PAL31 may play an important role as ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2009 | 108 | 1187–1197

doi: 10.1111/j.1471-4159.2008.05865.x

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*The Institute of Biochemistry and Molecular Biology, National Yang-Ming University, School of Life Sciences, Taipei, Taiwan Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan àCenter for Neural Regeneration, Department of Neurosurgery, Taipei Veterans General Hospital, Taipei, Taiwan §Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan

Abstract Functional regeneration in a complete T8 transection model Cheng et al. (1996) and most recently, acidic fibroblast growth factor (aFGF; also known as FGF-1) involved in the repair process of the spinal cord injury (SCI) rat Tsai et al. (2008) have been reported. To further reveal the mechanism of the repair process of SCI, in additionally, we have identified a 30 kDa specific protein kinase A substrate induced at 6 days after SCI. However, the induction of the transducing signal was reduced in samples treated with aFGF. The 30 kDa protein was purified and identified by mass spectrometry as a novel protein, PAL31. The results of immunohistochemical study showed that PAL31 is abundantly expressed in the epicenter of the injured spinal cord and colocalizes with ED1-positive cells (macrophages) and CD8 T

lymphocytes. Over-expression of PAL31 in RAW 264.7 cells resulted in the down-regulation of macrophage chemoattractant protein 1, inducible nitric oxide synthase, and signal transducer and activator of transcription-1. However, knockdown of PAL31 by small interfering RNA seems to lead to apoptosis when the cells were treated with inflammatory inducers. These experimental results suggest that PAL31 may involve in the modulation of the inflammatory response and, at the same time, prevent apoptosis process of macrophage after SCI. Keywords: apoptosis, inducible nitric oxide synthase, inflammation, macrophage chemoattractant protein 1, PAL31, signal transducer and activator of transcription-1, spinal cord injury. J. Neurochem. (2009) 108, 1187–1197.

Spinal cord injury (SCI) is a serious clinical problem that has irreversible impacts and results in functional lost. Landmark experiments over 20 years ago demonstrated that the neurological deficits are not because of an intrinsic inability of CNS neurons to regenerate, but rather to the unfavorable CNS environment (David and Aguayo 1981). At present, transplantation with various cell types including macrophages and stem cells (Lazarov-Spiegler et al. 1996; Deshpande et al. 2006) as well as the application of trophic factors such as brain-derived growth factor and nerve growth factor (Kim et al. 1996) are the major strategies used to help improve regeneration. Inflammatory responses are a major component of secondary injury and play a central role in regulating the pathogenesis of acute and chronic SCI (Trivedi et al. 2006; Fitch and Silver 2008). It has been reported that reducing inflammation decreases secondary degeneration and the functional deficit after SCI (Gonzalez et al. 2003; Beattie

2004). These responses seem to play a pivotal role in nerve injury and contribute to the control of the regenerative response; nevertheless, the nature of their functional roles is a matter of debate. Received October 29, 2008; revised manuscript received December 9, 2008; accepted December 11, 2008. Address correspondence and reprint requests to Kin-Fu Chak, Institute of Biochemistry and Molecular Biology, National Yang, Ming University, Taipei 11221, Taiwan, China. E-mail: kfchak@ym.edu.tw Abbreviations used: aFGF, acidic fibroblast growth factor; Anp, acidic nuclear protein; ED1, ectodermal dysplasia 1; GFP, Green Fluorescent Protein; IEF, isoelectric focusing; IFN-c, interferon-c; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; IpG, immobilized pH gradient; LPS, lipopolysaccharide; MCP-1, macrophage chemoattractant protein 1; NO, nitric oxide; NOS, nitric oxide synthase; PAL31, proliferation related acidic leucine-rich protein; PKA, protein kinase A; SCI, spinal cord injury; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; siRNA, small interfering RNA; STAT-1, signal transducer and activator of transcription-1; TNF-a, tumor necrosis factor-a.

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1187–1197

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1188 | L.-F. Shen et al.

