Correspondence: Masahiko Satoh (E-mail: masahiko@dpc.agu.ac.jp)

Involvement of ubiquitin-coding genes in cadmium-induced protein ubiquitination in human proximal tubular cells

Jin-Yong Lee1, Maki Tokumoto1, Yasuyuki Fujiwara1,2 and Masahiko Satoh1

1Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan

2Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

(Received October 4, 2015; Accepted October 21, 2015)

ABSTRACT — Cadmium (Cd) is a toxic heavy metal with a long half-life in humans. It causes disorders of various tissue systems, including the kidney, and is associated with protein aggregation. Our previous study demonstrated Cd-induced suppression of the UBE2D gene family, one of the ubiquitin-conjugating enzyme families. However, the precise role of ubiquitin-coding genes in Cd toxicity remains to be under-stood. In this study, we investigated the effect of Cd on expression of the ubiquitin-coding genes UBB, UBC, UBA80, and UBA52 in HK-2 human proximal tubular cells. Prior to the appearance of Cd toxici-ty, the UBB, UBC, and UBA80 expression levels increased following Cd treatment. Knockdown of UBB by siRNA transfection significantly decreased Cd cytotoxicity. Notably, Cd induces ubiquitinated pro-tein levels in HK-2 cells, and knockdown of UBB blocked this process. These results suggest that UBB is involved in Cd-induced increase of protein ubiquitination, and that accumulation of ubiquitinated proteins through increased UBB expression may contribute to Cd toxicity in HK-2 cells.

Key words: Cadmium, HK-2 cells, Renal toxicity, Ubiquitinated protein, Ubiquitin-coding genes

INTRODUCTION

Cadmium (Cd) is an environmental contaminant that induces severe clinical symptoms in various organs, including the kidney (Järup et al., 1998). Cd ingested oral-ly accumulates in the body, particularly in the kidney, and exhibits a long biological half-life (10-30 years) (Järup, 2002; Järup and Akesson, 2009). Overaccumulated Cd in the kidney damages the proximal tubules (Järup et al., 1998). Although Cd binds to the metal-binding protein metallothionein for detoxification, high concentrations of unbound Cd trigger adverse cellular functions (Järup et al., 1998; Klaassen et al., 2009). At the molecular level, Cd exposure is linked to multiple cytotoxic effects such as oxidative stress, enzyme inhibition, and disruption of gene expression (Fujiwara et al., 2012). However, the precise mechanism of Cd-induced renal toxicity remains to be clarified.

Recently, several studies have suggested that Cd tox-icity is associated with perturbation of the ubiquitin (Ub) proteasome system (Figueiredo-Pereira et al., 2002; Yu et al., 2008, 2011; Tokumoto et al., 2011). Our pre-

vious study has demonstrated that Cd decreases expres-sion of Ub proteasome system (UPS)-related genes in rat proximal tubular cells and mouse kidney (Tokumoto et al., 2011). In addition to Cd, inorganic arsenic and inorganic mercury decrease gene expression of UPS-re-lated proteins, such as Ube2d1, Ube2d2, and Ube2d4 (Tokumoto et al., 2013). Kim et al. (2015) demonstrat-ed that inorganic arsenic upregulated Ubc in mouse embryonic fibroblasts. Recently, UPS-regulated proteins involved in defense against methylmercury toxicity were identified in yeast cells (Lee et al., 2015).

The UPS is a highly conserved pathway that plays a critical role in adjusting protein levels (Vabulas, 2007). Furthermore, many UPS-regulated proteins are involved in DNA repair, cell cycle control and differentiation, tran-scription, and apoptosis (Vabulus, 2007; Hamer et al., 2010; Salomons et al., 2010). The main event of UPS is the conjugation of Ub, a 76-amino-acid highly conserved protein, to the substrate protein. A diverse set of proteins is needed for Ub conjugation. Initially, Ub is activated by an E1 Ub activating-enzyme. Subsequently, the activated Ub is transferred to a second protein, an E2 Ub-conjugat-

Original Article

The Journal of Toxicological Sciences (J. Toxicol. Sci.)Vol.40, No.6, 901-908, 2015

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ing enzyme, which transfers Ub to the target protein with the aid of E3 Ub ligase (Ravid and Hochstrasser, 2008). Classically, protein ubiquitination is a signal for pro-tein degradation. However, it also affects substrate pro-tein activity and cellular localization (Mukhopadhyay and Riezman, 2007). This critical process dependent on Ub imposes the necessity for cells to maintain adequate sup-plies of available Ub.

