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TOXICOLOGY
Regulates Tumor Necrosis Factor-
-Induced Stannin Gene Expression
Department of Pharmacology (B.E.R., C.D., M.L.B., J.Y.) and Jake Gittlen Cancer Research Institute (J.Y.), Penn State College of Medicine, Hershey, Pennsylvania
Received January 27, 2005; accepted March 25, 2005.
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Abstract |
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(TNF) production and that TNF treatment induces Snn gene expression in human umbilical vein endothelial cells (HUVECs). In this study, we investigated a signaling mechanism by which Snn gene expression is regulated by TMT and demonstrated that TNF stimulates Snn gene expression in a protein kinase C
-dependent manner in HUVECs in response to TMT exposure. Supporting this, we show that TMT-induced toxicity is significantly blocked by pretreatment with an anti-TNF antibody in HUVECs. Using a quantitative real-time polymerase chain reaction assay, we also show that the level of Snn gene expression is significantly increased in HUVECs in response to either TMT or TNF treatment. This TNF-induced Snn gene expression is blocked when HUVECs were pretreated with bisindolylmaleimide I, an inhibitor of protein kinase C (PKC). In contrast, when HUVECs were treated with phorbol 12-myristate 13-acetate, a PKC activator, we observed a significant increase in Snn gene expression. Using isotype-specific siRNA against PKC, we further show that knockdown of PKC, but not PKC
or PKC
, significantly blocked TNF-induced Snn gene expression. Together, these results indicate that TNF-induced, PKC-dependent Snn expression may be a critical factor in TMT-induced cytotoxicity.
; Gunasekar et al., 2001). Although organotins serve several useful functions, they are problematic to use since they are also potent neurotoxicants. This toxicity resulted in the 1995 banning of organotin use in most commercial applications worldwide (Champ, 2000). The organotin trimethyltin (TMT) causes a highly specific, reproducible pattern of selective damage to human and vertebrate nervous systems (Balaban et al., 1988). Exposure to TMT produces a consistent constellation of symptoms, including disorientation, increased aggression, and seizures (Gunasekar et al., 2001). In addition, TMT exposure causes cellular degeneration in lymphocytic tissues, kidney, and spleen (Balaban et al., 1988; Philbert et al., 2000). The selective toxicity of TMT suggests a common factor underlying organotin sensitivity.
Subtractive hybridization studies indicated that this common factor was the protein stannin (Snn); Snn was detected in a range of TMT-sensitive tissues (Krady et al., 1990; Dejneka et al., 1997). Previous studies have shown that Snn is necessary but not sufficient for TMT toxicity in vitro (Thompson et al., 1996). Snn is an 88-amino acid protein that is highly conserved throughout vertebrate evolution (Table 1; Dejneka et al., 1998). Rat and mouse Snn amino acid sequences are 100% identical, and human Snn differs by only two amino acids at the C terminus. Furthermore, mouse and human Snn nucleotide sequences are 90% identical (Dejneka et al., 1998). Such a highly conserved nature implies an important role for Snn in normal cellular function.
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Tumor necrosis factor- (TNF) is a pleiotropic cytokine known to have diverse cellular actions, including mediation of the inflammatory and immune responses (Leeuwenberg et al., 1995;Yoshizumi et al., 2004). TNF has also been linked to TMT toxicity in neuronal and mixed glial/neuronal cultures (Harry et al., 2003; Viviani et al., 2003). Specifically, these groups showed that TNF is up-regulated after TMT administration in mixed glial/neuronal cultures. Furthermore, Harry et al. (2003) observed some protection against TMT-induced cell death when cells were pretreated with a neutralizing TNF antibody. Using differential gene display, Horrevoets et al. (1999) showed that TNF treatment of human umbilical vein endothelial cells (HUVEC) induced several gene products, including Snn, indicating a possible regulatory role of TNF in the mediation of Snn expression in HUVECs. In addition, studies showed that both TNF and Snn were present in similar tissues, including the nervous and immune systems of embryonic mice (Dejneka et al., 1997; Pan et al., 1997; Yeh et al., 1998). This pattern of expression raised the possibility that the two proteins are coexpressed in specific cell types and tissues and that Snn may be a downstream component of a TNF-mediated cell-signaling pathway.
