Colonocyte keratins stabilize mitochondria and contribute to mitochondrial energy metabolism.

Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding, and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8-/-) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation, and blunted mitochondrial ketogenesis, the role of K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8-/- colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8-/- colonocyte mitochondria in vivo are smaller and rounder and that mitochondrial motility is increased in K8-/- Caco-2 cells. Furthermore, K8-/- Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared with controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both in vitro in K8-/- Caco-2 cells and in vivo in K8-/- mouse colonocytes, and reexpression of K8 into K8-/- Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca2+ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca2+. In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca2+ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies.NEW & NOTEWORTHY In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca2+ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology.

Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression: IMPLICATIONS FOR THYROID CANCER CELL MIGRATION AND PROLIFERATION.

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 (S1P1/S1P3), 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 S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 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, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α 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 G1 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 S1P3 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.

A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²âº microdomain in the caveolar compartment.

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 ([Ca²âº]cav). In HeLa cells, Cav1-Aeq reported different [Ca²âº] as compared to the plasma membrane [Ca²âº] in general (reported by SNAP25-Aeq) or as compared to the cytosolic [Ca²âº] (reported by cyt-Aeq). The Ca²âº signals detected by Cav1-Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-ß-cyclodextrin, suggesting that the caveolae are specific targets for Ca²âº signaling. HeLa cells overexpressing SK1 showed increased [Ca²âº]cav during histamine-induced Ca²âº mobilization in the absence of extracellular Ca²âº as well as during receptor-operated Ca²âº entry (ROCE). The SK1-induced increase in [Ca²âº]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca²âº]cav during ROCE. S1P treatment stimulated the [Ca²âº]cav upon ROCE. The Ca²âº responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca²âº signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Canonical transient receptor potential channel 2 (TRPC2) as a major regulator of calcium homeostasis in rat thyroid FRTL-5 cells: importance of protein kinase C δ (PKCδ) and stromal interaction molecule 2 (STIM2).

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCß1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca(2+)-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca(2+)-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.

Severe neonatal MEGDHEL syndrome with a homozygous truncating mutation in SERAC1.

In the diagnostic work-up of a newborn infant with a metabolic crisis, lethal multiorgan failure on day six of life, and increased excretion of 3-methylglutaconic acid, we found using whole genome sequencing a homozygous SERAC1 mutation indicating MEGDHEL syndrome (3-methylglutaconic aciduria with deafness-dystonia, hepatopathy, encephalopathy, and Leigh-like syndrome). The SERAC1 protein is located at the contact site between mitochondria and the endoplasmic reticulum (ER) and is crucial for cholesterol trafficking. Our aim was to investigate the effect of the homozygous truncating mutation on mitochondrial structure and function. In the patient fibroblasts, no SERAC1 protein was detected, the mitochondrial network was severely fragmented, and the cristae morphology was altered. Filipin staining showed uneven localization of unesterified cholesterol. The calcium buffer function between cytoplasm and mitochondria was deficient. In liver mitochondria, complexes I, III, and IV were clearly decreased. In transfected COS-1 cells the mutant protein with the a 45-amino acid C-terminal truncation was distributed throughout the cell, whereas wild-type SERAC1 partially colocalized with the mitochondrial marker MT-CO1. The structural and functional mitochondrial abnormalities, caused by the loss of SERAC1, suggest that the crucial disease mechanism is disrupted interplay between the ER and mitochondria leading to decreased influx of calcium to mitochondria and secondary respiratory chain deficiency.

Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes.

Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.

Sphingolipid-mediated calcium signaling and its pathological effects.

Metabolites of sphingomyelin, as well as calcium ion fluxes, have a profound role in cellular signaling in almost all cell types. In addition, metabolites of sphingomyelin often modulate calcium signaling, either directly or indirectly. This is an interesting aspect on how lipids may wield their physiological role, as calcium is probably one of the most versatile signaling molecules in the cell, and as modulation of calcium signaling has profound effects on cellular physiology. The aim of this review is to discuss the mechanisms by which metabolites of sphingomyelin, especially the sphingolipids sphingosine and sphingosine 1-phosphate (S1P), modulate calcium fluxes, and how this may affect cellular function. In addition, the pathological aspects of sphingolipid-evoked modulation of calcium fluxes will be discussed.

The influence of mineral particles on fibroblast behaviour: A comparative study.

Minerals are versatile tools utilised to modify and control the physical-chemical and functional properties of substrates. Those properties include ones directing cell fate; thus, minerals can potentially provide a direct and inexpensive method to manipulate cell behaviour. This paper shows how different minerals influence human dermal fibroblast behaviour depending on their properties. Different calcium carbonates, calcium sulphates, silica, silicates, and titanium dioxide were characterised using TEM, ATR-FTIR, and zeta potential measurements. Mineral-cell interactions were analysed through MTT assay, LDH assay, calcein AM staining, live cell imaging, immunofluorescence staining, western blot, and extra/intracellular calcium measurements. Results show that the interaction of the fibroblasts with the minerals was governed by a shared period of adaptation, followed by increased proliferation, growth inhibition, or increased toxicity. Properties such as size, ion release and chemical composition had a direct influence on the cells leading to cell agglomeration, morphological changes, and the possible formation of protein-mineral complexes. In addition, zeta potential and FTIR measurements of the minerals showed adsorption of the cell culture media onto the particles. This article provides fundamental insight into the mineral-fibroblast interactions, and makes it possible to arrange the minerals according to the time-dependent cellular response.

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.

FTY720 (Fingolimod) attenuates basal and sphingosine-1-phosphate-evoked thyroid cancer cell invasion.

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 (S1P1,3) 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 S1P1, protein kinase Cα(PKCα), PKCßI, 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.

Significance of the transient receptor potential canonical 2 (TRPC2) channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion.

Mammalian transient receptor potential (TRP) channels are involved in many physiologically important processes. Here, we have studied the significance of the TRPC2 channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion, using stable TRPC2 (shTRPC2) knock-down cells. In the shTRPC2 cells, proliferation was decreased due to a prolonged G1/S cell cycle phase. The tumor suppressor p53 and the cyclin-dependant kinase inhibitors p27 and p21 were upregulated. Cell invasion, adhesion and migration were also attenuated in shTRPC2 cells, probably due to decreased activity of both Rac and calpain, and a decreased secretion and activity of matrix metalloproteinase 2. The attenuated proliferation, migration, invasion and ATP-evoked calcium entry was mimicked by overexpressing a non-conducting, truncated TRPC2 (TRPC2-DN) in wild type cells, and was reversed by overexpression of TRPC2-GFP in shTRPC2 cells. In conclusion, TRPC2 is an important regulator of rat thyroid cell function.

Complexation of c6-ceramide with cholesteryl phosphocholine - a potent solvent-free ceramide delivery formulation for cells in culture.

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.

Communication between the calcium and cAMP pathways regulate the expression of the TSH receptor: TRPC2 in the center of action.

Transient receptor potential (TRP) cation channels are widely expressed and function in many physiologically important processes. Perturbations in the expression or mutations of the channels have implications for diseases. Many thyroid disorders, as excessive growth or disturbed thyroid hormone production, can be a result of dysregulated TSH signaling. In the present study, we found that of TRP canonicals (TRPCs), only TRPC2 was expressed in Fischer rat thyroid low-serum 5% cells (FRTL-5 cells). To investigate the physiological importance of the channel, we developed stable TRPC2 knockdown cells using short hairpin RNA (shTRPC2 cells). In these cells, the ATP-evoked entry of calcium was significantly decreased. This led to increased cAMP production, because inhibitory signals from calcium to adenylate cyclase 5/6 were decreased. Enhanced cAMP signaling projected to Ras-related protein 1-MAPK kinase 1 (MAPK/ERK kinase 1) pathway leading to phosphorylation of ERK1/2. The activated ERK1/2 pathway increased the expression of the TSH receptor. In contrast, secretion of thyroglobulin was decreased in shTRPC2 cells, due to improper folding and glycosylation of the protein. We show here a novel role for TRPC2 in regulating thyroid cell function.