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.

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.

The bioactive lipid sphingosine-1-phosphate (S1P) is a potent inducer of ML-1 thyroid cancer cell migration and invasion. It evokes migration and invasion by activating S1P receptor 1 and 3 (S1P 1,3 ) and downstream signaling intermediates as well as through cross-communication with vascular endothelial growth factor receptor 2 (VEGFR2). However, very little is known about the role of S1P receptors in thyroid cancer. Furthermore, the currently used treatments for thyroid cancer have proven to be rather unsuccessful. Thus, due to the insufficiency of the available treatments for thyroid cancer, novel and targeted therapies are needed. The S1P receptor functional antagonist FTY720, an immunosuppressive drug currently used for treatment of multiple sclerosis, has shown promising effects as an inhibitor of cancer cell proliferation and invasion. In this study, we investigated the effect of FTY720 on invasion and proliferation of several thyroid cancer cell lines. We present evidence that FTY720 attenuated basal as well as S1P-evoked invasion of these cell lines. Furthermore, FTY720 potently downregulated S1P 1 , protein kinase C α (PKCα), PKCβI, and VEGFR2. It also attenuated S1P-evoked phosphorylation of ERK1/2. Our results also showed that FTY720 attenuated S1P-induced MMP2 intracellular expression, S1P-induced secretion of MMP2 and MMP9, and decreased basal MMP2 and MMP9 activity. Moreover, in FTY720-treated cells, proliferation was attenuated, p21 and p27 were upregulated, and the cells were arrested in the G1 phase of the cell cycle. FTY720 attenuated cancer cell proliferation in the chick embryo chorioallantoic membrane assay. Thus, we suggest that FTY720 could be beneficial in the treatment of thyroid cancer.

Context : Central precocious puberty (CPP) results from premature activation of the hypothalamic–pituitary–gonadal axis. Loss-of-function mutations in makorin ring finger 3 ( MKRN3 ), a maternally imprinted gene, have been recognized as the most common genetic cause of CPP. More recently, a complex defect (deletion and duplication) in delta-like 1 homolog ( DLK1 ), another maternally imprinted gene, was associated with CPP and increased body fat in a family with several affected girls. Single nucleotide polymorphisms in or near the MKRN 3 and DLK1 genes have been associated with age of menarche when the variant is inherited from the father. Objectives: To investigate the prevalence of mutations in MKRN3 and DLK1 in a cohort of 50 patients (3 boys) with CPP. Design : The 5’ untranslated region (5’UTR) and coding regions of MKRN3 and DLK1 were amplified and sequenced by Sanger sequencing. Family members of patients with identified MKRN3 or DLK1 variants were included for genetic analysis when DNA was available. Results: We identified five mutations in MKRN3 : four novel missense (p.Tyr117Cys, p.Ile461Phe, p.Met126Val and p.Cys364Phe), and one rare frameshift mutations (p.Ala288Profs*108, gnomADE frequency 8e-06). These mutations were identified in five girls, two of whom had a family history of CPP. The Cys364Phe mutation, located in a key cysteine residue in the E3 ligase RING finger domain of MKRN3, was identified in a proband and her paternal cousin with CPP. Ala288Profs*108 was identified in a proband and in two affected relatives, her sister and a paternal cousin. The patient’s father also harbored the mutation; he did not have known early pubertal development. The fathers of the two girls with Ile461Phe and Met126Val mutations without a known family history of CPP were sequenced and harbored the corresponding mutations. In addition, one homozygous variant was identified in the 5’UTR of DLK1 , c.-217G>T, in an overweight girl without a family history of CPP. This variant has been reported in the heterozygous state at a frequency of 3e-05 in gnomADE. Her father was not available for genetic studies. Interestingly, her mother was homozygous for the wild type allele, suggesting that the mother did not transmit a DLK1 allele to the patient in this region. All patients with mutations had classical features of CPP. Conclusions: In our cohort of CPP, 10% of subjects had MKRN3 mutations. Familial segregation analysis was performed in four cases and in all cases the mutations were paternally inherited. A nucleotide change was identified in the 5’UTR of ...