Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.     

The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc

TRPC6 are plasma membrane cation channels. By means of live-cell imaging and spectroscopic methods, we found that HEK cells expressing TRPC6 channels (HEK-TRPC6) are enriched in zinc and sulphur and have a reduced copper content when compared to HEK cells and HEK cells expressing TRPC3 channels (HEK-TRPC3). Hence, HEK-TRPC6 cells have larger pools of mobilizable Zn2+ and are more sensitive to an oxidative stress. Synchrotron X-ray fluorescence experiments showed a higher zinc content in the nuclear region indicating that the intracellular distribution of this metal was influenced by the over-expression of TRPC6 channels. Their properties were investigated with the diacylglycerol analogue SAG and the plant extract hyperforin. Electrophysiological recordings and imaging experiments with the fluorescent Zn2+ probe FluoZin-3 demonstrated that TRPC6 channels form Zn2+-conducting channels. In cortical neurons, hyperforin-sensitive channels co-exist with voltage-gated channels, AMPA and NMDA receptors, which are known to transport Zn2+. The ability of these channels to regulate the size of the mobilizable pools of Zn2+ was compared. The data collected indicate that the entry of Zn2+ through TRPC6 channels can up-regulate the size of the DTDP-sensitive pool of Zn2+. By showing that TRPC6 channels constitute a Zn2+ entry pathway, our study suggests that they could play a role in zinc homeostasis.     

Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells

Endogenously expressed canonical transient receptor potential (TRPC) homologs were investigated for their role in forming store-operated, 1-oleoyl-2-acetyl-sn-glycerol-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba(2+) entry indicated that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by small interfering RNA (siRNA) techniques, dramatically inhibited (52-68%) store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 had no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gave only slightly more inhibition of SOCE (74-78%) than seen with suppression of TRPC1 alone (68%), suggesting that these three TRPC homologs work in tandem to mediate a large component of SOCE. Evidence from co-immunoprecipitation experiments indicates that a TRPC1-TRPC3-TRPC7 complex, predicted from siRNA results, does exist. The suppression of either TRPC3 or TRPC7, but not TRPC1, induced a high Ba(2+) leak flux that was inhibited by 2-APB and SKF96365, suggesting that the influx is via leaky store-operated channels. The high Ba(2+) leak flux is eliminated by co-suppression of TRPC1-TRPC3 or TRPC1-TRPC7. For 1-oleoyl-2-acetyl-sn-glycerol-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba(2+) entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6, and TRPC7. CCh-stimulated Ba(2+) entry, on the other hand, could be inhibited by suppression of any of the five endogenously expressed TRPC homologs, with the degree of inhibition being consistent with CCh stimulation of both store-operated and receptor-operated channels. In summary, endogenous TRPC1, TRPC3, and TRPC7 participate in forming heteromeric store-operated channels, whereas TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels.     

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells

Background information. TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. Results. Stably transfected HEK‐293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK‐TrxR15 and HEK‐TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK‐TrxR15 and HEK‐TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F‐actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCδ by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCδ, and PKCδ siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. Conclusions. The overexpression of TrxR1 inhibits migration of HEK‐293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCδ activation.     

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.     

Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha 1-subunit in HEK-293 cells

Insulin stimulates K⁺ uptake andNa⁺ efflux via the Na⁺-K⁺ pump inkidney, skeletal muscle, and brain. The mechanism of insulin action inthese tissues differs, in part, because of differences in the isoformcomplement of the catalytic -subunit of theNa⁺-K⁺ pump. To analyze specifically the effectof insulin on the ₁-isoform of the pump, we have studiedhuman embryonic kidney (HEK)-293 cells stably transfected with the ratNa⁺-K⁺ pump ₁-isoform tagged onits first exofacial loop with a hemagglutinin (HA) epitope. The plasmamembrane content of ₁-subunits was quantitated bybinding a specific HA antibody to intact cells. Insulin rapidly increased the number of ₁-subunits at the cell surface.This gain was sensitive to the phosphatidylinositol (PI) 3-kinaseinhibitor wortmannin and to the protein kinase C (PKC) inhibitorbisindolylmaleimide. Furthermore, the insulin-stimulated gain insurface -subunits correlated with an increase in the binding of anantibody that recognizes only the nonphosphorylated form of₁ (at serine-18). These results suggest that insulinregulates the Na⁺-K⁺ pump in HEK-293 cells, atleast in part, by decreasing serine phosphorylation and increasingplasma membrane content of ₁-subunits via a signalingpathway involving PI 3-kinase and PKC.

