Periodontics

The human ether-a-go-go related gene (HERG) potassium channel has elicited intense scientific interest due to its role in cardiac repolarization and its association with arrhythmia and sudden cardiac death. Increasing evidence indicates the involvement of HERG channels in the pathophysiology of cancer. In the present study we investigated the expression of HERG protein in MDA-MB-435S melanoma cells, and its importance in regulating cell proliferation and migration. Our results showed that HERG was expressed on protein and mRNA levels in MDA-MB-435S melanoma cells. In these cells blockade of HERG channels with the HERG blockers E 4301 or cisapride attenuated both proliferation and migration of the cells. Activation of HERG with PD118057 stimulated cell migration. Furthermore, HERG small interfering (si) RNA attenuated the proliferation and migration of the cells. Incubation of MDA-MB-435S cells with E 4301 decreased the phosphorylation of mitogen-activated protein (MAP) kinase and the expression of the c-fos transcription factor. In control experiments, overexpression of HERG channels in HEK-293 cells dramatically increased the proliferation and migration of the cells and blocking HERG in these cells attenuated both proliferation and migration. Our results indicate that MDA-MB-435S cells express HERG channels and blockade of HERG results in the attenuation of both proliferation and migration by a mechanism dependent, at least in part, on an inhibition of the MAP kinase/c-fos pathway.

The identity of calcium channels in the thyroid is not defined. Results: TRPC2 functions as major regulator of calcium homeostasis in rat thyroid cells. TRPC2 appears receptor-regulated and participates in regulating ER calcium content. Conclusion: We have defined a novel physiological role for TRPC2 channels. Significance: TRPC2, by regulating STIM2, PKC expression and SERCA activity, regulates calcium homeostasis in thyroid cells.

The identity of calcium channels in the thyroid is not defined. Results: TRPC2 functions as major regulator of calcium homeostasis in rat thyroid cells. TRPC2 appears receptor-regulated and participates in regulating ER calcium content. Conclusion: We have defined a novel physiological role for TRPC2 channels. Significance: TRPC2, by regulating STIM2, PKC expression and SERCA activity, regulates calcium homeostasis in thyroid cells.

The initial step in a synthesis of thyroid hormones is the uptake of iodide from the circulation. Iodide (I À ) is transported into thyroid cells via a Na þ /I À symporter (NIS), which is electrogenic and thus sensitive to alterations in membrane potential (V m ). I À is then released to the lumen of thyroid follicles where the hormones are synthesised and stored. The mechanisms of I À release to follicle lumen are poorly characterised. Our whole-cell voltage clamp recordings revealed the presence of a Ca 2þ activated Cl À current (CaCC) in Fisher rat thyroid cell line 5 (FRTL-5). Transcripts of anoctamin 1 (ANO1) and anoctamin 10 (ANO10), putative molecular constituents of CaCC, were detected. The anion channels underlying CaCC are highly permeable to I À . Both niflumic acid (NFA) and 2-aminoethyl diphenylborinate (2-APB), antagonists of CaCC and transient receptor potential channels, respectively, inhibited CaCC. Canonical transient receptor potential channel 2 (TRPC2) is the only TRPC member present in FRTL-5 cells. The activation rate of CaCC was markedly slower in shTRPC2 knock-down cells, indicating that Ca 2þ entry via TRPC2 contributes to CaCC activation. The uptake of iodide was enhanced and the resting V m was more depolarised in TRPC2 knock-down cells. We suggest that the interplay between TRPC2 and ANO1 may have dual effects on iodide transport, modulating I À release via ANO channels and I À uptake via the V m sensitive NIS. ORIGINAL RESEARCH ARTICLE 814 J o u r n a l o f J o u r n a l o f Cellular Physiology Cellular Physiology ß 2 0 1 2 W I L E Y P E R I O D I C A L S , I N C .

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca 2+ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca 2+ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides. Citation: Sukumaran P, Lö nnfors M, Långvik O, Pulli I, Tö rnquist K, et al. (2013) Complexation of C6-Ceramide with Cholesteryl Phosphocholine -A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture. PLoS ONE 8(4): e61290.

