Molecular and Cellular Endocrinology 411 (2015) 67–74
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e
Vitamin D-binding protein is required for the protective effects of vitamin D in renal fibroblasts and is phosphorylated in diabetic rats Chi-Hsien Chou a, Lea-Yea Chuang b,*, Chi-Yu Lu b, Jinn-Yuh Guh c,d,** a
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Taiwan Department of Internal Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan d Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Taiwan b c
A R T I C L E
I N F O
Article history: Received 11 January 2015 Received in revised form 28 March 2015 Accepted 13 April 2015 Available online 21 April 2015 Keywords: Diabetic nephropathy Vitamin D-binding protein High glucose Phosphorylation NRK-49F cells
A B S T R A C T
Serum vitamin D is bound to vitamin D-binding protein (DBP). We studied the roles of DBP in streptozotocindiabetic rats and high glucose (HG)-cultured cells. In diabetic rat sera, there was one upregulated (with a lower isoelectric point [pI], phosphorylated at S268, S270, S464 and T269) and one downregulated (with a higher pI, phosphorylated at S454 and S457) DBP. DBP levels with lower pI were increased in diabetic rat kidney and liver. HG (30 mM) increased DBP protein expression in NRK-49F cells and Clone-9 hepatocytes. HG decreased pI of DBP in Clone-9 hepatocytes. Moreover, DBP short hairpin ribonucleic acid attenuated 1,25-(OH)2D3-induced attenuation of HG-induced renin (but not collagen IV and fibronectin) protein expression in NRK-49F cells. Thus, DBP level is increased whereas DBP is phosphorylated in diabetic rat serum. HG increased DBP protein expression in renal fibroblasts and hepatocytes. Moreover, DBP is required for vitamin D-induced attenuation of HG-induced renin in NRK-49F cells. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Skin cholecalciferol (vitamin D3) is hydroxylated to 25(OH)-D3 by the liver and hydroxylated to the active 1,25-(OH)2D3 by the kidney or extra-renal tissues (Chun et al., 2014). Diabetic nephropathy (DN) is associated with a high prevalence of vitamin D deficiency (Dusso and Tokumoto, 2011) while vitamin D therapy reduces proteinuria in chronic kidney diseases including DN (de Borst et al., 2013). Moreover, vitamin D synergized with angiotensin blockade to ameliorate mouse DN (Vaidya and Williams, 2012; Zhang et al., 2008). Most of the lipophilic serum 25(OH)-D3 (and 1,25-(OH)2D3, with a lower affinity) is bound to vitamin D-binding protein (DBP, encoded by the group-specific component globulin gene), and albumin to a
Abbreviations: DN, diabetic nephropathy; DBP, vitamin D-binding protein; HG, high glucose; pI, isoelectric point; shRNA, short hairpin ribonucleic acid; DTT, dithiothreitol; DMEM, Dulbecco’s modified Eagle’s medium; 2D-DIGE, twodimensional difference gel electrophoresis; IPG, immobilized pH gradient; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel; MS/MS, tandem mass spectrometry; ESI–Q-TOF, electrospray ionization quadrupole-time of flight; UPLC, ultra performance liquid chromatography. * Corresponding author. Department of Biochemistry, Kaohsiung Medical University, 100 Zihyou 1st Rd., Kaohsiung, Taiwan 807. Tel.: +886 7 3121101; fax: +886 7 3218309. E-mail address: [email protected] (L.-Y. Chuang). ** Corresponding author. Department of Internal Medicine, Kaohsiung Medical University, 100 Zihyou 1st Rd., Kaohsiung, Taiwan 807. Tel.: +886 7 3121101; fax: +886 7 3218309. E-mail address: [email protected] (J.-Y. Guh). http://dx.doi.org/10.1016/j.mce.2015.04.012 0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.
