Oxysterol-binding protein related-proteins (ORPs) 5 and 8 regulate calcium signaling at specific cell compartments

Oxysterol-binding protein related-protein 5 and 8 (ORP5/8) localize to the membrane contact sites (MCS) of the endoplasmic reticulum (ER) and the mitochondria, as well as to the ER-plasma membrane (PM) MCS. The MCS are emerging as important regulators of cell signaling events, including calcium (Ca2+) signaling. ORP5/8 have been shown to interact with phosphatidylinositol-4,5-bisphosphate (PIP2) in the PM, and to modulate mitochondrial respiration and morphology. PIP2 is the direct precursor of inositol trisphosphate (IP3), a key second messenger responsible for Ca2+-release from the intracellular Ca2+ stores. Further, mitochondrial respiration is linked to Ca2+ transfer from the ER to the mitochondria. Hence, we asked whether ORP5/8 would affect Ca2+ signaling in these cell compartments, and employed genetically engineered aequorin Ca2+ probes to investigate the effect of ORP5/8 in the regulation of mitochondrial and caveolar Ca2+. Our results show that ORP5/8 overexpression leads to increased mitochondrial matrix Ca2+ as well as to increased Ca2+ concentration at the caveolar subdomains of the PM during histamine stimulation, while having no effect on the cytoplasmic Ca2+. Also, we found that ORP5/8 overexpression increases cell proliferation. Our results show that ORP5/8 regulate Ca2+ signaling at specific MCS foci. These local ORP5/8-mediated Ca2+ signaling events are likely to play roles in processes such as mitochondrial respiration and cell proliferation.Peer reviewe

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells

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.Peer reviewe

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells

Sphingosine kinase 1 (SKI) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (SIP). SW binds to G-protein-coupled receptors (S1PR(1-5)) 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 SKI augments agonist-induced Ca2+ release from the ER resulting in increased mitochondria] 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.Peer reviewe

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells

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

Salmonellan leviäminen suomalaisille sika- ja nautatiloille

Hankkeen tavoitteina oli tunnistaa nauta- ja sikatilojen salmonellatartuntojen lähteitä sekä toimintatapoja, joilla saneeraus ja tartuntojen ehkäisy tiloilla parhaiten onnistuu. Lukuisista tartuntalähteistä tärkeimmiksi arvioimme yhdyskuntalinnut ja muut haittaeläimet sekä turkistuotanto mahdollisena alkulähteenä haittaeläinten välittämille tartunnoille. Viime vuosina nauta- ja sikatiloille tehtyjen saneerausten aineistosta arvioimme seikkoja, jotka johtivat saneerauksen onnistumiseen tai pitkittymiseen tai tartunnan uusimiseen. Saneeraus pitkittyi, jos tartunta oli alkutilanteessa levinnyt laajalle, tartuntalähdettä ei saatu heti selville, salmonellapositiivisia eläimiä ei poistettu ajoissa, puhdistus-, pesu- ja desinfiointitoimet eivät olleet riittäviä tai käytettävissä oleva työpanos ei riittänyt. Saneerauksen onnistumista edistivät hyvä työnjohto, systemaattisuus, saneeraussuunnitelma sekä suunnitelman ja muiden ohjeiden noudattaminen. Tarkastelimme tilan salmonellatartunnan ja sen saneerauksen vaatimuksia myös työturvallisuuden suhteen. Salmonellatartunnan ehkäisy edellyttää tilatason tautisuojausta, jonka on oltava jokapäiväistä ja kohdistuttava tilan koko toimintaan ottaen huomioon haittaeläimistä aiheutuva tartuntavaara. Hankkeessa syntyi tietopääomaa salmonellan epidemiologiseen seurantaan, saneerausneuvontaan ja suosituksia tukemaan salmonellatorjuntaa.Tämä julkaisu on toteutettu osana valtioneuvoston selvitys- ja tutkimussuunnitelman toimeenpanoa. (tietokayttoon.fi) Julkaisun sisällöstä vastaavat tiedon tuottajat, eikä tekstisisältö välttämättä edusta valtioneuvoston näkemystä