Ca 2+ entry is essential for regulating vital physiological functions in all neuronal cells. Although neurons are engaged in multiple modes of Ca 2+ entry that regulates variety of neuronal functions, we will only discuss a subset of specialized Ca 2+ -permeable non-selective Transient Receptor Potential Canonical (TRPC) channels and summarize their physiological and pathological role in these excitable cells. Depletion of endoplasmic reticulum (ER) Ca 2+ stores, due to G-protein coupled receptor activation, has been shown to activate TRPC channels in both excitable and non-excitable cells. While all seven members of TRPC channels are predominately expressed in neuronal cells, the ion channel properties, mode of activation, and their physiological responses are quite distinct. Moreover, many of these TRPC channels have also been suggested to be associated with neuronal development, proliferation and differentiation. In addition, TRPCs also regulate neurosecretion, long-term potentiation and synaptic plasticity. Similarly, perturbations in Ca 2+ entry via the TRPC channels have been also suggested in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of TRPCs in neuronal function and in neurodegenerative conditions would presumably unveil avenues for plausible therapeutic interventions for these devastating neuronal diseases.

The identity of calcium channels in the thyroid is not defined. Results: TRPC2 functions as major regulator of calcium homeostasis in rat thyroid cells. TRPC2 appears receptor-regulated and participates in regulating ER calcium content. Conclusion: We have defined a novel physiological role for TRPC2 channels. Significance: TRPC2, by regulating STIM2, PKC expression and SERCA activity, regulates calcium homeostasis in thyroid cells.

The human ether-ago go related gene (HERG) potassium channel has elicited intense scientific interest due to its role in cardiac repolarization and its association with arrhythmia and sudden cardiac death. Increasing evidence indicates the involvement of HERG channels in the pathophysiology of cancer. In the present study we investigated the expression of HERG protein in MDA-MB-435S melanoma cells, and its importance in regulating cell proliferation and migration. Our results showed that HERG was expressed on protein and mRNA levels in MDA-MB-435S melanoma cells. In these cells blockade of HERG channels with the HERG blockers E 4301 or cisapride attenuated both proliferation and migration of the cells. Activation of HERG with PD118057 stimulated cell migration. Furthermore, HERG small interfering (si) RNA attenuated the proliferation and migration of the cells. Incubation of MDA-MB-435S cells with E 4301 decreased the phosphorylation of mitogen-activated protein (MAP) kinase and the expression of the c-fos transcription factor. In control experiments, overexpression of HERG channels in HEK-293 cells dramatically increased the proliferation and migration of the cells and blocking HERG in these cells attenuated both proliferation and migration. Our results indicate that MDA-MB-435S cells express HERG channels and blockade of HERG results in the attenuation of both proliferation and migration by a mechanism dependent, at least in part, on an inhibition of the MAP kinase/c-fos pathway.

Activation of Epithelial-to-Mesenchymal Transition (EMT) is important for tumor metastasis. Although growth factors such as TGFβ and EGF have been shown to induce EMT in breast epithelial cells, the mechanism resulting in migration is not well understood. Herein, we provide evidence that Ca 2+ entry into the cell, especially upon store-depletion, plays an important role in TGFβ-induced EMT by promoting cellular migration and potentially leading to metastasis. The increased migration by TGFβ in non-cancerous cells was due to the loss of E-cadherin along with a subsequent increase in N-cadherin levels. Importantly, TGFβ-treatment increases store-mediated Ca 2+ entry, which was essential for the activation of calpain leading to the loss of E-cadherin and MMP activation. Inhibition of Ca 2+ entry by using Ca 2+ channel blocker SKF-96365, significantly decreased Ca 2+ entry, decreased TGFβinduced calpain activation, and suppressed the loss of E-cadherin along with inhibiting cell migration. Furthermore, TRPC1 function as an endogenous Ca 2+ entry channel and silencing of either TRPC1 or its activator, STIM1, significantly decreased TGFβ induced Ca 2+ entry, inhibited TGFβ-mediated calpain activation and cell migration. In contrast, overexpression of TRPC1 showed increased Ca 2+ entry and promoted TGFβmediated cell migration. Moreover, increased TRPC1 expression was observed in ductal carcinoma cells. Together these results suggest that disrupting Ca 2+ influx via TRPC1/STIM1 mechanism reduces calpain activity, which could restore intercellular junction proteins thereby inhibiting EMT induced motility.

