Role of GLIS3 in thyroid development and in the regulation of gene expression in thyroid specific Glis3KO mice

Loss of GLI-Similar 3 (GLIS3) function in mice and humans causes congenital hypothyroidism (CH). In this study, we demonstrate that GLIS3 protein is first detectable at E15.5 of murine thyroid development, a time when GLIS3 target genes, such as Slc5a5 (Nis), become also expressed. We further show that Glis3KO mice do not display any major changes in prenatal thyroid gland morphology indicating that CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. Analysis of thyroid-specific Glis3 knockout (Glis3-Pax8Cre) mice fed either a normal or low-iodine diet (ND or LID) revealed that, in contrast to ubiquitous Glis3KO mice, thyroid follicular cell proliferation and the expression of cell cycle genes were not repressed suggesting that the inhibition of thyroid follicular cell proliferation in ubiquitous Glis3KO mice is related to loss of GLIS3 function in other cell types. However, the expression of several thyroid hormone biosynthesis-, extracellular matrix (ECM)-, and inflammation-related genes was still suppressed in Glis3-Pax8Cre mice particularly under conditions of high blood levels of thyroid stimulating hormone (TSH). We further demonstrate that treatment with TSH, protein kinase A (PKA) or adenylyl cyclase activators or expression of constitutively active PKA enhances GLIS3 protein and activity, suggesting that GLIS3 transcriptional activity is regulated in part by TSH/TSHR-mediated activation of the PKA pathway. This mechanism of regulation provides an explanation for the dramatic increase in GLIS3 protein expression and the subsequent induction of GLIS3 target genes, including several thyroid hormone biosynthetic genes, in thyroid follicular cells of mice fed a LID.

Krüppel-like zinc nger protein, GLI-Similar 3 (GLIS3), was recently identi ed as a critical regulator of thyroid follicular cell functions [14,15]. Loss-of-function mutations in human GLIS3 cause a syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) [16][17][18][19][20][21][22][23][24], while single nucleotide polymorphisms in GLIS3 are associated with thyroid dysfunction and increased risk of CH [25][26][27][28][29][30][31][32]. Ubiquitous Glis3 knockout (Glis3KO) mice exhibit a very similar phenotype as human NDH patients, including the development of neonatal diabetes and CH [33,34]. TH biosynthesis in thyroid follicular cells is greatly suppressed in postnatal Glis3KO mice [15,33]. The development of CH in Glis3KO is at least in part related to dyshormonogenesis due to the transcriptional repression of several TH biosynthesis-related genes [33]. However, whether GLIS3 also plays a role in the regulation of murine thyroid gland development has not yet been clearly established. In this study, we provide evidence indicating that GLIS3 does not play a major role in mouse embryonic thyroid morphogenesis suggesting that the development of CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis.
In this study, we further demonstrate that in contrast to ubiquitous Glis3KO mice, proliferation of thyroid follicular cells was not inhibited in thyroid-selective Glis3 knockout mice fed a LID (Glis3-Pax8Cre(LID)), whereas the expression of TH biosynthesis-and extracellular matrix (ECM)-related genes was still repressed. These observations indicate that the inhibition of proliferation and cell cycle-related genes in ubiquitous Glis3KO thyroid is related to changes in gene expression in other GLIS3 target tissues that indirectly affect thyroid follicular cell proliferation. Studies showing that GLIS3 is particularly critical for the transcriptional regulation of several genes induced by TSH [14,15,35], raised the question whether GLIS3 activity itself is controlled by TSH signaling. This hypothesis is supported by data showing that GLIS3 protein is greatly induced in thyroid follicular cells of mice fed a LID and that the level of GLIS3 protein and its transcriptional activity is signi cantly enhanced in PCCl3 cells by TSH, cAMP or the expression of constitutively active PKA. These ndings are consistent with the hypothesis that GLIS3 protein level and transcriptional activity are controlled by the TSH-TSHR signaling pathway, at least in part through the activation of PKA. This mechanism would explain the dramatic induction of GLIS3 target genes, including several thyroid hormone biosynthetic genes, in thyroid follicular cells when TSH levels are highly elevated, such as in LID conditions. Targeted expression or regulation of GLIS3 in thyroid follicular cells may provide new therapeutic strategies to manage certain subtypes of CH.