less extent, to be transported to the target tissues to bind to the vitamin D receptor (Brown and Coyne, 2012; Chun et al., 2014). Serum DBP is secreted by the liver (Malik et al., 2013). DBP-bound 25(OH)-D3 in the glomerular ultrafiltrate is endocytosed by megalin to be activated by 1α-hydoxylase in the proximal tubule (Chun et al., 2014). Thus, megalin-knockout mice develop vitamin-D deficiency (Chun et al., 2014). Interestingly, serum DBP level was found to be unchanged (Thrailkill et al., 2011), increased (Cho et al., 2007) or decreased (Blanton et al., 2011) whereas urine DBP was increased (Thrailkill et al., 2011) in diabetic patients. However, the role of serum or tissue DBP in DN or high glucose (HG)-induced effects remains unknown. Thus, in order to study the roles of DBP in DN, we measured diabetic rat serum, kidney and liver DBP levels and characterize phosphorylations of serum and tissue DBP by proteomic methods. Moreover, the role of DBP in HG-induced effects in vitro was studied by DBP short hairpin ribonucleic acid (shRNA). 2. Materials and methods 2.1. Materials 1,25(OH) 2 D 3 was purchased from Biovision Company (San Francisco, CA). Dithiothreitol (DTT) was obtained from AnaSpec Inc. (Fremont, CA). Acetonitrile was purchased from Merck Millipore (Billerica, MA). Sequencing grade modified trypsin was obtained from Promega Corp. (Madison, WI). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
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C.-H. Chou et al./Molecular and Cellular Endocrinology 411 (2015) 67–74
2.2. Cell culture NRK-52E cells (CRL-1571, rat renal proximal tubular cells), NRK49F cells (CRL-1570, rat renal interstitial fibroblasts) and LLC-PK1 cells (CRL-1392, porcine kidney epithelial cells) (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/ streptomycin (Gibco, Grand Island, NY) and 5% (or 10% for LLCPK1 cells) fetal bovine serum (FBS). MES13 cells (CRL-1927, mouse mesangial cells, American Type Culture Collection, Manassas, VA) were cultured in 3:1 DMEM/F12 supplemented with 1% penicillin/ streptomycin (Gibco, Grand Island, NY) and 5% FBS. Clone-9 rat hepatocytes (CRL-1493, American Type Culture Collection, Manassas, VA) were culture in Ham’s F12K supplemented with 10% FBS. Cells were cultured in a 5% CO2 incubator at 37 °C and starved (1% FBS) for 24 hours before experiments. 2.3. Sample preparation Serum protein concentration of the rats was measured by the Bradford method. Briefly, 100 μg serum proteins of each normal/ diabetic rat was pooled and precipitated with 1 mL ice-cold acetone/ 10 % w/v trichloroacetic acid (TCA)/20 mM DTT for a minimum of 30 min at −20 °C. The precipitate was washed twice with 1 mL icecold acetone containing 20 mM DTT, and then air-dried to remove the residual acetone. Protein pellet was resolubilized in the twodimensional difference gel electrophoresis (2D-DIGE) solubilizing buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris base). Finally, the protein concentration was measured by the Bradford method. Kidney and liver proteins were extracted with the T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc., San Jose, CA). After extraction, the sample was centrifuged (12,000 rpm for 20 min at 4 °C) and the concentration of protein solution was also measured by the Bradford method. 2.4. 2D-DIGE Briefly, a total of 50 μg of serum proteins from normal and diabetic rats was labeled with the Cy3 and Cy5 fluorescence dyes according to the manufacturer’s protocol (GE Healthcare, Little Chalfont, UK) and the two samples were pooled and mixed with
an equal volume of the sample buffer containing 5 M urea, 2 M thiourea, 3% w/v CHAPS, 1% immobilized pH gradient (IPG) buffer with a nonlinear pH of 3–10, 100 mM DeStreak reagent, and a trace of bromophenol blue. The 13 cm Immobiline DryStrips with a nonlinear pH of 3–10 were rehydrated overnight for 15 hours at room temperature in 250 μL rehydration buffer containing 7 M urea, 2 M thiourea, 3% w/v CHAPS, 20 mM DTT, 0.5% IPG buffer and a trace of bromophenol blue. The mixed sample was cup-loaded near the anode of the IPG strips using the Ettan IPGphor cup-loading (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s protocol. Protein focusing was achieved using the following IEF parameters: 