In this study, we examined the signaling events involved in TMT toxicity. We demonstrate that endothelial cells are vulnerable to TMT damage and that this damage is mediated, in part, by TNF and requires Snn. Furthermore, we showed that TMT increased Snn gene expression prior to inducing cell death. We also demonstrated that PKC was a critical modulator of TNF-induced Snn mRNA expression in both HUVECs and Jurkat T-cells. These data indicate that stannin can be induced by TNF and may further enhance cell sensitivity to TMT via PKC-mediated induction of the stannin gene product.
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Materials and Methods |
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Reagents. TNF and interleukin-1
(IL-1; Roche, Indianapolis, IN) were dissolved in sterile PBS and administered at concentrations of 200 and 10 ng/ml, respectively. The TNF neutralizing antibody (BD PharMingen, San Diego, CA) was used at 3 µg/ml, a concentration expected to neutralize 60 to 70% of TNF in culture (per manufacturer's literature). The -synuclein antibody was also used at 3 µg/ml (Santa Cruz Biochemicals, Santa Cruz, CA). Trimethyltin was a gift from James O'Callaghan (National Institute for Occupational Safety and Health, Morgantown, WV). Bisindolylmaleimide I (Calbiochem, San Diego, CA) was used at a final concentration of 10 nM and was dissolved in dimethyl sulfoxide (DMSO). Gö6976 (Calbiochem) was used at a concentration of 8 nM and was dissolved in DMSO. Phorbol 12-myristate 13-acetate (PMA; Roche) was used at a final concentration of 100 nM and was dissolved in DMSO.
Cell Viability. The trypan blue exclusion test was used as a measure of cell viability. HUVECs were incubated in 0.2% trypan blue (Sigma-Aldrich, St. Louis, MO), diluted in phosphate-buffered saline (PBS), and then subsequently washed once with PBS. The number of normal and blue-stained, dead cells were counted in four independent microscopic fields per culture, with three independent cultures being used for each condition (12 fields total). The percentage of viable cells was compared in each treatment condition with that of the untreated control condition, which was considered 100% for the purposes of relative viability.
RNA Isolation/cDNA Synthesis. RNA isolation was accomplished using the RNeasy kit, according to the protocol recommended by the manufacturer (Qiagen, Valencia, CA). Briefly, HUVECs were harvested using 0.05% trypsin-EDTA and pelleted at 2000g. Cells were then resuspended in 350 µl of RLT cell lysis buffer and homogenized using QIAshredder homogenization columns (Qiagen). The homogenized mixture was combined with an equal volume of 70% ethanol and added to an RNeasy RNA isolation column and spun at 8000g for 15 s. The RNA bound on the column was washed three times and finally eluted with RNase-free water as indicated by the manufacturer. The synthesis of cDNA was carried out using the First Strand cDNA Synthesis Kit (MBI Fermentas, Hanover, MD). This kit employs a standard M-MLV reverse transcriptase reaction and was used according to the recommendations of the manufacturer.
Quantitative Real-Time PCR. The cDNA templates from HUVECs were normalized based on their relative expression of -actin. To detect human Snn, the following primers and probe were used to amplify a 100-bp product corresponding to bases 222 to 322 of the mRNA: forward primer, 5'–TTG TCA TCC TCA TTG CCA TC–3'; reverse primer, 5'–GCT CTC CTC GTC CTC TGA CT–3'; and probe, 5'–CCT GGG CTG CTG GTG CTA CCT–3'. -Actin was detected using a predeveloped 20x primer-probe assay kit (Applied Biosystems, Foster City, CA).
