Regular Article
Comprehensive Analysis of BIRC Family Gene Expression Changes by Mercury Compounds and Arsenic Exposure in Neuroblastoma, Kidney, and Hepatic Cells
2018 Volume 1 Issue 1 Pages 20-24
Details
2018 Volume 1 Issue 1 Pages 20-24
Several metal(loid) toxicants can trigger cytotoxicity by causing apoptosis. Our previous study demonstrated that cadmium induces apoptosis by suppressing expression of the apoptosis inhibitor, BIRC3 [Baculoviral inhibition of apoptosis (IAP) protein repeat containing 3], in human proximal tubular cells (HK-2 cells). BIRC3 is a member of the BIRC family, which consists of eight family members in human, and seven in mouse. The observed suppression of BIRC3 gene expression was mostly specific to cadmium and to HK-2 cells. In this study, we examined whether methylmercury, inorganic mercury, or arsenic may affect the gene expression of other BIRC family members in several different cultured cells. Methylmercury decreased the level of BIRC2 mRNA and increased the level of BIRC5 mRNA in human IMR-32 neuroblastoma cells. Methylmercury increased the mRNA levels of BIRC2, BIRC5, and BIRC8 and decreased the mRNA levels of BIRC6 in HK-2 cells. Inorganic mercury increased the mRNA levels of BIRC2 and BIRC5 in HK-2 cells. Finally, arsenic increased the levels of Birc1 and Birc7 and decreased the levels of Birc2, Birc4, Birc5, and Birc6 mRNA in mouse normal hepatic AML-12 cells. Taken together, these results indicate that each metal(loid) toxicant may regulate the gene expression of BIRC family members in different manners. Therefore, each BIRC family member may play distinct roles when various tissues are exposed to toxic heavy metals or metalloid toxicants.
Toxic heavy metals and metalloids such as cadmium, mercury, and arsenic can induce apoptotic cell death in their target tissues.1-4) Our previous study demonstrated that cadmium, which causes chronic renal toxicity, induced apoptosis in human proximal tubular cells (HK-2 cells) and in the mouse kidney.5,6) Strikingly, cadmium-induced apoptosis involves BIRC3 gene suppression.5) BIRC3 is a member of the BIRC family of apoptosis inhibitors, which consists of eight family members in human and seven family members in mouse.7-9) In HK-2 cells, cadmium induces apoptosis by reducing BIRC3 expression through transcription suppression.5) The BIRC family includes BIRC1/NAIP, BIRC2/cIAP1, BIRC3/cIAP2, BIRC4/XIAP, BIRC5/Survivin, BIRC6/Apollon, BIRC7/ML-IAP, and BIRC8/ILP.7-9) Interestingly, of the eight BIRC family members, cadmium only reduced BIRC3 mRNA levels in HK-2 cells.5) BIRC family members possess one or more baculovirus IAP repeat (BIR) domain that selectively inhibits the activity of caspase-9, -3, or -7.7-9) The activation of caspase-3 is involved in cadmium-induced BIRC3-mediated apoptosis.5) We examined the effect of mercury compounds and arsenic, which are known to induce apoptosis, on the gene expression of BIRC3 (Birc3) and caspase-3 activity in various cultured cells.5) Those results showed that suppression of BIRC3 gene expression was mostly specific to cadmium and to proximal tubular cells. In addition to BIRC3, other BIRC family members are involved in apoptosis inhibition. This suggests that although BIRC3 may not be involved in apoptosis induced by mercury compounds and arsenic, different BIRC family members may be involved in regulating the apoptosis induced by these toxins. In this study, we examined weather methylmercury, inorganic mercury, and arsenic affect the gene expression levels of BIRC family members using HK-2, IMR-32 (human neuroblastoma), and AML-12 (mouse hepatic) cells.
HK-2 cells were purchased from ATCC (Manassas, MA, USA). HK-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 25 U/mL penicillin (DS Pharm, Osaka, Japan), 25 µg/mL streptomycin (DS Pharm), 1% Insulin-Transferrin-Selenium-X (Gibco), 10 ng/mL EGF (epidermal growth factor; Sigma-Aldrich), and 5 ng/mL hydrocortisone at 37°C in a humidified incubator containing 5% CO2.
IMR-32 cells were kindly provided by Dr. Katsuhiko Muraki (Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi Gakuin University, Japan). IMR-32 cells were cultured in DMEM supplemented with 10% FBS, 25 U/mL penicillin, 25 µg/mL streptomycin, and 1% MEM non-essential amino acid solution (Sigma-Aldrich) at 37°C in a humidified incubator containing 5% CO2.