350 V, step and hold 3.5 h; 650 V, gradient, 1 h; 1100 V, gradient, 1 h; 8000 V, gradient, 1.5 h; 8000 V, step and hold for 3 h, giving a total of 14,000 Vh. The first dimension isoelectric focusing IPG strips was equilibrated by gentle shaking for 15 min in 10 mL equilibration buffer (50 mM Tris-base, pH = 8.8, 6 M urea, 30% v/v glycerol, 0.2% w/v sodium dodecyl sulfate [SDS] and 1% w/v DTT), followed by 10 mL of the same solution containing 2.5% w/v iodoacetamide instead of DTT for 15 min. Then, the strip was transferred on top of the 12 % SDS–polyacrylamide gel (PAGE). The second dimension separation was performed with a constant voltage of 75 V for 0.5 h, and 100 V for 16 h. The 2D-DIGE image was detected with the Typhoon 9410 scanner (Amersham Biosciences, Uppsala, Sweden). The spots were compared and quantified by using the DeCyder Differential Analysis Software, Version 5.0 (Amersham Biosciences, Uppsala, Sweden). 2.5. Immunoblotting of 2D gel-separated proteins After 2D gel separation, the proteins were transferred to the polyvinylidine difluoride membranes. The membrane was blocked with 5% (w/v) skimmed milk in the TBST buffer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.05% Tween-20) and the membranes were incubated with primary antibodies (diluted 1/1000) at 4 °C for 16 hours. The membrane was washed with the TBST buffer for five times and incubated with the secondary antibody (diluted 1/5000) at room temperature for 1 hour. The protein bands were detected by using the chemiluminescence ECL reagent and visualized on Fuji SuperRX film. The gel was stained with the Pro-Q diamond reagent (Invitrogen, Molecular Probes, Carlsbad, CA) for the phosphorylated proteins according to the manufacturer’s protocol.
Fig. 1. Two dimensional difference gel electrophoresis (2D-DIGE) of rat serum proteins. Serum proteins of normal rats (n = 8) were extracted and labeled with Cy3 dye (green color) and serum proteins of diabetic rats (n = 20) were labeled with Cy5 dye (red color) according to the manufacturer’s protocol. Total proteins of both groups of rats were mixed and analyzed by 2D-DIGE. (A) 2D-DIGE of pooled rat serum proteins. (B) The magnified pattern of DBP protein (spots S3, S4, C2, C3). Three independent experiments were performed with similar results.
C.-H. Chou et al./Molecular and Cellular Endocrinology 411 (2015) 67–74
2.6. In-gel digestion The gel pieces were washed with distilled water and dehydrated with 100% acetonitrile and evaporated to dryness in a SpeedVac evaporator. The dried gel pieces were rehydrated with 25 mM ammonium bicarbonate buffer (pH = 8.5) containing the sequencegrade modified trypsin (0.01 μg/μL) and incubated at 37 °C for 16 hours. The tryptic peptides in gel pieces were extracted twice with 50% acetonitrile containing 5% formic acid by sonication for 25 min. The extracted solutions were evaporated to dryness in a SpeedVac evaporator and then redissolved in 20 μL 0.1% formic acid/10% acetonitrile. 2.7. Protein identification and quantitation by electrospray ionization–quadrupole-time of flight tandem mass spectrometry (ESI–Q-TOF–MS/MS) Briefly, the differentially expressed 2D gel protein samples were subjected to the nanoflow liquid chromatography and Waters-Micromass ESI–Q-TOF for protein identification (Waters, Manchester, UK) as described in our previous study (Feng et al., 2010). The MS/MS spectra of individual fragments obtained for each of the precursors were processed by using MassLynx 4.0 software (Manchester, UK) to obtain the corresponding peak lists. The peak list files were then uploaded to an in-house Mascot server for protein identification. 2.8. Protein phosphosite identification by the nano ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) Briefly, the differentially expressed DIGE protein samples were subjected to the nano UPLC system (nanoACQUITY UPLC) consisted of the desalting column (Symmetry C18, 5 μm, 180 μm × 20 mm) and the analytical column (BEH C18, 1.7 μm, 75 μm × 150 mm) was purchased from Waters (Milford, MA). The MS/MS consisting of the LTQ Orbitrap Discovery hybrid Fourier transform mass spectrometer (Thermo Fisher Scientific, Inc., Bremen, Germany) at a resolution of 30,000 coupled with a nanospray source was carried out in the positive ion mode. The peptide solutions were injected into the nano UPLC system and detected by the LTQ Orbitrap. Briefly, peptides were
