Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β

TGF-β₁ stimulation for 24 h increased the immuno-reactivity for α-SMA in human embryonic lung fibroblasts (HFL-1) cells, which was accompanied by a change in phenotype as shown by immunofluorescence (Figure 1A). These results were further confirmed by western blot that showed a significant two-fold increase of α-SMA upon TGF-β₁ stimulation for 48 h (P < 0.05) (Figure 1B). Following TGF-β₁ stimulation HFL-1 cells also had an increased immuno-reactivity for prolyl 4-hydroxylase (Figure 1C), which catalyzes post-translational formation of proline to 4-hydroxyproline in collagens. These results could not fully be confirmed by western blot, which showed a two-fold increase after 48 h (P < 0.06) (Figure 1D).

TGF-β₁ significantly increased the expression of both fibronectin and EDA fibronectin. However, following TGF-β₁ stimulation the relative ratio between EDA fibronectin and fibronectin was significantly increased over time as shown by q-PCR (Figure 2A). This was confirmed by western blot showing that TGF-β₁ increased the relative expression of EDA fibronectin compared to fibronectin from 269% at 24 h to (P < 0.01) to 526% at 48 h (P < 0.001) (Figure 2B). These data show that TGF-β₁ may induce myofibroblast differentiation and this is accompanied by an altered splicing pattern, exemplified by the increase in alternatively spliced isoforms of fibronectin.

To address the question whether TGF-β₁ stimulation affects nuclear proteins that are involved in the splicing process, we employed isotope coded affinity tag (ICAT^TM) reagent labeling and tandem mass spectrometry. Our experiments showed that the expression of EDA fibronectin increased from 6 to 48 h of TGF-β₁ stimulation (Figure 2A), which indicates that an alternative splice site selection was induced after 24 h. We therefore decided to examine the expression of nuclear proteins at this time point. Figure 3 shows a schematic overview of the workflow used for the identification and relative quantification of proteins in the nuclear fractions [30‐32]. Around 2500 proteins were identified in multiple LC-MS/MS experiments. This list was reduced to approximately 2000 unique proteins after consolidating the peptide-protein assignments. However, approximately 1,700 of these could be quantified after the proper normalization of the relative abundances of ‘heavy’ and ‘light’ ICAT^TM reagent labeled peptides. After classifying the proteins based on gene ontology [33], 76 proteins involved in pre-mRNA splicing and RNA processing were selected and divided into 13 functional groups as shown in Figure 4 and in Additional file 1: Table S1. The most abundant group was classified as heterogeneous ribonucleoproteins and contained 19 proteins (hnRNP). The second most abundant group (12 proteins) was proposed to be involved in splice site selection and the third most abundant group, containing 10 proteins, was classified as splicing factors. Nineteen proteins were subunits of the snRNPs or associated with the U1, U5, or U4/U6 snRNPs. In addition, helicases and peptidyl-prolyl cis-isomerases were detected. The relative standard deviation (RSD) was calculated from all ratios of the total list of identified proteins matched by more than one peptide and the average of this error was found to be 11% +/− 8. The proteome wide error was less than 20%, and in the case of the splicing factors the majority of RSDs was less than 20%.

Additional file 1: Table S1 show the relative expression levels (defined as TGF-β₁ stimulated/control) of the individual splicing related proteins calculated from four unique experiments from independent cell cultures and ICAT workflow runs.

Interestingly, TGF-β₁ altered the expression of splicing factors and additional proteins involved in splice site selection. The expression of splicing factor U2 small nuclear ribonucleoproteins auxiliary factor (65 kDa) (U2AF), and SRp20 were significantly increased by TGF-β₁ (1.09-fold and 1.24-fold, respectively) (Additional file 1: Table S1). Furthermore, splicing factor SRp30c was significantly repressed (0.79-fold) following TGF-β₁ stimulation. The relative expression of all proteins involved in splice site selection (grouped as splice site selection and splicing factors in Additional file 1: Table S1) was further statistically analyzed as shown in Figure 5. The data show that TGF-β₁ significantly repressed the expression of SRp30C labeled ‘2d’ compared to all other splicing factors except SRp 9 G8 (2b). SRp20 (2c) was significantly increased compared to SRp1 (2a) and SRp30c (2d). When comparing the levels of SRp20 with SRp30c, the actual difference between these two splicing factors was 1.6-fold. Several other proteins were significantly changed upon TGF-β₁ stimulation, such as RNA-binding region containing proteins, U5 snRNP 100 kDa protein, U5 small nuclear ribonucleoprotein 200 kDa, CGI-59 protein, and Cisplatin resistance-associated over expressed protein. In addition the expression of the helicase Nucleolar RNA helicase II (Nucleolar RNA helicase Gu) was also significantly different.

Phosphorylated SR proteins from TGF-β₁-stimulated HFL-1 cells were analyzed by western blots using an antibody as previously described [34, 35]. This antibody detects a conserved phospho-epitope on SR proteins, which does not coincide with absolute protein levels. We detected a complete array of SR proteins, including SRp75, SRp55, SRp40, SRp20, and an additional band that corresponds to proteins that co-migrate around 30 kDa (ASF/SF2, SC35, or SRp30c) [35] (Figure 6A and Table 1). TGF-β₁ induced an increase of all the detected SR proteins. A moderate increase was observed at 6 h but at 24 and 48 h the increase was more pronounced (Figure 6A and B). Moreover, TGF-β₁ triggered a 3.2-fold increase of the SRp75 at 24 h (P < 0.05) that was preserved at 48 h. SRp55 was 3.0-fold increased at 24 h (P < 0.05) and then slightly decreased at 48 h. SRp40 was continuously increased over time from a 3.5-fold elevation at 24 h (P < 0.05) to a 4.2-fold increase at 48 h. There was 3.4-fold increase of the band that was detected at 30 kDa at 24 h (P < 0.05) and the same increase was retained at 48 h. SRp20 was elevated 8.1-fold compared to untreated cells at 24 h (P < 0.05) which declined to 6.7-fold at 48 h (Figure 6B). To verify the TGF-β₁-induced increase of SRp20 we performed separate western blots using an antibody that recognizes the actual protein and does not discriminate between phosphorylated and non-phosphorylated variants (Figure 6C). With this antibody there was a 1.8-fold increase of SRp20 at 24 h (P < 0.05) and a 2.4-fold increase at 48 h (P < 0.05), which supports the results from Additional file 1: Table S1.

Next, we investigated how TGF-β₁ stimulation influenced the cellular localization of SR proteins and SRp20 by immunofluorescence. The staining pattern of the splicing factors, using the anti-phospho SR proteins antibody, was exclusively nuclear (Figure 7). The staining was more intense at 48 h but this could surprisingly not be observed at 24 h of TGF-β₁ stimulation compared to untreated cells. In addition the staining at 48 h was more speckled but not to the same extent at 24 h TGF-β₁ stimulation. The staining when using the SRp20 antibody was mainly nuclear in untreated cells and after 24 h of TGF-β₁ stimulation although some faint extra-nuclear staining also could be observed ( 8). However, after 48 h TGF-β₁ stimulation SRp20 was both located intra- and extra-nuclear and in addition the staining was more speckled and intense.

Human embryonic lung fibroblasts (HFL-1) (ATCC, Manassas, VA, USA) were sub-cultured in Eagles minimum essential medium (EMEM) supplemented with 1% glutamine and 10% fetal calf serum (FCS) and PEST. Before experiments cells were grown to confluency and were starved overnight in Dulbecco’s modified Eagle medium (DMEM) with 0.4% FCS. Cells were washed in PBS and were then incubated with or without TGF-β₁ (10 ng/mL) (R&D Systems Abingdon, UK) for the time-points indicated in the figures. Experiments were performed in passage 17–22.

Cells were grown under standardized conditions with or without TGF-β₁ (10 ng/mL) for 6, 24, or 48 h and whole cell lysates were prepared using lysis buffer (50 mM Tris–HCl, 500 mM NaCl, 1% NP-40, 10% glycerol, 10 mM MgCl2, pH 7.4) containing the protein inhibitor cocktail complete mini (1 mM PMSF, 1 μg/mL Aprotinin, 1 μg/mL Pepstatin, 1 μg/mL Leopeptin, Roche, Manheim, Germany). Samples were solubilized in Laemmli’s buffer and equal amounts of total protein (10 μg) were loaded and separated by electrophoresis on 4–12% Bis-Tris Gels (Invitrogen, Gibro, Carlsbad, CA, USA). The proteins were blotted to PVDF membranes (Immobilon-P Transfer Membrane, Millipore Corporation, Billerica, MA, USA). Membranes were incubated with antibodies against: a-SMA (Abcam, Cambridge, UK), prolyl 4-hydroxylase (Acris antibodies, Hiddenhausen, Germany), fibronectin (Dako, Glostrup, Denmark), EDA-fibronectin (Abcam, Cambridge, UK), GAPDH (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), Phospho-SR proteins (Zymed Laboratories, San Francisco, CA, USA), and SRp20 (Zymed Laboratories, San Francisco, CA, USA). Bound antibodies were visualized by peroxidase-conjugated secondary antibodies and enhanced luminescence (Amersham^TM western blotting Detection Reagents, GE Healthcare, Uppsala, Sweden) with exception for the blot against phospho-SR proteins where Dy-light 800 nm conjugated secondary antibodies (Cell Signaling Technology Inc., Boston, MA, USA) was used. The luminescence/fluorescence signal was detected on Odyssey® FC imaging system (LI-COR Biosciences, Lincoln, NE, USA). Exposure times were standardized so that all samples were treated the same way for each antibody. Individual bands were quantified with densitometry using the Quantity One software version 4.6.1 (BIORAD Laboratories, Hercules, CA, USA). Data are based on four individual sets of experiments.