Measurement of serum and tissue TSH and TH levels
Serum T3, T4, and TSH levels were measured by radioimmunoassay as described in detail previously [33,40].

QRT-PCR analysis
The expression of mRNA from thyroid gland was examined by QRT-PCR analysis using TaqMan and SYBR system. RNA from thyroid glands was extracted using RNAqueous-Micro total RNA isolation kit and reverse-transcribed using the high-capacity cDNA reverse transcription kit (both from ThermoFisher). QRT-PCR reaction in triplicate for each sample was carried out using StepOnePlus Real-time PCR system (Applied Biosystem). Primer sequences are listed in Supplementary Table 1.

Western blot analysis
PCCl3-pIND20-Glis3 cells, expressing a doxycycline (Dox)-inducible GLIS3 tagged with Flag and HA at the N-and C-terminus, respectively, were generated by infection of pIND20-Flag-Glis3-HA lentivirus [45]. To evaluate the expression of GLIS3, PCCl3-pIND20-Glis3 cells were treated with 100 ng/ml Dox for 72-96 h in the absence of TSH and subsequently with TSH for 24 h or as indicated. Nuclear extracts were prepared as described [46] and GLIS3-HA examined by Western blot analysis with an HA antibody (#3724, Cell Signaling). β-actin (MA5-15739, ThermoFisher) was used as internal control.
Statistical analysis P values were calculated by one-way ANOVA.

Expression of GLIS3 during thyroid folliculogenesis
Previously, we reported that GLIS3 protein is expressed in postnatal murine thyroid follicular cells and that it functions as a critical transcriptional regulator for several genes required for thyroid hormone biosynthesis [33]; however, whether GLIS3 plays a role in the regulation of mouse thyroid organogenesis, has not been clearly established. To study this, we monitored the expression of GLIS3 protein in Glis3-EGFP mice at different stages of embryonic development in comparison to PAX8, which is critical for early thyroid organogenesis, and NIS, a marker of differentiated thyroid follicular cells [4]. In contrast to the PAX8 protein, GLIS3 protein was undetectable in the thyroid gland at embryonic day 13.5 (E13.5) ( Supplementary Fig. 1). Weak expression of GLIS3 protein was rst observed at E15.5 in the nucleus of PAX8 + cells (Fig. 1A) and the intensity of GLIS3 immunostaining was signi cantly increased at E16.5.
Next, we examined whether loss of GLIS3 function had any major effect on thyroid gland morphology and folliculogenesis in Glis3KO1, in which the coding sequence of the 5th zinc nger was deleted [36], and Glis3KO2, in which the mCherry coding sequence with stop codon was inserted into exon 3 [33,37]. H&E histochemical staining of sections of E17.5 WT and Glis3KO1 thyroid glands revealed no signi cant differences in thyroid gland morphology ( Fig. 2A) and showed a comparable pattern of PAX8 and NKX2.  Fig. 2F). Together, these observations indicate that GLIS3 does not play a major role in the regulation of thyroid organogenesis and thyroid folliculogenesis during mouse embryonic development. This is consistent with the hypothesis that in GLIS3-de cient mice the development of congenital hypothyroidism is due to dyshormonogenesis rather than thyroid dysgenesis [33].