     

Imaging of intracellular calcium stores in single permeabilized lens cells

Intracellular Ca²⁺ storesin permeabilized sheep lens cells were imaged with mag-fura 2 tocharacterize their distribution and sensitivity toCa²⁺-releasing agents. Inositol1,4,5-trisphosphate (IP₃) orcyclic ADP-ribose (cADPR) releasedCa²⁺ from intracellularCa²⁺ stores that were maintainedby an ATP-dependent Ca²⁺ pump. TheIP₃ antagonist heparin inhibitedIP₃- but not cADPR-mediated Ca²⁺ release, whereas the cADPRantagonist 8-amino-cADPR inhibited cADPR- but notIP₃-mediatedCa²⁺ release, indicating thatIP₃ and cADPR were operatingthrough separate mechanisms. ACa²⁺ store sensitive toIP₃, cADPR, and thapsigarginappeared to be distributed throughout all intracellular regions. Insome cells a Ca²⁺ storeinsensitive to IP₃, cADPR,thapsigargin, and 2,4-dinitrophenol, but not ionomycin, was present ina juxtanuclear region. We conclude that lens cells containintracellular Ca²⁺ stores that aresensitive to IP₃, cADPR, andthapsigargin, as well as a Ca²⁺store that appears insensitive to all these agents.     

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.¹ In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca²⁺ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca²⁺ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca²⁺ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation. calcium signaling; nonselective cation channels     

Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.     

Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells

The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidneyHEK-293 cells, and the transport characteristics were measured in cellstransfected with a vector containing NaPi-3 or with the vector alone(sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na andPO4 with K Na1/2 of 128 mM(PO4 = 0.1 mM) andK PO41/2of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfectedcells expressing the transporter. Transfection had no effect on theNa-independent 32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenateinhibited flux with an inhibition constant of 0.4 mM. The phosphatetransport in sham- and NaPi-3-transfected cells has nearly the sametemperature dependence in the absence and presence of extracellularNa. The Na-dependent phosphate flux decreased with pH insham-transfected cells but was pH independent in transfected cells. TheNa-dependent32PO4influx was inhibited byp-chloromercuriphenylsulfonate,phosphonoformate, phloretin, vanadate, and5-(N-methyl-N-isobutyl)-amiloridebut not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior ofNaPi-3 homologues in the renal tubule of other species and, thus,demonstrate the fidelity of this transfection system for the study ofthis protein. Commensurate with the increased functional expression,there was an increase in the amount of NaPi-3 protein by Westernanalysis.

     

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer’s disease. However, its physiological function remains elusive. Cu²⁺ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu²⁺ reduction and ⁶⁴Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu²⁺ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu²⁺ ions. Moreover, wild-type cells exposed to both Cu²⁺ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu²⁺ reductase activity and increased ⁶⁴Cu uptake. We conclude that Cu²⁺ reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.     

Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells

A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed. Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B). The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated. The 293H cells accumulate 4-5 microg trehalose/mg dry weight and this concentration increases to 7-10 microg trehalose/mg dry weight if trehalose is included in the growth medium. The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome. When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected. Inclusion of 0.3 microg/ml tetracycline in the growth medium results in the accumulation of 11-14 microg trehalose/mg dry weight, a value which increases to 19-20 microg trehalose/mg dry weight if trehalose is included in the growth medium. The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline. This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration.     

Enhancement of intracellular sphingosine-1-phosphate production by inositol 1,4,5-trisphosphate-evoked calcium mobilisation in HEK-293 cells: endogenous sphingosine-1-phosphate as a modulator of the calcium response

Sphingosine-1-phosphate (S1P) regulates many cellular functions, such as migration, differentiation and growth. The effects of S1P are thought to be primarily mediated by G-protein coupled receptors, but an intracellular function as a calcium releasing second messenger has also been proposed. Here we show that in HEK-293 cells, exogenous S1P mobilises sequestered calcium by a mechanism primarily dependent on the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) pathway, and secondarily on the subsequent synthesis of intracellular S1P. Stimulating HEK-293 cells exogenously with S1P increased the production of both inositol phosphates and intracellular S1P. The calcium response was inhibited in cells treated with 2-APB, caffeine or U73122, showing that the PLC/IP3 pathway for calcium release is activated in response to exogenous S1P. The calcium response was partially inhibited in cells treated with the sphingosine kinase inhibitor DMS and in cells expressing a catalytically inactive sphingosine kinase, showing that endogenously produced S1P is also involved. Importantly, 2-APB and U73122 inhibited the S1P-evoked production of intracellular S1P. S1P is therefore not likely a major calcium releasing second messenger in HEK-293 cells, but rather a secondary regulator of calcium mobilisation.     

