Bisphenol A and Its Derivatives Induce Degradation of HIF-1alpha via the Lysosomal Pathway in Human Hepatocarcinoma Cell Line, Hep3B

Abstract

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane), one of the phenolic compounds widely used in the manufacture of plastic and epoxy resins, is known as an endocrine disruptor. In a previous study, we found that BPA induced hypoxia inducible factor-1alpha (HIF-1alpha) degradation by dissociation from heat shock protein 90 (Hsp90). In this study, to investigate the structural requirements for degradation of HIF-1alpha, we estimated the effect of BPA derivatives (BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP)) on HIF-1alpha protein degradation, using human hepatocarcinoma cell line, Hep3B. BPB, DMBDP, BHCH, and MBBP decreased HIF-1alpha protein levels more efficiently than BPA, but BPE, BPF, and EHDP did not affect HIF-1alpha protein levels. BPA degraded HIF-1alpha even in the presence of MG132, a proteasome inhibitor. In this study, we found that ammonium chloride (NH₄Cl), a lysosomal enzyme inhibitor, efficiently restored the decrease in HIF-1alpha protein levels by BPA. Recent studies indicated that HIF-1alpha is degraded by the lysosomal pathway as well as the proteasomal pathway. Therefore, we investigated the levels of heat shock cognate 70 kDa protein (HSC70) protein after treatment with BPA. We found that BPA induced HSC70 protein and overexpression of HSC70 enhanced HIF-1alpha degradation in Hep3B cells. These results suggested that BPA causes the degradation of HIF-1alpha by induction of HSC70, leading lysosomal degradation of HIF-1alpha.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane) is used in polycarbonate plastics, food cans, dental sealants and baby bottles and is found in lakes and rivers. BPA has been detected in human brain, liver, serum, urine, breast milk, amniotic fluid and adipose tissue.^1–7) BPA acts as a xenoestrogen and an estrogen receptor (ER) agonist, and several studies have indicated that BPA exposure influences reproductive function, sperm concentration, sexual differentiation, and abortion.⁸⁾ There are many reports of BPA target factors, such as estrogen related receptor gamma,⁹⁾ constitutive androstane receptor,¹⁰⁾ pregnane X receptor,¹¹⁾ and peroxisome proliferator-activated receptor gamma.¹²⁾ We found that BPA binds to presenilin, a component of gamma-secretase, leading to inhibition of Notch signaling and neural differentiation in Xenopus laevis.¹³⁾ Protein disulfide isomerase was also identified as a target of BPA in rat brain.¹⁴⁾ BPA inhibits the activity of thyroid hormone receptor by antagonization of triiodothyronine, which is related to ordinary brain development.¹⁵⁾

BPA derivatives with a similar structure are used in plastic products, and their productions are increasing annually. BPA derivatives have been detected in beverages and human serum and urine, indicating that these chemical compounds exist in the environment. Although it is estimated that the effect of BPA derivatives on human health is similar to BPA, further study is required to determine the effects of these chemicals.^16–18) Bisphenol F (BPF), which lacks the two central methyl groups of BPA, has estrogen activity and show no association with serum thyroid stimulating hormone.¹⁹⁾ BPAF, in which the central methyl groups of BPA are replaced by two trifluoromethyl groups, acted as an ERbeta antagonist.²⁰⁾ BPA was more effective in assays of cell proliferation of LM8 cells than Bisphenol E (BPE), which lacks one methyl group of BPA, and BPF. BPB, which has a longer alkyl group in the central position compared with BPA, increased the cell proliferation potential of LM8 cells.²¹⁾