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separated by the nano UPLC system. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The liquid chromatography gradient conditions were as follows: base on time (t) set at the mobile phase: t = 0–10 minutes, hold% B = 10; t = 10–60 minutes, %B from 10 to 75; and t = 60–70 minutes, %B from 75 to 100. The peptide eluate from the column was subjected to the nanospray source, and the MS/MS was acquired with a mass spectrometer operated in data-dependent mode. The raw data files were processed with Mascot Distiller (Version 2.2, Matrix Science Inc., Boston, MA) software and then submitted to the inhouse Mascot server (Version 2.2, Matrix Science Inc., Boston, MA) to get the corresponding protein identity and phosphosites. 2.9. Immunoblotting Briefly, cells were lysed with RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodiumdeoxycholate, and protease inhibitor cocktail (Calbiochem, La Jolla, CA), and the concentration of the soluble protein was measured by the Bradford method. A total of 60 μg protein was separated by 10% SDS–PAGE and the proteins were transferred to polyvinylidine difluoride membranes. After blocking, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this study were DBP (1:1000), β-actin (1:2000), glyceraldehyde-3phosphate dehydrogenase (GAPDH) (1:2000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), renin (1:1000, AnaSpec Inc., San Jose, CA), collagen IV (col4α1, 1:2000, Abgent, Inc., San Diego, CA), and fibronectin (1:5000, Merck Millipore, Billerica, MA). After washing with phosphate-buffered saline containing 0.05% Tween 20 five times, the membranes were incubated with horseradish peroxidaseconjugated secondary antibody (1:5000, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 hour at room temperature. Further, the protein bands were detected by using enhanced chemiluminescence ECL reagent (Amersham Corp, Arlington Heights, IL) and visualized on a Fuji SuperRX film. 2.10. DBP silencing DBP shRNA plasmids were purchased from the National RNAi Core Facility Platform (Academia Sinica, Taipei, Taiwan). The plasmids were
Fig. 2. Two dimensional gel electrophoresis of serum phosphorylated proteins in normal and diabetic rats. In a 2D gel, the first dimension consisted of isoelectric focusing and the second dimension consisted of SDS–PAGE. After 2D gel separation, the gel was stained with the Pro-Q diamond reagent (Molecular Probes, Carlsbad, CA) for the phosphorylated proteins according to the manufacturer’s protocol. (A) Pro-Q diamond reagent-stained 2D gel of a normal rat serum. (B) Pro-Q diamond reagent-stained 2D gel of a diabetic rat serum. Three independent experiments were performed with similar results.
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Fig. 3. Ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) identification of phosphorylated sites of DBP. Spots 7 and 8 in Fig. 2 were cut from the gels stained with Pro-Q diamond reagent and analyzed by the nano UPLC–MS/MS system. The nano UPLC consisted of the desalting column and the analytical column. The MS/MS consisted of the LTQ Orbitrap Discovery hybrid Fourier transform mass spectrometer. Peptides were separated by the nano UPLC system and subjected to analysis by the LTQ Orbitrap in the MS/MS mode. (A) Consistently identified phosphosites of spot 7 and spot 8 in three independent experiments. (B and C) The LTQ Orbitrap mass spectra of S270 and S454. Three independent experiments were performed with similar results.
transfected into NRK-49F cells by using FuGENE transfection reagents (Promega Corp., Madison, WI) according to the manufacturer’s instructions. After 16 hours transfection, the serum-free medium was added and the cells were treated with HG (30 mM) or vitamin D (1 × 10−8 M) at the indicated conditions for 48 hours. 2.11. Diabetic rats Male Sprague–Dawley rats weighing 200–300 g (BioLASCO Co., Taipei, Taiwan) received a single peritoneal injection of 60 mg/kg streptozotocin (Sigma-Aldrich Co., St Louis, MO) in 0.1 M citrate buffer (diabetic, N = 20) or citrate buffer (control, N = 8). Diabetic rats received Lantus insulin (Sanofi Aventis, Paris, France) to achieve nonfasting blood glucose levels between 19.4 and 27.8 mmol/L. Rats were anesthetized with sodium pentobarbital (Abbott Laboratories; Chicago, IL) on week 8 and perfused. Serum was obtained and the kidneys and the liver were removed for immunoblotting. All animal procedures were approved and done in accordance with the