Analysis of Glis3 -Pax8Cre thyroid gland phenotype In addition to hypothyroidism, ubiquitous Glis3 knockout mice exhibit other abnormalities, including severe hyperglycemia and hypoinsulinemia, due to defects in pancreatic β cell development and insulin production [15,38,39]. Insulin-like growth factors (IGFs) and insulin have been reported to play a critical role in the regulation of thyroid follicular cell proliferation and thyroid gene expression, including Slc5a5 [51][52][53][54][55][56][57]. To investigate whether changes related to other tissues, such as hypoinsulinemia, contributed to the Glis3KO thyroid phenotype, we analyzed the thyroid gland phenotype in thyroid-selective Glis3 knockout mice, Glis3-Pax8Cre (referred as conditional knockout or CKO in the Figures), in which Pax8Cre e ciently (> 90%) deleted exon 5 in Glis3 in the thyroid gland, but not in the pancreas ( Fig. 3A and Supplementary Fig. 3A). We demonstrated that in contrast to ubiquitous Glis3KO mice, pancreatic insulin expression and non-fasting blood glucose levels were not changed in Glis3-Pax8Cre mice ( Supplementary  Fig. 3B, C) con rming that these mice did not develop hypoinsulinemia/hyperglycemia [58]. However, serum T4 levels were still signi cantly decreased (44%) in Glis3-Pax8Cre(ND) mice, while levels of serum TSH and T3 were slightly elevated (Fig. 3B). Serum T4 and T3 levels were greatly decreased in both WT(LID) and Glis3-Pax8Cre(LID) mice fed a LID, whereas TSH was greatly increased but to a signi cantly greater extent in Glis3-Pax8Cre(LID) mice (Fig. 3B).
It is well established that elevated TSH levels, as under LID conditions, cause a dramatic increase in thyroid follicular cell proliferation [54,57,59]. We previously reported [33] that in contrast to WT(LID) mice, thyroid follicular cell proliferation and mTOR activation was not increased in ubiquitous Glis3KO2(LID) mice and that thyroid gland hypertrophy was not observed. In contrast, Glis3-Pax8Cre(LID) mice did develop thyroid gland hypertrophy and the percentage of EdU + PAX8 + cells was similar to that in WT(LID) (Fig. 3C, D) indicating that thyroid follicular cell proliferation was not repressed in Glis3-Pax8Cre(LID) mice. This is consistent with data showing that mTOR activation (pRSP6 staining), a major signaling pathway driving TSH-induced proliferation of thyroid follicular cells, was increased to a similar extent in both Glis3-Pax8Cre(LID) and WT(LID) thyroid glands (Fig. 3E). This increase in thyroid follicular cell proliferation correlated with the development of thyroid gland hypertrophy in both male and female Glis3-Pax8Cre(LID) mice (Fig. 3F). In fact, thyroid hypertrophy was more pronounced in Glis3-Pax8Cre(LID) than in WT(LID) mice as indicated by the larger increase in thyroid weight (Fig. 3F). No signi cant difference in total body weights was observed between Glis3-Pax8Cre and WT mice (Fig. 3F). Together, these results suggested that GLIS3 does not play a direct role in the regulation of TSHstimulated thyroid follicular cell proliferation and that the repression of cell proliferation in ubiquitous Glis3KO(LID) mice appears to be related to changes in other cell types that indirectly affect thyroid follicular cell proliferation.
Analysis of the Glis3 -Pax8Cre thyroid gland transcriptome To obtain insights into the differences in gene expression between thyroid glands from WT and Glis3-Pax8Cre mice, we performed RNA-Seq analysis (Supplementary Table 2). KEGG analysis of genes downregulated in Glis3-Pax8Cre(LID) thyroids compared to those of WT(LID) (fold change > 2; FDR < 0.01), identi ed ECM-receptor interaction, focal adhesion, and thyroid hormone synthesis among the top pathways, while analysis of up-regulated genes identi ed transcriptional-misregulation-in-cancer, calcium and AMPK signaling among the top pathways ( Fig. 4A and Supplementary Table 3). The expression of several thyroid hormone biosynthetic and TSH-induced genes, including Slc5a5, Slc26a4, Adm2, Sod3, Cdh13, Duox2, and Duoxa2 that were greatly induced in WT(LID) compared to WT(ND), were signi cantly repressed in thyroids from Glis3-Pax8Cre(LID) mice (Table 1 and Fig. 4B), consistent with our previous study of ubiquitous Glis3KO mice [33]. NIS protein expression was dramatically increased in WT(LID), but not in Glis3-Pax8Cre(LID) thyroids (Fig. 4C). Similarly, the increase in the expression of several ECMrelated and in ammatory genes, including Col18a1, Col6a2, Col4a1, Ccl7, Ccl2, and Itga2, observed in WT(LID) thyroids was signi cantly suppressed in Glis3-Pax8Cre(LID) thyroid glands (Table 1 and Fig. 5A). The increase in collagen mRNA expression in WT(LID) and its suppression in Glis3-Pax8Cre(LID) thyroids correlated with the level of immuno uorescent staining for pan-collagen (Fig. 5B).