In a previous study, we identified hypoxia inducible factor-1alpha (HIF-1alpha) as a novel BPA target protein.²²⁾ HIF-1alpha, a transcription factor, is well known as a key factor in hypoxia response. In normoxic conditions, proline (Pro)-402 and -564 of HIF-1alpha are modified by prolyl hydroxylase domain containing protein, inducing the ubiquitination of HIF-1alpha by von Hippel–Lindau tumor suppressor protein (pVHL) and its degradation via the proteasomal pathway.²³⁾ HIF-1alpha is stabilized under hypoxia, which commonly occurs in cancers, and binds to the hypoxia response element of target genes such as vascular endothelial growth factor, erythropoietin (EPO), and glucose transporter, resulting in the promotion of angiogenesis, glucose intake, alteration of energy metabolism, cell proliferation and cell motility.²³⁾

In our previous study, we found that BPA decreased HIF-1alpha protein levels, leading to the decrease of EPO mRNA expression, but BPF did not, suggesting that two methyl groups on the central carbon atoms are required for HIF-1alpha degradation by BPA. Moreover, BPA promoted the degradation of HIF-1alpha protein by dissociation of HIF-1alpha from heat shock protein 90 (Hsp90). HIF-1alpha is usually degraded via the proteasomal pathway, but MG132, a proteasome inhibitor, could not inhibit HIF-1alpha degradation induced by BPA. Thus, the mechanism behind HIF-1alpha degradation by BPA remains unclear.

In this study, we investigated that the effect of structural differences of BPA derivatives on HIF-1alpha protein degradation and the mechanism behind HIF-1alpha degradation caused by BPA and its derivatives.

MATERIALS AND METHODS

Materials

Fetal bovine serum (FBS) and penicillin–streptomycin solution was purchased from Sigma Chemical (St. Louis, MO, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), NH₄Cl, and anti-DYKDDDDK (Flag) antibody were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). An anti-heat shock cognate 70 kDa protein (HSC70) antibody was purchased from Novus Biologicals. MG132 was purchased from Peptide Institute (Osaka, Japan), Isogen from Nippon Gene (Toyama, Japan), and RevertAidTM M-MuLV Reverse Transcriptase from MBI Fermentas (Vilnius, Lithuania). BPA, BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP), whose structures were shown in Fig. 1 were purchased from Wako Pure Chemical Industries, Ltd.

Fig. 1. Chemical Structures of BPA and BPA Derivatives

Isolation of HSC70 and Hsp90alpha cDNAs and Preparation of Constructs

The cDNA of human HSC70 was isolated from Hep3B cells by PCR. The primers for human HSC70 (Acc No. NM_006597.5) were 5′-ATTGGATCCGCAACCATGTCCAAGGGAC-3′ (forward primer for HSC70; underline, BamHI site; double underline, start codon), 5′-TTTCTAGATTAATCAACCTCT-3′ (reverse primer for HSC70; underline, XbaI site; double underline, stop codon). PCR was performed with KOD Plus Neo: denaturation at 94°C for 2 min, and then 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 1 min. The amplified HSC70 cDNA was inserted into a pcDNA3.1 vector using BamHI and XbaI. The cDNA of human Hsp90alpha was isolated from Hep3B cells by PCR. The primers for human Hsp90alpha (Acc No. NM_005348.3) were 5′-AAGGATCCATGCCTGAGGAAACCCAGAC-3′ (forward primer for Hsp90alpha; underline, BamHI site; double underline, start codon), 5′-AAAGGATCCTTGTAGAGTACTGAACAGGT-3′ (reverse primer for Hsp90alpha; underline, BamHI site; stop codon is upstream of this primer). PCR was performed with KOD Plus Neo: denaturation at 94°C for 2 min, 40 cycles of 94°C for 30 s, 50°C for 30 s and 70°C for 3 min. The amplified HSC70 cDNA was inserted into pQE80L vector using BamHI.

Cell Culture

A human hepatocarcinoma cell line, Hep3B, was obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging, and Cancer of Tohoku University (Sendai, Japan). Hep3B cells were cultured in DMEM containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL) and maintained at 37°C in 5% CO₂ and 95% air (normoxia). For hypoxic stimulation, the cells were cultured in a multi-gas incubator, APM-30D (Astec, Shizuoka, Japan), with 1% O₂ and 5% CO₂ (hypoxia) for 6 h. Hep3B cells were treated with BPA or BPA derivatives (50–200 µM) under normoxia or hypoxia for 6 h.