It has been shown that cAMP is critical to neuron regeneration (Cai et al. 2001) and this regenerative effect is overwhelmingly dependent on the activity of protein kinase A (PKA). And an increase in intracellular cAMP concentration is able to mimic the effect of neurotrophin priming, which results in the activation of PKA (Cai et al. 1999). But it is likely that other parallel pathways exist that activate cell survival or overcome the inhibition of neural regeneration. Therefore, it hints us to find other PKA downstream factors probably participated in the regeneration pathway. In addition to our recent report on the involvement of acidic fibroblast growth factor (aFGF) in the repair process of SCI (Tsai et al. 2008), we further identified a novel phospho-motif in PKA substrates that correlates with SCI rat spinal cord regeneration. We then used protein purification and mass spectrometry analysis to identify a novel phospho-protein, proliferation related acidic leucine-rich protein (PAL31), which surprisingly was substantially reduced in experimental rats treated with aFGF. PAL31, a nuclear protein, has been recently identified (Mutai et al. 2000). In the nervous system, expression of PAL31 gradually declines along with the developmental process and it is rarely expressed in the adult nervous system, including the spinal cord. In addition to this function in proliferation, Sun et al. (2006) has further shown that PAL31 acts as a caspase 3 inhibitor. In addition, its expression is induced by prolactin and interleukin-3 (IL-3) in Nb2 cells (a pre-T lymphoma cell line) (Sun et al. 2001). Furthermore, in this study, we found that the results of over-expression and the small interfering RNA (siRNA) knockdown of PAL31 in RAW 264.7 cells suggested that the function of PAL31 might negatively regulated the expression of macrophage chemoattractant protein 1 (MCP-1) and signal transducer and activator of transcription-1 (STAT-1) and at the same time, it may rescue macrophages from apoptosis during an inflammatory response. Hence, we reported for the first time an additional novel function for PAL31 as an inflammatory response protein during SCI. Interestingly, this specific response is substantially reduced in proportion to the degree of recovery present in SCI rats. Based on these observations, we would like to suggest that PAL31 is a modulator of inflammation during SCI and may play an important role in preventing macrophage apoptosis when this is encouraged by inflammatory stimulation.

Materials and methods Animal and cell Adult female rats (Sprague–Dawley) around 250 g were used in spinal cord ‘transection-model’ and ‘repair-model.’ RAW 264.7 macrophage cells (ATCC, Manassas, VA , USA) were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, USA)

supplemented with 2 mM glutamate, 100 U/mL penicillin, 100 lg/ mL streptomycin, and 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA). In the stimulation by inflammatory factors experiments, the cells were changed to serum-free medium for 24 h, and then treated with medium containing various different factors over a time course. The IL-1, tumor necrosis factor-a (TNFa), and interferon-c (IFN-c) used in this study were purchased from R&D (Minneapolis, MN, USA), and lipopolysaccharide (LPS) was purchased from Sigma (St Louis, MO, USA). A spinal cord ‘repair-model’ with nerve graft and slow release growth factor The detailed methods for spinal cord reconstruction have been previously reported (Cheng et al. 1996). Briefly, vertebrate T7–T10 were exposed and following T8 and T9 posterior laminectomies, a 5 mm T8 spinal cord segment was removed. Eighteen intercostal nerves from the same animal were used to reroute pathways between the spinal cord stumps. aFGF was mixed into fibrinogen plus aprotinin solution and this was used to form an aFGF-containing (2.1 lg/mL flue) glue casting in the engrafted area. Finally, the T7 and T10 processed spinal cord was fixed in dordiflexion using compressive S-shaped monofilament surgical steel (BS gauge 20) loops fastened to the spinal column with non-absorbent threads. Western blotting Spinal cord tissue blocks or cells were homogenized in lysis buffer containing 10 mM Tris, pH 7.4, 50 mM NaCl, 1% NP-40, 1x protease inhibitor (Roche, Indianapolis, IN, USA), 30 mM Na4P2O7, 1 mM Na3VO4, and 30 mM NaF. Protein concentration was assayed using a Bio-Rad DC kit (Hercules, CA, USA). The protein extract (20 lg/lane) was next separated on 12.5% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a polyvinylidene difluoride filter (Millipore, Bedford, MA, USA). Primary antibodies such as Phospho-(Ser/Thr)-PKA substrate antibody, phospho-(Ser) Akt substrate antibody, phospho(Ser) PKC substrate antibody, phospho-(Thr) mitogen-activated protein kinase)/cyclin-dependent kinases substrate antibody, phospho-STAT-1, STAT-1 were purchased from Cell Signaling (Beverly, MA, USA) and used at 1000-fold dilution. Inducible nitric oxide synthase (iNOS) (1 : 1000; R&D), b-actin (1 : 5000; Chemicon, Temecula, CA, USA), and glyceraldehyde-3-phosphate dehydrogenase (1 : 10 000; LabFronter, Seoul, Korea) antibodies were also used. Horseradish peroxidase-conjugated antibody was used as the secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein bands were visualized by enhanced chemiluminescence development (PerkinElmer Life Sciences, Waltham, MA, USA). The detailed procedures for the western blotting were the same as described in the New England Nuclear western blot manual, which is provided by the manufacturer. Q ionic column fractionation Tissue lysate from transected spinal cord tissue (50 mg) was suspended in 20 mM phosphate buffer, pH 7, containing 1x protease inhibitor (Roche). The tissue lysate was then separated by anion (Q Sepharose) exchange chromatography (GE Healthcare, Wauwatosa, WI, USA) using a NaCl gradient (from 0 to 1 M). Five protein fractions, Q1–Q5, were obtained. These protein fractions were concentrated by Centrion (Millipore).