In mammals, Ub-coding genes are found in two forms (Baker and Board, 1991). The first type of Ub-coding genes consists of UBB and UBC, which encode Ub tan-dem repeats (Baker and Board, 1991). The second type of Ub-coding genes, which are UBA52 and UBA80, encodes a monoUb fused to a tail protein (Baker and Board, 1991). UBB and UBC encode 3 and 9 tandem polyUbs, respec-tively (Baker and Board, 1987; Oh et al., 2013). UBA52 and UBA80 encode Ub fusion proteins characterized by differences in the protein tail lengths, 52- and 80-ami-no-acids, respectively (Baker and Board, 1991; Han et al., 2012). Ub protein levels are tightly controlled in cells (Vabulas, 2007). For example, genes coding for polyUb are reported to be stress-induced (Bond and Schlesinger 1986; Fornace et al., 1989).

Although several UPS-related genes have previous-ly been reported to mediate Cd toxicity, the role of Ub-coding genes in Cd renal toxicity remains poorly defined. Therefore, we sought to investigate the effect of Cd on transcription of Ub-coding genes in human proximal tubular cells (HK-2 cells). Moreover, the effect of Ub-coding genes on Cd toxicity was examined, using siRNA transfection in HK-2 cells.

MATERIALS AND METHODS

Cell culture and Cd treatmentHK-2 cells were purchased from ATCC (Manassas,

MA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s Nutrient Mixture F-12 (F-12) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 25 U/mL penicillin, 25 μg/mL streptomycin, 1% Insulin-Transferrin-Selenium-X (Gibco), 10 ng/mL EGF, and 5 ng/mL hydrocortisone, at 37°C in a humidi-fied incubator containing 5% CO2. HK-2 cells were trans-ferred to testing plates at a density of 2.5 × 104 cells/cm2 and cultured until confluent. The culture medium was discarded and cells treated with Cd (CdCl2) (Wako Pure Chemical Industries, Osaka, Japan) in serum-free culture medium for 3, 6, or 12 hr.

Cell viabilityAfter treatment, culture medium was changed to fresh

10% FBS-DMEM/F-12 containing 0.5 mg/mL MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-zolium bromide; DOJINDO Laboratories, Kumamoto, Japan] and incubated for another 4 hr at 37°C. After removing the medium, dimethyl sulfoxide (Wako Pure Chemical Industries) was added to MTT formazan. Absorbance at 570 nm was measured by the DTX880 multimode detector (Beckman Coulter Inc., Brea, CA, USA). Cell morphology was photographed using a trans-mitted-light microscope EVOS XL Core Imaging System (Life Technology, Carlsbad, CA, USA) at 100 × magnifi-cation.

RNA extraction and real-time RT-PCRCd-treated or siRNA-transfected HK-2 cells were

washed twice with ice-cold phosphate-based saline (PBS) (Nissui, Tokyo, Japan), and total RNA extract-ed with the Quick Gene RNA cultured cell kit S (Fujifilm, Tokyo, Japan) according to the manufactur-er’s protocol. RNA quantitation and purity were meas-ured using the BioSpec-nano (Shimadzu Biotech, Kyoto, Japan). cDNA was generated from total RNA using the PrimeScript reverse transcription (RT) Reagent Kit (Perfect Real Time) (Takara Bio, Shiga, Japan). Real-time PCR was performed with the SYBR Premix Ex Taq II (Perfect Real Time) (Takara Bio), and a Thermal Cycler Dice Real Time System (Takara Bio). PCR conditions were as follows: 10 sec hot-start at 95°C followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. Gene expres-sion was normalized to GAPDH mRNA levels. The oli-gonucleotide sequences of the primers were as follows: sense, 5′-GCACCGTCAAGGCTGAGAAC-3′, and anti-sense, 5′-TGGTGAAGACGCCAGTGGA-3′, for the human GAPDH; sense, 5′-TCACTCTGGAGGTGGA-3′, and anti-sense, 5′-CCCTCAGGCGCAGGAC-3′, for the human UBB; sense, 5′-AAAGAGTCCACCTTGCACCTG-3′, and antisense, 5′-ACCTCAAGGGTGATGGTCTTG-3′ for the human UBC; sense, 5′-TCGTGGTGGTGCTAAGAAAA-3′, and antisense, 5′-TCTCGACGAAGGCGACTAAT-3′ for the human UBA80; sense, 5′-AGGAGGGTATCCCACCTGAC-3′, and antisense, 5′-CAGGGTGGACTCTTTCTGGA-3′ for the human UBA52.