AML-12 cells were purchased from the ATCC, and cultured in DMEM/F-12, supplemented with 10% FBS, 1 mM sodium pyruvate (Gibco), 25 U/mL penicillin, 25 µg/mL streptomycin, 1% Insulin-Transferrin-Selenium-X, and 10 ng/mL EGF at 37°C in a humidified incubator containing 5% CO2.
HK-2 and AML-12 cells were grown in plates at a density of 250 cells/mm2 and cultured for 48 h. IMR-32 cells were grown in plates at a density of 500 cells/mm2 and cultured for 48 h. The culture medium was discarded, and the cells were treated with methylmercury (CH3HgCl; GL Sciences Inc., Tokyo, Japan), inorganic mercury (HgCl2; Wako Pure Chemical Industries, Osaka, Japan), or arsenic (NaAsO2; Wako Pure Chemical Industries) in serum-free culture medium.
RNA ExtractionCells were washed twice with ice-cold PBS (-) and total RNA was extracted with the PureLink™ RNA Mini Kit (Ambion, Grand Island, NY, USA) or QuickGene-810 (Fujifilm; Wako Pure Chemical Industries) following the manufacturer’s instructions. RNA was quantified and purity was determined using BioSpec-nano (Shimadzu, Kyoto, Japan).
Real Time RT-PCRTotal RNA was incubated with the PrimeScript reverse transcription (RT) Reagent Kit (Perfect Real Time) (Takara Bio, Shiga, Japan) to generate cDNA. Real-time PCR was performed with SYBR Premix Ex TaqII (Perfect Real Time) (Takara Bio) and the Thermal Cycler Dice Real Time System (Takara Bio). PCR conditions were as follows: 10 s hot-start at 95°C followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. Gene expression was normalized to GAPDH or ß-actin mRNA levels. The oligonucleotide sequences of the primers were as follows: sense, 5′-CATGTGTGTGGAGGGTGAAG-3′, and antisense, 5′-TTTAACAGGGGACAGCATCC-3′, for the human BIRC1 gene; sense, 5′-GCATTTTCCCAACTGTCCAT-3′, and antisense, 5′-ATTCGAGCTGCATGTGTCTG-3′, for the human BIRC2 gene; sense, 5′-TGGGGTTCAGTTTCAAGGAC-3′, and antisense, 5′-TGCAACCAGAACCTCAAGTG-3′, for the human BIRC4 gene; sense, 5′-GTTGCGCTTTCCTTTCTGTC-3′, and antisense, 5′-TCTCCGCAGTTTCCTCAAAT-3′, for the human BIRC5 gene; sense, 5′-TGACGCTTTCAACCTCACTG-3′, and antisense, 5′-GTGTCCGCTGGACCAGTTAT-3′, for the human BIRC6 gene; sense, 5′-TGGCCTCCTTCTATGACTGG-3′, and antisense, 5′-ACCTCACCTTGTCCTGATGG-3′, for the human BIRC7 gene; sense, 5′-AAGCCCGGCTCATTACTTTT-3′, and antisense, 5′-ATCTTCCTTGGGCTTCCAGT-3′, for the human BIRC8 gene; sense, 5′-GCACCGTCAAGGCTGAGAAC-3′, and antisense, 5′-TGGTGAAGACGCCAGTGGA-3′, for the human GAPDH gene; sense, 5′-CATGTGTGTGGAGGGTGAAG-3′ and antisense, 5′-TTTAACAGGGGACAGCATCC-3′ for the mouse Birc1 gene; sense, 5′-GCATTTTCCCAACTGTCCAT-3′ and antisense, 5′-ATTCGAGCTGCATGTGTCTG-3′ for the mouse Birc2 gene; sense, 5′-TGGGGTTCAGTTTCAAGGAC-3′ and antisense, 5′-TGCAACCAGAACCTCAAGTG-3′ for the mouse Birc4 gene; sense, 5′-GTTGCGCTTTCCTTTCTGTC-3′ and antisense, 5′-TCTCCGCAGTTTCCTCAAAT-3′ for the mouse Birc5 gene; sense, 5′-TGACGCTTTCAACCTCACTG-3′ and antisense, 5′-GTGTCCGCTGGACCAGTTAT-3′ for the mouse Birc6 gene; sense, 5′-TGGCCTCCTTCTATGACTGG-3′ and antisense, 5′-ACCTCACCTTGTCCTGATGG-3′ for the mouse Birc7 gene; sense, 5′-CCTAAGGCCAACCGTGAAAA-3′, and antisense, 5′-AGCCATACAGGGACAGCACA-3′, for the mouse ß-actin gene.
Statistical AnalysisStatistical analyses were performed using one-way ANOVA. When the F value was significant (P < 0.05), Bonferroni’s multiple t-test was performed for post-hoc comparison (P < 0.05).