Small Interfering RNA (siRNA) Experiment

Hep3B cells were transfected with the si-HSC70 and si-control (Qiagen, Hilden, Germany) using Screen Fect A (Wako Pure Chemical Industries, Ltd.) in accordance with the manufacturer’s instructions. The target sequence for human HSC70 was 5′-AAG GAC CTA AAT TCG TAG CAA-3′.

RNA Isolation and RT-PCR

Total RNA was extracted from cells using Isogen in accordance with the manufacturer’s instructions, and converted to cDNA by reverse transcription. Real-time PCR was performed using SYBR premix Ex Taq II in accordance with the manufacturer’s instructions. PCR was performed with a Thermal Cycler Dice Real Time System Single TP850 (TaKaRa Bio). The primers for human EPO (Acc No. NM_000799) were 5′-ATG TGG ATA AAG CCG TCA GTG G-3′ (forward) and 5′-CTG GAG TGT CCA TGG GAC AG-3′ (reverse). The primers for human beta-actin (Acc No. NM_001101) were 5′-CAA GAG ATG GCC ACG GCT GCT-3′ (forward) and 5′-TCC TTC TGC ATC CTG TCG GCA-3′ (reverse).

To detect human HIF-1alpha and beta-actin mRNAs, PCR was done with 10 pmol of each primer, 100 ng of cDNA, and Go Taq Green Master Mix (Promega, Madison, WI, U.S.A.), and its condition was as follows: first 2 min at 94°C and then 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The number of cycles for human HIF-1alpha were 23 cycles and that for human beta-actin were 18 cycles. The primers for HIF-1alpha (NM_001530) were 5′-CCT AAC GTG TTA TCT GTC GC-3′ (forward) and 5′-GTC AGC TGT GGT AAT CCA CT-3′ (reverse).

Preparation of Antibodies

pQE80L vector including Hsp90alpha cDNA was transfected to Escherichia coli strain, BL21. The expressed Hsp90alpha was purified using nickel–nitrilotriacetic acid (Ni–NTA) agarose (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions, and used for the preparation of an antibody in rabbit using the method described previously.²⁴⁾ All experiments were conducted in accordance with guidelines on the welfare of experimental animals and with the approval of the Ethics Committee on the use of animals of Kwansei Gakuin University.

Western Blotting

Whole homogenate of cells was subjected to immunoblotting with anti-HIF-1alpha, anti-Hsp90, or anti-beta-actin antibody. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The membranes were incubated with the primary antibodies. Each protein was visualized using horseradish peroxidase conjugated to goat anti-rabbit immunoglobulin G (IgG) (secondary antibody) and with 4-chloro-1-naphthol. Band intensity was quantified using the NIH Image program.

Calculation of Hydrophobicity of BPA and Its Derivatives

Hydrophobicity of BPA and its derivatives was calculated using Molinspiration program.

RESULTS

Decrease of HIF-1alpha Levels and EPO mRNA Expression by BPA Derivatives under Hypoxia