Most importantly and in contrast to ubiquitous Glis3KO(LID) mice [33], the expression of cell cycle regulatory genes, including Ccnb1, Ccnb2, and Cdca2, was not suppressed in Glis3-Pax8Cre(LID) thyroid glands (Table 1; Fig. 5C) consistent with our data showing little difference in the percentage of PAX8 + EdU + cells (Fig. 3D). These data supported the concept that the inhibition of thyroid follicular cell proliferation in ubiquitous Glis3KO mice appears to be related to changes in gene expression in other cell types that subsequently affect the proliferation of these cells. Together, these observations indicate that in contrast to TH biosynthetic genes, which transcription is directly regulated by GLIS3, GLIS3 does not play a major role in the direct transcriptional regulation of cell proliferation-regulatory genes in thyroid follicular cells in mice fed a LID. These ndings are consistent with the concept that TSH regulates proliferation and TH-biosynthesis in thyroid follicular cells through activation of different signaling pathways [7].
Analysis of thyroid TF expression showed that Pax8 expression, but not that of Nkx2.1 and Foxe1, was increased in both Glis3-Pax8Cre(ND) and Glis3-Pax8Cre(LID) mice compared to that of WT(ND) and WT(LID) (Supplementary Fig. S4). However, the expression of Pax8, Nkx2.1, and Foxe1 was not signi cantly different between thyroids from Glis3-Pax8Cre(ND) and Glis3-Pax8Cre(LID) mice indicating that the suppression of gene expression in Glis3-Pax8Cre(LID) thyroid is independent of the changes in the expression of these thyroid TFs.
The transcriptome and QRT-PCR analyses were carried out with thyroid glands from female mice, analysis of the expression of several genes, including cell cycle, ECM, in ammation, and TF genes in thyroids from male mice showed a very similar pattern as that of female mice ( Supplementary Fig. 5).

Correlation between GLIS3 protein and TSH levels
Our studies demonstrated that GLIS3 plays a critical role particularly in the transcriptional regulation of several thyroid hormone biosynthetic and TSH-inducible genes under conditions when TSH levels are elevated, such as in LID (Table 1 and Fig. 4B) [33,60]. This led to the hypothesis that GLIS3 protein levels and/or activity itself might be controlled by a TSH/TSHR-dependent signaling pathway. This concept was supported by observations showing a correlation between the decrease in GLIS3 and TSH levels during the rst 2 postnatal months. Both the intensity of GLIS3 protein staining in follicular cells and the percentage of PAX8 + GLIS3 + cells in Glis3-EGFP mice steadily decreased during this period (Fig. 6A, B) and was accompanied with a similar decline in postnatal blood TSH levels (Fig. 6C). The correlation between GLIS3 protein and TSH levels was strengthened by observations showing that GLIS3 staining and the percentage of PAX8 + GLIS3 + cells were greatly increased in Glis3-EGFP mice fed a LID, a condition in which blood TSH level is greatly elevated (Fig. 6D, E). These changes in GLIS3 protein did not strongly correlate with alterations in Glis3 mRNA expression ( Supplementary Fig. 6) suggesting that the higher levels of GLIS3 protein expression under conditions of elevated TSH, might be due to an increase in protein stability or rate of translation rather than increased transcription. The increase in GLIS3 protein might be part of the mechanism by which GLIS3 induces the transcriptional activation of target genes, such as Slc5a5, in thyroid follicular cells of LID mice.

