Molecular analysis of tyrosine-and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli

Listeriolysin O (LLO) is a pore-forming cytolysin secreted by the pathogen Listeria monocytogenes and is required for its intracellular survival. We recently demonstrated that in endothelial cells, LLO activates the NF-kappaB signalling pathway. In this work, we studied the LLO-induced molecular cascade of NF-kappaB activation with a cellular model extensively used to analyse the signalling pathway of NF-kappaB activation, i.e. the human embryonic kidney HEK-293 cell line and its derivatives (transfectants or mutants). When the stably transfected derivative HEK-293 cells expressing IL-1RI were exposed to LLO, a strong NF-kappaB activation was detected, contrasting with other cell lines (HEK-293 wild type, HEK-293.T and COS) expressing a very low level of IL-1RI. Although a delayed kinetics of LLO-dependent NF-kappaB activation suggests an autocrine or paracrine IL-1-dependent pathway, we found that LLO-dependent NF-kappaB activation did not require the IL-1 protein synthesis nor the interaction with the IL-1RI specific receptor. Herein, we demonstrated that LLO-dependent NF-kappaB activation requires the activation of the IkappaB kinase beta (IKKbeta) subunit of IKK complex to phosphorylate and degrade cytoplasmic IkappaBalpha, a natural inhibitor of NF-kappaB. The activation induced by LLO does not require the adapters MyD88 and IL-1R-associated kinase (IRAK). We suggested that LLO induces a distinct signalling pathway from that of IL-1 and its receptor.     

FLASH coordinates NF-kappa B activity via TRAF2

FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-alpha-induced activation of NF-kappaB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-kappaB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-kappaB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKalpha, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856-1191 of FLASH activated NF-kappaB as efficiently as the full-length and could directly bind to TRAF2 in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-kappaB activity via a TRAF2-dependent pathway in the TNF-alpha signaling.     

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

Abstract.   Objectives : Polycystin-1 (PC1), a signalling receptor regulating Ca²⁺-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca²⁺ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca²⁺ entry (NCCE) and Ca²⁺ oscillations, with downstream effects on cell proliferation. Results and discussion : By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca²⁺ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca²⁺ oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca²⁺ oscillations and a molecular mechanism to explain the association between abnormal Ca²⁺ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.     

Myocilin Regulates Cell Proliferation and Survival

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Deficiency of polycystic kidney disease-1 gene (PKD1) expression increases A3 adenosine receptors in human renal cells: Implications for cAMP-dependent signalling and proliferation of PKD1-mutated cystic cells

Cyst growth and expansion in autosomal dominant polycystic kidney disease (ADPKD) has been attributed to numerous factors, including ATP, cAMP and adenosine signalling. Although the role of ATP and cAMP has been widely investigated in PKD1-deficient cells, no information is currently available on adenosine-mediated signalling. Here we investigate for the first time the impact of abnormalities of polycystin-1 (PC1) on the expression and functional activity of adenosine receptors, members of the G-protein-coupled receptor superfamily. Pharmacological, molecular and biochemical findings show that a siRNA-dependent PC1-depletion in HEK293 cells and a PKD1-nonsense mutation in cyst-derived cell lines result in increased expression of the A₃ adenosine receptor via an NFkB-dependent mechanism. Interestingly, A₃ adenosine receptor levels result higher in ADPKD than in normal renal tissues. Furthermore, the stimulation of this receptor subtype with the selective agonist Cl-IB-MECA causes a reduction in both cytosolic cAMP and cell proliferation in both PC1-deficient HEK293 cells and cystic cells. This reduction is associated with increased expression of p21^waf and reduced activation not only of ERK1/2, but also of S6 kinase, the main target of mTOR signalling. In the light of these findings, the ability of Cl-IB-MECA to reduce disease progression in ADPKD should be further investigated. Moreover, our results suggest that NFkB, which is markedly activated in PC1-deficient and cystic cells, plays an important role in modulating A₃AR expression in cystic cells.     

Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.     

Human Nogo-C overexpression induces HEK293 cell apoptosis via a mechanism that involves JNK-c-Jun pathway

The neurite outgrowth inhibitor protein Nogo-A has been identified as an inhibitor of axonal regeneration, and Nogo-B as a regulator of vasculature remodeling, but the additional roles of Nogo isoforms, especially Nogo-C, have obtained little attention. Nogo-C is weakly expressed in liver and kidney compared to the high expression in skeletal muscle. Here we detected the weak expression of Nogo-C in human embryonic kidney cell line HEK293, and found that Nogo-C expressed in HEK293 could induce cell apoptosis. Further experiments demonstrated the activation of JNK/SAPK and c-Jun, but not p38 in Nogo-C expressed cells. And JNK-specific inhibitor SP600125 could reduce cell apoptosis induced by Nogo-C. Furthermore, the activation of caspase-3 and PARP, the expression and phosphorylation of p53 were also detected. The data first revealed Nogo-C expressed in HEK293 confers apoptosis by inducing caspase-3 and p53 activation through the JNK-c-Jun-dependent pathway.     

Glycogen synthase kinase 3 beta is a natural activator of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1)

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1.     

The regulation of p53, p38 MAPK,JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.     