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siRNA Construction. All siRNA except the siRNA specific to PKC was constructed using the Silencer siRNA Construction Kit (Ambion, Austin, TX). The following oligonucleotides were used to construct siRNA (only the sense strand is given, shown without T7 adapter sequence): control siRNA, 5'–AAA GGC ACT TAG GAC CCA GGG–3'; Snn siRNA 1, 5'–AAG GAA CCC TTC CTG CTG GTG–3'; Snn siRNA 2, 5'–AAG GGA CCG TGC GTG GAG AGA–3'; PKC siRNA 1, 5'–GCC CCT AAA GAC AAT GAA GTT–3'; PKC siRNA 2, 5'–CTT CAT TGT CTT TAG GGG CTT–3'; PKC siRNA 1, 5'–GAT GAA GGA GGC GCT CAG TT–3'; and PKC siRNA 2, 5'–GGC TGA GTT CTG GCT GGA CTT–3'.
The procedure for constructing the Snn siRNA was as outlined by Ambion. In brief, sense and antisense DNA oligonucleotides, each containing an eight-nucleotide sequence complementary to the T7 promoter, were separately hybridized to a T7 promoter and made double-stranded with Exo- Klenow DNA polymerase. Each reaction was mixed with a T7 RNA polymerase to generate the siRNA templates. Both the sense and antisense reactions were combined and incubated to form dsRNA. Finally, each double-stranded siRNA was purified and eluted into nuclease-free water. The sequences for the PKC siRNA were obtained from Irie et al. (2002), and the sequences for the PKC-specific siRNA were obtained from Yoshida et al. (2003). PKC was knocked down using SMARTpool siRNA from Upstate Cell Signaling Solutions (Charlottesville, VA).
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Contributes to Trimethyltin Toxicity. Previous work has implicated TNF as a contributing factor in TMT toxicity in neurons and glia (Harry et al., 2003; Viviani et al., 2003). To determine whether HUVECs were vulnerable to TMT, and whether TNF played a role in such toxicity, HUVECs were exposed to 10 µM TMT ± 3 µg/ml of TNF neutralizing antibodies or an -synuclein antibody, as a control, for 48 h. As shown in Fig. 1, exposure of HUVECs to 10 µM TMT resulted in 60% (±4.01) cell death. In contrast, when HUVECs were pretreated with a neutralizing anti-TNF antibody, a significant decrease (20%) in cell death was observed, implicating TNF as a factor contributing to TMT toxicity in HUVECs. Although the magnitude of cell death was less (
20%), a similar trend was observed when HUVECs were exposed to 5 µM TMT (data not shown). This trend has also been observed in other studies involving mixed neuronal/glial cocultures (Harry et al., 2002). Together, these results indicate that TNF may be an important factor in TMT-induced cytotoxicity.
Trimethyltin Induces Stannin Gene Expression in HUVECs. Trimethyltin-mediated cell death is known to require Snn (Thompson et al., 1996). However, the effect of TMT on the expression of Snn in vulnerable cell types has not been previously assayed. To characterize the genetic response of HUVECs to TMT exposure, a 5-µM dose of TMT was added to the culture medium for 1.5 to 24 h, and levels of Snn mRNA were assayed using quantitative real-time PCR. A significant increase in Snn mRNA was found in HUVECs after 1.5 h of exposure to 5 µM TMT, with a more robust induction of Snn observed after 6 h (approximately 4-fold; Fig. 2A). This induction appears to be maintained for an extended period of time, as the levels of Snn mRNA were still 2-fold higher than the level of unexposed control cultures 24 h after exposure to TMT (Fig. 2A). It is possible that the increase in Snn mRNA observed after TMT treatment is mediated by TNF. To demonstrate that TMT administration does induce the expression of TNF-responsive genes, interleukin (IL)-6, a known TNF-inducible gene, was examined for induction after 5 µM TMT administration. Significant up-regulation of IL-6 mRNA was observed after 3, 6, and 9 h of TMT exposure (Fig. 2B), which corresponds to published literature examining IL-6 levels after TMT administration (Harry et al., 2002). These results indicate that TNF-induced Snn mRNA expression may be an important component of TMT's toxic activity in HUVECs.