Loss of dopaminergic (DA) neurons leads to Parkinson's disease; however, the mechanism(s) for the vulnerability of DA neurons is(are) not fully understood. We demonstrate that TRPC1 regulates the L-type Ca(2+) channel that contributes to the rhythmic activity of adult DA neurons in the substantia nigra region. Store depletion that activates TRPC1, via STIM1, inhibits the frequency and amplitude of the rhythmic activity in DA neurons of wild-type, but not in TRPC1(-/-), mice. Similarly, TRPC1(-/-) substantia nigra neurons showed increased L-type Ca(2+) currents, decreased stimulation-dependent STIM1-Cav1.3 interaction, and decreased DA neurons. L-type Ca(2+) currents and the open channel probability of Cav1.3 channels were also reduced upon TRPC1 activation, whereas increased Cav1.3 currents were observed upon STIM1 or TRPC1 silencing. Increased interaction between Cav1.3-TRPC1-STIM1 was observed upon store depletion and the loss of either TRPC1 or STIM1 led to DA cell death, whi...

Alterations in Ca2+ homeostasis affect neuronal survival. However, the identity of Ca2+ channels and the mechanisms underlying neurotoxin-induced neuronal degeneration are not well understood. In this study, the dopaminergic neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl -4-phenylpyridium ions (MPP+/MPTP), which mimic Parkinson's disease (PD), induced neuronal degeneration by decreasing store-mediated Ca2+ entry. The function of the transient receptor potential canonical (TRPC)-1 channel was decreased upon exposure to the neurotoxins, followed by a decrease in TRPC1 expression. Similar to neurotoxins, samples from patients with PD exhibited attenuated TRPC1 expression, which was accompanied by a decrease in autophagic markers and a subsequent increase in apoptosis markers. Furthermore, exposure to neurotoxins attenuated PKC phosphorylation, decreased expression of autophagic markers, and increased apoptosis in SHSY-5Y neuroblastoma cells, which was again dependent on TRPC1....

The transient receptor potential canonical channel-1 (TRPC1) is a Ca2+-permeable channel found in key metabolic organs and tissues, including the hypothalamus, adipose tissue, and skeletal muscle. Loss of TRPC1 may alter the regulation of cellular energy metabolism resulting in insulin resistance thereby leading to diabetes. Exercise reduces insulin resistance, but it is not known whether TRPC1 is involved in exercise-induced insulin sensitivity. The role of TRPC1 in adiposity and obesity-associated metabolic diseases has not yet been determined. Our results show that TRPC1 functions as a major Ca2+ entry channel in adipocytes. We have also shown that fat mass and fasting glucose concentrations were lower in TRPC1 KO mice that were fed a high-fat (HF) (45% fat) diet and exercised as compared with WT mice fed a HF diet and exercised. Adipocyte numbers were decreased in both subcutaneous and visceral adipose tissue of TRPC1 KO mice fed a HF diet and exercised. Finally, autophagy marke...

Macrophage plasticity is essential for innate immunity, but in-depth signaling mechanism(s) regulating their functional phenotypes are ill-defined. Here we report that interferon (IFN) γ priming of naive macrophages induces store-mediated Ca entry and inhibition of Ca entry impairs polarization to M1 inflammatory phenotype. In vitro and in vivo functional analyses revealed ORAI1 to be a primary contributor to basal Ca influx in macrophages, whereas IFNγ-induced Ca influx was mediated by TRPC1. Deficiency of TRPC1 displayed abrogated IFNγ-induced M1 inflammatory mediators in macrophages. In a preclinical model of peritonitis by Klebsiella pneumoniae infection, macrophages showed increased Ca influx, which was TRPC1 dependent. Macrophages from infected TRPC1 mice showed inhibited expression of M1-associated signature molecules. Furthermore, in human patients with systemic inflammatory response syndrome, the level of TRPC1 expression in circulating macrophages directly correlated with ...