Impact of Cell Type and Epitope Tagging on Heterologous Expression of G Protein-Coupled Receptor: A Systematic Study on Angiotensin Type II Receptor

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.     

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.     

Zoledronic acid is synergic with vinblastine to induce apoptosis in a multidrug resistance protein-1 dependent way: an in vitro study

We have explored the action of zoledronic acid, which has an apoptotic effect and is used as an agent for treating skeletal metastases and osteoporosis, in the presence of vinblastine, and whether this effect is associated with MRP-1 (multidrug resistance protein-1) expression. HEK (human embryonic kidney) 293 cells were transfected to form the multidrug resistant cell line designated 293MRP (MRP-1 expressing HEK293 cells). Both lines were treated with varying concentrations of vinblastine and zoledronic acid. Apoptosis was determined by the TUNEL (deoxyuridine triphosphate nick end-labeling) method. The type of treatment, MRP-1 expression status, and the type of treatment with respect to MRP-1 expression status significantly affected (P < 0.001) the degree of apoptosis. The largest increase in cytotoxicity was noted in HEK293 cells, when 100 micromol zoledronic acid was added to 4 microg/ml vinblastine (an increment of 80.3%, P < 0.001). This preliminary work shows that zoledronic acid acts synergistically with vinblastine to induce apoptosis in an MRP-1 dependent way.     

Response of HEK293 and CHO cells overexpressing fusiogenic syncytin-1 to mitochondrion-mediated apoptosis induced by antimycin A

Apoptosis is essential for the regulation of cellular homeostasis in the placenta and is also involved in the pathophysiology of pregnancy-related diseases such as pre-eclampsia and intrauterine growth restriction (IUGR). Syncytin-1, a fusiogenic glycoprotein of endogenous-retroviral origin expressed in human trophoblasts, facilitates placental syncytium formation and is found reduced in pre-eclamptic placentas. We focus here on the mitochondrial apoptotic pathway and investigate whether the overexpression of syncytin-1 in HEK293-52 (human embryonic kidney cells) and CHO-52 cells influences the apoptotic response to the mitochondrial inhibitor antimycin A (AA). After the induction of apoptosis by 5 microM AA and incubation for up to 36 h in the absence of serum, the mean apoptotic rate was reduced by 15-30% in syncytin-1 transfected cells compared with mock-transfectants. After 12 h of challenge with AA we found lower cytochrome c levels in the cytoplasmic protein fraction and higher amounts in the mitochondrial fraction in syncytin-1 transfectants compared with mock-transfectants. We observed a decreased Mitotracker Red staining of mitochondria following AA challenge for 24 h in mock-treated CHO cells, in particular, compared with syncytin-1 transfectants. Moreover, we found a reduced activation of caspase 9 in syncytin-1 transfected HEK293-52 cells after 48 h of apoptotic challenge compared to mock-transfectants. However, a high expression of anti-apoptotic Bcl-x(L) was found in both cell types. Using syncytin-1 transfected HEK293-52 cells and CHO-52 cells, we provide initial evidence that syncytin-1 may exert its anti-apoptotic function at the mitochondrial level. A reduced release of cytochrome c followed by a diminished activation of caspase 9 is a possible mechanism.     

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

Fas ligand induces cell-autonomous NF-kappaB activation and interleukin-8 production by a mechanism distinct from that of tumor necrosis factor-alpha

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-kappaB- and AP-1 reporter constructs indicated that an FasL-induced NF-kappaB and AP-1 activity are required for maximal promoter activity. FasL induced NF-kappaB activation with slower kinetics than did TNF-alpha, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-kappaB activation by FasL. A dominant-negative mutant of IKKgamma inhibited the FasL-induced NF-kappaB activation. However, TRADD and RIP, which are essential for the TNF-alpha-induced NF-kappaB activation, were not involved in the FasL-induced NF-kappaB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-alpha-induced NF-kappaB activation. These results show that FasL induces NF-kappaB activation and IL-8 production by a novel mechanism, distinct from that of TNF-alpha. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-kappaB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-kappaB activation.     

The role of thiol reduction in hydroquinone-induced apoptosis in HEK293 cells

Hydroquinone (HQ) is a chemical used as a reducing agent, antioxidant, polymerization inhibitor, and chemical intermediate. It has a minor use as a bleaching agent in dermatologic preparations. HQ also occurs as a main metabolite of benzene. In the present study, HQ-induced apoptosis was evaluated by cell morphology changes, determination of phosphatidylserine (PS) externalization and analysis of sub-G1 cells. The effect of HQ on intracellular thiol concentration, including glutathione and protein thiol, and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on HQ-induced apoptosis were investigated. The results showed that HQ was able to induce typical apoptosis in HEK293 cells (human embryonic kidney cells) in a dose-dependent manner. Intracellular thiol, including glutathione and protein thiol, was decreased following treatment with HQ. NAC, a precursor of intracellular GSH synthesis, significantly inhibited HQ-induced apoptosis. However, BSO, a specific inhibitor of intracellular GSH synthesis, enhanced HQ-induced apoptosis significantly. Taken together, the present study demonstrates that HQ is able to induce apoptosis in HEK293 cells, most probably through depletion of intracellular thiol. The results also suggest that, at least in HEK293 cells, the control of intracellular redox homeostasis has a central role in the regulation of cell death induced by HQ.