Stannin Knockdown Rescues HUVECs from TMT Toxicity. To effectively knock down Snn in HUVECs, we designed siRNA specific for human Snn. To validate the Snn siRNA, we transfected HUVECs with Snn siRNA using the siPORT reagent (Ambion) and allowed the cells to incubate for 48 h. The cells were then harvested, and quantitative real-time PCR (QRT-PCR) was used to assess the expression levels of Snn mRNA. As shown in Fig. 3A, each species of siRNA resulted in a significant level of knockdown (
60%) of Snn mRNA expression after 48 h. Although this was taken as a positive indication of the siRNA's ability to knock Snn mRNA expression down, a further verification was required to determine whether this results in decreased Snn protein expression as well. Currently, there are no specific anti-Snn antibodies available. Therefore, we validated our Snn siRNA by performing a functional assay. Earlier studies on Snn have shown that the Snn protein is required for TMT-induced cytotoxicity (Thompson et al., 1996); if the Snn siRNA knocks down the Snn protein, a protection from TMT toxicity should be observed. Indeed, this protective effect was observed in HUVECs transfected with Snn siRNA (Fig. 3B); a 20-nM mix of the two siRNA species (50:50 ratio) completely protected the cells from TMT-induced toxicity. These data indicate that Snn is required for the action of TMT in HUVECs.
Induction of Snn mRNA by TNF. To determine the level of Snn mRNA expression in HUVEC cells following treatment with TNF, QRT-PCR was used. Figure 4A illustrates the temporal characteristics of this induction, with significant induction of Snn mRNA occurring by 1.5 h of TNF treatment and reaching a maximum of 8-fold (±0.32) induction above control levels after 3 h of TNF treatment. By 6 and 9 h, the level of Snn mRNA expression was 5-fold (±1.53) and 3.4-fold (±1.14) above control levels, respectively. After 24 h of TNF exposure, the level of Snn mRNA in HUVEC cells returned to basal levels. Figure 4B shows a more direct representation of TNF-mediated induction of Snn mRNA expression via reverse transcription PCR (RT-PCR) followed by agarose gel electrophoresis. As a control to determine whether up-regulation of Snn mRNA was a TNF-specific effect, HUVECs were also treated with IL-1. The specificity of TNF-induced Snn gene expression was examined using IL-1, since IL-1 and TNF are both intimately involved in the inflammatory and immune responses and are capable of up-regulating some of the same gene products (Apte and Voronov, 2002). As shown in Fig. 1A, IL-1 did not significantly up-regulate Snn mRNA levels at any of the time points examined. Together, these results show that TNF specifically induces Snn mRNA expression in HUVEC cells with a relatively rapid time of induction.
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Protein Kinase C (PKC) Is Required for TNF-Induced Snn mRNA Expression. PKC is known to be vital for TMT cytotoxicity in some cell types and is activated in response to TNF signaling in HUVECs (Pavlakovic et al., 1995; Basu et al., 2002). Based on these observations, we hypothesized that PKC might mediate TNF-induced Snn gene expression. To test this hypothesis, we first used pharmacological inhibitors of PKC to determine the role of PKC in TNF's induction of Snn mRNA expression. For all experiments, QRT-PCR was used to determine levels of Snn mRNA expression. When HUVECs were pretreated with bisindolylmaleimide I, an inhibitor of the I, II, 
, , and isoforms of PKC at 10 nM (Gekeler et al., 1996), TNF-induced up-regulation of Snn mRNA expression was completely blocked (Fig. 5A). In contrast, pretreatment of HUVECs with Gö6976, an inhibitor of the and I isoforms of PKC at 8 nM (Martiny-Baron et al., 1993; Gschwendt et al., 1996), showed no significant effect on TNF-induced Snn mRNA expression (Fig. 5A). These results may indicate that one or more of the II, , , or isoforms of PKC may be responsible for the observed induction of Snn mRNA by TNF in HUVECs. To determine whether activation of PKC is sufficient to induce Snn mRNA expression in HUVECs in the absence of TNF, PMA was used as a direct activator of the classical and novel subfamilies of PKC. Exposure to 100 nM PMA for 30 min resulted in a significant up-regulation of Snn mRNA (5-fold, ±1.08) above control levels (Fig. 5B). Together, these results indicate that stimulation of one or more PKC isoforms is sufficient to induce Snn mRNA expression.
