RELT induces cellular death in HEK 293 epithelial cells

RELT is a recently identified Tumor Necrosis Factor Receptor that possess two homologues in humans named RELL1 and RELL2. We investigated whether RELT and its homologues could induce cellular death when transiently transfected into HEK 293 epithelial cells. Transfection of RELT family members into HEK 293 epithelial cells induced cell death characterized by rounding and lifting of cells accompanied by DNA fragmentation, characteristics that are consistent with the activation of an apoptotic pathway. Overexpression of RELT in COS-7 cells resulted in cell rounding and lifting without DNA fragmentation, suggesting that the effects of RELT signaling may vary among different cell types. In summary, we report that overexpression of RELT or its homologues RELL1 and RELL2 in HEK 293 epithelial cells results in cell death with morphological characteristics consistent with the activation of an apoptotic pathway.     

Critical role of calbindin-D28k in calcium homeostasis revealed by mice lacking both vitamin D receptor and calbindin-D28k

Calbindin (CaBP)-D28k and CaBP-D9k are cytosolic vitamin D-dependent calcium-binding proteins long thought to play an important role in transepithelial calcium transport. However, recent genetic studies suggest that CaBP-D28k is not essential for calcium metabolism. Genetic ablation of this gene in mice leads to no calcemic abnormalities. Genetic inactivation of the vitamin D receptor (VDR) gene leads to hypocalcemia, secondary hyperparathyroidism, rickets, and osteomalacia, accompanied by 90% reduction in renal CaBP-D9k expression but little change in CaBP-D28k. To address whether the role of CaBP-D28k in calcium homeostasis is compensated by CaBP-D9k, we generated VDR/CaBP-D28k double knockout (KO) mice, which expressed no CaBP-D28k and only 10% of CaBP-D9k in the kidney. On a regular diet, the double KO mice were more growth-retarded and 42% smaller in body weight than VDRKO mice and died prematurely at 2.5-3 months of age. Compared with VDRKO mice, the double KO mice had higher urinary calcium excretion and developed more severe secondary hyperparathyroidism and rachitic skeletal phenotype, which were manifested by larger parathyroid glands, higher serum parathyroid hormone levels, much lower bone mineral density, and more distorted growth plate with more osteoid formation in the trabecular region. On high calcium, high lactose diet, blood-ionized calcium levels were normalized in both VDRKO and the double KO mice; however, in contrast to VDRKO mice, the skeletal abnormalities were not completely corrected in the double KO mice. These results directly demonstrate that CaBP-D28k plays a critical role in maintaining calcium homeostasis and skeletal mineralization and suggest that its calcemic role can be mostly compensated by CaBP-D9k.     

Expression of calbindin-D 28k in developing and growing chick testes

Summary Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

Expression of calbindin-D28k in developing and growing chick testes.

Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells

Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0 ± 0.1 °C) for 2, 4, 8, or 12 h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4 h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.     

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells

Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.     

Corticosterone regulates calbindin-D28k mRNA and protein levels in rat hippocampus

Corticosterone was administered to normal and bilaterally adrenalectomized rats (250-300 g), and hormonal regulation of brain calbindin-D28k (CaBP28k) levels was investigated by radioimmunoassay for CaBP28k protein and by slot and Northern blot analyses for CaBP28k mRNA. The specificity of the changes observed in CaBP28k mRNA levels was tested by reprobing blots with calmodulin and B-actin cDNAs. Rats were either adrenalectomized, adrenalectomized treated with corticosterone, intact, or intact treated with corticosterone. Chronic corticosterone administration (subcutaneous injection for 7 days, 10 mg/day) to normal intact rats significantly increased levels of CaBP28k immunoreactivity (43%) and mRNA (125%) in the hippocampus. Adrenalectomy (animals were killed 7 days after adrenalectomy) produced a significant decrease in hippocampal CaBP28k immunoreactivity (85%) and mRNA (80%) compared with intact controls. Immunocytochemical analysis of tissue sections inducated a marked depletion of CaBP28k immunoreactivity in the dentate gyrus of the hippocampus 2 weeks after adrenalectomy. When adrenalectomized rats were treated with corticosterone (10 mg/day for 7 days), CaBP28k protein and mRNA levels in hippocampus were restored to levels observed in intact controls. No changes in CaBP28k protein and mRNA in kidney, cerebellum, striatum, or cerebral cortex were noted in adrenalectomized rats or in intact rats treated with corticosterone when compared with controls, indicating the specificity of the effect on CaBP28k for the hippocampus. These studies present the first evidence of a regulator of CaBP28k gene expression in the brain.     

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.     