The novel role of PAL31 in the spinal cord injury | 1189

Analysis of Q ionic fractions containing either PKAS1 or PAL31 protein using 2D-PAGE and western blot The Q4 fraction containing the 30 kDa protein was subject to 2DPAGE, isoelectric focusing (IEF) and SDS–PAGE according to the method described by manufacturer’s manual (Amersham Biosciences) with minor modifications. For the first-dimension IEF, pH 3.5– 4.5 linear range of immobilized pH gradient (IPG) strips (18 cm) were rehydrated with 350 lL of solubilized sample for 12 h before the sample was separated by IEF at 150 V for 2 h, 500 V for 1 h, 1000 V for 1 h, 4000 V for 1 h, and finally 8000 V for 7 h. Prior to the second-dimension SDS–PAGE, the IPG strips were equilibrated with equilibration buffer, consisting of 50 mM Tris–HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 50 mM dithiothreitol, and 0.01% bromophenol blue at 25C for 15 min, followed by equilibration in 50 mM Tris–HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 25% iodoacetamide and 0.01% bromophenol blue at 25C for 15 min. The second-dimensional SDS–PAGE used a 12.5% separating gel was performed. The 2D-PAGE was then performed the western blot analysis. Over-expression and knockdown of pal31 gene cell cloning The construction of over-expression clone of pal31 Full length-PAL31 cDNA was ligated into the EcoRI and SalI site of pEGFP-C1 vector (BD Biosciences Clontech, San Jose, CA, USA) and 5 lg of the recombinant plasmid was mixed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) for transfection into RAW 264.7 cells. After 4 h, the cells were transferred to culture medium for 24 h, which was then replaced with medium containing G418 (Sigma). Finally, the mix population of transfected clones was used in this experiment. The construction of the knockdown clone of pal31 The pSilencer 3.1-H1 neo vector (Ambion, Austin, TX, USA) was linearized with BamHI and HindIII to facilitate directional cloning. We designed three oligonucleotides as pal31 gene hairpin siRNA, the sequences used were shown in Table S1. The synthesis step was performed by the DHARMACON (Lafayette, CO, USA). The cloning sites for BamHI and HindIII were also synthesized into the hairpin of siRNA-pal31. Next, the genes were inserted into the pSilencer 3.1-H1 neo vector. Recombinant plasmid (5 lg) was mixed with Lipofectamine 2000 reagent for transfection into RAW 264.7 cells. Subsequently, the transfected cells were cultured in culture medium containing G418 for 10–14 days. Single clones were harvested by diluted the cells during cell subculture. Screening of proinflammatory protein After stimulation with inflammatory factors, cell culture medium was incubated in RayBio Mouse Inflammation Antibody Array I from RayBiotech Inc. (Norcross, GA, USA) and developed according to the manufacturer’s instructions. Blot densities were assayed using IMAGEQUANT software version 5.2 (Amersham Biosciences) and normalized with the positive controls incorporated into the membranes. The method of immunoprecipitation, in-gel digestion and mass spectrometric analysis, immunohistochemistry and immunocytochemistry, nitrate assays, real-time PCR, caspase 3 activity assay, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay are available in the Supporting information.

Results Detection of a novel differentially expressed protein in the injured spinal cord partitions of an animal model To identify the expression pattern of PKA substrate(s), we collected spinal cord samples from the ‘transection-model’ and ‘repair-model’ at various time points after the operation. Western blotting of the proteins extracts from these samples was analyzed using a specific antibody raised against the motifs (RXXpS/T or RXpS/T) found in PKA substrates. As showed in Fig. 1a, it was found that a signal situated at 30 kDa was induced in injured samples compared with the control. The induced signal was designated PKAS1 and the differential expression of this signal increased with time after injury. Surprisingly, we observed that the induced signal of PKAS1 in the ‘repair-model’ was substantially reduced compared with that in the ‘transection-model’ (Fig. 1a). In addition, we found that the signal could be detected along the spinal cord starting from T4 reaching to T12 (Fig. 1b). However, the most abundant signal was detected at the stump ends around T8. Thus, these results clearly demonstrated that the appearance of PKAS1 would seem to correlate with the degree of severity of spinal cord damage. A protein fractionation strategy was therefore developed to purify this protein signal. Eventually, five fractions, Q1–Q5 were collected. Western blot data indicated that only the Q4 fraction contained the 30 kDa PKAS1 transducing signal (Fig. 1c, lane 6). This protein signal was then extracted from gel and analyzed by Quadric-Tof spectrometry. The Mass spectrometry results were processed through MASCOT (Matrix Science; http://www.matrixscience.com) and this pinpointed the novel protein PAL31 as the only potential candidate for PKAS1 (Fig. S1 and Table S2). The densitometric quantitative analysis of the PKA-phosphorylated form of PAL31 (PKAS1) and PAL31 from the western blot data shown in Fig. 1a indicated that expression of both PKAS1 and PAL31 were indeed slightly reduced in the repaired model (Fig. S2). Furthermore, the same set of gels shown in Fig. 1a (upper panel) were re-probed with antibody raised against PAL31 (Fig. 1a, middle panel). The result clearly indicated that the changes in PAL31 protein at various time points were correlated with the pattern as detected by PKA-phosphosubstrates antibody (Fig. 1a, upper panel). Thus, it is highly likely that the increase in intensity of the PAL31 phosphorylation signal is due purely to an increase in the amount of protein. PAL31/PKAS1 is a specific PKA substrate Furthermore, an experiment was also carried out to show whether PKAS1 responded in the same way as PAL31 with respective to specific PKA kinase. Four different antibodies against substrate of kinases (mitogen-activated protein kinase-S, PKC-S, Akt-S, and PKA-S) were used to react



