”}). We observed a decrease in cAMP level in cells treated with L-T4 or T3 after long-term exposure (16–18 h) to these hormones, demonstrating the transient effect of the hormones on the cAMP signaling pathway ([Supplementary Fig. 4C](#S1){ref-type=”supplementary-material”}).

Regulate Thyroid Hormone-Sensitive Genes

In addition, L-T4 or T3 are known to regulate thyroid hormone-sensitive genes in human and rodent cells[@b45]. To elucidate whether AC stimulation was related to TSHR-dependent gene regulation, we examined the impact of TSH, L-T4 and T3 on mRNA expression of the TSHR-target genes, *TG, TPO*, and *D2*. In the absence of TSH or L-T4/T3, *TG* and *TPO* mRNA expression levels were very low (data not shown).

However, stimulation with TSH, L-T4 and T3 induced a significant increase in *TG* and *TPO* mRNA expression in WT and mutant TSHR-CHO cells ([Fig. 1C,D](#f1){ref-type=”fig”}). Stimulation of L-T4 or T3 did not significantly increase the expression level of TSHR ([Supplementary Fig. 4D](#S1){ref-type=”supplementary-material”}). However, we have observed that a long-term treatment (16–18 h) of cells with the TSHR activators L-T4 or T3 down-regulates expression level of *TSHR* mRNA ([Supplementary Fig. 4E](#S1){ref-type=”supplementary-material”}). The results show that cAMP-dependent AC stimulation is linked to the regulation of genes in the TSH-induced signalling pathway. Moreover, our results show that the TSHR-AC-cAMP-TSHR-TRAF2-JNK signalling pathway is involved in regulating the expression of the *TG* and *TPO* genes. Expression of cAMP-dependent transcription factors ————————————————– To gain more insight into the cAMP-dependent transcriptional regulation of *TG* and *TPO*, we examined the level of cAMP-dependent transcription factors after thyroid hormone treatment.

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To assess the potential effect of thyroid hormones on the activity of transcription factors involved in thyroid hormone response, WT TSHR-CHO cells were transiently transfected with a luciferase reporter plasmid containing the *TG* promoter (p-*TG*-luc) and treated with TSH, L-T4 or T3 for different time periods. We observed that thyroid hormones increased the activity of this reporter in a dose- and time-dependent manner, which is similar to that observed for the endogenous *TG* and *TPO*