national guidelines and the guidelines by the Kaohsiung Medical University Animal Experiment Committee which were equivalent to the NIH Guide for the Care and Use of Laboratory Animals. 2.12. Statistics Data are expressed as the means ± standard errors of the mean (SEM). Unpaired Student’s t-test was used for the comparison of two groups. P values
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e
Vitamin D-binding protein is required for the protective effects of vitamin D in renal fibroblasts and is phosphorylated in diabetic rats Chi-Hsien Chou a, Lea-Yea Chuang b,*, Chi-Yu Lu b, Jinn-Yuh Guh c,d,** a
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Taiwan Department of Internal Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan d Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Taiwan b c
A R T I C L E
I N F O
Article history: Received 11 January 2015 Received in revised form 28 March 2015 Accepted 13 April 2015 Available online 21 April 2015 Keywords: Diabetic nephropathy Vitamin D-binding protein High glucose Phosphorylation NRK-49F cells
A B S T R A C T
Serum vitamin D is bound to vitamin D-binding protein (DBP). We studied the roles of DBP in streptozotocindiabetic rats and high glucose (HG)-cultured cells. In diabetic rat sera, there was one upregulated (with a lower isoelectric point [pI], phosphorylated at S268, S270, S464 and T269) and one downregulated (with a higher pI, phosphorylated at S454 and S457) DBP. DBP levels with lower pI were increased in diabetic rat kidney and liver. HG (30 mM) increased DBP protein expression in NRK-49F cells and Clone-9 hepatocytes. HG decreased pI of DBP in Clone-9 hepatocytes. Moreover, DBP short hairpin ribonucleic acid attenuated 1,25-(OH)2D3-induced attenuation of HG-induced renin (but not collagen IV and fibronectin) protein expression in NRK-49F cells. Thus, DBP level is increased whereas DBP is phosphorylated in diabetic rat serum. HG increased DBP protein expression in renal fibroblasts and hepatocytes. Moreover, DBP is required for vitamin D-induced attenuation of HG-induced renin in NRK-49F cells. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Skin cholecalciferol (vitamin D3) is hydroxylated to 25(OH)-D3 by the liver and hydroxylated to the active 1,25-(OH)2D3 by the kidney or extra-renal tissues (Chun et al., 2014). Diabetic nephropathy (DN) is associated with a high prevalence of vitamin D deficiency (Dusso and Tokumoto, 2011) while vitamin D therapy reduces proteinuria in chronic kidney diseases including DN (de Borst et al., 2013). Moreover, vitamin D synergized with angiotensin blockade to ameliorate mouse DN (Vaidya and Williams, 2012; Zhang et al., 2008). Most of the lipophilic serum 25(OH)-D3 (and 1,25-(OH)2D3, with a lower affinity) is bound to vitamin D-binding protein (DBP, encoded by the group-specific component globulin gene), and albumin to a
Abbreviations: DN, diabetic nephropathy; DBP, vitamin D-binding protein; HG, high glucose; pI, isoelectric point; shRNA, short hairpin ribonucleic acid; DTT, dithiothreitol; DMEM, Dulbecco’s modified Eagle’s medium; 2D-DIGE, twodimensional difference gel electrophoresis; IPG, immobilized pH gradient; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel; MS/MS, tandem mass spectrometry; ESI–Q-TOF, electrospray ionization quadrupole-time of flight; UPLC, ultra performance liquid chromatography. * Corresponding author. Department of Biochemistry, Kaohsiung Medical University, 100 Zihyou 1st Rd., Kaohsiung, Taiwan 807. Tel.: +886 7 3121101; fax: +886 7 3218309. E-mail address: [email protected] (L.-Y. Chuang). ** Corresponding author. Department of Internal Medicine, Kaohsiung Medical University, 100 Zihyou 1st Rd., Kaohsiung, Taiwan 807. Tel.: +886 7 3121101; fax: +886 7 3218309. E-mail address: [email protected] (J.-Y. Guh). http://dx.doi.org/10.1016/j.mce.2015.04.012 0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.