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Fig. 1 Differential expression of PKAS1 (PAL31) in the rat transection and repair models of the injured spinal cord. (a) The upper and middle panels show the results of western blots, respectively, against the phosphorylation motif of PKA (anti-PKAS) and pan-PAL31. Arrows show the PKA-phosphorylated form of PKAS1 (PAL31) and the panprotein of PAL31, respectively. Samples were taken at 2–14 days after the operation. Protein samples designated ‘P’ were prepared from a 0.5-cm long spinal cord segment rostral to the injury site and protein samples designated ‘D’ were prepared from a 1-cm long spinal cord segment caudal to injury site. T: transection, R: repair. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal control. (b) Eight days transected spinal cord tissue was divided into eight

sections corresponding to the thoracic vertebra number. PKAS1 (PAL31) was mainly found in the lesion stump of the injured spinal cord as detected by the anti-PKAS antibody with b-actin used as internal control. (c) Protein samples were fractionated by Q-ionic column. PKAS1 (PAL31) was found to be located at the fourth fraction (Q4) (lane 6). Lane 1, 6-day transected spinal cord was used as the control; lane 2, the fraction collected from the unbound solution; and lanes 3–7, five fractions harvested at different salt concentrations of eluting buffer (Q1–Q5). All samples were separated by 12.5% SDS– PAGE. The results shown here are obtained from three independent experiments.

with samples containing PKAS1 and PAL31. Our result confirmed that the PKAS1 extracted from the transected spinal cord and PAL31 extracted from the cerebellum were both specific substrates of PKA (Fig. S3a). To further confirm whether PKAS1 is really identical to PAL31, we performed an antibody pull-down assay to capture the protein from the injured spinal cord tissue using anti-PAL31 antibody. The result was analyzed by western blotting using antibody raised against PKA-phospho-substrates. The pull-down fraction gave a positive signal with a molecular mass similar to PKAS1 (Fig. S3b). To investigate whether PAL31 was undergone phosphorylation, the Q4 fraction (Fig. 1c, lane 6) was isolated and performed IEF

using an IPG strip ranging from pI 3.5 to 4.5 (pI of PAL31 is 3.87) followed by 2-D PAGE analysis. Subsequently, the 2-D PAGE was performed western blot using antibodies raised against PKA phospho-substrates and PAL31, respectively. Our results indicated one protein band stretched over the pI value around 3.87 when detected by antibody raised against PKA phospho-substrates (Fig. 2 upper panel). Interestingly, two protein bands slightly differed from each other on their pI and molecular mass were identified when detected by antiPAL31 antibody (Fig. 2 lower panel). It was known that the phosphorylated form of protein is slightly more acidic and higher in molecular mass than that of the unphosphorylated form of protein. Therefore, our experimental results clearly



pI 3.5

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Anti-PKAS

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Anti-PAL31 Fig. 2 PAL31/PKAS1 is a specific PKA substrate. Q4 fraction (SDS– PAGE of the protein is shown in Fig. 1c) was analyzed by isoelectric focusing with IPG strip (pI 3.5–4.5) followed by 2-D PAGE and then performed immunoblotting using antibodies against anti-PKAS and PAL31, respectively. Two protein bands were observed on the western blot against anti-PAL31 antibody (lower panel), and only one band was identified on western blot against anti-PKAS (upper panel). Note that the protein band with lower pI value and higher molecular mass shown on the lower panel was found to be localized in the similar position of the protein band shown on the upper panel.

indicated that PAL31 is indeed a PKA phospho-protein. Thus, all the results support the idea that PKAS1 is indeed identical to PAL31. Therefore, we would like to rename PKAS1 as PAL31 from this point onwards. Localization of PAL31 protein over 6 days after transaction of the spinal cord tissue As shown in Fig. 3a, the localization of PAL31 in the 6-day transected spinal cord tissue was basically detectable in the region of stump adjacent to the lesion epicenter (the relative position designated B enclosed in the large rectangle of Fig. 3a). The caudal end of the spinal cord expressed much more PAL31 than the rostral end. This result was in agreement with the data shown in Fig. 1b. Amplified images captured from the lesion epicenter (Fig. 3b and c) show the magnified morphology of a PAL31-positive cell. In addition, we also observed that a higher cell density of PAL31producing cells was found in regions where there was lesion cavitation compared with other regions (Fig. 3d). If we compare the positive signal in the lesion stump with the rostral end far from the lesion epicenter (Fig. 3e) and the control normal spinal cord tissue (Fig. 3f), the latter two samples show a negative signal for PAL31. Note that the relative positions of Fig. 3d and e have been indicated in Fig. 3a. It is noteworthy that PAL31 was not detected using the pre-immune serum of PAL31 (Fig. 3g). Subcellular localization of PAL31 in macrophages and CD8 T lymphocytes at day 6 after transaction of the spinal cord To investigate whether production of PAL31 protein correlated with the cellular inflammatory response, the subcellular localization of PAL31 in macrophages and T lymphocytes present in the injured spinal cord was performed. Figure 4a shows a magnified image of immunohistochemical analysis

using double staining for ectodermal dysplasia 1 (ED1) (a known cellular marker of activated macrophages) and PAL31. Our results indicated that both ED1 and PAL31 were colocalized in the macrophages. Apparently, ED1 is localized in the cytosol and PAL31 is present in the nucleus (Fig. 4a). Our histochemical results also indicated that various cell types are able to produced PAL31 (Fig. 3b). It was noted that the morphology of some of the cell types was very similar to T lymphocytes. Probably, at this stage, T lymphocytes are able to infiltrate to the lesion site; therefore, antibodies against CD4 (T-helper lymphocytes) and CD8 (Tcytotoxic lymphocytes) were used to identify the existence of T lymphocytes. Our results indicated that only CD8-positive cells (Fig. 4b) but not CD4-positive cell (data not shown) were detected. Most interestingly, many CD8-positive cells also showed colocalization with PAL31 (Fig. 4b). In addition, an image taken using a confocal microscopy (Fig. 4c and d) confirmed that the ED1 and CD8 signal are really colocalized with PAL31 in one cell type. Down-regulation of MCP-1, iNOS, and STAT-1 in RAW 264.7 cells over-expressing PAL31 To further study the function of PAL31 in immune cells, a PAL31 constitutively over-expressed clone was constructed. Fig. S4a showed the protein of expressed-PAL31 [Green Fluorescent Protein (GFP)–PAL31] and endogenous PAL31 in PAL31-over-expressed and GFP vector control RAW 264.7 cells stimulated with IL-1, TNF-a, and IFN-c for 24 h. The inflammatory antibody array for screening the effected protein was used to investigate the possible functional role of PAL31 in the inflammatory response. RAW 264.7 cells were treated with factors known as the cytokine induced in SCI (IFN-c, TNF-a, and IL-1). Noted that only twofolds changes or above of the effected protein were selected for further discussion in this experiment. Interestingly, this experimental result indicated that production of the MCP-1 in PAL31-over-expressed cells was around 2.7-fold less than that of the control (Fig. 5a). It was well documented that iNOS is a typical marker of inflammatory response. Surprisingly, in the PAL31-overexpressed RAW 264.7 cells, stimulation of the cells with some inflammatory factors (Fig. 5b) resulted in a substantial down-regulation of expression of the iNOS protein compared with cells containing the GFP vector. Furthermore, secretion of nitrate from the RAW 264.7 cells containing over-expressed PAL31 under the stimulation of various inflammatory factors was substantially reduced (Fig. 5c). Transcriptional expression of the iNOS gene by the RAW 264.7 cells under various experimental conditions was also investigated using quantitative RT-PCR. It was found that under the stimulation of these cells with inflammatory factors (IFN-c, TNF-a, and IL-1), expression of iNOS mRNA in the cells containing over-expressed PAL31 was only 6.69% of the expression level in cells containing the GFP vector



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Fig. 3 Histological analysis of the localization pattern of the PAL31 protein in the 6-day transected spinal cord tissue. The expression pattern of PAL31 in 6-day transected spinal cord tissue was studied by immunohistochemistry (IHC) using antibody raised against PAL31. (a) Overview of the IHC image of the transected spinal cord. The big rectangle indicates the lesion epicenter area. (b) Localization of PAL31 was mainly in the stump and the region adjacent to the lesion epicenter (the relative position of this region is indicated in a small rectangle designated B enclosed in the big rectangle in (a). The magnified

image was taken from the cross section of the region. (c) The magnified image relative to the region in (b) shows clearly the presence of PAL31-positive cells. (d) A high density of PAL31 producing cells were found localized in the cavity regions, which are magnified from the region indicated in (a). (e) The rostral end, shows only a weak PAL31 signal (magnified region was away from the left-hand side of the lesion epicenter as showed in a). (f) The IHC pattern in the normal spinal cord tissue shows a negative signal for PAL31. (g) Pre-immune serum of PAL31 was used for the negative control. Scale bar, 50 lm (b–g).

(Fig. S4b). Thus, correlation of all these experimental results indicated that over-expression of PAL31 in RAW 264.7 cells probably either directly or indirectly controls the downregulation of the transcriptional expression of the iNOS gene in response to stimulation by inflammatory factors.

It was known that Jak-stat-1 plays an important role in regulating the production of iNOS under stimulation by IFN-c. In PAL31 over-expressed RAW 264.7 cells, our western blot analysis of the expression of phospho-STAT-1 and STAT-1 in cells stimulated with IFN-c indicated that



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Fig. 4 Immunohistochemical analysis demonstrated that PAL31 colocalized with ED1 (macrophages) and CD8-positive T lymphocytes in a section of 6-day transected spinal cord. (a) In the cavity region, large numbers of macrophage have infiltrated. The arrows indicate double staining cells. ED1 (purple) is localized in the cytosol and PAL31 (brown) occupies the nucleus. (b) Double staining of CD8 and PAL31 captured from the stump area. The arrow points to the round morphology of the CD8+ lymphocytes (purple), which colocalize with PAL31 (brown). (c and d) The immunofluorescent image captured using a laser scanning confocal microscope shows the colocalization pattern of ED1 or CD8 (red) with PAL31 (green). The arrows indicate double staining cells. ED1 + macrophages are labeled for ED1 (Cy3) and PAL31 (Alexa Fluor 488) in (c), CD8+ lymphocyte are labeled for CD8 (Cy3), and PAL31 (Alexa Fluor 488) in (d). Scale bar, 20 lm (a, b, and c) and 13.48 lm (d).

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expression of these two proteins were significantly reduced in the PAL31 over-expressed cells compared with cells containing the GFP vector (Fig. 5d, lanes 2 and 4). Moreover, we also found that this down-regulation of phospho-STAT-1 and STAT-1 in RAW 264.7 cells without stimulation with inflammatory factor under normal culture conditions was similar to that of the cells treated with IFNc (Fig. 5d, lane 6). Furthermore, transcriptional level of STAT-1 in response to the inflammatory factors (IFN-c, TNF-a, and IL-1) detected by RT-PCR showed that the mRNA level of STAT-1 in PAL31-over-expressed RAW 264.7 cells was only reduced to 65.3% when compared with the control (Fig. S4c). Probably, our experimental results implied that PAL31 might be either directly or indirectly involved in the regulation of transcriptional level of STAT-1 and this might result in the down-regulation of the expression of iNOS. Knockdown of PAL31 in RAW 264.7 resulted in a higher percentage of apoptotic cells after treatment with an inflammatory inducer To further reveal the function of PAL31, we constructed a PAL31 knockdown clone in the RAW 264.7 cell line. In Fig. S5, it was clearly shown that the expression level of PAL31 protein was inhibited completely in clone 1-3, and is substantially reduced in clone 3-23 compared with the negative control clones 4-3 and 4-4. To investigate the

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knockdown clone’s response to inflammatory factor stimulation, we treated the cells with IFN-c or LPS to induce inflammation. Our results demonstrated that the morphology of the 4-3 clone stimulated with inflammatory factors was similar to that of untransfected cells (data not shown). However, a high percentage of cells from the 1-3 clone, after stimulation, became apoptotic (our unpublished data). Therefore, further apoptotic experiments were carried out. Since PAL31 had been reported to be a caspase 3 inhibitor (Sun et al. 2006), we detected caspase 3 activity after stimulation with LPS or IFN-c. The result indicated that the 1-3 clone showed higher caspase 3 activity (Fig. 6a). In addition, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay for labeling the DNA strand breaks showed that the PAL31 knockdown clone produced a higher percentage of apoptotic cells after stimulation (Fig. 6b). These results probably suggested that PAL31 might exert an anti-apoptotic effect on cells in response to inflammatory factor stimulation.

Discussion In this study, we have successfully identified for the first time that a novel protein, PAL31, is induced after SCI. Up to the present, no report has identified PAL31 present in an injured spinal cord. As the expression level of PAL31 is very low, it was necessary to enrich the protein using ionic column



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Fig. 5 Over-expression of PAL31 in RAW 264.7 cells resulted in a substantial reduction in MCP-1, iNOS, and STAT-1 protein expression. (a) Cells were treated with IFN-c, TNF-a, and IL-1 for 24 h. The conditional medium was used for the assay of inflammation antibody array. MCP-1 is marked by rectangle, and the positive controls are marked by circles. The results are normalized with positive control. (b) Under stimulation by IFN-c (lane 2), the mixture of three inflammatory factors (lane 3) and LPS/IFN-c (lane 6), the amount of iNOS was found to be substantially lower in the PAL31 over-expressed clones than control vector only. Note that the cell lysates were separated by 12.5% SDS–PAGE and subsequently analyzed by western blot using antibodies raised against iNOS or b-actin which was used for the internal control. (c) The amount of nitrate produced was substantially reduced

in clones with over-expressed PAL31 than that of the control when challenged with different combination of inflammatory factors. Note that no detectable amount of nitrate was found in cells without inflammatory factor challenge. (d) Over-expression of PAL31 (lanes 2, 4, and 6) resulted in less STAT-1 in cells either treated with IFN-c (lanes 2–4) or without IFN-c under common culture condition (lane 6). Lanes 1, 3, and 5: cells containing the GFP expression vector as the control. Anti-phospho-STAT-1 and anti-STAT-1 were used to detect the phosphorylation signal of the STAT-1 and the total STAT-1 protein, respectively. The concentration of inflammatory factors used was as follows: IFN-c, 25 ng/mL; IL-1, 10 ng/mL; TNF-a, 10 ng/mL; LPS, 1 lg/mL.

chromatography. Furthermore, using this purified protein, we were able to show that PAL31 is a substrate of PKA kinase. PAL31 is a nuclear protein expressed and colocalized with Proliferating Cell Nuclear Antigen in the developing brain and this protein is also found in the highly proliferative tissues, such as spleen, placenta, and testis (Mutai et al. 2000). In this report, an additional function of PAL31 is proposed. In contrast to previous findings, we found that both native PAL31 and its phosphorylated form were detected in damaged spinal cord. Thus far, the signal for activation of PAL31 is not known; however, it seems likely that induction of PAL31 may be acting as a sensor for the signal induced by the damage to the spinal cord. Our previous animal behavior

studies indicated that the degree of recovery of the SCI rat correlated to the presence of aFGF (Cheng et al. 1996; Tsai et al. 2008). In this study, we found that amount of PAL31 detected was substantially reduced in the presence of aFGF (Fig. 1a). Probably, these experimental results implied that the PAL31 might be correlated with the SCI repair process. Post-traumatic inflammatory reactions play an important role in secondary injury after SCI. It has been reported that infiltration by macrophages and T lymphocytes reaches a peak 7 days after SCI (Bethea and Dietrich 2002). In this study, coincidently, the occurrence of PAL31 in traumatic SCI also reached a peak 6–10 days after injury. PAL31 was highly expressed in the injured rats compared with its



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(b)

Fig. 6 A higher percentage of apoptotic cells were found in the PAL31 knockdown clones. A stable clone of PAL31 knockdown cells was constructed using the RAW 264.7 cell line. (a and b), cells from the 1-3 clone and the 4-3 clone were stimulated with LPS (1 lg/mL) or IFN-c (25 ng/mL) for 24 h. (a) Caspase 3 activity was found to be higher in 13 clone than in the 4-3 clone after challenge with LPS or IFN-c. (b) A higher percentage of apoptotic cells were identified by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay in 1-3 clone than in the 4-3 clone when the cells were treated with LPS or IFN-c. The percentage of cell death was calculated as 100 · [number of FITC-positive cells]/[number of Hochest-positive cells]. The results represent the mean ± SE (n = 3). Student’s t-test was performed to analyze significance of differences between 1-3 and 4-3 clones; *p < 0.05.

expression in the repaired rats. Thus, we infer that the highly proliferative activity of macrophages and T lymphocytes may produce a larger amount of PAL31 in those inflammatory cells. Therefore, lower PAL31 expression in the rat with aFGF treatment can be attributed to a lower inflammatory reaction. This result probably indicates that the effect of the repair process induced by aFGF might be beneficial to the regulation of the inflammatory reaction and hence improved neural regeneration. Moreover, in this study, a siRNA knockdown clone of PAL31 produced large numbers of apoptotic RAW 264.7 cells in response to inflammatory factors. This result probably indicates that the presence of PAL31 might improve the survival of inflammatory cells in the traumatic injury animal model. Therefore, the function of PAL31 may be to confer an antiapoptotic effect on macrophages to modulate the cellular inflammatory response during the repair process of the

damaged spinal cord. Thus, we would like to postulate that PAL31 might behave as an inflammatory modulator during the SCI regeneration process. Over-expression of PAL31 in RAW 264.7 cells surprisingly resulted in a down-regulation of the iNOS protein expression and its mRNA level. STAT-1 is one of the known positive transcription factor of iNOS gene (Aktan 2004). In addition, phosphorylation of STAT-1 is a prerequisite for its ubiquitination (Kim and Maniatis 1996). Thus, reduction in the phosphorylated form of STAT-1 in a PAL31-overexpression clone might be because of the post-translational regulatory of protein degradation via ubiquitination. Besides, the mRNA level of stat-1 was also assayed which showed a minor reduction of stat-1 mRNA when compared with the control (68.97%). Thus, over-expression of PAL31 influenced the STAT-1 in transcriptional level. Based on these observations, we suggest that the decreasing of STAT-1 mRNA and protein in the PAL31-over-expression clone might be involved in the suppression of iNOS gene expression. Nitric oxide (NO) is the molecule catalyzed by NO synthase (NOS). It has strongly evident that NO is involved in the mechanisms of neurotoxicity after ischemic and traumatic injuries to the CNS (Conti et al. 2007). The inhibitor of iNOS applied to the SCI rat showed the neuroprotective effect (Sharma et al. 2005; Lee et al. 2008). Thus, NO is indeed involved in secondary detrimental mechanisms that aggravate the primary physical injury to the CNS. On the other hand, apart from iNOS, we also found that expression of another inflammatory cytokine MCP-1 was down-regulated to around 2.7-fold of the control. In the CNS after focal cerebral ischemia, the up-regulation of MCP1 and subsequent appearance of macrophages has been reported (Kim et al. 1995). The expression of MCP-1 has been observed within CNS also after mechanical trauma (Berman et al. 1996). Noticeably, down-regulation of inflammatory factors could result in a better neuroprotective effect of the damaged spinal cord. Based on this observation, we strongly believe that PAL31 may play a positive role in the SCI repair processes. Therefore, we would like to postulate that PAL31 might play a role in modulating the inflammatory effect to suit the entire repair processes. Thus, differential expression of PAL31 in SCI either with or without the treatment of aFGF should characterize its modulating role in fine-tuning the inflammatory effect in various situations. Regulatory expression of inflammatory factors such as iNOS and MCP-1 should be tightly controlled by a multiple factor. Thus, PAL31 might only play in part of the regulatory processes with respect to inflammatory response of the SCI. Unlike other cell type (such as fibroblasts), inflammatory inducer will cause cell apoptosis. On the contrary, macrophage shows the specific feature of anti-apoptosis in the presence of the inducers. Nevertheless, the molecular



mechanism for the activation-induction resistance of macrophages to death remains obscure at present (Lakics and Vogel 1998). Surprisingly, in this study, we revealed that the PAL31 knockdown clone showed a higher degree of apoptosis when the cells were treated with IFN-c or LPS. Thus, this result implied that PAL31 probably might perform as an antiapoptotic agent in response to stimulation by inflammatory inducers. Based on these experimental results, we postulated that PAL31 might play a dual role to maintain the viability of the macrophage cells at such devastating condition and at the time, modulate the production of inflammatory factors such as iNOS and MCP-1, resulting in attenuation of the formation of secondary injury of the damaged spinal cord. PAL31 is a member of the acidic nuclear protein (Anp32) family and is classified as ANP32B (Matilla and Radrizzani 2005). The Anp32 protein family is also classified as phospho-proteins, which includes SET, ANP32A, and ANP32E. It is known that phosphorylation of ANP32A (mapmodulin) is requires for effective interaction with microtubule-associated proteins (Ulitzur et al. 1997). However, little is known about what kind of kinase regulates their post-translational modification. Only recently PP32 (a member of Anp32) was found to be phosphorylated by casein kinase II (Hong et al. 2004). The phosphorylation of PAL31 has not been study so far and its physiological function of the protein is still waiting to be resolved. In conclusion, this study has identified a novel protein, PAL31, as being induced after SCI and the expression of PAL31 was colocalized in inflammatory response cells such as macrophages and CD8 positive cells. Alleviation of this damage-induced signal in the repair-model SCI rat showed a good correlation with better recovery of damage spinal cords. PAL31 may behave like an inflammatory modulator in response to the regeneration process in SCI rats. Most interestingly, knockdown of PAL31 in macrophages triggered apoptosis in cells stimulated with IFN-c or LPS, which suggests that PAL31 may play an important role in maintaining the survival of macrophage in the presence of inflammatory stress.

Acknowledgements We are grateful to National Science Council of Taiwan under the National Research Project for Genomic Medicines (NSC92-3112-B010-002, NSC93-3112-B-010-024, and NSC94-3112-B-010-001) for supporting this study. We also acknowledge the support from the Yen Tjing Ling Medical Foundation (CI-95-10), Veterans General Hospital-University System of Taiwan Grant (VGHUST97-P6-20), and a research grant from the Aim for the Top University Plan for Nation Yang Ming University. Protein identification was performed in the Proteomic Research Center at National Yang Ming University and Core Facilities for Proteomic Research at the Institute of Biological Chemistry, Academia Sinica,Taipei, Taiwan. Finally, we thank Dr. K. Shiota for sending us the PAL31 antibody for our pilot experiment.

Supporting information Additional Supporting information may be found in the online version of this article: Fig. S1 The mass spectrometry result of PKAS1. Fig. S2 Quantitative analysis of the protein expression level of PKAS1 and PAL31 using the western blot result shown in Fig. 1a for densitometry. Fig. S3 PKAS1 (PAL31) was found to be a specific substrate of PKA when the results were analyzed by western blot using antibody against the phospho-substrate motif of the kinase. Fig. S4 (a) The protein expression level of PAL31 in the GFP–PAL31-over-expression and GFP-over-expressed RAW 264.7 cells stimulated with IL-1, TNF-a, and IFN-c for 24 h. (b) Under stimulation by a combination of inflammatory factors (IL-1, TNF-a and IFN-c), repression of iNOS transcriptional expression was found in clones with overexpressed PAL31; this was detected by Q-RT-PCR. (c) Reduction of STAT-1 transcriptional expression was found in PAL31-overexpression RAW 264.7 cells. Fig. S5 The 1-3, 3-23, and 3-24 are the PAL31 knockdown clones, among them 1-3 showed the best efficiency of knockdown effect as detected by western blotting against PAL31 antibodies Table S1 The sequence of hairpin siRNA of pal31. Table S2 The mass spectrometry result of PKAS1. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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