To examine the effect of BPA and BPA derivatives on HIF-1alpha expression, Hep3B cells were treated with BPA or BPA derivatives under hypoxia. HIF-1alpha was induced under hypoxia, and BPA decreased HIF-1alpha protein levels. However, BPE, which lacks one methyl group, and BPF, which lacks two methyl groups, did not decrease HIF-1alpha protein levels (Fig. 2A). We used BPE and BPF to compare their efficiency and hydrophobicity with those of other BPA derivatives although the efficiency of BPE and BPF on HIF-1alpha degradation was already reported by Kubo et al. These results were consistent with findings of Kubo et al.²²⁾ Hep3B cells were treated with BPA, BPB, DMBDP, or EHDP which have the longer alkyl groups on the central carbon atom between the two phenyl rings. BPA, BPB, and DMBDP decreased expression of HIF-1alpha but EHDP did not (Figs. 2B, C). Hep3B cells were treated with BPA, BHCH, in which a methyl group is replaced with cyclohexane, or MBBP, in which a methyl group is replaced with a benzene ring, inhibited the expression of HIF-1alpha (Figs. 2D, E). DMBDP, EHDP, and MBBP showed toxicity against Hep3B cells at a concentration of 200 µM. The concentration of 100 µM was adopted in this experiment with DMBDP, EHDP, and MBBP, and 100 µM BPA was also used in this experiment. We also investigated the effect of BPA and BPA derivatives on EPO mRNA expression. BPA decreased EPO mRNA expression; however, BPE and BPF did not (Fig. 3A). These results were consistent with findings of Kubo et al.²²⁾ BPB and DMBDP but not EHDP decreased EPO mRNA expression (Figs. 3B, C). HIF-1alpha protein disappeared by treatment with BPB and DMBDP but BPB and DMBDP did not inhibit EPO mRNA expression completely. The discrepancy between HIF-1alpha protein levels and EPO mRNA expression may be caused by specificity of these compounds to HIF-2alpha degradation because HIF-1alpha and HIF-2alpha may contribute to EPO mRNA expression.²⁵⁾ BHCH and MBBP also decreased EPO mRNA expression (Figs. 3D, E). These results suggested that methyl groups are important for the inhibition of HIF-1alpha and EPO mRNA expression. We calculated log P (Partition Coefficient) for BPA and its derivatives (Table 1). BPE and BPF are less hydrophobic (lower log P value) than BPA. Because the log P values of BPB, DMBDP, BHCH, and MBBP is larger than that of BPA, the hydrophobicity of BPB, DMBDP, BHCH, and MBBP is higher than BPA. EHDP showed the highest hydrophobicity in BPA derivatives, however, EHDP cannot affect HIF-1alpha expression. These results suggested that hydrophobicity is important for BPA and BPA derivatives in exerting their effect on HIF-1alpha expression.

Fig. 2. Decrease of HIF-1alpha Levels by BPA Derivatives under Hypoxia

Hep3B cells were cultured for 6 h under hypoxia in the presence of BPA or BPA derivatives at a concentration of 100 µM (B and D) or 200 µM (A, C and E). HIF-1alpha or beta-actin were detected by Western blotting with anti-HIF-1alpha or -beta-actin antibodies. The graphs indicate the relative intensity of the HIF-1alpha band normalized by beta-actin. The control value under hypoxia was set at 1.0. Values are the mean±standard deviation (S.D.) for three separate experiments. * p<0.05, ** p<0.01, significantly different from the control. #, less than 0.05. N, Normoxia; Hyp, Hypoxia; Ctrl, Control.

Fig. 3. Decrease of EPO mRNA Levels Related to the Structure of BPA Derivatives under Hypoxia

Hep3B cells were cultured for 6 h under hypoxia in the presence of BPA or BPA derivatives 100 µM (B and D) or 200 µM (A, C and E). Total RNA was isolated from cells in three different culture plates, and real-time PCR was performed. The control value under hypoxia was set at 1.0. Values are the mean±S.D. for three separate experiments. * p<0.05, ** p<0.01, significantly different from the control. #, less than 0.05. N, Normoxia; Hyp, Hypoxia; Ctrl, Control.