siRNA transfectionSilencer Select Pre-designed siRNAs were purchased

from Ambion (Grand Island, NY, USA). The I.D. of siR-NAs were as follows: s337 (Silencer® Select Validated siRNA), for human UBB; s14559 and s14560 (Silencer® Select Pre-designed siRNA), for human UBC; s52245

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and s12339 (Silencer® Select Pre-designed siRNA), for human UBA80; s14556 and s14557 (Silencer® Select Pre-designed siRNA), for human UBA52. siRNA trans-fection was performed using Lipofectamine RNAiMAX (Invitrogen, Grand Island, NY, USA). After the siRNA mixture was incubated for 15 min with Lipofectamine RNAiMAX and Opti-MEM® I Reduced Serum Medium (Opti-MEM; Gibco), HK-2 cells were transfected with the siRNA mixture (1 nM siRNA/sequece, 0.2% Lipofectamine RNAiMAX, 10% Opti-MEM) for 48 hr.

Western blottingAfter treatment, cells were washed twice with ice-cold

PBS and harvested in RIPA buffer [25 mM Tris (pH7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxychola-te 0.1% SDS; Thermo Fisher Scientific, Waltham, MA, USA]. Protein concentrations were measured using the BCA protein assay kit (Thermo Fisher Scientific). Protein was electrophoresed on gradient (4-15%) SDS-polyacry-lamide gels (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (ATTO, Tokyo, Japan), and probed with antibodies against Ub (P4D1) (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), or β-actin (1:1,000; American Research Products, Waltham, MA, USA). Membranes were subsequently probed with horseradish peroxidase-conjugating (HRP) secondary antibodies (1:10000; GE Healthcare, Tokyo, Japan), and protein detected by enhanced chemiluminescence using ImmunoStar® Zeta (Wako Pure Chemical Industries). Chemiluminescent images were obtained using the Image Quant LAS 500 (GE Healthcare) device.

RESULTS

Cd increases Ub transcription prior to the appearance of cytotoxicity

The degree of Cd toxicity on HK-2 cells is shown in Fig. 1. HK-2 cells treated with 40 μM Cd for 6 hr exhib-ited 90% cell viability, whereas a 3 hr treatment did not induce cytotoxicity. Therefore, we investigated the effect of Cd on expression of 4 Ub-coding genes (UBB, UBC, UBA80, UBA52) with 40 μM Cd for 1, 3, and 6 hr. The 3 hr treatment induced expression of UBB, UBC, and UBA80 with the exception of UBA52 compared with non-treated cells (Fig. 2). Notably, UBB and UBA80 expres-sion increased prior to signs of cytotoxicity (Figs. 2A, C). Additionally, UBC expression increased even when cell viability decreased following Cd treatment (Fig. 2B). These results suggest that Cd induces expression of Ub-coding genes prior to the appearance of cytotoxicity.

