Background: Defects in thyroid hormone synthesis at birth lead to congenital hypothyroidism (CH). Recently, pathogenic variants in the SLC26A7 gene have been linked to dyshormonogenetic goitrous CH. This anion transporter is highly expressed in the thyroid and is involved in thyroid hormone synthesis; however, its exact function and cellular localization remain unclear. In this study, we investigated SLC26A7 variants in Finnish patients with CH, characterized the phenotypes, and analyzed thyroid-specific gene expression.Methods: SLC26A7 variants were identified from a clinical CH cohort (n = 139) using exome sequencing, and the FinnGen database (R12 release) was screened for disease associations. Thyroid histology and thyroid-specific gene expression were analyzed in six human samples (including two homozygous SLC26A7 pathogenic variant carriers, patients with goitrous and hyperactive thyroids, and normal controls) and in thyroids from different mouse models (including hypo- and hyperthyroid mice, thyroid-specific G-protein deficient, and Slc26a7-knockout mice).Results: Four CH patients from four novel families carried the homozygous SLC26A7 (c.1893delT, p.F631Lfs*8) pathogenic variant. Two had large trachea-compressing goiters, requiring thyroidectomy already at birth. In addition, one homozygous participant with normal CH screening results developed hypothyroidism at age 16, and one patient with heterozygous SLC26A7 pathogenic variant had permanent CH at birth. Dentofacial abnormalities were frequently noted, including enamel hypoplasia (in four carriers), pro- or retrognathia, and malocclusion requiring orthodontic treatment (in 8/24 carriers). Thyrocyte hypertrophy with large colloid aggregates was a hallmark of homozygous patients. FinnGen screening revealed a 75-fold enrichment of the variant in the Finnish population, identifying a few other homozygous and seven heterozygous cases with early-onset hypothyroidism and dentofacial abnormalities. In human thyrocytes, SLC26A7 was localized to the basolateral membrane, with intense staining in hyperthyroid samples, while in mouse thyroid models, its expression pattern depended on dietary iodide levels, thyrotropin signaling, and GNAS activity.Conclusions: We describe variable phenotypes associated with the SLC26A7 pathogenic variant, ranging from severe CH with large congenital goiters to delayed onset hypothyroidism and dentofacial abnormalities. SLC26A7 shows thyrotropin-, GNAS-, and dietary iodine-dependent basolateral localization, suggesting their role in phenotypic variations.

BACKGROUND:Prostate cancer (PrCa) is a significant health concern, ranking as the second most common cancer in males globally. Genetic factors contribute substantially to PrCa risk, with up to 57% of the risk being attributed to genetic determinants. A major challenge in managing PrCa is the early identification of aggressive cases for targeted treatment, while avoiding unnecessary interventions in slow-progressing cases. Therefore, there is a critical need for genetic biomarkers that can distinguish between aggressive and non-aggressive PrCa cases. Previous research, including our own, has shown that germline variants in ANO7 are associated with aggressive PrCa. However, the function of ANO7 in the prostate remains unknown. METHODS:We performed RNA-sequencing (RNA-seq) on RWPE1 cells engineered to express ANO7 protein, alongside the analysis of a single-cell RNA-sequencing (scRNA-seq) dataset and RNA-seq from prostate tissues. Differential gene expression analysis and gene set enrichment analysis (GSEA) were conducted to identify key pathways. Additionally, we assessed oxidative phosphorylation (OXPHOS), glycolysis, and targeted metabolomics. Image analysis of mitochondrial morphology and lipidomics were also performed to provide further insight into the functional role of ANO7 in prostate cells. RESULTS:ANO7 expression resulted in the downregulation of metabolic pathways, particularly genes associated with the MYC pathway and oxidative phosphorylation (OXPHOS) in both prostate tissue and ANO7-expressing cells. Measurements of OXPHOS and glycolysis in the ANO7-expressing cells revealed a metabolic shift towards glycolysis. Targeted metabolomics showed reduced levels of the amino acid aspartate, indicating disrupted mitochondrial function in the ANO7-expressing cells. Image analysis demonstrated altered mitochondrial morphology in these cells. Additionally, ANO7 downregulated genes involved in fatty acid metabolism and induced changes in lipid composition of the cells, characterized by longer acyl chain lengths and increased unsaturation, suggesting a role for ANO7 in regulating lipid metabolism in the prostate. CONCLUSIONS:This study provides new insights into the function of ANO7 in prostate cells, highlighting its involvement in metabolic pathways, particularly OXPHOS and lipid metabolism. The findings suggest that ANO7 may act as a key regulator of cellular lipid metabolism and mitochondrial function in the prostate, shedding light on a previously unknown aspect of ANO7's biology.