less extent, to be transported to the target tissues to bind to the vitamin D receptor (Brown and Coyne, 2012; Chun et al., 2014). Serum DBP is secreted by the liver (Malik et al., 2013). DBP-bound 25(OH)-D3 in the glomerular ultrafiltrate is endocytosed by megalin to be activated by 1α-hydoxylase in the proximal tubule (Chun et al., 2014). Thus, megalin-knockout mice develop vitamin-D deficiency (Chun et al., 2014). Interestingly, serum DBP level was found to be unchanged (Thrailkill et al., 2011), increased (Cho et al., 2007) or decreased (Blanton et al., 2011) whereas urine DBP was increased (Thrailkill et al., 2011) in diabetic patients. However, the role of serum or tissue DBP in DN or high glucose (HG)-induced effects remains unknown. Thus, in order to study the roles of DBP in DN, we measured diabetic rat serum, kidney and liver DBP levels and characterize phosphorylations of serum and tissue DBP by proteomic methods. Moreover, the role of DBP in HG-induced effects in vitro was studied by DBP short hairpin ribonucleic acid (shRNA). 2. Materials and methods 2.1. Materials 1,25(OH) 2 D 3 was purchased from Biovision Company (San Francisco, CA). Dithiothreitol (DTT) was obtained from AnaSpec Inc. (Fremont, CA). Acetonitrile was purchased from Merck Millipore (Billerica, MA). Sequencing grade modified trypsin was obtained from Promega Corp. (Madison, WI). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
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C.-H. Chou et al./Molecular and Cellular Endocrinology 411 (2015) 67–74
2.2. Cell culture NRK-52E cells (CRL-1571, rat renal proximal tubular cells), NRK49F cells (CRL-1570, rat renal interstitial fibroblasts) and LLC-PK1 cells (CRL-1392, porcine kidney epithelial cells) (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/ streptomycin (Gibco, Grand Island, NY) and 5% (or 10% for LLCPK1 cells) fetal bovine serum (FBS). MES13 cells (CRL-1927, mouse mesangial cells, American Type Culture Collection, Manassas, VA) were cultured in 3:1 DMEM/F12 supplemented with 1% penicillin/ streptomycin (Gibco, Grand Island, NY) and 5% FBS. Clone-9 rat hepatocytes (CRL-1493, American Type Culture Collection, Manassas, VA) were culture in Ham’s F12K supplemented with 10% FBS. Cells were cultured in a 5% CO2 incubator at 37 °C and starved (1% FBS) for 24 hours before experiments. 2.3. Sample preparation Serum protein concentration of the rats was measured by the Bradford method. Briefly, 100 μg serum proteins of each normal/ diabetic rat was pooled and precipitated with 1 mL ice-cold acetone/ 10 % w/v trichloroacetic acid (TCA)/20 mM DTT for a minimum of 30 min at −20 °C. The precipitate was washed twice with 1 mL icecold acetone containing 20 mM DTT, and then air-dried to remove the residual acetone. Protein pellet was resolubilized in the twodimensional difference gel electrophoresis (2D-DIGE) solubilizing buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris base). Finally, the protein concentration was measured by the Bradford method. Kidney and liver proteins were extracted with the T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc., San Jose, CA). After extraction, the sample was centrifuged (12,000 rpm for 20 min at 4 °C) and the concentration of protein solution was also measured by the Bradford method. 2.4. 2D-DIGE Briefly, a total of 50 μg of serum proteins from normal and diabetic rats was labeled with the Cy3 and Cy5 fluorescence dyes according to the manufacturer’s protocol (GE Healthcare, Little Chalfont, UK) and the two samples were pooled and mixed with
an equal volume of the sample buffer containing 5 M urea, 2 M thiourea, 3% w/v CHAPS, 1% immobilized pH gradient (IPG) buffer with a nonlinear pH of 3–10, 100 mM DeStreak reagent, and a trace of bromophenol blue. The 13 cm Immobiline DryStrips with a nonlinear pH of 3–10 were rehydrated overnight for 15 hours at room temperature in 250 μL rehydration buffer containing 7 M urea, 2 M thiourea, 3% w/v CHAPS, 20 mM DTT, 0.5% IPG buffer and a trace of bromophenol blue. The mixed sample was cup-loaded near the anode of the IPG strips using the Ettan IPGphor cup-loading (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s protocol. Protein focusing was achieved using the following IEF parameters: 350 V, step and hold 3.5 h; 650 V, gradient, 1 h; 1100 V, gradient, 1 h; 8000 V, gradient, 1.5 h; 8000 V, step and hold for 3 h, giving a total of 14,000 Vh. The first dimension isoelectric focusing IPG strips was equilibrated by gentle