Effectors of the frequency of calcium oscillations in HEK-293 cells: wavelet analysis and a computer model

Oscillations of the intracellular concentration of Ca²⁺ in cultured HEK-293 cells, which heterologously expressed the calcium-sensing receptor, were recorded with the fluorophore Fura-2 using fluorescence microscopy. HEK-293 cells are extremely sensitive to small perturbations in extracellular calcium concentrations. Resting cells were attached to cover slips and perifused with saline solution containing physiologically relevant extracellular Ca²⁺ concentrations in the range 0.5–5 mM. Acquired digitized images of the cells showed oscillatory fluctuations in the intracellular Ca²⁺ concentration over the time course, and were processed as a function of the change in Fura-2 excitation ratio and frequency at 12–37°C. Newly developed data processing techniques with wavelet analysis were used to estimate the frequency at which the rectified sinusoidal oscillations occurred; we estimated ~4 min⁻¹ under normal conditions. Temperature variations revealed an Arrhenius relationship in oscillation frequency. A critical Ca²⁺ concentration of ~2 mM was estimated, below which oscillations did not occur. These data were used to develop a kinetic model of the system that was simulated using Mathematica; kinetic parameter values were adjusted to match the experimentally observed oscillations of intracellular Ca²⁺ concentration as a function of extracellular Ca²⁺ concentration, and temperature; and from these, limit cycles were obtained and control coefficients were estimated for all parameters.     

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

There is an imperative need for expression systems allowing the efficient and robust manufacturing of high quality glycoproteins. In the present work, HEK-293 cells stably expressing interferon-α2b were further engineered with the insertion of the yeast pyruvate carboxylase 2 gene. In batch cultures, marked reductions in lactate and ammonia production were observed compared to the parental cell clone. Although the maximum specific growth rate remained unchanged, the altered metabolism led to a 2-fold increase in maximum cell density and 33% increase in the integral of viable cell concentration and interferon production yield. The underlying metabolic changes were further investigated using various ¹³C-labeled substrates and measuring the resulting lactate mass isotopomer distributions. Simultaneous metabolite and isotopomer balancing allowed the accurate determination of key intracellular fluxes. Such detailed and quantitative knowledge about the central carbon metabolism of the cells is instrumental to further support the development of high-yield fed-batch processes.     

Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis

HEK-293 is the most extensively used human cell line for the production of viral vectors and is gaining increasing attention for the production of recombinant proteins by transient transfection. To further improve the metabolic characterization of this cell line, we have performed cultures using ¹³C-labeled substrates and measured the resulting mass isotopomer distributions in lactate by LC/MS. Simultaneous metabolite and isotopomer balancing allowed improvement and validation of the metabolic model and quantification of key intracellular pathways. We have determined the amounts of glucose carbon channeled through the PPP, incorporated into the TCA cycle for energy production and lipids biosynthesis, as well as the cytosolic and mitochondrial malic enzyme fluxes. Our analysis also revealed that glutamine did not significantly contribute to lactate formation. An improved and quantitative understanding of the central carbon metabolism is greatly needed to pursue the rational development of engineering approaches at both the cellular and process levels.     

TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells

Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca(2+)](i)) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca(2+)](i), which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCgamma1 by RNA interference inhibited the Epo-stimulated increase in [Ca(2+)](i) in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCgamma, and substitution of predicted PLCgamma Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCgamma and inhibited the rise in [Ca(2+)](i). Substitution of Tyr(226) alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca(2+)](i) but not the association of PLCgamma with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP(3)). To determine whether IP(3) is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP(3) receptor (IP(3)R) binding domains. In cells expressing TRPC3 with mutant IP(3)R binding sites and Epo receptor, interaction of IP(3)R with TRPC3 was abolished, and Epo-modulated increase in [Ca(2+)](i) was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCgamma-mediated process that requires interaction of PLCgamma and IP(3)R with TRPC3. They also show that TRPC3 Tyr(226) is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.     

Release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.