The clinical spectrum of thyrotropin receptor-mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T-knockin model revealed that the phenotype was dependent on the mutation's signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. In PCa the expression of anoctamin 7 (ANO7) has been shown to diminish as the cancer progresses. We have previously linked single-nucleotide polymorphisms in the ANO7 gene to the risk of aggressive prostate cancer and shown that in homozygous carriers of rs77559646, the variant leads to a total loss of ANO7 protein. ANO7 is a prostate-specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. To uncover the cellular functions of ANO7 has proven challenging because ANO7 is not expressed in commercially available cell lines. To study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and gene set enrichment analysis. Interestingly, we found enrichment and downregulation of mitochondrial genes participating in oxidative phosphorylation in ANO7 cells. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the basal and maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells. Furthermore, the glycolysis stress assay showed that glycolytic capacity is increased in ANO7 overexpressing cells. In addition, a targeted metabolite screening revealed that aspartate is decreased in ANO7 cells, which again points towards perturbed mitochondrial function as aspartate is produced from a tricarboxylic acid cycle intermediate. By inhibiting the uptake of extracellular aspartate, we measured slower proliferation of ANO7 expressing cells, while inhibiting aspartate had no effect on control cells. Interestingly, preliminary results also suggest ANO7's involvement in regulation of phospholipid acyl chain length and saturation, offering insights into altered signaling observed in ANO7 cells. Taken together, this study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism, which could explain why it is beneficial for cancer cells to lose ANO7 expression. Citation Format: Christoffer Löf, Nasrin Sultana, Neha Goel, Gudrun Wahlström, Johanna Schleutker. The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7056.

Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. We have previously linked single-nucleotide polymorphisms in the anoctamin 7 (ANO7) gene to the risk of aggressive prostate cancer and shown that carriers of a rs77559646 variant have improved treatment response to docetaxel. The expression of ANO7 is lost when prostate cancer progresses and is completely lacking in prostate cancer metastases. ANO7 is a prostate specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. Whether ANO7 has any of these capabilities is unclear. To uncover the cellular functions of ANO7 has proven challenging because the expression of ANO7 at protein level is either minimal or nonexistent in all commercially available cell lines. To overcome this and to study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. The characterization of the RWPE-1 cells overexpressing ANO7 showed that the viability and proliferation was increased both in 2D and 3D. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and pathway analysis. Interestingly, we found enrichment of mitochondrial genes participating in oxidative phosphorylation in cells overexpressing ANO7. The genes expressed from the mitochondrial genome were upregulated and the nuclear genes encoding for proteins involved in mitochondrial functions were mostly downregulated. This suggests that the mitochondrial metabolism has changed. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced compared to control cells. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells as there were no differences in maximal respiratory capacity when we treated the cells with the UK5099 inhibitor, which inhibits pyruvate import to mitochondria. Furthermore, preliminary results from the glycolysis stress assay indicates that glycolysis is increased in ANO7 overexpressing cells. In addition, electron microscopy study suggests that ANO7 overexpressing cells have less cristae compared to control. This study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism. Citation Format: Nasrin Sultana, Christoffer Lof, Neha Goel, Johanna Schleutker. Unraveling the role of ANO7 in prostate cancer metabolism [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A020.

Background: The human adrenal cortex undergoes several rapid remodeling steps during its lifetime. In rodents, similar remodeling occurs postnatally in the "X-zone" layer through unknown mechanisms. Furthermore, little is known regarding the impact of thyroid hormone (TH) on adrenal glands in humans.Methods: To investigate the impact of TH on adrenal pathophysiology, we created two genetic murine models mimicking human nonautoimmune hypothyroidism and hyperthyroidism. Moreover, we analyzed serum thyrotropin (TSH) and steroid hormone concentrations in patients diagnosed with congenital hypothyroidism and premature adrenarche (PA).Results: We found that TH receptor beta-mediated hypertrophy of the X-zone significantly elevated the adrenal weights of hyperthyroid women. In the hypothyroid model, the X-zone was poorly developed in both sexes. Moreover, large reciprocal changes in the expression levels of genes that regulate adrenal cortical function were observed with both models. Unexpectedly, up- and downregulation of several genes involved in catecholamine synthesis were detected in the adrenal glands of the hypothyroid and hyperthyroid models, respectively. Furthermore, TSH and adrenal steroid concentrations correlated positively in pediatric patients with congenital hypothyroidism and PA.Conclusions: Our results revealed that congenital hypothyroidism and hyperthyroidism functionally affect adrenal gland development and related steroidogenic activity, as well as the adrenal medulla.