Mg 2+ homeostasis is essential for cell survival and the loss of this regulation has been associated with many neurodegenerative diseases, including loss of dopaminergic neurons. Although the neurotoxin-mediated loss of dopaminergic neurons in Parkinson disease models is extensively studied, the ion channel(s) that regulate Mg 2+ homeostasis and thus could prevent neuronal cell death is not yet identified. Here, we show that TRPM7 (transient receptor potential melastatin 7) is involved in regulating Mg 2+ homeostasis in dopaminergic cells. Importantly, transient loss of TRPM7 decreased intracellular Mg 2+ levels and decreased dopaminergic cells/neurons survival. We provide further evidence that both increases in extracellular Mg 2+ or transiently increasing TRPM7 levels protected dopaminergic SH-SY5Y cells against neurotoxin-mediated cell death. Neurotoxin treatment significantly decreased TRPM7 levels in both SH-SY5Y cells and the substantia nigra pars compacta region of mice, along with a decrease in Mg 2+ influx. Moreover, Mg 2+ supplementation showed a concentration-dependent decrease in caspase-3 activity, an increase in cell survival, restored mitochondrial membrane potential, and increase TRPM7 levels in neurotoxin-treated cells. In contrast, transient silencing of TRPM7 inhibited the positive effect of Mg 2+ supplementation in protecting against neurotoxins. Whereas, TRPM7 overexpression not only maintained Mg 2+ homeostasis but also inhibited caspase 3 activity that induced cell survival. Overall, these results suggest a significant role of TRPM7 channels in Mg 2+ homeostasis and the survival of neurotoxininduced loss of dopaminergic cells.

Intracellular calcium (Ca 2+) levels play a vital role in regulating cellular fate. The coordination and interrelation among the cellular organelles, mainly the intracellular Ca 2+ stores in endoplasmic reticulum (ER), are crucial in maintaining cytosolic Ca 2+ levels and in general cellular homeostasis. Moreover, maintaining Ca 2+ homeostasis is essential for regulating diverse and sometimes opposing processes such as cell survival and cell death in disease conditions such as, neurodegeneration, cancer and aging. Ca 2+ is able to regulate opposing functions by either regulating the cellular "self-eating" phenomenon of autophagy to promote cell survival or by regulating the programmed cell death process of apoptosis. Autophagy is also important for cell survival especially after induction of ER stress and association between ER stress and autophagy may have relevance to numerous diseases. Moreover, a multitude of evidence is emerging that the functional regulation of TRP channels, their unique localization, and their interaction with other Ca 2+-sensing elements define these diverse regulatory pathways. It is this unique function which allows individual TRP channels to contribute differently in the regulation of cell fate and, in turn, determines the precise effect of modulating Ca 2+ signaling via the particular channel. Thus, in this review we have focused on the aspects of TRP channel localization and function (Ca 2+ signaling) that affects the ER stress and autophagic process.

Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca(2+) is suggested to have an important role in cell survival, the ion channel(s) involved in autophagy have not been identified. Here we demonstrate that increase in intracellular Ca(2+) via transient receptor potential canonical channel-1 (TRPC1) regulates autophagy, thereby preventing cell death in two morphologically distinct cells lines. The addition of DMOG or DFO, a cell permeable hypoxia-mimetic agents, or serum starvation, induces autophagy in both epithelial and neuronal cells. The induction of autophagy increases Ca(2+) entry via the TRPC1 channel, which was inhibited by the addition of 2APB and SKF96365. Importantly, TRPC1-mediated Ca(2+) entry resulted in increased expression of autophagic markers that prevented cell death. Furthermore, hypoxia-mediated autophagy also increased TRPC1, but not STIM1 or Orai1, expression. Silencing of ...

Calcium is a ubiquitous second messenger that modulates most of the cellular functions including gene expression and cellular homeostasis, 1,2 neurotransmitter release and neuronal function, 3,4 and modulation of metabolism and cell survival. 5 The known molecular regulators of cell calcium homeostasis, such as calcium release-activated calcium channel (ORAI), stromal interaction molecule 1 (STIM1) and TRPC channels are all implicated in modulating Ca 2+ entry in both excitable and nonexcitable cells. Importantly, TRPC and ORAI channels have been