Calcium Signaling in the Thyroid: Friend and Foe

Calcium signaling participates in a vast number of cellular processes, ranging from the regulation of muscle contraction, cell proliferation, and mitochondrial function, to the regulation of the membrane potential in cells. The actions of calcium signaling are, thus, of great physiological significance for the normal functioning of our cells. However, many of the processes that are regulated by calcium, including cell movement and proliferation, are important in the progression of cancer. In the normal thyroid, calcium signaling plays an important role, and evidence is also being gathered showing that calcium signaling participates in the progression of thyroid cancer. This review will summarize what we know in regard to calcium signaling in the normal thyroid as, well as in thyroid cancer.Non peer reviewe

Sphingosine 1-phosphate attenuates MMP2 and MMP9 in human anaplastic thyroid cancer C643 cells: Importance of S1P2.

In anaplastic thyroid cancer C643 cells, sphingosine 1-phosphate (S1P) attenuates migration by activating the S1P2 receptor and the Rho-ROCK pathway. In the present study, we show that stimulating C643 cells with S1P decreases the expression, secretion and activity of matrix metalloproteinase-2 (MMP2), and to a lesser extent MMP9. Using receptor-specific antagonists, and S1P2 siRNA, we showed that the inhibition of expression of MMP2 is mediated through S1P2. Furthermore, S1P inhibited calpain activity, and inhibiting calpain pharmacologically, inhibited the effect of S1P on MMP2 expression and activity, and on MMP9 activity. S1P treatment increased Rho activity, and by incubating cells with the Rho inhibitor C3 transferase or the ROCK inhibitor Y27632, the S1P-induced inhibition of invasion and MMP2 expression and activity was abolished. We conclude that S1P attenuates the invasion of C643 cells by activating S1P2 and the Rho-ROCK pathway, by decreasing calpain activity, and by decreasing the expression, secretion and activity of MMP2 and, to a lesser extent, MMP9. Our results thus unveil a novel function for the S1P2 receptor in attenuating thyroid cancer cell invasion

S1P-evoked inhibition of MMP2 is mediated through the Rho-ROCK pathway.

<p>(A) Time-dependent effect of S1P on Rho activity in C643 cells. (B) Stimulatory effect of the Rho inhibitor C3-transferase or the ROCK inhibitor Y-27632 on C643 cell invasion. (C) C3-transferase or Y-27632 attenuated the S1P-evoked inhibition of MMP2 activity. (D and E) Lack of an effect of pretreatment with C3-transferase or Y-27632 on the expression of MMP2 but S1P-evoked-attenuation of MMP2 expression was abolished. The representative blots are shown. β-Actin was used as loading control, and the normalized results are presented in graphs. The data in the graphs are the mean ±S.E.M, n = 3. Asterisks (*) denote the statistically significant differences compared with respective control. (¤) indicates comparisons between S1P effects. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001; ¤ <i>P</i> < 0.05; ¤¤ <i>P</i> < 0.01).</p

S1P inhibits invasion and affects expression, secretion and activity of MMP2 and MMP9 in C643 cells.

<p>(A) Cells were treated with S1P (100 nM for 2 h, 4 h 6 h and 8 h). The representative western blots show the expression of MMP2 and MMP9. β-Actin was used as loading control and the normalized data are presented in the graphs. (B) Cells were treated with S1P (100 nM for 2 h, 4 h, 6 h and 8 h). MMP2 and MMP9 mRNA expression was measured with quantitative real-time PCR. (C) Cells were treated with S1P (100 nM for 2 h, 4 h, 6 h and 8 h). The representative western blots show the secretion of MMP2 and MMP9. (D) Cells were treated with S1P (100 nM for 2 h, 4 h, 6 h and 8 h). The representative zymography blot shows the activity of MMP2 and MMP9. The data were normalized to the protein content on the plates and are presented in respective graphs. (E) C643 cells were allowed to invade through collagen IV towards 5% lipid-stripped FBS in the presence or absence of S1P (100 nM for 2 h, 4 h, 6 h and 8 h). Representative images of invasion inserts are shown. The scale bar is 50 μm. The normalized data in the graphs are the mean ± S.E.M, n = 3–6. Asterisks (*) denote the statistically significant differences compared with respective control. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001).</p