Our previous report demonstrated that treatment with 6 µM methylmercury for 3 h and treatment with 4 µM methylmercury for 6 h slightly, but significantly, decreased the viability of IMR-32 cells.5) Moreover, 6 h methylmercury treatment increased activated caspase-3 levels. However, we also showed that BIRC3 mRNA levels were not altered following methylmercury treatment.5) In this study, we examined the mRNA levels of seven BIRC family genes in IMR-32 cells treated with various concentrations of methylmercury for 6 h. The mRNA levels of BIRC1, BIRC4, BIRC6, BIRC7, and BIRC8 did not change following methylmercury treatment (Figs. 1A, C, E, F, G). However, the mRNA levels of BIRC5 and BIRC2 were significantly increased and decreased, respectively, by methylmercury (Figs. 1D, B).
mRNA levels of BIRC family genes in IMR-32 cells treated with methylmercury for 6 h
IMR-32 cells were seeded onto 6-well plates at a density of 500 cells/mm2 and cultured for 48 h. The culture medium was discarded, and the cells were treated with methylmercury (CH3HgCl) in serum-free medium for 6 h. BIRC family gene mRNA levels were examined using real-time RT-PCR. (A) BIRC1. (B) BIRC2. (C) BIRC4. (D) BIRC5. (E) BIRC6. (F) BIRC7. (G) BIRC8. Expression levels were normalized to that of GAPDH. Values are means ± S.D. (n = 3). *Significantly different from the control group, P < 0.05.
Methylmercury treatment for 3 h significantly decreased the viability of HK-2 cells, increased activated caspase-3, but had no effect on BIRC3 mRNA levels.5) Here, we examined the effect of methylmercury on the mRNA levels of seven BIRC family genes in HK-2 cells. In HK-2 cells, the mRNA levels of BIRC1, BIRC4, and BIRC7 showed no change following methylmercury treatment (Figs. 2A, C, F). However, methylmercury significantly increased the mRNA levels of BIRC2, BIRC5, and BIRC8 (Figs. 2B, D, G) and significantly decreased the mRNA levels of BIRC6 mRNA (Fig. 2E) in HK-2 cells.
mRNA levels of BIRC family genes in HK-2 cells treated with methylmercury for 3 h
HK-2 cells were seeded onto 6-well plates at a density of 250 cells/mm2 and cultured for 48 h. The culture medium was discarded, and the cells were treated with methylmercury (CH3HgCl) in serum-free medium for 3 h. BIRC family gene mRNA levels were examined using real-time RT-PCR. (A) BIRC1. (B) BIRC2. (C) BIRC4. (D) BIRC5. (E) BIRC6. (F) BIRC7. (G) BIRC8. Expression levels were normalized to that of GAPDH. Values are means ± S.D. (n = 3). *Significantly different from the control group, P < 0.05.
We previously demonstrated that although the viability was almost the same as in the control group, inorganic mercury decreased BIRC3 mRNA level in HK-2 cells. When inorganic mercury treatment reduced the viability of HK-2 cells, cleaved caspase-3 level was unchanged.5) In this study, the effects of inorganic mercury on the expression levels of the remaining seven BIRC family members were examined in HK-2 cells. Inorganic mercury did not affect the mRNA levels of BIRC1, BIRC4, BIRC6, BIRC7, and BIRC8 in HK-2 cells (Figs. 3A, C, E, F, G). However, inorganic mercury treatment resulted in a significant increase in BIRC2 and BIRC5 mRNA levels HK-2 cells (Figs. 3B, D).
mRNA levels of BIRC family genes in HK-2 cells treated with inorganic mercury for 3 h
HK-2 cells were seeded onto 6-well plates at a density of 250 cells/mm2 and cultured for 48 h. The culture medium was discarded, and the cells were treated with inorganic mercury (HgCl2) in serum-free medium for 3 h. BIRC family gene mRNA levels were examined using real-time RT-PCR. (A) BIRC1. (B) BIRC2. (C) BIRC4. (D) BIRC5. (E) BIRC6. (F) BIRC7. (G) BIRC8. Expression levels were normalized to that of GAPDH. Values are means ± S.D. (n = 3). *Significantly different from the control group, P < 0.05.