Prostate cancer is among the most common cancers in men, with a large fraction of the individual risk attributable to heritable factors. A majority of the diagnosed cases does not lead to a lethal disease, and hence biological markers that can distinguish between indolent and fatal forms of the disease are of great importance for guiding treatment decisions. Although over 300 genetic variants are known to be associated with prostate cancer risk, few have been associated with the risk of an aggressive disease. One such variant is rs77559646 located in ANO7. This variant has a dual function. It constitutes a missense mutation in the short isoform of ANO7 and a splice region mutation in full-length ANO7. In this study, we have analyzed the impact of the variant allele of rs77559646 on ANO7 mRNA splicing using a minigene splicing assay and by performing splicing analysis with the tools IRFinder (intron retention finder), rMATS (replicate multivariate analysis of transcript splicing) and LeafCutter on RNA sequencing data from prostate tissue of six rs77559646 variant allele carriers and 43 non-carriers. The results revealed a severe disruption of ANO7 mRNA splicing in rs77559646 variant allele carriers. Immunohistochemical analysis of prostate samples from patients homozygous for the rs77559646 variant allele demonstrated a loss of apically localized ANO7 protein. Our study is the first to provide a mechanistic explanation for the impact of a prostate cancer risk SNP on ANO7 protein production. Furthermore, the rs77559646 variant is the first known germline loss-of-function mutation described for ANO7. We suggest that loss of ANO7 contributes to prostate cancer progression.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1–5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.

The thyroid gland produces thyroid hormones (TH), which are essential regulators for growth, development and metabolism. The thyroid is mainly controlled by the thyroid-stimulating hormone (TSH) that binds to its receptor (TSHR) on thyrocytes and mediates its action via different G protein-mediated signaling pathways. TSH primarily activates the Gs-pathway, and at higher concentrations also the Gq/11-pathway, leading to an increase of intracellular cAMP and Ca2+, respectively. To date, the physiological importance of other G protein-mediated signaling pathways in thyrocytes is unclear. Congenital hypothyroidism (CH) is defined as the lack of TH at birth. In familial cases, high-throughput sequencing methods have facilitated the identification of novel mutations. Nevertheless, the precise etiology of CH yet remains unraveled in a proportion of cases. Genetically modified mouse models can reveal new pathophysiological mechanisms of thyroid diseases. Here, we will present an overview of genetic mouse models for thyroid diseases, which have provided crucial insights into thyroid gland development, function, and growth with a special focus on TSHR and microRNA signaling.

Background: Constitutively active thyrotropin receptor (TSHR) mutations are the most common etiology of non-autoimmune hyperthyroidism (NAH). Thus far, the functionality of these mutations has been tested in vitro, but the in vivo models are lacking. Methods: To understand the pathophysiology of NAH, the patient-derived constitutively active TSHR D633H mutation was introduced into the murine Tshr by homologous recombination. Results: In this model, both subclinical and overt hyperthyroidism was observed, depending on the age, sex, and genotype. Homozygous mice presented hyperthyroidism at two months of age, while heterozygous animals showed only suppressed thyrotropin. Interestingly, at six months of age, thyroid hormone concentrations in all mutant mice were analogous to wild-type mice, and they showed colloid goiter with flattened thyrocytes. Strikingly, at one year of age, nearly all homozygous mice presented large papillary thyroid carcinomas. Mechanistically, this papillary thyroid carcinoma phenotype was associated with an overactive thyroid and strongly increased stainings of proliferation-, pERK-, and NKX2-1 markers, but no mutations in the hot-spot areas of common oncogenes (Braf, Nras, and Kras) were found. Conclusions: This is the first study to reveal the dynamic age-, sex-, and genotype-dependent development of NAH. Furthermore, the study shows that a constitutively active TSHR can trigger a malignant transformation of thyrocytes.