Link between PKA and GLIS3 transcriptional activity
To obtain further support for the hypothesis that GLIS3 activity is regulated by TSH signaling, we examined the effect of TSH on GLIS3-mediated transcriptional activation of a GLISBS-dependent luciferase reporter in rat thyrocyte PCCl3 cells. Addition of TSH increased GLIS3-mediated transcriptional activation of the reporter 2-to 3-fold (Fig. 7A, B) without causing a change in the level of Glis3 mRNA expression (Fig. 7C). In the absence of GLIS3, TSH did not increase GLISBS-dependent activation (data not shown). It is well-established that interaction of TSH with TSHR induces the activation of several protein kinases, including PKA, phospholipase C (PLC), PI3K, mTOR, ERK, and Ca ++ -mediated signaling [7,52,61,62]. To examine whether any of these downstream kinase pathways are involved in the regulation of GLIS3 activity, we analyzed the effect of several kinase inhibitors on GLIS3-mediated transcriptional activation. Addition of the PKA inhibitor H89 signi cantly suppressed the increase in GLIS3-mediated transactivation by TSH (Fig. 7A), whereas inhibition of the mTOR, Ca ++ or ERK pathways by, respectively, rapamycin, FK506 or trametinib, had little effect, while transactivation was slightly reduced by the PKC inhibitor Gö6976 (Fig. 7A). A role for PKA activation in the regulation of GLIS3-mediated transcriptional activation by TSH was further supported by data showing that co-expression of a constitutively active form of PKA and treatment with 8BrcAMP or the adenylyl cyclase agonist, forskolin, enhanced GLIS3dependent transcriptional activation and that this increase was signi cantly inhibited by H89 (Fig. 7B). Together, these results are consistent with the concept that activation of PKA is part of the mechanism by which by TSH enhances GLIS3 transcriptional activation of target genes.
Since TSH treatment did not signi cantly change Glis3 mRNA levels (Fig. 7C), one possible mechanism by which TSH enhances GLIS3 transcriptional activity is increasing GLIS3 protein stability. To study this, we examined the effect of TSH on GLIS3 protein in PCCl3 cells expressing doxycycline (Dox)-inducible GLIS3-HA (PCCl3-pIND20-Glis3) and examined GLIS3 protein expression by immuno uorescence staining and Western blot analysis. These analyses showed that addition of TSH signi cantly increased GLIS3 protein expression in Dox-treated PCCl3-pIND20-Glis3 cells without changing Glis3 mRNA expression (Fig. 7C-E). We further showed that treatment with the proteasome inhibitor MG132 enhanced GLIS3 protein stability (Fig. 7F). Together, these data are consistent with the hypothesis that the stimulation of GLIS3 transcriptional activity by TSH is at least in part mediated via a PKA-dependent increase in GLIS3 protein stability (Fig. 8).

Discussion
Loss of GLIS3 function in both humans and mice causes CH [15,16,18,33]. However, whether this is related to thyroid dysgenesis or dyshormonogenesis has not been clearly established. Analysis of GLIS3 protein expression during mouse thyroid development demonstrated that GLIS3 protein was rst detectable in thyroid follicular cells at E15.5, a time during which thyroid follicles are being formed ( Fig. 1 and Supplementary Fig. 1) [4,8]. We further show that thyroid gland morphology and the formation of thyroid follicles are not greatly altered in E17.5 and neonatal Glis3KO mice ( Fig. 2A, Supplementary  Fig. 2). These data indicate that GLIS3 is not required for early thyroid development in mice and that the development of CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. This contrasts the role of glis3 in zebra sh, in which glis3 has been shown to play an important role in thyroid development [63]. Moreover, unlike glis3 knockdown in zebra sh, the expression of NKX2.1 and PAX8 was not impaired in thyroid gland in Glis3KO mice ( Fig. 2B and C). Whether the development of CH in human patients with GLIS3-de ciency is related to thyroid dysgenesis or dyshormonogenesis has been inconclusive and shown to vary among patients [16, 18, 20-22, 24, 25, 29, 64, 65]. This variability might be attributed to the highly oligogenic nature of CH [27, 29,32]. Our study further shows that the onset of GLIS3 expression is distinct from that of the thyroid TFs, PAX8, NKX2.1, FOXE1, and HHEX, which are critical for early thyroid gland development and which loss of function causes thyroid dysgenesis or athyreosis [3,4,6,9,10,12,66]. Several of these TFs also play a role postnatally in the regulation of several thyroid functions, including TH biosynthesis. We recently reported that PAX8 and NKX2.1 are bound to regulatory regions of the Glis3 gene, which would be consistent with the concept that these factors have a role in the transcriptional activation of Glis3 during thyroid development [33,60]. Interestingly, the time interval at which Glis3 expression is induced parallels that of a major GLIS3 target gene, Slc5a5, which expression is induced during E15.5 and 16.5 and greatly repressed in E17.5 Glis3KO thyroids ( Fig. 1B and 2F). Impairment in iodide transport in NIS knockout mice was reported to cause severe hypothyroidism [67]. Together, these ndings are consistent with our conclusion that CH in Glis3KO mice is due to dyshormonogenesis.
We previously demonstrated that under LID conditions not only the activation of thyroid hormone biosynthetic genes was suppressed in ubiquitous Glis3-de cient mice, but also the induction of thyroid follicular cell proliferation, the expression of cell cycle genes, and the activation of the mTOR pathway [15,33]. We also reported that, in addition to congenital hypothyroidism, these mice develop neonatal diabetes and hypoinsulinemia [39,58]. IGF-1 and insulin, together with TSH, have been reported to play a critical role in the regulation of gene expression and proliferation in thyroid follicular cells [51][52][53][54][55][56] raising the possibility that the pancreatic phenotype, including hypoinsulinemia, and conceivably changes in other tissues in ubiquitous Glis3-de cient mice might in uence the function and gene expression in thyroid follicular cells. This hypothesis was supported by observations showing that in contrast to ubiquitous Glis3-KO(LID) mice, thyroid follicular cell proliferation, expression of cell cycle genes, including Ccnb1, Ccnb2, and Cdca2, and activation of the mTOR, a pathway that promotes cell proliferation, were not repressed in Glis3-Pax8Cre(LID) mice but induced to a similar degree as in WT(LID) mice (Fig. 3C -E and Fig. 5C). We further observed that proliferation of thyroid follicular cells (EdU + cells) and the expression of the cell cycle genes were slightly higher in Glis3-Pax8Cre(ND) mice than in WT(ND) mice. This might be due to the 3-fold higher level of TSH in Glis3-Pax8Cre(ND) mice compared to WT(ND) mice (Fig. 3B). These data indicate that GLIS3 does not play a major role in the regulation of thyroid follicular cell proliferation and suggest the suppression of thyroid follicular cell proliferation in ubiquitous Glis3de cient mice does not involve direct transcriptional regulation of cell cycle-related genes by GLIS3, but is related to abnormalities in other tissues, such as hypoinsulinemia that indirectly affect thyroid follicular cell proliferation. This conclusion is consistent with our cistrome analysis showing that GLIS3 binds to very few cell cycle-related genes [60]. In contrast to cell proliferation-regulatory genes, the expression of several TH-inducible genes, including Slc5a5, Slc26a4, Adm2, Sod3, and Cdh13, remain dramatically suppressed in Glis3-Pax8Cre(LID) thyroid as we observed in ubiquitous Glis3-de cient mice, consistent with the conclusion that their transcription is directly regulated by GLIS3 [60]. In addition, these ndings support the concept that TSH regulates proliferation and TH-biosynthesis in thyroid follicular cells through different protein kinase signaling pathways [7].
The largest repression of GLIS3 target genes was observed in thyroids from Glis3KO and Glis3-Pax8Cre mice under LID conditions when TSH levels are highly elevated (Fig. 3B) [33]. The induction of the transcriptional activation of GLIS3 target genes in thyroid follicular cells in mice fed a LID coincides with the signi cant increase in GLIS3 protein expression (Fig. 6). These observations indicated a possible link between the TSH/TSHR signaling pathway, the regulation of GLIS3 protein, and the activation of GLIS3 target genes. This was supported by data showing that TSH enhanced GLIS3-mediated transcriptional activation and GLIS3 protein expression in PCCl3 cells (Fig. 7A, D). This induction was not due to a change in the level of Glis3 mRNA expression (Fig. 7C) suggesting that it might be due to increased GLIS3 protein stability or rate of translation. We provided evidence indicating that the stimulation of GLIS3 transcriptional activity is at least in part due to increased GLIS3 protein stability (Fig. 7E, F). GLIS3 protein stability and GLIS3-dependent transcriptional activation of target genes might be controlled by posttranslational modi cation(s) of GLIS3 that are mediated by TSH/TSHR-induced activation of (a) downstream kinase pathway(s) [11,52,59,68]. Study of the effect of several kinase inhibitors on TSHinduced stimulation of GLIS3 transcriptional activity revealed that the PKA antagonist H89 suppressed this increase, but that inhibition of several other kinase pathways (ERK, PKC, mTOR, Ca2 + ) had relatively little effect. A role for PKA in mediating the effect of TSH was supported by data showing that the PKA agonist 8BrcAMP and the adenylyl cyclase agonist, forskolin, similarly enhanced GLIS3-mediated transcriptional activation and that this stimulation was inhibited by H89. A role for PKA was further strengthened by observations demonstrating that expression of a constitutively active PKA stimulated GLIS3-mediated transcriptional activation (Fig. 7B). Further studies are needed to identify the amino acid(s) within GLIS3 that are phosphorylated by PKA and are critical for regulating GLIS3 protein stability and activity. Together, our study reveals a link between TSH signaling and its regulation of GLIS3 protein activity and transcriptional activation GLIS3 target genes, including several TH biosynthesis-related genes. We further provide evidence for a role of the PKA signaling pathway in mediating the transcriptional regulation of several TSH-induced genes by GLIS3 in thyroid follicular cells (Fig. 8).

Figure 5
The expression of ECM-and in ammation-related genes, but not those of cell cycle-related genes, was suppressed in thyroid glands from Glis3-Pax8Cre(LID) mice. A QRT-PCR analysis of the expression of TSH and PKA agonists enhanced GLIS3-mediated transcriptional activation. A GLISBS-dependent transcriptional activation of the Luc reporter by GLIS3 was examined in PCCl3 cells as described in Materials and Methods. Addition of TSH stimulated GLIS3-mediated transcriptional activation. This increase was inhibited by the PKA inhibitor H89, whereas treatment with the mTOR inhibitor rapamycin (Rapa), the calcium signaling inhibitor FK506, the PKC inhibitor Gö6976, or the ERK inhibitor Trametinib Schematic showing a model of the mechanistic link between TSH signaling and its regulation of GLIS3 transcriptional activity. Binding of TSH to TSHR leads to activation of several protein kinase pathways and increased GLIS3 protein stability and transcriptional activity. TSH, the PKA activator 8BrcAMP, the adenylyl cyclase activator forskolin, and expression of constitutively active PKA enhance GLIS3 transcriptional activity at least in part by stabilizing GLIS3 protein. The PKA antagonist H89 suppressed the TSH-mediated increase, whereas inhibition of mTOR, PKC, Ca ++ or ERK pathways by, respectively, rapamycin, Gö6976, FK506 or trametinib, had little effect. The stimulation in GLIS3 transcriptional activity by the TSH-TSHR-PKA pathway appears to be at least in part due to increased GLIS3 protein stability. This mechanism of regulation provides an explanation for the dramatic increase in GLIS3 protein expression and the subsequent induction of GLIS3 target genes, including several thyroid hormone biosynthetic genes, in thyroid follicular cells of mice fed a LID.

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