In AML-12 cells, arsenic decreased cell viability, and significantly increased the mRNA level of Birc3.5) Both caspase-3 and activated caspase-3 levels were slightly increased by arsenic treatment.5) Here, we examined the effect of arsenic treatment on the mRNA levels of six Birc family genes in AML-12 cells. The mRNA levels of Birc1 and Birc7 were significantly elevated by arsenic treatment (Figs. 4A, F). However, the mRNA levels of Birc2, Birc4, Birc5, and Birc6 were significantly reduced by arsenic (Figs. 4B, C, D, E).
mRNA levels of Birc family genes in AML-12 cells treated with arsenic for 6 h
AML-12 cells were seeded onto 6-well plates at a density of 250 cells/mm2 and cultured for 48 h. The culture medium was discarded, and the cells were treated with arsenic (NaAsO2) in serum-free medium for 6 h. Birc family gene mRNA levels were examined using real-time RT-PCR. (A) Birc1. (B) Birc2. (C) Birc4. (D) Birc5. (E) Birc6. (F) Birc7. Expression levels were normalized to that of ß-actin. Values are means ± S.D. (n = 3). *Significantly different from the control group, P < 0.05.
Our results indicate that methylmercury, inorganic mercury, and arsenic change the expression of BIRC family genes in various patterns in cultured cells. In HK-2 cells, cadmium markedly decreased BIRC3 gene expression levels and significantly increased the levels of activated caspase-3.5) However, cadmium did not induce significant changes in the expression of other BIRC family members in HK-2 cells.5) Although methylmercury activated caspase-3 in IMR-32 and HK-2 cells,5) methylmercury increased and decreased mRNA levels of BIRC family member genes in IMR-32 and HK-2 cells (Figs. 1, 2). Moreover, although arsenic treatment resulted in slightly increased activated caspase-3 levels,5) it altered the expression of all Birc family member genes in AML-12 cells (Fig. 4). Taken together, each metal(loid) toxicant may regulate the expression of BIRC family member genes in different manners. Therefore, each BIRC family member gene may play a distinct role following the exposure of various tissues to toxic heavy metal or metalloid toxicants.
Methylmercury causes central nervous system disorders via apoptosis.2) In this study, methylmercury markedly increased BIRC5 mRNA levels in IMR-32 cells, even though caspase-3 was profoundly activated by methylmercury. Inorganic mercury, in addition to cadmium, is also known to cause kidney cell damage.3) Our previous study demonstrated that inorganic mercury induced apoptosis signals, such as DNA fragmentation, in rat proximal tubular NRK-52E cells.10) In HK-2 cells, inorganic mercury did not activate caspase-3,5) but significantly increased BIRC5 mRNA levels (Fig. 3). BIRC5 is an anti-apoptotic factor also known as Survivin, and has been reported to suppress apoptosis in nerve cells.11) Therefore, BIRC5 may act as a protective factor against inorganic mercury-induced human kidney damage and may perform a different role in the case of methylmercury toxicity. Arsenic exposure can cause liver and skin disorders, Bowen's disease, skin cancer, and apoptosis.9,12,13) Previously, arsenic markedly increased Birc3 expression in AML-12 cells.5) However, we showed here that Birc2 expression was significantly suppressed by arsenic in AML-12. Furthermore, slight activation of caspase-3 was observed in AML-12 cells treated with arsenic.5) Therefore, changes in Birc family gene expression following arsenic treatment may be associated with apoptosis in mouse hepatic cells.
BIRC4, known as XIAP (X-linked IAP), is the most studied member of the BIRC family.14) The BIR2 and BIR3 regions of BIRC4 interact with the active-site pocket of caspases to suppress apoptosis.15) Therefore, it was proposed that most BIRC family members neutralize caspase activities in the same manner. However, recent reports indicate that BIRC family members have non-equivalent functions and regulate the activities of caspases through distinct mechanisms.16-18) BIRC2, BIRC3, and BIRC4 contain a C-terminal RING zinc finger domain with E3 ubiquitin ligase activity, which mediates proteasomal degradation of target proteins as well as themselves.19) BIRC2 independently interacts with caspase-7 at the active-site pocket.16) Moreover, BIRC2 suppresses apoptosis by ubiquitinating activated caspase-3 and -7 to mediate their proteasomal degradation.17)
Our previous study demonstrated that cadmium suppresses BIRC3 gene expression by inhibiting the activity of the ARNT (aryl hydrocarbon receptor nuclear translocator) transcription factor.5) We used DNA microarrays to screen ARNT knockdown cells for the down-stream targets of ARNT, and identified BIRC3 among these targets. It has been suggested that the PI3K/Akt signaling pathway regulates BIRC2 expression.20) Therefore, methylmercury, inorganic mercury, or arsenic may affect other transcription factors in addition to ARNT.
Future detailed studies investigating the involvement of the BIRC family in the toxicity of various harmful heavy metals will clarify the mechanisms of toxicity and/or defense against toxicity.
We sincerely thank Mr. T. Ieda, Mr. H. Shinkai, and Mr. Y. Noda for their excellent experimental support. This research was supported by JSPS KAKENHI (grant number JP16K00563).
Conflict of InterestThe authors declare no conflict of interest.