shaking for 15 min in 10 mL equilibration buffer (50 mM Tris-base, pH = 8.8, 6 M urea, 30% v/v glycerol, 0.2% w/v sodium dodecyl sulfate [SDS] and 1% w/v DTT), followed by 10 mL of the same solution containing 2.5% w/v iodoacetamide instead of DTT for 15 min. Then, the strip was transferred on top of the 12 % SDS–polyacrylamide gel (PAGE). The second dimension separation was performed with a constant voltage of 75 V for 0.5 h, and 100 V for 16 h. The 2D-DIGE image was detected with the Typhoon 9410 scanner (Amersham Biosciences, Uppsala, Sweden). The spots were compared and quantified by using the DeCyder Differential Analysis Software, Version 5.0 (Amersham Biosciences, Uppsala, Sweden). 2.5. Immunoblotting of 2D gel-separated proteins After 2D gel separation, the proteins were transferred to the polyvinylidine difluoride membranes. The membrane was blocked with 5% (w/v) skimmed milk in the TBST buffer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.05% Tween-20) and the membranes were incubated with primary antibodies (diluted 1/1000) at 4 °C for 16 hours. The membrane was washed with the TBST buffer for five times and incubated with the secondary antibody (diluted 1/5000) at room temperature for 1 hour. The protein bands were detected by using the chemiluminescence ECL reagent and visualized on Fuji SuperRX film. The gel was stained with the Pro-Q diamond reagent (Invitrogen, Molecular Probes, Carlsbad, CA) for the phosphorylated proteins according to the manufacturer’s protocol.
Fig. 1. Two dimensional difference gel electrophoresis (2D-DIGE) of rat serum proteins. Serum proteins of normal rats (n = 8) were extracted and labeled with Cy3 dye (green color) and serum proteins of diabetic rats (n = 20) were labeled with Cy5 dye (red color) according to the manufacturer’s protocol. Total proteins of both groups of rats were mixed and analyzed by 2D-DIGE. (A) 2D-DIGE of pooled rat serum proteins. (B) The magnified pattern of DBP protein (spots S3, S4, C2, C3). Three independent experiments were performed with similar results.
C.-H. Chou et al./Molecular and Cellular Endocrinology 411 (2015) 67–74
2.6. In-gel digestion The gel pieces were washed with distilled water and dehydrated with 100% acetonitrile and evaporated to dryness in a SpeedVac evaporator. The dried gel pieces were rehydrated with 25 mM ammonium bicarbonate buffer (pH = 8.5) containing the sequencegrade modified trypsin (0.01 μg/μL) and incubated at 37 °C for 16 hours. The tryptic peptides in gel pieces were extracted twice with 50% acetonitrile containing 5% formic acid by sonication for 25 min. The extracted solutions were evaporated to dryness in a SpeedVac evaporator and then redissolved in 20 μL 0.1% formic acid/10% acetonitrile. 2.7. Protein identification and quantitation by electrospray ionization–quadrupole-time of flight tandem mass spectrometry (ESI–Q-TOF–MS/MS) Briefly, the differentially expressed 2D gel protein samples were subjected to the nanoflow liquid chromatography and Waters-Micromass ESI–Q-TOF for protein identification (Waters, Manchester, UK) as described in our previous study (Feng et al., 2010). The MS/MS spectra of individual fragments obtained for each of the precursors were processed by using MassLynx 4.0 software (Manchester, UK) to obtain the corresponding peak lists. The peak list files were then uploaded to an in-house Mascot server for protein identification. 2.8. Protein phosphosite identification by the nano ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) Briefly, the differentially expressed DIGE protein samples were subjected to the nano UPLC system (nanoACQUITY UPLC) consisted of the desalting column (Symmetry C18, 5 μm, 180 μm × 20 mm) and the analytical column (BEH C18, 1.7 μm, 75 μm × 150 mm) was purchased from Waters (Milford, MA). The MS/MS consisting of the LTQ Orbitrap Discovery hybrid Fourier transform mass spectrometer (Thermo Fisher Scientific, Inc., Bremen, Germany) at a resolution of 30,000 coupled with a nanospray source was carried out in the positive ion mode. The peptide solutions were injected into the nano UPLC system and detected by the LTQ Orbitrap. Briefly, peptides were
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separated by the nano UPLC system. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The liquid chromatography gradient conditions were as follows: base on time (t) set at the mobile phase: t = 0–10 minutes, hold% B = 10; t = 10–60 minutes, %B from 10 to 75; and t = 60–70 minutes, %B from 75 to 100. The peptide eluate from the column was subjected to the nanospray source, and the MS/MS was acquired with a mass spectrometer operated in data-dependent mode. The raw data files were processed with Mascot Distiller (Version 2.2, Matrix Science Inc., Boston, MA) software and then submitted to the inhouse Mascot server (Version 2.2, Matrix Science Inc., Boston, MA) to get the corresponding protein identity and phosphosites. 2.9. Immunoblotting Briefly, cells were lysed with RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodiumdeoxycholate, and protease inhibitor cocktail (Calbiochem, La Jolla, CA), and the concentration of the soluble protein was measured by the Bradford method. A total of 60 μg protein was separated by 10% SDS–PAGE and the proteins were transferred to polyvinylidine difluoride membranes. After blocking, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this study were DBP (1:1000), β-actin (1:2000), glyceraldehyde-3phosphate dehydrogenase (GAPDH) (1:2000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), renin (1:1000, AnaSpec Inc., San Jose, CA), collagen IV (col4α1, 1:2000, Abgent, Inc., San Diego, CA), and fibronectin (1:5000, Merck Millipore, Billerica, MA). After washing with phosphate-buffered saline containing 0.05% Tween 20 five times, the membranes were incubated with horseradish peroxidaseconjugated secondary antibody (1:5000, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 hour at room temperature. Further, the protein bands were detected by using enhanced chemiluminescence ECL reagent (Amersham Corp, Arlington Heights, IL) and visualized on a Fuji SuperRX film. 2.10. DBP silencing DBP shRNA plasmids were purchased from the National RNAi Core Facility Platform (Academia Sinica, Taipei, Taiwan). The plasmids were
Fig. 2. Two dimensional gel electrophoresis of serum phosphorylated proteins in normal and diabetic rats. In a 2D gel, the first dimension consisted of isoelectric focusing and the second dimension consisted of SDS–PAGE. After 2D gel separation, the gel was stained with the Pro-Q diamond reagent (Molecular Probes, Carlsbad, CA) for the phosphorylated proteins according to the manufacturer’s protocol. (A) Pro-Q diamond reagent-stained 2D gel of a normal rat serum. (B) Pro-Q diamond reagent-stained 2D gel of a diabetic rat serum. Three independent experiments were performed with similar results.
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Fig. 3. Ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) identification of phosphorylated sites of DBP. Spots 7 and 8 in Fig. 2 were cut from the gels stained with Pro-Q diamond reagent and analyzed by the nano UPLC–MS/MS system. The nano UPLC consisted of the desalting column and the analytical column. The MS/MS consisted of the LTQ Orbitrap Discovery hybrid Fourier transform mass spectrometer. Peptides were separated by the nano UPLC system and subjected to analysis by the LTQ Orbitrap in the MS/MS mode. (A) Consistently identified phosphosites of spot 7 and spot 8 in three independent experiments. (B and C) The LTQ Orbitrap mass spectra of S270 and S454. Three independent experiments were performed with similar results.
transfected into NRK-49F cells by using FuGENE transfection reagents (Promega Corp., Madison, WI) according to the manufacturer’s instructions. After 16 hours transfection, the serum-free medium was added and the cells were treated with HG (30 mM) or vitamin D (1 × 10−8 M) at the indicated conditions for 48 hours. 2.11. Diabetic rats Male Sprague–Dawley rats weighing 200–300 g (BioLASCO Co., Taipei, Taiwan) received a single peritoneal injection of 60 mg/kg streptozotocin (Sigma-Aldrich Co., St Louis, MO) in 0.1 M citrate buffer (diabetic, N = 20) or citrate buffer (control, N = 8). Diabetic rats received Lantus insulin (Sanofi Aventis, Paris, France) to achieve nonfasting blood glucose levels between 19.4 and 27.8 mmol/L. Rats were anesthetized with sodium pentobarbital (Abbott Laboratories; Chicago, IL) on week 8 and perfused. Serum was obtained and the kidneys and the liver were removed for immunoblotting. All animal procedures were approved and done in accordance with the
national guidelines and the guidelines by the Kaohsiung Medical University Animal Experiment Committee which were equivalent to the NIH Guide for the Care and Use of Laboratory Animals. 2.12. Statistics Data are expressed as the means ± standard errors of the mean (SEM). Unpaired Student’s t-test was used for the comparison of two groups. P values