Human glutamylcysteine synthetase protects HEK293 cells against UV-induced cell death through inhibition of c-Jun NH2-terminal kinase

Human glutamylcysteine ligase catalytic subunit (GCLC) is the rate-limiting enzyme for glutathione synthesis. The heavy subunit possesses all the catalytic activities. UV irradiation (UV-C, 30 J/m(2)) induced apoptosis in HEK293 cells, but the morphological changes were inhibited significantly by expression of GCLC. MTS assay and flow cytometry results also indicated that GCLC and JNK1(APF) expression enhanced cellular resistance to UV irradiation. Western blotting showed that irradiation strongly activated the c-Jun NH(2)-terminal kinases (JNKs) and caspase-3 as well as p38 in HEK293 cells. Interestingly, existing data show that GCLC blocks JNK1 phosphorylation but does not affect p38 phosphorylation. Therefore, overexpression of GCLC protected HEK293 cells against UV irradiation-induced cell death by inhibiting the phosphorylation and activation of JNK1, concomitantly with the inhibition of caspase-3 activation and p21(WAF1)-luciferase activity downstream of JNK.     

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.     

Bad plays a more significant role than Bid and Bim in mediating cell death signals in batch cultures of HEK 293 cells

The expression of three BH3-only proteins, Bad, Bid and Bim, were knocked down in HEK 293 cells using vectors that express corresponding siRNAs. When cultured in the presence of 10% (v/v) serum and a diminished glucose/nutrients environment, cells lacking any one of these BH3-only proteins showed delayed cell death compared to wild type cells. Remarkably, the culture life of Bad (−) cells was extended for an additional 5 days compared to WT HEK 293 cells. In the absence of serum, the suppression of either Bad, Bim or Bid expression delayed cell death under several stress conditions. Results presented in this paper offer an insight into the functions of BH3-only proteins in mediating the death signals under different stress conditions. Anup Padmanabhan and Sen Liu contributed equally to this work.     

Calcium transport genes are differently regulated in maternal and fetal placenta in the knockout mice of calbindin-D(9k) and -D(28k)

Calbindin-D(9k) (CaBP-9k) and -D(28k) (CaBP-28k) are cytosolic proteins with EF-hand motifs that have a high affinity for calcium ions. Many types of calcium channels and intracellular calcium binding proteins, such as sodium/calcium exchangers (NCXs) and transient receptor potential cation channels (TRPVs), have been detected in the placenta. In this study, the expression of calcium channels involved in maternal-fetal calcium transport were investigated in wild-type mice versus CaBP-9k, CaBP-28k, and CaBP-9k/28k double knockout (KO) mouse models. The expression of calcium transport genes in three dissected sections of the placenta (maternal, central, and fetal) was examined on gestational day 19 (GD 19). The expression of CaBP-9k, TRPV6, TRPV5, and NCX1 mRNA was high in fetal compared to maternal placenta, while CaBP-28k was abundant in the maternal placenta. CaBP-9k was enhanced in all sections of placenta in CaBP-28k KO mice, whereas CaBP-28k was reduced in CaBP-9k KO mice. The expression of TRPV6, TRPV5, and NCX1 were induced in both maternal and fetal placentas in CaBP-9k KO mice, but were upregulated in maternal and central placentas of CaBP-28k KO mice. The levels of these proteins showed similar patterns with those of their mRNA. Placental CaBP-9k, TRPV6, TRPV5, and NCX1 proteins were abundantly expressed in the intraplacental yolk sac located in the fetal placenta. CaBP-28k did not colocalize with other calcium transport genes, although it was enriched in the placental trophoblasts of the decidual zone in the maternal placenta. These results indicate that placental TRPV6, TRPV5, and NCX1 compensate for CaBPs in CaBP-9k and/or CaBP-28k KO mice, and may take over the roles of CaBP-9k and CaBP-28k to transfer calcium ions in the placenta. Taken together, these results indicate that TRPV6, NCX1, and CaBP-9k in the fetal placenta and CaBP-28k in the maternal placenta may play key roles in controlling calcium transport across the placenta during pregnancy.     

Contribution of K(ir)2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca2(+) concentration ([Ca2(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca2(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca2(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ～15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba2(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba2(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba2(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca2(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.     

Prestin-prestin and prestin-GLUT5 interactions in HEK293T cells

The remarkable hearing sensitivity and frequency selectivity in mammals is attributed to cochlear amplifier in the outer hair cells (OHCs). Prestin, a membrane protein in the lateral wall of OHC plasma membrane, is required for OHC electromotility and cochlear amplifier. In addition, GLUT5, a fructose transporter, is reported to be abundant in the plasma membrane of the OHC lateral wall and has been originally proposed as the OHC motor protein. Here we provide evidence of interactions between prestin/prestin and prestin/GLUT5 in transiently transfected HEK293T cells. We used a combination of techniques: (1) membrane colocalization by confocal microscopy, (2) fluorescence resonance energy transfer (FRET) by fluorescence activated cell sorting (FACS), (3) FRET by acceptor photobleaching, (4) FRET by fluorescence lifetime imaging (FRET-FLIM), and (5) coimmunoprecipitation. Our results suggest that homomeric and heteromeric prestin interactions occur in native OHCs to facilitate its electromotile function and that GLUT5 interacts with prestin for its elusive function.     

Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells

Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D(28k), a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+ concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D(28k) against hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D(28k) cDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D(28k) in the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 microM) and glucose deprivation for 30-120 min. The transfection of calbindin-D(28k) reduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D(28k) cDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30-120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D(28k) gene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i.