Valproate and Bone Loss: iTRAQ Proteomics Show that Valproate Reduces Collagens and Osteonectin in SMA Cells Heidi R. Fuller,†,‡ Nguyen Thi Man,† Le Thanh Lam,† Vladimir A. Shamanin,§ Elliot J. Androphy,§ and Glenn E. Morris*,†,‡ Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK, Institute for Science and Technology in Medicine, Keele University, and Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, LRB 328, Worcester, Massachusetts 01605-2324 Received May 26, 2010
Valproate is commonly used as an anticonvulsant and mood stabilizer, but its long-term side-effects can include bone loss. As a histone deacetylase (HDAC) inhibitor, valproate has also been considered for treatment of spinal muscular atrophy (SMA). Using iTRAQ labeling technology, followed by twodimensional liquid chromatography and mass spectrometry analysis, a quantitative comparison of the proteome of an SMA cell line, with and without valproate treatment, was performed. The most striking change was a reduction in collagens I and VI, while over 1000 other proteins remained unchanged. The collagen I alpha-chain precursor was also reduced by more than 50% suggesting that valproate affects collagen I synthesis. The collagen-binding glycoprotein, osteonectin (SPARC, BM-40) was one of the few other proteins that were significantly reduced by valproate treatment. Collagen I is the main protein component of bone matrix and osteonectin has a major role in bone development, so the results suggest a possible molecular mechanism for bone loss following long-term exposure to valproate. SMA patients may already suffer bone weakness as a result of SMN1 gene deletion, so further bone loss would be undesirable. Keywords: Valproate • bone • spinal muscular atrophy • iTRAQ • histone deacetylase inhibitor • collagen • osteonectin • SMN • Valproate and bone loss
Introduction Valproate (valproic acid, VPA) has been in clinical use for over 40 years.1-3 It is now widely used as an anticonvulsant in epilepsy and as a mood stabilizer to control manic episodes in bipolar disorder.4 It has also been used to treat a variety of other conditions, such as migraine headaches,5 and has been suggested as a possible treatment for spinal muscular atrophy (SMA).6,7 For SMA treatment, there has been particular interest in drugs that are already licensed for human use, including histone deacetylase (HDAC) inhibitors, like valproate,6,7 butyrate,8 phenylbutyrate,9 and trichostatin,10,11 as well as drugs that act by different mechanisms, such as aclarubicin,12 indoprofen13 and hydroxyurea.14 Valproate has been shown to up-regulate SMN expression in cell lines and SMA patients.7,15,16 Encouraging initial results from mouse models17 and clinical trials with SMA patients18 were followed, however, by reservations about the use of valproate for SMA treatment.19,20 Bone weakness and bone loss have been frequently reported as side-effects of long-term valproate treatment for epilepsy, though the mechanism for this effect is not known.21,22 As * To whom correspondence should be addressed. E-mail: Glenn.morris@ rjah.nhs.uk. Telephone: 44-1691-404155. Fax: 44-1691-404170. † RJAH Orthopaedic Hospital. ‡ Keele University. § University of Massachusetts Medical School.
4228 Journal of Proteome Research 2010, 9, 4228–4233 Published on Web 06/22/2010
noted by Verrotti et al22 in 2010, “Many studies have shown a significant reduction in bone mineral density in patients treated with classic (phenobarbital, carbamazepine, valproate, etc.) and with new (oxcarbazepine, gabapentin) antiepileptic drugs. In spite of data about the possible effects of the antiepileptic drugs on calcium metabolism, the mechanisms of this important side effect remain to be defined. The abnormalities of calcium metabolism were thought to result from the cytochrome P450 enzyme-inducing properties of some antiepileptic drugs and the resultant reduction in vitamin D levels, but the effect of many medications (e.g., valproate) cannot be readily explained by vitamin D metabolism”. As an approach to the study of possible side-effects of valproate, we adopted a quantitative proteomic approach that examines over 2000 cellular proteins and determines whether any are significantly increased or decreased by valproate treatment in vitro. Our proteomic study has revealed that treatment of cultured cells with valproate causes reduced production of both collagen, the main protein in bone, and osteonectin, which is required for maintenance of bone mass. This is a possible mechanism for valproate-induced bone loss.
Experimental Section Cell Culture and Drug Treatment. Skin fibroblasts from a type I SMA patient (Cat No. GM03813) were obtained from the Coriell Cell Bank (Camden., NJ) and immortalised by infection 10.1021/pr1005263
2010 American Chemical Society
Valproate and Bone Loss with pBabe-puro-hTERT retrovirus produced using LinXA amphotropic retrovirus packaging cells. At 48 h postinfection, cells were selected with puromycin (0.5 µg/mL) for 6 days. Cells were serially passaged for 18 population doublings by which time the parental 3813 cells senesced. Immortalisation enabled cloning to remove myogenic cells and all subsequent studies were done with a fibroblastic subclone, B3. Cells were dispensed into T75 tissue-culture flasks at a density of 60 000 cells per cm2 in DMEM-Glutamax I with 10% heat-inactivated fetal calf serum (1 mL per 100 000 cells) and incubated at 37 °C for 24 h with or without 0.5 mM sodium valproate (Sigma-Aldrich) (3 flasks of each). We have shown elsewhere that valproate increases SMN levels without affecting growth rates under these short-term and high-density conditions.23 Cell Extraction. Fibroblast cell pellets were extracted in 10 volumes of extraction buffer (w/v) containing 6 M Urea, 2 M thiourea, 2% CHAPS and 0.5% SDS in HPLC-grade water (Sigma Chromasolv plus). The extracts were sonicated briefly and left on ice for 10 min, followed by centrifugation at 13 000× g for 10 min at 4 °C to pellet any insoluble material. The proteins were precipitated in 6 volumes of ice cold acetone overnight at -20 °C. The acetone precipitates were pelleted by centrifugation at 13 000× g for 10 min at 4 °C and the supernatant was carefully remove and discarded. The pellets were allowed to air-dry, followed by resuspension in 6 M Urea in 50 mM TEAB. The protein concentration in each group was determined using a Bradford assay.24 Sample Preparation for Mass Spectrometry Analysis. Reduction, alkylation and digestion steps were performed using the reagents and according to the recommendations in the iTRAQ labeling kit (Applied Biosystems). The extracts were diluted with 50 mM TEAB so that the urea concentration was less than 1M, before the addition of trypsin and overnight incubation at 37 °C. The digests were then dried down in a vacuum centrifuge and iTRAQ labeling was carried out according to the instructions in the iTRAQ labeling kit. The iTRAQ tags were assigned to samples as follows: 114suntreated SMA cells (i), 115suntreated SMA cells (ii), 116svalproate-treated SMA cells (i), 117svalproate-treated SMA cells (ii) (where (i) and (ii) represent duplicate cultures of untreated and valproatetreated SMA cells). Each tag was incubated with 85 µg of protein (as determined by a Bradford protein assay). iTRAQ-labeled peptides were pooled and made up to a total volume of 2.4 mL in SCX buffer A (10 mM phosphate, pH 3 in 20% acetonitrile (Romil, U.K.)). The pooled peptides (2.4 mL) were then separated by strong cation-exchange chromatography (SCX) using a polysulfethyl A column, 300A, 5uM (PolyLC)) at a flow rate of 400 µl/minute. Following sample injection, the column was washed with SCX buffer A until the baseline returned. The gradient was run as follows: 0-50% SCX buffer B (10 mM phosphate, 1 M NaCl, pH3 in 20% acetonitrile) over 25 min followed by a ramp up from 50 to 100% SCX buffer B over 5 min. The column was then washed in 100% SCX buffer B for 5 min before equilibrating for 10 min with SCX buffer A. Fractions were collected (400 µL) during the elution period and dried down completely in a vacuum centrifuge. Protein Identification and Quantification by Mass Spectrometry. The iTRAQ tryptic peptide fractions were each resuspended in 30 µL of RP buffer A (2% acetonitrile, 0.05% TFA in water (Sigma Chromasolv plus). Prior to mass spectrometry analysis, fractions were first separated by liquid chromatography (Dionex Ultimate 3000) on a Pepmap C18 column, 200 µm × 15 cm (LC Packings) at a flow rate of 3 µL/minute. Fractions
research articles were injected by full-loop injection (20 µL) and the order of loading was randomized to minimize effects from carry-over. The eluants used were: A. 0.05% TFA in 2% acetonitrile in water and B. 0.05% TFA in 90% acetonitrile in water. The gradient was run as follows: 10 min isocratic prerun at 100% A, followed by a linear gradient from 0-30% B over 100 min, followed by another linear gradient from 30-60% over 35 minutes. The column was then washed in 100% B for a further 10 min, before a final equilibration step in 100% A for 10 min. During the elution gradient, sample was spotted at 10 s intervals using a Probot (LC Packings) with R-cyano-4-hydroxycinnamic acid (CHCA) at 3 mg/mL (70% MeCN, 0.1% TFA) at a flow rate of 1.2 µL/min. Both MS and MS/MS analysis was performed on the fractionated peptides using an Applied Biosystems 4800 MALDI TOF/TOF mass spectrometer. The mass spectrometer was operated under control of 4000 Series Explorer v3.5.2 software (Applied Biosystems). A total of 1000 shots per MS spectrum (no stop conditions) and 2500 shots per MS/MS spectrum (no stop conditions) were acquired. The following MS/MS acquisition settings were used: 2 KV operating mode with CID on and precursor mass window resolution set to 300.00 (fwhm). Peak lists of MS and MS/MS spectra were generated using 4000 Series Explorer v3.5.2 software and the following parameters were used after selective labeling of monoisotopic mass peaks: MS peak lists: S/N threshold 10, Savitzky Golay smoothing (3 points across peak (fwhm)), no baseline correction, MS/MS peak lists: S/N threshold 14; smoothing algorithm: Savitzky Golay, smoothing (7 points across peak (fwhm)). An automated database search was run using GPS Explorer v3.6 (Applied Biosystems). MASCOT was used as the search engine to search the NCBI nonredundant database using the following search parameters: precursor ion mass tolerance of 100 ppm, MS/MS fragment ion mass tolerance of 0.3 Da, iTRAQ fragment ion mass tolerance of 0.2 Da, the taxonomy was selected as human, oxidation of methionine residues were allowed as variable modifications and N-term (iTRAQ), lysine (iTRAQ) and MMTS modification of cysteine residues were set as fixed modifications. Quantification of the iTRAQ peptides was performed using 114 (untreated SMA cells (i)) as the reference mass. The identification criterion was at least 2 peptides by MS/MS with the most stringent search settings (peptide rank 1 and total ion score confidence intervals of at least 95%). iTRAQ Ratios were normalized using the following formula: iTRAQ Ratio ) Ratio/(median iTRAQ Ratio of all found pairs) that was applied in GPS Explorer software. SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. Protein extracts were prepared by extracting cells in SDS extraction buffer (2% sodium dodecyl sulfate-SDS, 5% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8), brief sonication followed by boiling for 2 min. Proteins were subjected to SDSPAGE using 12% polyacrylamide gels and transferred to nitrocellulose membranes (either by electroblot or diffusion blotting). After blocking nonspecific sites with 4% powdered milk solution, membranes were incubated with primary antibodies (mouse antiSMN (MANSMA1)25 and mouse antiLamin A/C (MANLAC1)26) at a dilution of 1/100, mouse antibeta-actin (Abcam) at a dilution of 1/1000 and rabbit antiSPARC (Santa Cruz) at a dilution of 1/200), diluted in dilution buffer (PBS, 1% fetal bovine serum, 1% horse serum and 0.1% BSA). Antibody reacting bands were visualized by development with either peroxidase-labeled goat antimouse Ig, or peroxidiseJournal of Proteome Research • Vol. 9, No. 8, 2010 4229
research articles
Fuller et al. Immunohistochemistry. Skin fibroblast cells were grown on glass coverslips in DMEM with 10% fetal bovine serum and fixed with 50:50 acetone/methanol. Coverslips were incubated with antipro-collagen I monoclonal antibody at a dilution of 1/10 (developed by McDonald, J.A. and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, The University of Iowa, Department of Biology, Iowa City, IA) for 1 h at 20 °C. Coverslips were then washed four times and incubated for 1 h with 5 µg mL-1 goat antimouse ALEXA 488 (Molecular Probes, Eugene, OR) diluted in PBS containing 1% horse serum, 1% fetal bovine serum and 0.1% BSA. DAPI (diamidinophenylindole 200 ng mL-1) was added for the final 5 min of incubation to counterstain nuclei. After washing, coverslips were mounted on slides in Hydromount (Merck). High magnification images were obtained using a Leica SP5 confocal microscope with a 40× oil immersion objective.
Results
Figure 1. SMN levels increase in Type I SMA fibroblast cells after treatment with valproate (VPA). Protein extracts from type I SMA skin fibroblasts were subjected to SDS-PAGE and transferred to nitrocellulose by Western blotting. The blot was cut into horizontal strips and probed with antibodies against SMN25 (at 1/100 dilution) and a Lamin A/C[26] (at 1/100 dilution) as a loading control. After visualization using a chemiluminescent system, the integrated density of the bands was measured using ImageJ software.
labeled swine antirabbit Ig (1 µg/mL in dilution buffer) and a chemiluminescent detection system (West Femto, Pierce).
An SMA human skin fibroblast cell line from the Coriell Cell Bank (GM03813) was used for this study since we used it previously to study upregulation of SMN protein by valproate in SMA cells.23 It is important for a proteomics study to have a control protein that is known to be upregulated to establish that the valproate treatment was effective and, indeed, an upregulation of SMN by approximately 40% under these conditions was confirmed by Western blot (Figure 1). The SMA cells were grown with or without valproate at very high density (confluent) for a short period (24 h) to minimize any possible effects of the drug on cell division. These cell culture conditions are known to reveal SMN up-regulation by valproate23 and serum levels of 50-100 mg/L are used for treatment of epilepsy and bipolar disorder corresponding to 0.3-0.6 mM sodium valproate, so our 0.5 mM is within the clinically effective range.27 To determine which proteins are up-regulated or downregulated by valproate treatment, without prejudging what
Table 1. Proteins Altered by Treatment with Valproate after Strict Filtering of the Raw Dataa protein name
accession number
peptide count
total ion score C.I. %
avg iTRAQ ratio (115/114)
avg iTRAQ ratio (116/114)
avg iTRAQ ratio (117/114)
p value
Collagen alpha-1(I) chain Collagen alpha-2(I) chain Collagen alpha -3 (VI) chain Collagen alpha 1(VI) chain pro-alpha-1 collagen type 1 Collagen alpha-1(III) chain Osteonectin collagen alpha 1(V) chain
gi|62088774 gi|124056488 gi|55743098 gi|87196339 gi|179629 gi|124056490 gi|2624793 gi|219510
23 19 26 10 3 4 3 3
100 100 100 100 100 100 100 100
1.03 ( 0.17 [25] 1.00 ( 0.18 [20] 0.96 ( 0.21 [28] 1.03 ( 0.18 [11] 1.07 ( 0.10 [4] 1.15 ( 0.29 [4] 1.04 ( 0.09 [3] 0.93 ( 0.17 [3]
0.51 ( 0.20 [25] 0.67 ( 0.18 [20] 0.79 ( 0.15 [28] 0.80 ( 0.23 [11] 0.43 ( 0.1 [4] 0.77 ( 0.19 [4] 0.75 ( 0.16 [3] 0.64 ( 0.2 [3]
0.46 ( 0.2 [25] 0.59 ( 0.21 [20] 0.74 ( 0.17 [28] 0.73 ( 0.34 [11] 0.41 ( 0.08 [4] 0.69 ( 0.22 [4] 0.75 ( 0.07 [3] 0.48 ( 0.06 [3]
8.41 × 10-15 4.75 × 10-8 4.27 × 10-6 2.27 × 10-5 3.39 × 10-4 0.022 0.04 0.048
TCTP unnamed PHB hypothetical melanoma antigen D, 2 unnamed eIF 2 beta caveolin 2 isoform a and b hypothetical protein SBBI88
gi|114794484 gi|194381290 gi|49456373 gi|13276691 gi|19387846 gi|14041989 gi|29826335 gi|4557413 gi|6942315
3 8 4 3 3 2 2 2 2
100 100 100 100 100 100 100 100 100
1.09 ( 0.04 [3] 0.88 ( 0.08 [8] 1.10 ( 0.19 [4] 0.10 ( 0.34 [3] 0.85 ( 0.12 [3] 0.85 ( 0.27 [2] 0.96 ( 0.16 [2] 0.90 ( 0.27 [2] 1.09 ( 0.15 [2]
0.92 ( 0.07 [3] 0.77 ( 0.2 [8] 1.43 ( 0.30 [4] 0.74 ( 0.27 [3] 0.86 ( 0.13 [3] 0.59 ( 0.17 [3] 0.81 ( 0.02 [2] 0.87 ( 0.02 [2] 1.31 ( 0.26 [2]
0.60 ( 0.28 [3] 0.83 ( 0.11 [8] 1.25 ( 0.24 [4] 0.83 ( 0.28 [3] 0.66 ( 0.18 [3] 0.79 ( 0.02 [2] 0.76 ( 0.13 [2] 0.66 ( 0.37 [2] 1.48 ( 0.12 [2]
0.08 0.1 0.116 0.13 0.23 n/a n/a n/a n/a
a First eight proteins showed significant decreases in valproate-treated cells, while the remaining proteins showed changes that did not achieve significant p values. iTRAQ tags were assigned as follows: 114 and 115, untreated SMA fibroblasts; 116 and 117, valproate-treated SMA fibroblasts. The designated reference for calculating ratios from raw peak intensities was 114, so the 114/115 ratio should be close to unity. iTRAQ values for 115/114 were compared with the average of 116/114 and 117/114 for each peptide (see Supplementary file II, Supporting Information), using Student’s pairwise t-test to determine the significance of the iTRAQ changes for each protein. Significant changes with p values