Disruption of UBB affects the sensitivity of HK-2 cells to Cd

We employed siRNA transfection to target each Ub-coding gene to investigate whether expression changes in Ub-coding genes may affect the sensitivity of HK-2 cells to Cd. Transfection of UBB siRNA markedly decreased UBB mRNA level (Fig. 3A). Interestingly, UBB siR-NA transfection decreased the sensitivity of HK-2 cells to Cd (Fig. 3B). Transfection of UBA80 siRNA mark-edly decreased UBA80 mRNA level (Fig. 3C). How-ever, in contrast to UBB siRNA treatment, UBA80 siR-NA-transfected cells exhibited the same Cd sensitivity as control siRNA (Fig. 3D). On the other hand, UBC and UBA52 siRNA caused severe damage to HK-2 cells, with most cells floating and exhibiting a changed morphology (Fig. 4A). The viability of UBC or UBA52 siRNA-trans-fected cells was low compared with control siRNA-trans-fected cells (Fig. 4B). Taken together, these data suggest that disruption of UBB leads to resistance against Cd-in-duced toxicity in HK-2 cells.

Disruption of UBB blocks the increase of Cd-induced protein ubiquitination in HK-2 cells

In the present study, Cd treatment induced expression of Ub-coding genes. Others have shown that an intracel-

Fig. 1. The viability of HK-2 cells after Cd treatment. HK-2 cells were grown in 96-well plates at 2.5 × 104 cells/cm2 and cultured for 48 hr. Culture medium was discarded and cells treated with Cd in serum-free culture me-dium for 3, 6, or 12 hr. Cell viability was measured by MTT assay. Values are means ± S.D. (n = 3). *Significantly different from the corresponding control group, P < 0.05.

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lular increase in Ub protein levels is necessary for pro-tein ubiquitination (D'Andrea and Pellman, 1998). Our recent study reported that Cd increased chaperon-related protein expression in HK-2 cells (Lee et al., 2013), and Cd is known to trigger ER-stress (Fujiwara et al., 2012). Together, these observations suggest that Cd may increase levels of unfolded protein, which should be degraded by the UPS. Therefore, we examined the effect of Cd on the level of intracellular ubiquitinated proteins, which increased following 6 hr treatments (Fig. 5A). Because UBB disruption eliminated Cd toxicity in HK-2 cells, we examined whether UBB siRNA-treatment might affect the elevation of ubiquitinated proteins by Cd. Although the level of ubiquitinated proteins following UBB siRNA

treatment alone was the same as for control siRNA treat-ments, Cd-induced protein ubiquitination in control siR-NA treated cells was eliminated by UBB siRNA treatment (Fig. 5). These results suggest that UBB regulates Cd-in-duced increase of protein ubiquitination, and that accumu-lation of ubiquitinated protein through UBB-induced gene expression may result from Cd toxicity in HK-2 cells.

DISCUSSION

Ub is a small, highly conserved protein that plays an important role in not only ubiquitination of proteins for proteasomal degradation but also various cellular sig-naling pathways (Vabulus, 2007; Hamer et al., 2010;

Fig. 2. Effects of Cd on mRNA levels of ubiquitin-coding genes in HK-2 cells. Real-time RT-PCR of UBB (A), UBC (B), UBA80 (C) and UBA52 (D) gene expression. HK-2 cells were grown in 6-well plates at 2.5 × 104 cells/cm2 and cultured for 48 hr. Culture medium was discarded and cells treated with Cd in serum-free culture medium for the indicated times. mRNA lev-els were normalized to GAPDH. Values are means ± S.D. (n = 3). *Signifi cantly different from the control group, P < 0.05.

A B

C D

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Salomons et al., 2010). In eukaryotic cells, Ub protein levels exist in a dynamic equilibrium between Ub conju-gates and free Ub monomer (Wilkinson, 1997). Although Ub supplies are maintained by deubiquitinase activi-ty (D'Andrea and Pellman, 1998), they are also man-aged by synthesis at the transcriptional level (Ryu et al., 2008). Although Ub is encoded by two different types of Ub genes (Baker and Board, 1991), under stress or nor-mal conditions the contribution of polyUb genes in main-

taining Ub protein levels is significant (Ryu et al., 2007). The present study clearly demonstrates that Cd induc-es expression of polyUb genes, UBB and UBC in HK-2 cells. Moreover, UBB expression levels play a role in Cd toxicity. Disruption of the UBB gene suppresses the Cd-induced increase of ubiquitinated protein, as well as weakens Cd toxicity. Yu et al. (2011) reported that Cd increased accumulation of high molecular weight-polyUb conjugates in mouse embryonic fibroblast cells. There-