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Knockdown of PKC Epsilon via siRNA Prevents TNF-Mediated Snn Up-Regulation. A search of the literature to date indicates that only the , , , and isoforms of PKC are expressed in HUVECs and that TNF activates the , , and isoforms of PKC in HUVECs (Ross and Joyner, 1997; Mehta et al., 2001; Basu et al., 2002; Satoh et al., 2004). Therefore, we next examined the role of specific PKC isotypes as putative mediators of TNF-induced Snn mRNA expression. To examine the regulatory roles of PKC, PKC, and PKC, we used isotype-specific PKC siRNA to selectively knock down these enzymes in HUVECs. Figure 6A shows that siRNA targeted to PKC blocks TNF-stimulated induction of Snn mRNA expression, with a maximal inhibition after 48 h of exposure to 200 nM siRNA. In contrast, treatment of cells with PKC siRNA, PKC siRNA, or a control siRNA did not block TNF-induced Snn mRNA expression. Together, these results demonstrate that PKC, but not PKC or PKC, modulates TNF-induced increased Snn mRNA expression in HUVECs. Figure 6B shows a representative Western blot of PKC knockdown after siRNA treatment in HUVECs. To assess the cell-type specificity of PKC-mediation of TNF-induced Snn expression, we examined Jurkat T-cells, which have been shown to express Snn (Thompson et al., 1996). As shown in Fig. 7, Jurkat T-cells also showed an increase in Snn mRNA in response to TNF treatment in a PKC-dependent manner. Based on these results involving two different Snn-expressing cell lines responding to TNF with a PKC-mediated increase in Snn mRNA expression, it is suggested that this might be a common mechanism of TMT-induced toxicity in Snn-expressing cell types.
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; Thompson et al., 1996). Snn's high degree of sequence conservation across diverse vertebrate species suggests an important role in cell function (Table 2). As a result, antibodies specific for Snn have been difficult to develop. To date, very few single nucleotide polymorphisms have been characterized in the coding portion of the Snn gene, suggesting that mutations to Snn are detrimental (http://www.ncbi.nlm.nih.gov). Although the only known function for Snn involves mediating TMT toxicity, it is obvious that Snn has not been conserved across vertebrates for this purpose. However, it is possible that levels of Snn can lead to enhanced or reduced sensitivity to TMT toxicity.
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In this study, we presented the first evidence of TMT toxicity in endothelial cells and showed that Snn is required for TMT toxicity. Moreover, we demonstrated that TNF--induced Snn gene expression is regulated by protein kinase C, thus providing insight as to how TMT exposure may result in cell death. Our work showed that although Snn is required for TMT toxicity in HUVECs, blocking TNF- does not completely rescue HUVECs. However, siRNA knockdown of Snn completely protects HUVECs from TMT toxicity. There are at least two possible reasons for this observation. 1) The level of neutralization of TNF- expected using the anti-TNF- antibody was most likely not complete; it is possible that the remaining unbound TNF- was able to effectively up-regulate Snn enough to allow TMT toxicity in some HUVECs. 2) It is possible that Snn is regulated by additional signaling pathways. In either case, it is clear from our work and that of others (Harry et al., 2003) that TNF, although involved, is not a causal part of the TMT signaling cascade.