Thyroid function is controlled by thyroid-stimulating hormone (TSH), which binds to its G protein-coupled receptor [thyroid-stimulating hormone receptor (TSHR)] on thyrocytes. TSHR can potentially couple to all G protein families, but it mainly activates the G(s)- and G(q/11)-mediated signaling cascades. To date, there is a knowledge gap concerning the role of the individual G protein cascades in thyroid pathophysiology. Here, we demonstrate that the thyrocyte-specific deletion of G(s) protein subunit (G(s)) in adult mice [tamoxifen-inducible G(s) protein subunit deficient (iTG(s)KO) mice] rapidly impairs thyrocyte function and leads to hypothyroidism. Consequently, iTG(s)KO mice show reduced food intake and activity. However, body weight and the amount of white adipose tissue were decreased only in male iTG(s)KO mice. Unexpectedly, hyperplastic follicles and papillary thyroid cancer-like tumor lesions with increased proliferation and slightly increased phospho-ERK1/2 staining were found in iTG(s)KO mice at an older age. These tumors developed from nonrecombined thyrocytes still expressing G(s) in the presence of highly elevated serum TSH. In summary, we report that partial thyrocyte-specific G(s) deletion leads to hypothyroidism but also to tumor development in thyrocytes with remaining G(s) expression. Thus, these mice are a novel model to elucidate the pathophysiological consequences of hypothyroidism and TSHR/G(s)/cAMP-mediated tumorigenesis.Patyra, K., Jaeschke, H., Lof, C., Jannari, M., Ruohonen, S. T., Undeutsch, H., Khalil, M., Kero, A., Poutanen, M., Toppari, J., Chen, M., Weinstein, L. S., Paschke, R., Kero, J. Partial thyrocyte-specific G(s) deficiency leads to rapid-onset hypothyroidism, hyperplasia, and papillary thyroid carcinoma-like lesions in mice.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Background: Congenital hypothyroidism (CH) is defined as the lack of thyroid hormones at birth. Mutations in at least 15 different genes have been associated with this disease. While up to 20% of CH cases are hereditary, the majority of cases are sporadic with unknown etiology. Apart from a monogenic pattern of inheritance, multigenic mechanisms have been suggested to play a role in CH. The genetics of CH has not been studied in Finland so far. Therefore, multigenic sequencing of CH candidate genes was performed in a Finnish patient cohort with both familial and sporadic CH. Methods: A targeted next-generation sequencing (NGS) panel, covering all exons of the major CH genes, was applied for 15 patients with sporadic and 11 index cases with familial CH. Results: Among the familial cases, six pathogenic mutations were found in the TPO, PAX8, and TSHR genes. Furthermore, pathogenic NKX2.1 and TG mutations were identified from sporadic cases, together with likely pathogenic variants in the TG, NKX2.5, SLC26A4, and DUOX2 genes. All identified novel pathogenic mutations were confirmed by Sanger-sequencing and characterized in silico and/or in vitro. Conclusion: In summary, the CH panel provides an efficient, cost-effective, and multigenic screening tool for both known and novel CH gene mutations. Hence, it may be a useful method to identify accurately the genetic etiology for dyshormogenic, familial, or syndromic forms of CH.

Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1–Aequorin fusion protein (Cav1–Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca2+]cav). In HeLa cells, Cav1–Aeq reported different [Ca2+] as compared to the plasma membrane [Ca2+] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca2+] (reported by cyt-Aeq). The Ca2+ signals detected by Cav1–Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca2+]cav during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+ as well as during receptor-operated Ca2+ entry (ROCE). The SK1-induced increase in [Ca2+]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca2+]cav during ROCE. S1P treatment stimulated the [Ca2+]cav upon ROCE. The Ca2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Thyroid stimulating hormone (TSH) is the main physiological regulator of the thyroid gland. TSH controls thyroid function by activating a receptor, which belongs to the family of the G-protein coupled receptors. The TSH receptor (TSHR) is able to couple not only to the G-protein GS resulting in stimulation of adenylyl cyclases, but also to Gq/G11 leading to an activation of phospholipase C, increase in inositol 1,4,5 triphosphate and subsequent release of intracellular Ca2+. Furthermore, in vitro studies, using human follicular thyroid cells, have shown that the TSHR also interacts with G12/G13, activating the Mitogen-activated protein kinase pathway. To date, the physiological significance of this unusual pleiotropic G-protein coupling is unclear.

Background: The identity of calcium channels in the thyroid is undefined. Results: TRPC1 functions as a major regulator of S1P and VEGF receptors via a calcium-dependent mechanism. This is important for cell migration. Conclusion: We have defined a novel physiological role for the TRPC1 channel. Significance: This study explains how TRPC1 regulates receptor expression and migration in thyroid cancer cells.The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P(1)/S1P(3)), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P(3) and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P(3) and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P(3) and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1 expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G(1) phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P(3) and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells.