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Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells Qun Wei a, Jun Zhang a, Le Su a, Xuan Zhao a, BaoXiang Zhao b, JunYing Miao a, ZhaoMin Lin c, * a b c
Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan, 250100, PR China Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, PR China Institute of Medical Sciences, The Second Hospital of Shandong University, Jinan, 250033, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2019 Accepted 1 February 2019 Available online xxx
Vascular endothelial cell (VEC) apoptosis takes part in the development of various cardiovascular diseases. Heat shock protein 90 (HSP90) regulates apoptosis through various apoptosis associated client proteins. In previous study, we identified a novel HSP90 inhibitor HCP1 induced apoptosis in A549 human lung cancer cells. Here, we found that low-concentration HCP1 (1 mM, 2 mM) suppressed VEC apoptosis caused by serum and fibroblast growth factor 2 (FGF-2) deprivation. HCP1 directly bound to glucose-regulated protein 94 (Grp94), an isoform of HSP90 located in endoplasmic reticulum, and HCP1 selectively inhibited Grp94 activity via binding to site 3. Overexpression of Grp94 inhibited the antiapoptotic effect of HCP1 in human umbilical vein endothelial cells. Therefore, we provided HCP1 as a new VEC apoptosis inhibitor which might be a potential compound in the treatment of VEC apoptosis related vascular diseases. And we provided new pieces of evidence to understand the role of Grp94 in VEC apoptosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Vascular endothelial cell Apoptosis inhibitors Glucose-regulated protein 94
1. Introduction Vascular endothelial cells (VECs) compose a single-layered endothelium located on the inner surface of blood vessels. VECs play an important role in the maintenance of vascular homeostasis. Under normal conditions, VECs secrete functional factors, regulate vascular tone, monitor plasma components transportation and play key roles in signal transduction [1]. VEC dysfunction is reported to lead to the development of various cardiovascular diseases. And apoptosis of VECs is the hallmark of cardiovascular diseases associated with pathological conditions toward thrombosis, vasoconstriction and inflammatory state [2]. For example, VEC apoptosis can accelerate lipid accumulation and the formation of atherosclerotic plaque [3,4]. Besides, endothelial cell apoptosis in plaque can result in its instability and rupture, which finally leads to thrombosis [5,6]. Thus, inhibition of VEC apoptosis is significant in the treatment of cardiovascular diseases. And it is necessary to develop novel inhibitors of VEC apoptosis.
* Corresponding author. E-mail address: [email protected] (Z. Lin).
Heat shock protein 90 (HSP90) is a highly abundant chaperone protein expressed by all eukaryotic cells [7]. HSP90 participates in the regulation of apoptosis. On one hand, HSP90 is a powerful antiapoptotic protein associated with various apoptotic factors and inhibition of HSP90 has emerged as a novel strategy for cancer therapy. On the other hand, low-concentration HSP90 inhibitors DPB and AUY-922 were reported to repress apoptosis in HUVECs [8]. In advanced eukaryotes, HSP90 paralogs include cytoplasmic Hsp90a and Hsp90b, endoplasmic reticulum (ER) isoform glucoseregulated protein 94 (Grp94) and the mitochondrial isoform Trap-1 [9]. Grp94 is an essential master chaperone mainly for secreted and membrane proteins [9]. Multiple receptors including Toll-like receptors, Wnt coreceptors, and integrins have been shown to be the client proteins of Grp94. It is reported that Grp94 is essential for the expression of integrin a2 within the hematopoietic system [10]. The protein level of integrin a2 could be reduced in tumor cells after Grp94 inhibition [11,12]. In addition, Grp94 also regulates Ca2þ homeostasis, controls protein quality in ER and participates in immune process through regulation of its immune client proteins. In our previous study, we synthesized and identified a novel coumarin pyrazoline derivative 3-(1,5-diphenyl-4,5-dihydro-1H-
https://doi.org/10.1016/j.bbrc.2019.02.003 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003
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pyrazol-3-yl)-7-hydroxy-2H-chromen-2-one (HCP1) as the inhibitor of HSP90. HCP1 could inhibit A549 lung cancer cell growth and induce apoptosis [13]. However, the impact of HCP1 on VEC apoptosis remains unclear. Deprivation of serum and fibroblast growth factor 2 (FGF-2) can lead to VEC apoptosis [14]. Therefore, we investigated the effects of HCP1 on serum and FGF-2 deprivation-induced VEC apoptosis as well as the underlying mechanism. 2. Materials and methods 2.1. Antibodies and reagents Antibodies for integrin a2 (ab181548), Grp94 (ab3674) and TLR9 (ab37154) were purchased from Abcam (Cambridge, UK). Antibodies against LAMP2 (sc-20004), NF-kB (p65) (Sc-109), Hsp70 (sc1060), GAPDH (sc-365062) and horseradish peroxidase conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Akt (4691) was from Cell Signaling Technology (USA). Antibody for Grp78 (11587-1-AP) was from Proteintech Group (USA). Antibody for ACTB (122M4782) was from Sigma-Aldrich (St. Louis, Missouri, USA). Secondary antibodies for immunofluorescence goat anti-rabbit Alexa 647, donkey anti-rabbit Alexa 546 and goat anti-mouse Alexa 488 were all from Invitrogen (Carlsbad, CA, USA). HCP1 was synthesized as described previously [13] and dissolved in DMSO (10 mM, Sigma-Aldrich, St. Louis, Missouri, USA) as a stock solution. Recombinant Grp94 (HSP90B1-132H) was purchased from Creative BioMart (New York, USA). Mito-tracker deep red (M22426) was obtained from Invitrogen (Carlsbad, CA, USA). 2.2. Cell culture
2.5. Western blot analysis Cells were lysed in western and IP buffer (Beyotime, P0013) containing 150 mM NaCl, 20 mM Tris-HCl (PH 7.5), 1% Triton X-100, and proteinase inhibitors mix. After centrifuging at 12000 g for 15 min under 4 C, the supernatant was collected. Protein samples (20 mg/lane) were loaded on 15% SDS-polyacrylamide gel, and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, IPVH00010). The membranes were incubated with primary antibodies, then horseradish peroxidase-linked secondary antibodies and detected using an enhanced chemiluminescence detection kit (Thermo Fisher, 34080). The relative quantity of proteins was analyzed by Image J and normalized to loading controls. 2.6. Surface plasmon resonance (SPR) SPR analysis was carried out with Biacore T200 (GE Healthcare, England) to determine the binding of HCP1 to full length Grp94. Grp94 was immobilized to the surface of a CM5 sensor chip using amine coupling. The immobilization has been performed according to the manufacturer's instructions. Binding experiments were performed with multiple concentrations of HCP1. The compound was flowed at a rate of 10 ml/min for 2 min to allow for association and followed by 600 s for dissociation over immobilized protein in phosphate-buffered saline (PBS) (in mM: 10 phosphate, 137 NaCl, 2.68 KCl, 0.1% dimethyl sulfoxide [v/v], pH 8.0) at 25 C. The sensor chip surface was regenerated using 10 mM NaOH. The binding responses were recorded as resonance units (RU). Data analyses using the Biacore T200 Evaluation Software were performed after blank subtraction. 2.7. Cell staining for immunofluorescence microscopy
37 C
All cell lines were maintained at under humidified conditions and 5% CO2. Human umbilical vein endothelial cells (HUVECs) were obtained from human umbilical cord veins as described previously [15]. HUVECs were cultured as routine on gelatin coated plastic dishes in M199 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone Lab, Inc, Tauranga, New Zealand) and 2 ng/mL FGF-2. All HUVECs involved in experiments were at no more than passage 10. A549 cells were grown in RPMI Medium 1640 (Gibco, Grand Island, NY) in the presence of 10% FBS. HEK293T cells were cultured as routine in DMEM medium (Gibco, Grand Island, NY) supplemented with 10% FBS. 2.3. Cell viability assay Cells were plated and treated in 96-well plates followed by precipitating in 100 ml 10% trichloroacetic acid (Shenggong Biotech, Shanghai, China) for 1 h at 4 C. Then cells were stained with 50 ml sulforhodamine B (SRB; Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min and the bounded dye was reconstituted in 100 ml of 10 mM Trisbase (pH 10.5). The optical density was read by a Spec-traMAX 190 microplate spectrophotometer (GMI Co., USA) at 540 nm. Cell viability (%) ¼ (OD of treated group/OD of control group) 100. 2.4. Hoechst 33258 staining Cells were seeded on gelatin coated 24-well plates for 24 h before deprivation of serum and FGF-2 and treated with HCP1 for 48 h. Then, Hoechst 33258 (Sigma-Aldrich, St. Louis, Missouri, USA) was used to stain cells for 10 min at 37 C. Cells were washed twice with PBS gently and photographed with Olympus (Japan) BH-2 fluorescence microscope. More than 400 cells were counted.
Cells were fixed in 4% paraformaldehyde (w/v) for 15 min at room temperature or ice-cold methanol at 20 C for 10 min and blocked in PBS, 0.01% Triton X-100 (v/v) and 5% donkey serum (v/v) for 60 min. Then cells were incubated with primary antibodies overnight at 4 C and washed in PBS three times followed by incubation with corresponding secondary antibodies for 1 h at 37 C. Fluorescence was detected by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). At least 3 fields of view were analyzed by the microscope for each sample, and representative results are shown. 2.8. RNA interference Grp94 siRNA (sc-35523) and scramble siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells at 70%e80% confluence were transfected with siRNA against Grp94 and scramble siRNA by use of Lipofectamin 2000 (Invitrogen, 11668e019) following the manufacturer's instructions. 2.9. Plasmids and overexpression The coding region of Grp94 was sub-cloned into the pCMV3-CMyc expression vector to produce the c-Myc-Grp94-wt construct. Mt1 mutant (Gly196, Val197, Phe199 and Tyr200 of Grp94 were mutated to alanine), mt2 mutant (Phe199, Ala202, Phe203, Val209 and Val211 of Grp94 were mutated to alanine), mt3 mutant (Val209, Val211, Ile247 and Leu249 of Grp94 were mutated to alanine) and mt4 mutant (Phe199, Ala202, Phe203, Ile247 and Leu249 of Grp94 were mutated to alanine) were sub-cloned into pCMV3-C-Myc expression vector as the mutated Grp94 cDNA. All of the constructs were confirmed by DNA sequencing. Cells, at 70%e 80% confluence, were transfected with the expression vectors for
Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003
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24 h by using Lipofectamine 2000 (Invitrogen, 11668e019) following the manufacturer's instructions. 2.10. Statistical analyses Results are reported as means ± SEM. All experiments were repeated at least three times independently. For statistical analysis, Graph Pad Prism software (version 5.0) was used. Statistical comparisons for data were performed using one-way analysis of variance (ANOVA) followed by Tukey: compare all pairs of columns test. Differences were regarded as statistically significant when p < 0.05. 3. Results
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detached from the dish gradually after withdraw of serum and FGF2 and HCP1 (1 mM) inhibited cell death (Fig. 1C). Furthermore, we detected the effects of HCP1 on HUVEC apoptosis. Results showed that HCP1 (1 mM) inhibited HUVEC apoptosis induced by serum and FGF-2 deprivation (Fig. 1D). 3.2. Low-concentration HCP1 did not affect Hsp90a/b activity We further investigated the underlying mechanism of which HCP1 inhibited HUVEC apoptosis caused by deprivation of serum and FGF-2. We determined the effects of low-concentration HCP1 on Hsp90a/b activity. Treatment with HCP1 at low concentrations (1 mM, 2 mM) did not influence protein levels of Akt, NFkB (p65) and Hsp70 (Supplementary Fig. 1). Thus, low-concentration HCP1 did not influence the activity of Hsp90a/b.
3.1. Low-concentration HCP1 inhibited HUVEC apoptosis induced by serum and FGF-2 deprivation
3.3. HCP1 bound to Grp94
First of all, we determined the effects of HCP1 on VEC apoptosis induced by serum and FGF-2 deprivation. Results showed that treatment with HCP1 at low concentrations (1 mM, 2 mM) increased HUVEC viability under serum and FGF-2 deprivation conditions both for 24 h and 48 h (Fig. 1AeB). Morphologically, HUVECs
Then, we detected the effects of HCP1 on Grp94 and Trap-1. The distribution of HCP1 in living cells showed that HCP1 co-localized with Grp94 in endoplasmic reticulum (Fig. 2AeB). And HCP1 did not located in mitochondria (Supplementary Fig. 2). Therefore, we further determined the interaction between HCP1 and Grp94.
Fig. 1. HCP1 inhibited HUVEC apoptosis induced by serum and FGF-2 deprivation. (A, B) Viabilities of HUVECs cultured under normal conditions (Control), deprived of serum and FGF-2 (Serum deprived) and treated with or without HCP1 of the indicated concentrations under serum and FGF-2 deprivation conditions (Treated) for 24 h and 48 h. Data are means ± SEM; (***p < 0.001 vs. Control; **p < 0.01 vs. Serum deprived; n ¼ 3). (C) Morphology of HUVECs cultured under normal conditions, deprived of serum and FGF-2 (Serum deprived) or treated with or without HCP1 (1 mM) for 24 h and 48 h under serum and FGF-2 deprivation conditions (magnification 100 ). Scale bar, 50 mm. (D) Hoechst 33258 staining and quantification of apoptotic HUVECs cultured under normal conditions, deprived of serum and FGF-2 (Serum deprived) and treated with or without HCP1 (1 mM) under serum and FGF-2 deprivation conditions for 24 h. Scale bar, 50 mm. Data are means ± SEM; (***p < 0.001 vs. Control; **p < 0.01 vs. Serum deprived; n ¼ 3).
Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003
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Fig. 2. HCP1 inhibited Grp94 activity. (A, B) HCP1 co-localized with Grp94. Immunofluorescence microscopy image of A549 cells treated with HCP1 (1 mM) for 3 h and then stained with Grp94 or Grp78. Nuclei were stained with 40 , 6-diamidino-2-phenylindole (DAPI). Bar charts show quantification of weighted co-localization coefficient (Pearson's coefficient) for HCP1 and Grp94/Grp78 Scale bar, 20 mm. (C) Interaction of HCP1 with Grp94 was measured by using SPR analysis. Various concentrations of HCP1 were injected onto Grp94immobilized chip and RU values were recorded. (D, E) Immunofluorescence microscopy image of TLR9 and LAMP2 in A549 cells after treatment with Grp94 siRNA or treated with HCP1 (1 mM) for 24 h. Nuclei were stained with DAPI. Bar charts show quantification of weighted co-localization coefficient for TLR9 and LAMP2. Scale bar, 10 mm. (F, G) Protein levels of integrin a2 treated with the indicated concentrations of HCP1 for 24 h in HEK293T cells. Data are means ± SEM; (*p < 0.05, **p < 0.01; n ¼ 3).
Typical sensorgrams of the interactions between HCP1 and Grp94 were obtained (Fig. 2C). The equilibrium dissociation constant (KD value) of HCP1/Grp94 interaction was 0.923 mM. These results indicated that HCP1 bound to Grp94 directly.
HEK293T cells after treatment with HCP1 for 24 h (Fig. 2FeG). These results showed that HCP1 inhibited Grp94 activity.
3.4. HCP1 inhibited Grp94 activity
Currently, paralog-selective Grp94 inhibitors have been reported to bind to two specific pockets adjacent to the adenosine triphosphate (ATP) binding cavity termed Site 2 and Site 3 [17]. To determine whether HCP1 could bind to these two sites in Grp94, potent binding amino acid residues were selected and plasmids of Grp94 mutant at these two sites were constructed: c-Myc-Grp94-
Next, we detected the effects of HCP1 on Grp94 activity. Results showed that HCP1 inhibited Toll-like receptor 9 (TLR9) trafficking which was mediated by Grp94 (Fig. 2DeE and Supplementary Fig. 3) [10,16]. The protein level of integrin a2 also decreased in
3.5. HCP1 bound to Site 3 in Grp94
Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003
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mt1 of Site 3 and c-Myc-Grp94-mt2, -mt3, -mt4 of Site 2 (Fig. 3A). Then HEK293T cells were transfected with plasmids coding for cMyc-Grp94-wt, -mt1, -mt2, -mt3 and -mt4 for 24 h followed by treatment with HCP1 for 24 h. Results revealed that integrin a2 protein level was elevated in HEK293T cells transfected with c-
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Myc-Grp94-mt1. While protein levels of integrin a2 in c-MycGrp94-mt2, -mt3 and -mt4 transfected cells showed no differences with that in c-Myc-Grp94-wt transfected cells (Fig. 3BeC). We further evaluated the effects of mt1 on the binding of HCP1 to Grp94. Results showed that co-localization coefficient of c-Myc-
Fig. 3. HCP1 suppressed HUVEC apoptosis through inhibition of Grp94 activity. (A) Mutation sites on Grp94 protein. The alignment shows the selected amino acid residues within the two hot spots targeted by small molecule compounds (site 2 and site 3). Different mutations were designed as indicated in the diagram. (B, C) Protein levels of integrin a2 in HEK293T cells transfected with expression vectors c-Myc and c-Myc-Grp94-wt, -mt1, -mt2, -mt3 and -mt4 for 24 h, then treated with HCP1 (1 mM) for 24 h. Data are means ± SEM; (*p < 0.05, ***p < 0.001; n ¼ 3). (D, E) Immunofluorescence microscopy image of A549 cells transfected with expression vectors c-Myc-Grp94-wt, -mt1 for 24 h, then treated with HCP1 (1 mM) for 3 h and stained with Grp94. Nuclei were stained with DAPI. Bar charts show quantification of weighted co-localization coefficient (Pearson's coefficient) for HCP1 and Grp94. Scale bar, 20 mm (F, G) Viabilities of HUVECs transfected with expression vectors c-Myc and c-Myc-Grp94-wt, -mt1 for 24 h, then cultured under normal conditions or deprived of serum and FGF-2 conditions treated with or without HCP1 (1 mM, 2 mM) for 24 h. Data are mean ± SEM; (*p < 0.05, **p < 0.01, ***p < 0.001; n ¼ 3).
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Grp94-mt1 with HCP1 was lower than that of c-Myc-Grp94-wt with HCP1 (Fig. 3DeE). Therefore, mt1 of Grp94 decreased the binding of HCP1 to Grp94 and HCP1 bound to Site 3 in Grp94. 3.6. Low-concentration HCP1 suppressed HUVEC apoptosis through inhibition of Grp94 activity Next, we determined the relationship between the effects of HCP1 on HUVEC apoptosis and Grp94 activity. Results showed that overexpression of c-Myc-Grp94-wt inhibited the protective effect of HCP1 on HUVEC apoptosis which suggested that HCP1 suppressed HUVEC apoptosis through inhibition of Grp94 activity (Fig. 3FeG). In addition, compared with c-Myc-Grp94-wt transfected group, overexpression of c-Myc-Grp94-mt1 further blocked the anti-apoptotic effects of HCP1 in HUVECs (Fig. 3FeG). These results showed that low-concentration HCP1 (1 mM, 2 mM) suppressed HUVEC apoptosis through inhibition of Grp94 activity via binding to site 3. 4. Discussion This study determined the anti-apoptotic effect of a coumarin pyrazoline derivative HCP1 in HUVECs. We found that lowconcentration HCP1 (1 mM, 2 mM) effectively suppressed serum and FGF-2 deprivation-induced HUVEC apoptosis through selective inhibition of Grp94. Grp94 is a canonical hallmark of ER stress. Grp94 as well as its clients’ chaperons participate in metabolic stress, and whose expressions are induced during ER stress [18]. It is reported that ER stress is involved in serum deprivation-induced apoptosis in hippocampal neuroblasts [19]. ER stress-mediated apoptosis could be inhibited in human breast cancer MCF-7 cells and hepatoblastoma HepG2 cells by pretreatment with FGF-2 [20]. Here, we found that low-concentration HCP1 (1 mM, 2 mM) selectively inhibited Grp94 activity and suppressed apoptosis induced by serum and FGF-2 deprivation in HUVECs. These results indicated that inhibition of Grp94 activity also suppressed apoptosis and provided us new pieces of evidence to understand the underlying mechanism of VEC apoptosis. Grp94 is closely associated with the pathogenesis of different diseases. Grp94 is reported to be up-regulated in tumor cells and plays a role in oncogenesis and metastasis [10]. In addition, Grp94 prevents misfolded myocilin degradation via autophagy which promotes the pathologies associated with glaucoma [21]. Because of the pro-apoptotic effects in VECs, Grp94 could be a potential therapeutic target in clinical treatment of vascular diseases. And HCP1 would become a valuable tool to study the pathogenesis of vascular diseases. Clinical HSP90 inhibitors inhibited all isoforms of HSP90 and exhibited various adverse side effects such as hepatotoxicity, hyponatremia, hypoglycemia, fatigue and diarrhea which limited their application in clinical treatment [22,23]. Therefore, it is necessary to improve the isoform selectivity of HSP90 inhibitors to minimize the side effects. The high homology in sequence and structure of HSP90 family members seriously restrained the development of HSP90 isoform selective inhibitors. Despite the high identity, two pockets distinct from other paralogs were identified in Grp94 which greatly facilitated the development of selective inhibitors [17]. These two pockets formed a halo of potential selectivity adjacent to the ATP binding pocket in Grp94 which were termed as Site 2 and Site 3. For example, NECA and resorcinilic scaffold-inhibitors target Site 3 [11,24,25] while purine derivatives bind to Site 2 in Grp94 [12,16,26]. The adenosine receptor agonist NECA bound site 3 of Grp94. However, the activation of the adenosine receptor pathway led to the off-target effects of NECA, which limited the use of NECA scaffold in the development of
Grp94 selective inhibitors. We found coumarin pyrazoline derivative HCP1 could inhibit Grp94 activity through binding to Site 3. Therefore, coumarin pyrazoline scaffold is a novel potential scaffold for Grp94 selective inhibitors bound to Site 3. In summary, we discovered coumarin pyrazoline derivative HCP1 as a novel inhibitor of VEC apoptosis. Low-concentration HCP1 (1 mM, 2 mM) inhibited apoptosis induced by deprivation of serum and FGF-2 in HUVECs through selectively inhibited Grp94 activity via binding to site 3. HCP1 was a useful tool to inhibit Grp94 and would be a potential candidate for developing new drugs against vascular diseases. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31870831, 91539105, 81502948, 81321061, 91313303) and Shandong Excellent Young Scientist Award Fund (No. BS2015YY031). We thank Zhifeng Li and Jing Zhu from Analysis & Testing Center of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for assistance in surface plasmon resonance technology of Biacore T200. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.003. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.003. References [1] E.M. Gleeson, J.S. O'Donnell, R.J. Preston, The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling, Cell. Mol. Life Sci. CM 69 (2012) 717e726. [2] S. Godo, H. Shimokawa, Endothelial functions, Arterioscler. Thromb. Vasc. Biol. 37 (2017) e108ee114. [3] L. Zhang, H.Y. Li, H. Li, J. Zhao, L. Su, Y. Zhang, S.L. Zhang, J.Y. Miao, Lipopolysaccharide activated phosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cells, J. Cell. Physiol. 226 (2011) 1694e1701. [4] B. Messner, I. Zeller, C. Ploner, S. Frotschnig, T. Ringer, A. Steinacher-Nigisch, A. Ritsch, G. Laufer, C. Huck, D. Bernhard, Ursolic acid causes DNA-damage, p53-mediated, mitochondria- and caspase-dependent human endothelial cell apoptosis, and accelerates atherosclerotic plaque formation in vivo, Atherosclerosis 219 (2011) 402e408. [5] A.V. Sima, C.S. Stancu, M. Simionescu, Vascular endothelium in atherosclerosis, Cell Tissue Res. 335 (2009) 191e203. [6] K. Kobayashi, K. Sato, T. Kida, K. Omori, M. Hori, H. Ozaki, T. Murata, Stromal cell-derived factor-1 alpha/C-X-C chemokine receptor type 4 axis promotes endothelial cell barrier integrity via phosphoinositide 3-kinase and Rac 1 activation, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 1716e1722. [7] X. Wang, M. Chen, J. Zhou, X. Zhang, HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy (Review), Int. J. Oncol. 45 (2014) 18e30. [8] S.Y. Bai, L.Q. Yao, L. Su, S.L. Zhang, B.X. Zhao, J.Y. Miao, Low-dose HSP90 inhibitors DPB and AUY-922 repress apoptosis in HUVECs, RSC Adv. 5 (2015) 75753e75755. [9] M. Marzec, D. Eletto, Y. Argon, GRP94: an HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum, Biochim. Biophys. Acta 1823 (2012) 774e787. [10] B.X. Wu, F. Hong, Y. Zhang, E. Ansa-Addo, Z. Li, GRP94/gp96 in cancer: biology, structure, immunology, and drug development, Adv. Cancer Res. 129 (2016) 165e190. [11] V.M. Crowley, A. Khandelwal, S. Mishra, A.R. Stothert, D.J. Huard, J. Zhao, A. Muth, A.S. Duerfeldt, J.L. Kizziah, R.L. Lieberman, C.A. Dickey, B.S. Blagg, Development of glucose regulated protein 94-selective inhibitors based on the BnIm and radamide scaffold, J. Med. Chem. 59 (2016) 3471e3488.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells Qun Wei a, Jun Zhang a, Le Su a, Xuan Zhao a, BaoXiang Zhao b, JunYing Miao a, ZhaoMin Lin c, * a b c
Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan, 250100, PR China Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, PR China Institute of Medical Sciences, The Second Hospital of Shandong University, Jinan, 250033, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2019 Accepted 1 February 2019 Available online xxx
Vascular endothelial cell (VEC) apoptosis takes part in the development of various cardiovascular diseases. Heat shock protein 90 (HSP90) regulates apoptosis through various apoptosis associated client proteins. In previous study, we identified a novel HSP90 inhibitor HCP1 induced apoptosis in A549 human lung cancer cells. Here, we found that low-concentration HCP1 (1 mM, 2 mM) suppressed VEC apoptosis caused by serum and fibroblast growth factor 2 (FGF-2) deprivation. HCP1 directly bound to glucose-regulated protein 94 (Grp94), an isoform of HSP90 located in endoplasmic reticulum, and HCP1 selectively inhibited Grp94 activity via binding to site 3. Overexpression of Grp94 inhibited the antiapoptotic effect of HCP1 in human umbilical vein endothelial cells. Therefore, we provided HCP1 as a new VEC apoptosis inhibitor which might be a potential compound in the treatment of VEC apoptosis related vascular diseases. And we provided new pieces of evidence to understand the role of Grp94 in VEC apoptosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Vascular endothelial cell Apoptosis inhibitors Glucose-regulated protein 94
1. Introduction Vascular endothelial cells (VECs) compose a single-layered endothelium located on the inner surface of blood vessels. VECs play an important role in the maintenance of vascular homeostasis. Under normal conditions, VECs secrete functional factors, regulate vascular tone, monitor plasma components transportation and play key roles in signal transduction [1]. VEC dysfunction is reported to lead to the development of various cardiovascular diseases. And apoptosis of VECs is the hallmark of cardiovascular diseases associated with pathological conditions toward thrombosis, vasoconstriction and inflammatory state [2]. For example, VEC apoptosis can accelerate lipid accumulation and the formation of atherosclerotic plaque [3,4]. Besides, endothelial cell apoptosis in plaque can result in its instability and rupture, which finally leads to thrombosis [5,6]. Thus, inhibition of VEC apoptosis is significant in the treatment of cardiovascular diseases. And it is necessary to develop novel inhibitors of VEC apoptosis.
* Corresponding author. E-mail address: [email protected] (Z. Lin).
Heat shock protein 90 (HSP90) is a highly abundant chaperone protein expressed by all eukaryotic cells [7]. HSP90 participates in the regulation of apoptosis. On one hand, HSP90 is a powerful antiapoptotic protein associated with various apoptotic factors and inhibition of HSP90 has emerged as a novel strategy for cancer therapy. On the other hand, low-concentration HSP90 inhibitors DPB and AUY-922 were reported to repress apoptosis in HUVECs [8]. In advanced eukaryotes, HSP90 paralogs include cytoplasmic Hsp90a and Hsp90b, endoplasmic reticulum (ER) isoform glucoseregulated protein 94 (Grp94) and the mitochondrial isoform Trap-1 [9]. Grp94 is an essential master chaperone mainly for secreted and membrane proteins [9]. Multiple receptors including Toll-like receptors, Wnt coreceptors, and integrins have been shown to be the client proteins of Grp94. It is reported that Grp94 is essential for the expression of integrin a2 within the hematopoietic system [10]. The protein level of integrin a2 could be reduced in tumor cells after Grp94 inhibition [11,12]. In addition, Grp94 also regulates Ca2þ homeostasis, controls protein quality in ER and participates in immune process through regulation of its immune client proteins. In our previous study, we synthesized and identified a novel coumarin pyrazoline derivative 3-(1,5-diphenyl-4,5-dihydro-1H-
https://doi.org/10.1016/j.bbrc.2019.02.003 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003
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pyrazol-3-yl)-7-hydroxy-2H-chromen-2-one (HCP1) as the inhibitor of HSP90. HCP1 could inhibit A549 lung cancer cell growth and induce apoptosis [13]. However, the impact of HCP1 on VEC apoptosis remains unclear. Deprivation of serum and fibroblast growth factor 2 (FGF-2) can lead to VEC apoptosis [14]. Therefore, we investigated the effects of HCP1 on serum and FGF-2 deprivation-induced VEC apoptosis as well as the underlying mechanism. 2. Materials and methods 2.1. Antibodies and reagents Antibodies for integrin a2 (ab181548), Grp94 (ab3674) and TLR9 (ab37154) were purchased from Abcam (Cambridge, UK). Antibodies against LAMP2 (sc-20004), NF-kB (p65) (Sc-109), Hsp70 (sc1060), GAPDH (sc-365062) and horseradish peroxidase conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Akt (4691) was from Cell Signaling Technology (USA). Antibody for Grp78 (11587-1-AP) was from Proteintech Group (USA). Antibody for ACTB (122M4782) was from Sigma-Aldrich (St. Louis, Missouri, USA). Secondary antibodies for immunofluorescence goat anti-rabbit Alexa 647, donkey anti-rabbit Alexa 546 and goat anti-mouse Alexa 488 were all from Invitrogen (Carlsbad, CA, USA). HCP1 was synthesized as described previously [13] and dissolved in DMSO (10 mM, Sigma-Aldrich, St. Louis, Missouri, USA) as a stock solution. Recombinant Grp94 (HSP90B1-132H) was purchased from Creative BioMart (New York, USA). Mito-tracker deep red (M22426) was obtained from Invitrogen (Carlsbad, CA, USA). 2.2. Cell culture
2.5. Western blot analysis Cells were lysed in western and IP buffer (Beyotime, P0013) containing 150 mM NaCl, 20 mM Tris-HCl (PH 7.5), 1% Triton X-100, and proteinase inhibitors mix. After centrifuging at 12000 g for 15 min under 4 C, the supernatant was collected. Protein samples (20 mg/lane) were loaded on 15% SDS-polyacrylamide gel, and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, IPVH00010). The membranes were incubated with primary antibodies, then horseradish peroxidase-linked secondary antibodies and detected using an enhanced chemiluminescence detection kit (Thermo Fisher, 34080). The relative quantity of proteins was analyzed by Image J and normalized to loading controls. 2.6. Surface plasmon resonance (SPR) SPR analysis was carried out with Biacore T200 (GE Healthcare, England) to determine the binding of HCP1 to full length Grp94. Grp94 was immobilized to the surface of a CM5 sensor chip using amine coupling. The immobilization has been performed according to the manufacturer's instructions. Binding experiments were performed with multiple concentrations of HCP1. The compound was flowed at a rate of 10 ml/min for 2 min to allow for association and followed by 600 s for dissociation over immobilized protein in phosphate-buffered saline (PBS) (in mM: 10 phosphate, 137 NaCl, 2.68 KCl, 0.1% dimethyl sulfoxide [v/v], pH 8.0) at 25 C. The sensor chip surface was regenerated using 10 mM NaOH. The binding responses were recorded as resonance units (RU). Data analyses using the Biacore T200 Evaluation Software were performed after blank subtraction. 2.7. Cell staining for immunofluorescence microscopy
37 C
All cell lines were maintained at under humidified conditions and 5% CO2. Human umbilical vein endothelial cells (HUVECs) were obtained from human umbilical cord veins as described previously [15]. HUVECs were cultured as routine on gelatin coated plastic dishes in M199 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone Lab, Inc, Tauranga, New Zealand) and 2 ng/mL FGF-2. All HUVECs involved in experiments were at no more than passage 10. A549 cells were grown in RPMI Medium 1640 (Gibco, Grand Island, NY) in the presence of 10% FBS. HEK293T cells were cultured as routine in DMEM medium (Gibco, Grand Island, NY) supplemented with 10% FBS. 2.3. Cell viability assay Cells were plated and treated in 96-well plates followed by precipitating in 100 ml 10% trichloroacetic acid (Shenggong Biotech, Shanghai, China) for 1 h at 4 C. Then cells were stained with 50 ml sulforhodamine B (SRB; Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min and the bounded dye was reconstituted in 100 ml of 10 mM Trisbase (pH 10.5). The optical density was read by a Spec-traMAX 190 microplate spectrophotometer (GMI Co., USA) at 540 nm. Cell viability (%) ¼ (OD of treated group/OD of control group) 100. 2.4. Hoechst 33258 staining Cells were seeded on gelatin coated 24-well plates for 24 h before deprivation of serum and FGF-2 and treated with HCP1 for 48 h. Then, Hoechst 33258 (Sigma-Aldrich, St. Louis, Missouri, USA) was used to stain cells for 10 min at 37 C. Cells were washed twice with PBS gently and photographed with Olympus (Japan) BH-2 fluorescence microscope. More than 400 cells were counted.
Cells were fixed in 4% paraformaldehyde (w/v) for 15 min at room temperature or ice-cold methanol at 20 C for 10 min and blocked in PBS, 0.01% Triton X-100 (v/v) and 5% donkey serum (v/v) for 60 min. Then cells were incubated with primary antibodies overnight at 4 C and washed in PBS three times followed by incubation with corresponding secondary antibodies for 1 h at 37 C. Fluorescence was detected by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). At least 3 fields of view were analyzed by the microscope for each sample, and representative results are shown. 2.8. RNA interference Grp94 siRNA (sc-35523) and scramble siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells at 70%e80% confluence were transfected with siRNA against Grp94 and scramble siRNA by use of Lipofectamin 2000 (Invitrogen, 11668e019) following the manufacturer's instructions. 2.9. Plasmids and overexpression The coding region of Grp94 was sub-cloned into the pCMV3-CMyc expression vector to produce the c-Myc-Grp94-wt construct. Mt1 mutant (Gly196, Val197, Phe199 and Tyr200 of Grp94 were mutated to alanine), mt2 mutant (Phe199, Ala202, Phe203, Val209 and Val211 of Grp94 were mutated to alanine), mt3 mutant (Val209, Val211, Ile247 and Leu249 of Grp94 were mutated to alanine) and mt4 mutant (Phe199, Ala202, Phe203, Ile247 and Leu249 of Grp94 were mutated to alanine) were sub-cloned into pCMV3-C-Myc expression vector as the mutated Grp94 cDNA. All of the constructs were confirmed by DNA sequencing. Cells, at 70%e 80% confluence, were transfected with the expression vectors for
Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003
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24 h by using Lipofectamine 2000 (Invitrogen, 11668e019) following the manufacturer's instructions. 2.10. Statistical analyses Results are reported as means ± SEM. All experiments were repeated at least three times independently. For statistical analysis, Graph Pad Prism software (version 5.0) was used. Statistical comparisons for data were performed using one-way analysis of variance (ANOVA) followed by Tukey: compare all pairs of columns test. Differences were regarded as statistically significant when p < 0.05. 3. Results
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detached from the dish gradually after withdraw of serum and FGF2 and HCP1 (1 mM) inhibited cell death (Fig. 1C). Furthermore, we detected the effects of HCP1 on HUVEC apoptosis. Results showed that HCP1 (1 mM) inhibited HUVEC apoptosis induced by serum and FGF-2 deprivation (Fig. 1D). 3.2. Low-concentration HCP1 did not affect Hsp90a/b activity We further investigated the underlying mechanism of which HCP1 inhibited HUVEC apoptosis caused by deprivation of serum and FGF-2. We determined the effects of low-concentration HCP1 on Hsp90a/b activity. Treatment with HCP1 at low concentrations (1 mM, 2 mM) did not influence protein levels of Akt, NFkB (p65) and Hsp70 (Supplementary Fig. 1). Thus, low-concentration HCP1 did not influence the activity of Hsp90a/b.
3.1. Low-concentration HCP1 inhibited HUVEC apoptosis induced by serum and FGF-2 deprivation
3.3. HCP1 bound to Grp94
First of all, we determined the effects of HCP1 on VEC apoptosis induced by serum and FGF-2 deprivation. Results showed that treatment with HCP1 at low concentrations (1 mM, 2 mM) increased HUVEC viability under serum and FGF-2 deprivation conditions both for 24 h and 48 h (Fig. 1AeB). Morphologically, HUVECs
Then, we detected the effects of HCP1 on Grp94 and Trap-1. The distribution of HCP1 in living cells showed that HCP1 co-localized with Grp94 in endoplasmic reticulum (Fig. 2AeB). And HCP1 did not located in mitochondria (Supplementary Fig. 2). Therefore, we further determined the interaction between HCP1 and Grp94.
Fig. 1. HCP1 inhibited HUVEC apoptosis induced by serum and FGF-2 deprivation. (A, B) Viabilities of HUVECs cultured under normal conditions (Control), deprived of serum and FGF-2 (Serum deprived) and treated with or without HCP1 of the indicated concentrations under serum and FGF-2 deprivation conditions (Treated) for 24 h and 48 h. Data are means ± SEM; (***p < 0.001 vs. Control; **p < 0.01 vs. Serum deprived; n ¼ 3). (C) Morphology of HUVECs cultured under normal conditions, deprived of serum and FGF-2 (Serum deprived) or treated with or without HCP1 (1 mM) for 24 h and 48 h under serum and FGF-2 deprivation conditions (magnification 100 ). Scale bar, 50 mm. (D) Hoechst 33258 staining and quantification of apoptotic HUVECs cultured under normal conditions, deprived of serum and FGF-2 (Serum deprived) and treated with or without HCP1 (1 mM) under serum and FGF-2 deprivation conditions for 24 h. Scale bar, 50 mm. Data are means ± SEM; (***p < 0.001 vs. Control; **p < 0.01 vs. Serum deprived; n ¼ 3).
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Fig. 2. HCP1 inhibited Grp94 activity. (A, B) HCP1 co-localized with Grp94. Immunofluorescence microscopy image of A549 cells treated with HCP1 (1 mM) for 3 h and then stained with Grp94 or Grp78. Nuclei were stained with 40 , 6-diamidino-2-phenylindole (DAPI). Bar charts show quantification of weighted co-localization coefficient (Pearson's coefficient) for HCP1 and Grp94/Grp78 Scale bar, 20 mm. (C) Interaction of HCP1 with Grp94 was measured by using SPR analysis. Various concentrations of HCP1 were injected onto Grp94immobilized chip and RU values were recorded. (D, E) Immunofluorescence microscopy image of TLR9 and LAMP2 in A549 cells after treatment with Grp94 siRNA or treated with HCP1 (1 mM) for 24 h. Nuclei were stained with DAPI. Bar charts show quantification of weighted co-localization coefficient for TLR9 and LAMP2. Scale bar, 10 mm. (F, G) Protein levels of integrin a2 treated with the indicated concentrations of HCP1 for 24 h in HEK293T cells. Data are means ± SEM; (*p < 0.05, **p < 0.01; n ¼ 3).
Typical sensorgrams of the interactions between HCP1 and Grp94 were obtained (Fig. 2C). The equilibrium dissociation constant (KD value) of HCP1/Grp94 interaction was 0.923 mM. These results indicated that HCP1 bound to Grp94 directly.
HEK293T cells after treatment with HCP1 for 24 h (Fig. 2FeG). These results showed that HCP1 inhibited Grp94 activity.
3.4. HCP1 inhibited Grp94 activity
Currently, paralog-selective Grp94 inhibitors have been reported to bind to two specific pockets adjacent to the adenosine triphosphate (ATP) binding cavity termed Site 2 and Site 3 [17]. To determine whether HCP1 could bind to these two sites in Grp94, potent binding amino acid residues were selected and plasmids of Grp94 mutant at these two sites were constructed: c-Myc-Grp94-
Next, we detected the effects of HCP1 on Grp94 activity. Results showed that HCP1 inhibited Toll-like receptor 9 (TLR9) trafficking which was mediated by Grp94 (Fig. 2DeE and Supplementary Fig. 3) [10,16]. The protein level of integrin a2 also decreased in
3.5. HCP1 bound to Site 3 in Grp94
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mt1 of Site 3 and c-Myc-Grp94-mt2, -mt3, -mt4 of Site 2 (Fig. 3A). Then HEK293T cells were transfected with plasmids coding for cMyc-Grp94-wt, -mt1, -mt2, -mt3 and -mt4 for 24 h followed by treatment with HCP1 for 24 h. Results revealed that integrin a2 protein level was elevated in HEK293T cells transfected with c-
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Myc-Grp94-mt1. While protein levels of integrin a2 in c-MycGrp94-mt2, -mt3 and -mt4 transfected cells showed no differences with that in c-Myc-Grp94-wt transfected cells (Fig. 3BeC). We further evaluated the effects of mt1 on the binding of HCP1 to Grp94. Results showed that co-localization coefficient of c-Myc-
Fig. 3. HCP1 suppressed HUVEC apoptosis through inhibition of Grp94 activity. (A) Mutation sites on Grp94 protein. The alignment shows the selected amino acid residues within the two hot spots targeted by small molecule compounds (site 2 and site 3). Different mutations were designed as indicated in the diagram. (B, C) Protein levels of integrin a2 in HEK293T cells transfected with expression vectors c-Myc and c-Myc-Grp94-wt, -mt1, -mt2, -mt3 and -mt4 for 24 h, then treated with HCP1 (1 mM) for 24 h. Data are means ± SEM; (*p < 0.05, ***p < 0.001; n ¼ 3). (D, E) Immunofluorescence microscopy image of A549 cells transfected with expression vectors c-Myc-Grp94-wt, -mt1 for 24 h, then treated with HCP1 (1 mM) for 3 h and stained with Grp94. Nuclei were stained with DAPI. Bar charts show quantification of weighted co-localization coefficient (Pearson's coefficient) for HCP1 and Grp94. Scale bar, 20 mm (F, G) Viabilities of HUVECs transfected with expression vectors c-Myc and c-Myc-Grp94-wt, -mt1 for 24 h, then cultured under normal conditions or deprived of serum and FGF-2 conditions treated with or without HCP1 (1 mM, 2 mM) for 24 h. Data are mean ± SEM; (*p < 0.05, **p < 0.01, ***p < 0.001; n ¼ 3).
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Grp94-mt1 with HCP1 was lower than that of c-Myc-Grp94-wt with HCP1 (Fig. 3DeE). Therefore, mt1 of Grp94 decreased the binding of HCP1 to Grp94 and HCP1 bound to Site 3 in Grp94. 3.6. Low-concentration HCP1 suppressed HUVEC apoptosis through inhibition of Grp94 activity Next, we determined the relationship between the effects of HCP1 on HUVEC apoptosis and Grp94 activity. Results showed that overexpression of c-Myc-Grp94-wt inhibited the protective effect of HCP1 on HUVEC apoptosis which suggested that HCP1 suppressed HUVEC apoptosis through inhibition of Grp94 activity (Fig. 3FeG). In addition, compared with c-Myc-Grp94-wt transfected group, overexpression of c-Myc-Grp94-mt1 further blocked the anti-apoptotic effects of HCP1 in HUVECs (Fig. 3FeG). These results showed that low-concentration HCP1 (1 mM, 2 mM) suppressed HUVEC apoptosis through inhibition of Grp94 activity via binding to site 3. 4. Discussion This study determined the anti-apoptotic effect of a coumarin pyrazoline derivative HCP1 in HUVECs. We found that lowconcentration HCP1 (1 mM, 2 mM) effectively suppressed serum and FGF-2 deprivation-induced HUVEC apoptosis through selective inhibition of Grp94. Grp94 is a canonical hallmark of ER stress. Grp94 as well as its clients’ chaperons participate in metabolic stress, and whose expressions are induced during ER stress [18]. It is reported that ER stress is involved in serum deprivation-induced apoptosis in hippocampal neuroblasts [19]. ER stress-mediated apoptosis could be inhibited in human breast cancer MCF-7 cells and hepatoblastoma HepG2 cells by pretreatment with FGF-2 [20]. Here, we found that low-concentration HCP1 (1 mM, 2 mM) selectively inhibited Grp94 activity and suppressed apoptosis induced by serum and FGF-2 deprivation in HUVECs. These results indicated that inhibition of Grp94 activity also suppressed apoptosis and provided us new pieces of evidence to understand the underlying mechanism of VEC apoptosis. Grp94 is closely associated with the pathogenesis of different diseases. Grp94 is reported to be up-regulated in tumor cells and plays a role in oncogenesis and metastasis [10]. In addition, Grp94 prevents misfolded myocilin degradation via autophagy which promotes the pathologies associated with glaucoma [21]. Because of the pro-apoptotic effects in VECs, Grp94 could be a potential therapeutic target in clinical treatment of vascular diseases. And HCP1 would become a valuable tool to study the pathogenesis of vascular diseases. Clinical HSP90 inhibitors inhibited all isoforms of HSP90 and exhibited various adverse side effects such as hepatotoxicity, hyponatremia, hypoglycemia, fatigue and diarrhea which limited their application in clinical treatment [22,23]. Therefore, it is necessary to improve the isoform selectivity of HSP90 inhibitors to minimize the side effects. The high homology in sequence and structure of HSP90 family members seriously restrained the development of HSP90 isoform selective inhibitors. Despite the high identity, two pockets distinct from other paralogs were identified in Grp94 which greatly facilitated the development of selective inhibitors [17]. These two pockets formed a halo of potential selectivity adjacent to the ATP binding pocket in Grp94 which were termed as Site 2 and Site 3. For example, NECA and resorcinilic scaffold-inhibitors target Site 3 [11,24,25] while purine derivatives bind to Site 2 in Grp94 [12,16,26]. The adenosine receptor agonist NECA bound site 3 of Grp94. However, the activation of the adenosine receptor pathway led to the off-target effects of NECA, which limited the use of NECA scaffold in the development of
Grp94 selective inhibitors. We found coumarin pyrazoline derivative HCP1 could inhibit Grp94 activity through binding to Site 3. Therefore, coumarin pyrazoline scaffold is a novel potential scaffold for Grp94 selective inhibitors bound to Site 3. In summary, we discovered coumarin pyrazoline derivative HCP1 as a novel inhibitor of VEC apoptosis. Low-concentration HCP1 (1 mM, 2 mM) inhibited apoptosis induced by deprivation of serum and FGF-2 in HUVECs through selectively inhibited Grp94 activity via binding to site 3. HCP1 was a useful tool to inhibit Grp94 and would be a potential candidate for developing new drugs against vascular diseases. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31870831, 91539105, 81502948, 81321061, 91313303) and Shandong Excellent Young Scientist Award Fund (No. BS2015YY031). We thank Zhifeng Li and Jing Zhu from Analysis & Testing Center of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for assistance in surface plasmon resonance technology of Biacore T200. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.003. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.003. References [1] E.M. Gleeson, J.S. O'Donnell, R.J. Preston, The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling, Cell. Mol. Life Sci. CM 69 (2012) 717e726. [2] S. Godo, H. Shimokawa, Endothelial functions, Arterioscler. Thromb. Vasc. Biol. 37 (2017) e108ee114. [3] L. Zhang, H.Y. Li, H. Li, J. Zhao, L. Su, Y. Zhang, S.L. Zhang, J.Y. Miao, Lipopolysaccharide activated phosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cells, J. Cell. Physiol. 226 (2011) 1694e1701. [4] B. Messner, I. Zeller, C. Ploner, S. Frotschnig, T. Ringer, A. Steinacher-Nigisch, A. Ritsch, G. Laufer, C. Huck, D. Bernhard, Ursolic acid causes DNA-damage, p53-mediated, mitochondria- and caspase-dependent human endothelial cell apoptosis, and accelerates atherosclerotic plaque formation in vivo, Atherosclerosis 219 (2011) 402e408. [5] A.V. Sima, C.S. Stancu, M. Simionescu, Vascular endothelium in atherosclerosis, Cell Tissue Res. 335 (2009) 191e203. [6] K. Kobayashi, K. Sato, T. Kida, K. Omori, M. Hori, H. Ozaki, T. Murata, Stromal cell-derived factor-1 alpha/C-X-C chemokine receptor type 4 axis promotes endothelial cell barrier integrity via phosphoinositide 3-kinase and Rac 1 activation, Arterioscler. Thromb. Vasc. Biol. 34 (2014) 1716e1722. [7] X. Wang, M. Chen, J. Zhou, X. Zhang, HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy (Review), Int. J. Oncol. 45 (2014) 18e30. [8] S.Y. Bai, L.Q. Yao, L. Su, S.L. Zhang, B.X. Zhao, J.Y. Miao, Low-dose HSP90 inhibitors DPB and AUY-922 repress apoptosis in HUVECs, RSC Adv. 5 (2015) 75753e75755. [9] M. Marzec, D. Eletto, Y. Argon, GRP94: an HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum, Biochim. Biophys. Acta 1823 (2012) 774e787. [10] B.X. Wu, F. Hong, Y. Zhang, E. Ansa-Addo, Z. Li, GRP94/gp96 in cancer: biology, structure, immunology, and drug development, Adv. Cancer Res. 129 (2016) 165e190. [11] V.M. Crowley, A. Khandelwal, S. Mishra, A.R. Stothert, D.J. Huard, J. Zhao, A. Muth, A.S. Duerfeldt, J.L. Kizziah, R.L. Lieberman, C.A. Dickey, B.S. Blagg, Development of glucose regulated protein 94-selective inhibitors based on the BnIm and radamide scaffold, J. Med. Chem. 59 (2016) 3471e3488.
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Please cite this article as: Q. Wei et al., Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.02.003