GNAS mutation inhibits growth and induces phosphodiesterase 4D expression in colorectal cancer cell lines.

Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.

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 for 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 to 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 re-expression 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.

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.

Transient Receptor Potential Canonical (TRPC) Channels as Modulators of Migration and Invasion.

Calcium (Ca2+) is perhaps the most versatile signaling molecule in cells. Ca2+ regulates a large number of key events in cells, ranging from gene transcription, motility, and contraction, to energy production and channel gating. To accomplish all these different functions, a multitude of channels, pumps, and transporters are necessary. A group of channels participating in these processes is the transient receptor potential (TRP) family of cation channels. These channels are divided into 29 subfamilies, and are differentially expressed in man, rodents, worms, and flies. One of these subfamilies is the transient receptor potential canonical (TRPC) family of channels. This ion channel family comprises of seven isoforms, labeled TRPC1-7. In man, six functional forms are expressed (TRPC1, TRPC3-7), whereas TRPC2 is a pseudogene; thus, not functionally expressed. In this review, we will describe the importance of the TRPC channels and their interacting molecular partners in the etiology of cancer, particularly in regard to regulating migration and invasion.

Comparison of Polydopamine-Coated Mesoporous Silica Nanorods and Spheres for the Delivery of Hydrophilic and Hydrophobic Anticancer Drugs.

Mesoporous silica nanoparticles (MSNs) have been widely studied as drug delivery systems in nanomedicine. Surface coating of MSNs have enabled them to perform efficiently in terms of bioavailability, biocompatibility, therapeutic efficacy and targeting capability. Recent studies have suggested the use of polydopamine (PDA) as a facilitative coating for MSNs that provides sustained and pH-responsive drug release, owing to the adhesive "molecular-glue" function of PDA. This further endows these hybrid MSN@PDA particles with the ability to carry large amounts of hydrophilic drugs. In this study, we expand the feasibility of this platform in terms of exploring its ability to also deliver hydrophobic drugs, as well as investigate the effect of particle shape on intracellular delivery of both a hydrophilic and hydrophobic anticancer drug. MSN@PDA loaded with doxorubicin (hydrophilic) and fingolimod (hydrophobic) was studied via a systematic in vitro approach (cellular internalization, intracellular drug distribution and cytotoxicity). To promote the cellular uptake of the MSN@PDA particles, they were further coated with a polyethylene imine (PEI)-polyethylene glycol (PEG) copolymer. Drug-loaded, copolymer-coated MSN@PDA showed effective cellular uptake, intracellular release and an amplified cytotoxic effect with both doxorubicin and fingolimod. Additionally, rods exhibited delayed intracellular drug release and superior intracellular uptake compared to spheres. Hence, the study provides an example of how the choice and design of drug delivery systems can be tuned by the need for performance, and confirms the PDA coating of MSNs as a useful drug delivery platform beyond hydrophilic drugs.

Sphingolipids inhibit vimentin-dependent cell migration.

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

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): old name-new games. Importance in regulating of rat thyroid cell physiology.

In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation.

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.

Functional coupling of TRPC2 cation channels and the calcium-activated anion channels in rat thyroid cells: implications for iodide homeostasis.

The initial step in a synthesis of thyroid hormones is the uptake of iodide from the circulation. Iodide (I(-)) is transported into thyroid cells via a Na(+)/I(-) symporter (NIS), which is electrogenic and thus sensitive to alterations in membrane potential (V(m)). I(-) is then released to the lumen of thyroid follicles where the hormones are synthesised and stored. The mechanisms of I(-) release to follicle lumen are poorly characterised. Our whole-cell voltage clamp recordings revealed the presence of a Ca(2+) activated Cl(-) 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(-). 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(2+) 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(-) release via ANO channels and I(-) uptake via the V(m) sensitive NIS.

Proliferation and migration of ML1 follicular thyroid cancer cells are inhibited by IU1 targeting USP14: role of proteasome and autophagy flux.