Fig. 3. Effects of UBB or UBA80 knockdown on Cd toxicity in HK-2 cells. (A) The knockdown efficiency of UBB in HK-2 cells following UBB siRNA treatment. UBB siRNA was added to HK-2 cells and cells incubated for 48 hr. UBB mRNA levels were measured using real-time RT-PCR. UBB mRNA levels were normalized to GAPDH mRNA levels. (B) Viability of UBB or control siRNA-transfected HK-2 cells following Cd treatment. Cells were grown in 96-well plates at 2.5 × 104 cells/cm2 with siRNA mixture and cultured for 48 hr. Culture medium was discarded and cells treated with Cd in serum-free cul-ture medium for 12 hr, and cell viability measured by MTT assay. (C) The knockdown efficiency of UBA80 in HK-2 cells following UBA80 siRNA treatment. UBA80 siRNA was added to HK-2 cells and cells incubated for 48 hr. UBA80 mRNA levels were measured using real-time RT-PCR after normalization to GAPDH mRNA levels. (D) Viability of UBA80 or control siRNA-transfected HK-2 cells following Cd treatment. Cells were grown in 96-well plates at 2.5 × 104 cells/cm2 with siRNA mixture and cultured for 48 hr. Culture medium was discarded and cells treated with Cd in serum-free culture medium for 12 hr. Cell viability was measured by MTT assay. (A-D) Values are means ± S.D. (n = 3). *Significantly differ-ent from the corresponding control siRNA group, P < 0.05.

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fore, it is suggested the increase of ubiquitinated pro-tein occurs through an UBB-increased supply of Ub, pro-viding what may be a partial clue to Cd toxicity. Protein ubiquitination is accompanied by the attachments of each Ub unit to a protein (Mukhopadhyay and Riezman, 2007). On the other hand, this present study implies that polyUb is involved in Cd toxicity rather than monoUb. Hence, it is strongly suggested that polyUb is preferably engaged in Cd-induced accumulation of ubiquitinated proteins in HK-2 cells. Recently, Ubc was reported to be involved in arsenite-induced toxicity in mouse embryonic fibroblasts (MEFs) (Kim et al., 2015), suggesting that polyUb genes may be critical to the mechanism of metal(loid) toxicity.

Cd causes the formation of protein inclusion bodies by promoting the accumulation of ubiquitinated proteins in aggresomes in HEK293 cells (Song et al., 2008), and it has been suggested that the Ub ligase FBXO6 reduces

Cd toxicity by expediting degradation of misfolded pro-teins by the UPS in HEK293 cells (Du et al., 2014). The present study also suggests that accumulation of ubiqui-tinated proteins may increase Cd toxicity in HK-2 cells. Therefore, disruption of the UPS may play a critical role in Cd-induced renal toxicity.

Recently, a few reports indicated that disruption of Ub genes has adverse effects on the proliferation of viable cells. Disruption of Ubc leads to defective proliferation of fetal liver epithelial progenitor cells in mice (Park et al., 2013). Disruption of the Ubb gene causes dysregula-tion of neural stem cell differentiation in mice (Ryu et al., 2014). In the present study, disruption of UBC or UBA52 genes by siRNA transfection induced cytotoxicity in HK-2 cells. However, the UBB knockdown did not affect cell viability. Therefore, the cellular level of Ub genes is associated with cellular functions and distinct specificity

Fig. 4. Viability of HK-2 cells following treatment with UBC or UBA52 siRNA. Cells were grown in 6-well plates at 2.5 × 104 cells/cm2 with siRNA mixture and cultured for 48 hr. (A) Cell morphology 48 hr after treatment with UBC or UBA52 siRNA. Images were obtained by light microscopy. The bars represent 400 μm. (B) Viability of HK-2 cells after treatment with UBC or UBA52 siRNA. Cell viability was measured by MTT assay. Values are means ± S.D. (n = 3). *Sig-nificantly different from the control siRNA group, P < 0.05.

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that is not observed under stress conditions such as Cd and arsenic exposure.

Conflict of interest---- The authors declare that there is no conflict of interest.

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