Since PKC has been implicated as having a regulatory role in both TNF-initiated signaling events and TMT toxicity (Pavlakovic et al., 1995; Basu et al., 2002), the functional role of PKC in Snn gene expression is not surprising. Studies have demonstrated that PKC is involved in several cellular processes, including cellular proliferation (Akita, 2002; Soh and Weinstein, 2003), cell death (Comalada et al., 2003; Jung et al., 2004), and the immune response (Aksoy et al., 2004). The apparent regulation of stannin by PKC is supported by the observation that stimulation of PKC in vitro results in cell death in response to lipopolysaccharide (Comalada et al., 2003) and oxidative stress (Jung et al., 2004). In addition, PKC is found in all the major TMT-sensitive tissues/cells, including neurons (Jung et al., 2004), glia (Xiao et al., 1994), B- and T-cells (Krappmann et al., 2001), and lung (Lang et al., 2004).
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-induced Snn gene expression is not yet known. In our previous study, we have identified 26 putative transcription factor-binding sites on the Snn gene promoter region (Dejneka et al., 1998). However, the functionality of these sites has not been determined. A review of the literature, together with the data presented in this report, implicates the human aryl hydrocarbon receptor nuclear translocator (hARNT) as a possible candidate for regulating Snn expression in HUVEC cells. Several characteristics of hARNT support this hypothesis. First, a hARNT consensus binding sequence is present in the Snn promoter region (Dejneka et al., 1998). Second, hARNT has been shown to be regulated by PKC (Long et al., 1999); this attribute provides a mechanism whereby Snn expression would be induced by PKC. Third, hARNT is active in endothelial cells and Jurkat cells (Adelman et al., 1999; Nagai et al., 2002).
In addition to transcription factors, it is possible that TNF-induced mRNA up-regulation is modulated by additional mechanisms. One such mechanism may involve mRNA stability. The Snn mRNA has a very long 3'-untranslated region (UTR; Dejneka et al., 1998); the 3'-UTR of mRNAs is a significant source of regulatory control (Ostareck-Ledrer et al., 1994; Mendez et al., 2000), and the binding of regulatory elements to this region of the mRNA may well regulate the stability of the message and the rate of translation in the cell. Analysis of the Snn mRNA sequence has uncovered nine candidate iron regulatory elements in the Snn mRNA. Several articles have been published regarding TNF's role in iron homeostasis in the cell as well as the effect of PKC on iron regulatory protein-iron responsive element binding in vitro (Kwak et al., 1995; Thomson et al., 2000; Xiong et al., 2003). Thus, it is possible that the pattern of Snn induction resulting from stimulation by TNF or PMA is due to a modulation of regulatory factors existing in the Snn mRNA's 3'-UTR.
We have previously shown that Snn is primarily localized to the mitochondria (Davidson et al., 2004). This localization implies that mitochondrial dysfunction may be an integral downstream event initiated by Snn in TMT toxicity. Supporting this, multiple studies have demonstrated that mitochondrial dysfunction occurs in vitro after TMT exposure (Stine et al., 1988; Skarning et al., 2002). In summary, we demonstrated that Snn is a critical signaling molecule that mediates TMT-induced cytotoxicity in a TNF-PKC-dependent manner. Based on evidence from our study and previous studies, we propose a model of TMT-induced cytotoxicity, which is illustrated in Fig. 8.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: TMT, trimethyltin; Snn, stannin; TNF, tumor necrosis factor-; HUVEC, human umbilical vein endothelial cell(s); PKC, protein kinase C; IL-1, interleukin-1; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase chain reaction; IL, interleukin; QRT-PCR, quantitative real-time PCR; RT-PCR, real-time PCR; hARNT, human aryl hydrocarbon receptor nuclear translocator; UTR, untranslated region.
Address correspondence to: Melvin Billingsley, Department of Pharmacology H078, 500 University Drive, Hershey, PA 17033. E-mail address: mlb8{at}psu.edu
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