Table 1. The Hydrophobicity of BPA and Its Derivatives

BPA derivatives	BPA	BPE	BPF	BPB	DMBDP	EHDP	BHCH	MBBP
Hydrophobicity (p value)	3.37	2.92	2.94	3.87	4.64	5.47	4.53	4.58

BPA Induces Degradation of HIF-1alpha via Lysosomal Enzymes

We investigated the effect of BPA and DMBDP on HIF-1alpha mRNA. BPA and DMBDP did not change HIF-1alpha mRNA level (Fig. 4A), suggesting that these compounds degrade HIF-1alpha in the protein level. Then, MG132, a proteasome inhibitor, used but it did not inhibit the degradation of HIF-1alpha by BPA. However, ammonium chloride (NH₄Cl), a lysosomal enzyme inhibitor inhibited the degradation of HIF-1alpha by BPA. The decrease of HIF-1alpha was completely recovered in the presence of both MG132 and NH₄Cl in hypoxic (Fig. 4B) or normoxic conditions (Fig. 4C). DMBDP also decreased HIF-1alpha protein in the presence of MG132 and the decrease was recovered in the presence of MG132 and NH₄Cl (Fig. 4C). These findings suggested that BPA and DMBDP accelerate the degradation of HIF-1alpha in a lysosome-dependent manner.

Fig. 4. BPA Induces Degradation of HIF-1alpha via Lysosomal Enzymes

Hep3B cells were cultured for 6 h under hypoxia in the presence of BPA (200 µM) or DMBDP (100 µM) (A). Total RNA was isolated from cells in three different culture plates, and RT-PCR was performed. The control value was set at 1.0. Values are the mean±S.D. for three separate experiments. Ctrl, control. Hep3B cells were cultured in the presence of MG132 (5 µM), a proteasome inhibitor, or NH₄Cl (10 mM), a lysosome inhibitor, for 8 h in the absence or presence of BPA (200 µM) or DMBDP (100 µM) under hypoxia (B) or normoxia (C). HIF-1alpha or beta-actin were detected by Western blotting with anti-HIF-1alpha or -beta-actin antibodies (B and C). The graphs indicate the relative intensity of HIF-1alpha band normalized by beta-actin. The control value under hypoxia was set at 1.0. Values are the mean±S.D. for three separate experiments. * p<0.05, ** p<0.01, significantly different from the control. #, less than 0.05.

Time-Dependent Degradation of HIF-1alpha Protein by BPA

We examined the time-dependency of HIF-1alpha degradation by BPA. The expression of HIF-1alpha was decreased to around 70% by treatment with BPA for 30 min in the presence of MG132 and Cycloheximide (CHX), and HIF-1alpha expression was decreased to less than 40% after 1 h of BPA exposure (Fig. 5A). NH₄Cl prolonged the degradation time of HIF-1alpha protein (Fig. 5B).

Fig. 5. Time-Dependent Degradation of HIF-1alpha Protein by BPA

Hep3B cells were pretreated with MG132 (A), and MG132 and NH₄Cl (B) for 6 h under hypoxia, and then were treated with CHX for 1 h. These cells were treated with BPA (200 µM) for additional 30–120 min. HIF-1alpha or beta-actin was detected by Western blotting with anti-HIF-1alpha or -beta-actin antibodies (A and B). The graphs indicate the relative intensity of HIF-1alpha band normalized by beta-actin. The control value under hypoxia was set at 1.0. Values are the mean±S.D. for three separate experiments. N, Normoxia; Hyp, Hypoxia; Ctrl, Control

Changes in HSC70 Levels by BPA Treatment

Hubbi et al. suggested that HIF-1alpha is degraded via chaperone mediated autophagy (CMA) as well as the proteasome pathway.²⁶⁾ HSC70 transports CMA target proteins to lysosomes, hence we investigated that the effect of BPA on the expression of HSC70 in Hep3B cells. The expression of HSC70 was increased by treatment with 200 µM BPA; however, Hsp90 expression did not change (Figs. 6A, B). Moreover, BPF did not change HSC70 expression, however DMBDP did (Fig. 6C). These results suggest that the degradation of HIF-1alpha by BPA is not due to a decrease in Hsp90 protein.

Fig. 6. Changes in HSC70 Levels by BPA Treatment

Hep3B cells were cultured for 6 h under hypoxia in the presence of BPA (50–200 µM), and HSC70 (A) or Hsp90 (B) were detected by Western blotting. Hep3B cells were cultured for 6 h under hypoxia in the presence of BPA (200 µM), BPF (200 µM), or DMBDP (100 µM). HSC70 and beta-actin were detected by Western blotting with anti-HSC70 or -beta-actin antibodies (C). Hep3B cells were overexpressed with HSC70/pcDNA3.1 and were cultured for 6 h under hypoxia in the presence or absence of NH₄Cl (10 mM). The inlet of Fig. 6D indicates the confirmation of HSC70 overexpression by Western blotting. HIF-1alpha and beta-actin were detected by Western blotting with anti-HIF-1alpha or -beta-actin antibodies (D). The graphs indicate the relative intensity of the HSC70, Hsp90, and HIF-1alpha bands normalized by beta-actin. The control (A and B) or mock (D) value under hypoxia was set at 1.0. Values are the mean±S.D. for three separate experiments. ** p<0.01, significantly different from the control. #, less than 0.05. N, Normoxia; Hyp, Hypoxia; Ctrl, Control; O.E., Overexpression.

Effects of HSC70 Protein Levels on HIF-1alpha Degradation

Because BPA induced HSC70 expression, we investigated that the effect of HSC70 on HIF-1alpha protein levels. Overexpression of HSC70 decreased HIF-1alpha expression, and the decrease was recovered by NH₄Cl (Fig. 6D). These results suggested that BPA induces HSC70 expression, resulting in the degradation of HIF-1alpha by lysosomal enzymes through HSC70. To investigate the role of HSC70 in the degradation of HIF-1alpha by BPA, we prepared HSC70 knockdown cells. BPA did not decrease HIF-1alpha in HSC70 knockdown cells, again suggesting HSC70 is important for BPA in exerting its effect on HIF-1alpha expression (Fig. 7).

Fig. 7. Effects of HSC70 Knockdown on the Degradation of HIF-1alpha by BPA

HSC70 in Hep3B cells was knocked down with si-HSC70, and these cells were cultured for 6 h under hypoxia in the presence of BPA (200 µM). The inlet of Fig. 6 indicates the confirmation of HSC70 knockdown by Western blotting. HIF-1alpha and beta-actin were detected by Western blotting with anti-HIF-1alpha or -beta-actin antibodies. The graph indicates the relative intensity of the HIF-1alpha bands normalized by beta-actin. The si-control value under hypoxia was set at 1.0. Values are the mean±S.D. for three separate experiments. ** p<0.01, significantly different from the control. #, less than 0.05. N, Normoxia; Hyp, Hypoxia; Ctrl, Control; K.D., Knockdown.

DISCUSSION

In this study, to investigate the effect of BPA derivatives on HIF-1alpha expression and activity, we investigated the effect of alkyl groups on carbon atoms between the two phenyl rings. BPE, which lacks one methyl group of BPA, and BPF, which lacks two methyl groups of BPA, did not alter HIF-1alpha expression. The hydrophobicity of BPA is higher than BPE and BPF. BPB and DMBDP, which have the longer alkyl groups than BPA, decreased HIF-1alpha levels more strongly than BPA. Additionally, the derivatives, with methyl groups replaced by a cyclohexyl group (derivative was designated BHCH) or benzene ring (derivative was designated MBBP), promoted degradation of HIF-1alpha compared with BPA. BPB, DMBDP, BHCH, and MBBP have higher hydrophobicity than BPA. These results suggested that the hydrophobicity is required for the inhibition of HIF-1alpha. However, EHDP, which is the most hydrophobic derivative used in this study, did not affect the expression of HIF-1alpha. EHDP has branched alkyl group on the central carbon, therefore not only the hydrophobicity of the side chain but also its structure is required for inhibition by BPA derivatives. In our previous study, we found BPA but not BPF dissociated HIF-1alpha from Hsp90 in vitro experiment,²²⁾ suggesting that the effect of BPA and its derivatives on HIF-1alpha expression may not be caused by difference in the membrane permeability of BPA derivatives.

Perez et al., suggested that longer alkyl groups between the two phenyl rings in BPA, resulted in higher estrogenicity on MCF7 human breast cancer.²⁷⁾ Moreover, the estrogenicity of BPA is determined not only by the length of alkyl groups but also by the distance between two oxygen atoms in two phenyl rings.²⁷⁾ These results suggest that the effect of BPA derivatives on HIF-1alpha is determined be several factors such as the nature and configuration of side chains on the central carbon.

In a previous study, we suggested that BPA degraded HIF-1alpha even in the presence of proteasome inhibitor.²²⁾ Moreover, our previous study suggested that BPA inhibits the interaction between HIF-1alpha and Hsp90.²²⁾ The geldanamycin derivative 17-AAG also inhibits the interaction HIF-1alpha and Hsp90, thereby inducing the degradation of HIF-1alpha via proteasomes.²⁸⁾ Barliya et al. found that a quinoid compound, hypericin, promoted poly-ubiquitination and degradation of Hsp90 via the proteasome pathway, resulting in interruption of the interaction between Hsp90 and HIF-1alpha.²⁹⁾ Hypericin reduces intracellular pH, inducing the activation of cathepsin-B, a lysosomal protease, and the degradation of HIF-1alpha via the lysosomal pathway. In this study, we found BPA induced the degradation of HIF-1alpha via the lysosomal pathway; however, BPA did not degrade Hsp90. BPA may degrade HIF-1alpha via a different pathway from hypericin.

Hubbi et al. identified that HIF-1alpha is target protein of CMA, one of the lysosomal degradation pathways. HSC70 interacts with HIF-1alpha, and overexpression of HSC70 decreased HIF-1alpha levels.²⁶⁾ Recently, it was indicated that the expression of HSC70 and lysosome associated membrane protein type2, key factors in CMA, increased in hippocampus of rats treated with BPA.³⁰⁾ In the present study, we found that BPA and DMBDP induced the expression of HSC70 in Hep3B cells but not BPF. Moreover, we determined that the overexpression of HSC70 decreased HIF-1alpha expression and the decrease was recovered by NH₄Cl. We found that HSC70 knockdown efficiently restored the decrease in HIF-1alpha protein levels by BPA. These results suggested that BPA increases the expression of HSC70, inducing the degradation of HIF-1alpha via the lysosomal pathway.

Wang et al. found that HSC70 levels also increased in the testes of rats treated with BPA, however the mechanism remains unclear.³¹⁾ Papaconstantinou et al. suggested that BPA affects the transcription of heat shock protein 72 (Hsp72) through ER, inducing Hsp72 expression in mice uterine tissue.³²⁾ HSC70 and Hsp72 are included in the same HSPA group. However, Molina-Molina et al. suggested that not only BPA but also BPF activated ER in the estrogen-sensitive human breast cancer cell line MCF-7.³³⁾ We found that the decrease of HIF-1alpha by BPA was very fast, suggesting that degradation of HIF-1alpha by BPA derivatives may not depend on response of receptor. Therefore, ER may not be involved in the effect of BPA and its derivatives on HIF-1alpha expression. Moreover, HSC70 induced by BPA may be replaced with Hsp90 on HIF-1alpha, resulting in the degradation of HIF-1alpha via the lysosomal pathway. Cypher et al. indicated that BPA decreased the cardiovascular response under hypoxia in Danio rerio embryos.³⁴⁾ The effect of BPA on the mechanism of HIF-1alpha degradation via the lysosomal pathway and biological effects of BPA via HIF-1alpha require further study.

Acknowledgments

This study was partially supported by the Support Project to Assist Private Universities in Developing Bases for Research by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and was also supported by Grant-in-Aid from Kwansei Gakuin University.

Conflict of Interest

The authors declare no conflict of interest.

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