USP14 is a deubiquitinating enzyme involved in protein degradation by interacting with the proteasome and removal of poly-ubiquitin chains on target proteins. USP14 can influence cellular processes such as cell survival, DNA repair, ER stress, endocytosis, and the inflammatory response. USP14 further plays a role in tumor growth, and the inhibition of USP14 by compounds such as IU1 may affect cancer cell migration and invasion. Here we have studied the mechanisms for the action of IU1 in ML1 follicular thyroid cancer cells, comparing them with control, primary thyroid cells. Treatment with IU1 reduced proliferation of ML1 cells in a concentration-dependent manner, and more prominently than in control cells. IU1 decreased basal migration of ML1 cells, and after stimulation of cells with the bioactive compound, sphingosine-1-phosphate. The sphingosine-1-phosphate receptor 3 was increased in ML1 cells as compared with control thyroid cells, but this was not influenced by IU1. Further studies on the mechanism, revealed that IU1 enhanced the proteasome activity as well as LC3B-dependent autophagy flux in ML1 cells with an opposite effect on control thyroid cells. This indicates that IU1 elicits a cell-type dependent autophagy response, increasing it in ML1 cancer cells. The IU1-mediated stimulation of autophagy and proteasomes can likely contribute to the reduced cell proliferation and migration observed in ML1 cells. The precise set of proteins affected by IU1 in ML1 thyroid and other cancer cells warrant further investigations.

Suppression of Calcium Entry Modulates the Expression of TRß1 and Runx2 in Thyroid Cancer Cells, Two Transcription Factors That Regulate Invasion, Proliferation and Thyroid-Specific Protein Levels.

The thyroid hormone receptor beta 1 (TRß1) is downregulated in several human cancer cell types, which has been associated with development of an aggressive tumor phenotype and the upregulation of Runt-related transcription factor 2 (Runx2). In this study, we show that the expression of TRß1 protein is downregulated in human thyroid cancer tissues and cell lines compared with the normal thyroid tissues and primary cell line, whilst Runx2 is upregulated under the same conditions. In contrast, the expression of TRß1 is upregulated, whereas Runx2 is downregulated, in STIM1, Orai1 and TRPC1 knockdown cells, compared to mock transfected cells. To study the functional significance of Runx2 in follicular thyroid cancer ML-1 cells, we downregulated it by siRNA. This increased store-operated calcium entry (SOCE), but decreased cell proliferation and invasion. Moreover, restoring TRß1 expression in ML-1 cells decreased SOCE, basal and sphingosine 1-phosphate (S1P)-evoked invasion, the expression of the promigratory S1P3 receptor and pERK1/2, and at the same time increased the expression of the thyroid specific proteins thyroglobulin, thyroperoxidase, and thyroid transcription factor-1. In conclusion, we show that TRß1 is downregulated in thyroid cancer cells and that restoration of its expression can reverse the cancer cell phenotype towards a normal thyroid cell phenotype.

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.

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol.

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein-dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.

TRPC2: of mice but not men.

Relatively little is known in regard to the physiological significance of TRPC2 and its regulation or interaction with other calcium regulating signalling molecules. In rodents, however, the importance of TRPC2 is indisputable. In mice, transcripts for TRPC2 have been found in testis, sperm, in neurons in the vomeronasal organ, and both in the dorsal root ganglion and in the brain. In rats, TRPC2 is thought to be expressed exclusively in the vomeronasal organ. In mice, TRPC2 is of importance in regulating both sexual and social behaviour. In sperm, TRPC2 is of importance in the acrosome reaction. This review will summarize the known physiological effects of TRPC2 channels, and the regulation of the function of the channel. In addition, some new preliminary data on the role of TRPC2 in rat thyroid cells will be presented.

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.

Sphingolipids as Modulators of SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic with severe consequences for afflicted individuals and the society as a whole. The biology and infectivity of the virus has been intensively studied in order to gain a better understanding of the molecular basis of virus-host cell interactions during infection. It is known that SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) via its spike protein. Priming of the virus by specific proteases leads to viral entry via endocytosis and to the subsequent steps in the life cycle of SARS-CoV-2. Sphingosine and ceramide belong to the sphingolipid family and are abundantly present in cell membranes. These lipids were recently shown to interfere with the uptake of virus particles of SARS-CoV-2 into epithelial cell lines and primary human nasal cells in culture. The mechanisms of action were partly different, as sphingosine blocked, whilst ceramide facilitated viral entry. Acid sphingomyelinase (ASM) is vital for the generation of ceramide and functional inhibition of ASM by drugs like amitriptyline reduced SARS-CoV-2 entry into the epithelial cells. Recent data indicates that serum level of sphingosine-1-phosphate (S1P) is a prognostic factor for COVID-2 severity. Further, stimulation of sphingosine-1-phosphate receptor 1 (S1PR1) might also constrain the hyper-inflammatory conditions linked to SARS-CoV-2. Here, we review recent exciting findings regarding sphingolipids in the uptake of SARS-CoV-2 and in the course of COVID-19 disease. More studies are required on the mechanisms of action and the potential use of antidepressant drugs and sphingolipid modifiers in SARS-CoV-2 infections and in the treatment of the more serious and fatal consequences of the disease.

Transmembrane Prolyl 4-Hydroxylase is a Novel Regulator of Calcium Signaling in Astrocytes.

Prolyl 4-hydroxylases (P4Hs) have vital roles in regulating collagen synthesis and hypoxia response. A transmembrane P4H (P4H-TM) is a recently identified member of the family. Biallelic loss of function P4H-TM mutations cause a severe autosomal recessive intellectual disability syndrome in humans, but functions of P4H-TM are essentially unknown at cellular level. Our microarray data on P4h-tm-/- mouse cortexes where P4H-TM is abundantly expressed indicated expression changes in genes involved in calcium signaling and expression of several calcium sequestering ATPases was upregulated in P4h-tm-/- primary mouse astrocytes. Cytosolic and intraorganellar calcium imaging of P4h-tm-/- cells revealed that receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE) and calcium re-uptake by mitochondria were compromised. HIF1, but not HIF2, was found to be a key mediator of the P4H-TM effect on calcium signaling. Furthermore, total internal reflection fluorescence (TIRF) imaging showed that calcium agonist-induced gliotransmission was attenuated in P4h-tm-/- astrocytes. This phenotype was accompanied by redistribution of mitochondria from distal processes to central parts of the cell body and decreased intracellular ATP content. Our data show that P4H-TM is a novel regulator of calcium dynamics and gliotransmission.

Human adipocyte differentiation and composition of disease-relevant lipids are regulated by miR-221-3p.

MicroRNA-221-3p (miR-221-3p) is associated with both metabolic diseases and cancers. However, its role in terminal adipocyte differentiation and lipid metabolism are uncharacterized. miR-221-3p or its inhibitor was transfected into differentiating or mature human adipocytes. Triglyceride (TG) content and adipogenic gene expression were monitored, global lipidome analysis was carried out, and mechanisms underlying the effects of miR-221-3p were investigated. Finally, cross-talk between miR-221-3p expressing adipocytes and MCF-7 breast carcinoma (BC) cells was studied, and miR-221-3p expression in tumor-proximal adipose biopsies from BC patients analyzed. miR-221-3p overexpression inhibited terminal differentiation of adipocytes, as judged from reduced TG storage and gene expression of the adipogenic markers SCD1, GLUT4, FAS, DGAT1/2, AP2, ATGL and AdipoQ, whereas the miR-221-3p inhibitor increased TG storage. Knockdown of the predicted miR-221-3p target, 14-3-3γ, had similar antiadipogenic effects as miR-221-3p overexpression, indicating it as a potential mediator of mir-221-3p function. Importantly, miR-221-3p overexpression inhibited de novo lipogenesis but increased the concentrations of ceramides and sphingomyelins, while reducing diacylglycerols, concomitant with suppression of sphingomyelin phosphodiesterase, ATP citrate lyase, and acid ceramidase. miR-221-3p expression was elevated in tumor proximal adipose tissue from patients with invasive BC. Conditioned medium of miR-221-3p overexpressing adipocytes stimulated the invasion and proliferation of BC cells, while medium of the BC cells enhanced miR-221-3p expression in adipocytes. Elevated miR-221-3p impairs adipocyte lipid storage and differentiation, and modifies their ceramide, sphingomyelin, and diacylglycerol content. These alterations are relevant for metabolic diseases but may also affect cancer progression.