Fibroblasts (7000/well) were grown overnight on chamber slides and were then incubated with or without TGF-β₁ 24 and 48 h. Cells were then fixed in 4% formaldehyde for 15 min and permeabilized with 0.1% Triton X for 30 min. After blocking in 2% BSA-TBS containing 5% goat serum (Vector laboratories, Burlingame, CA, USA) for 30 min, cells were incubated with primary antibodies against: a-SMA (Abcam, Cambridge, UK), prolyl 4-hydroxylase (Acris antibodies, Hiddenhausen, Germany), Phospho-SR proteins (Zymed Laboratories, San Francisco, CA, USA), and SRp20 (Zymed Laboratories, San Francisco, CA, USA) and with secondary antibodies: Alexafluor 555-conjugated goat anti-mouse antibody and Alexafluor 555-conjugated goat anti-rabbit antibody (both from Molecular Probes Invitrogen, Eugene, OR, USA). To stain nuclei, cells were incubated with DAPI (Molecular Probes Invitrogen, Eugene, OR, USA). Glasses were mounted with mounting media (Dako, Glostrup, Denmark) and photographed using a TE2000-E fluorescence microscope (Nikon, Tokyo, Japan) equipped with a DXM1200C camera (Nikon).

Cells were grown under standardized conditions with or without TGF-β₁ (10 ng/mL) for 6, 24, or 48 h. Total RNA was isolated from cells using RNeasy (Qiagen, GmBH, Hilden, Germany) according to the manufacturer´s instructions. The quantity of RNA was measured by spectrophotometry using a NanoDrop ND-100 (Nano Drop Technologies, Delaware, MD, USA). Total RNA (1 μg) was reverse-transcribed using superscript II according to the manufacturer’s manual (Invitrogen, Carlsbad, CA, USA) and stored at −70°C.

Five μL of cDNA (diluted 1:250) was mixed with 15 μL SYBR-green mixture (PE Biosystems, Foster City, CA, USA) and amplified by real-time RT-PCR using Stratagene MX3005P QPCR system (Agilent Technologies, Santa Clara, CA, USA). Initially the samples were held for 2 min at 50°C then for 10 min at 95°C; they were then cycled for 40 cycles of 30 s at 95°C, 1 min at 58°C, and 1 min at 72°C. Each sample was analyzed in triplicate. Reactions were performed using MX3000P 96-well plates (Agilent Technologies, Santa Clara, CA, USA). All primers were constructed using the online Primer 3 program (http://​frodo.​wi.​mit.​edu/​primer3/​) and they were ordered from A/S DNA Technology (Risskov, Denmark). The following primers were used. Fibronectin forward: CGA TCA CTG GCT TCC AAG TT, and reverse: TCC GAG CAT TGT CAT TCA AG. Fibronectin-EDA forward: AAT CCA AGC GGA GAG AGT CA, and reverse: CGT AAA GGG CTC AGC TCA AG. S18 forward: CGA ACG TCT GCC CTA TCA AC, and reverse: TGC CTT CCT TGG ATG TGG TA. All primers were tested for specificity by sequence alignment in the PubMed nucleic acid database (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi). Data are based on three individual sets of experiments.

The nuclei were purified according from a protocol previously described [55]. Briefly, the cells were harvested in 60 mM KCl, 15 mM NaCl, 0.15 mM Spermine, 0.5 mM Spermidine, 15 mM Hepes, and 14 mM Mercaptoethanol supplemented with 0.2% (v/v) Nonidet p40, aprotinin (1 μg/mL), leupeptin (1 μg/mL), pepstatin A (1 μg/mL), PMSF (10 μg/mL) and 0.3 M sucrose. The cell suspension was then homogenized with 60 strokes at 2,000 rpm (Labortechnik, Berlin, Germany) and the homogenate was filtered with 100 μm filter paper (Schleicher&Schuell, Dassel, Germany). The nuclei were counted in a Bürker chamber and the purity was assessed and protein content was determined by Bradford protein reagent kit (Pierce, Rockford, IL, USA).

Fibroblast nuclei were lysed in a buffer containing 6 M Guanidium-HCl (pH 8.5), 1% Triton X-100, and 50 mM Tris HCl, followed by sonication. Starting with 500 μg for control and TGF-β₁ stimulated, each sample was reduced by adding 10 μl of 50 mM TCEP (Tris(2-carboxyethyl)phosphine) and boiled for 10 min. The samples were allowed to cool and the acid cleavable ICAT^TM reagent (all the materials in this section are from the ICATTM reagent kit Applied Biosystems, Framingham, MA, USA) was added: the light reagent to the control and heavy reagent containing nine ¹³ C to the TGF-β₁-treated sample. Alkylation was allowed to complete for 2 h, at 37°C. The two samples are combined and acetone precipitated in order to remove the guanidium-HCl and the unreacted ICAT^TM reagent. The pellet was dissolved in a buffer consisting of 50 mM Tris (pH 8.5), 5 mM CaCl₂, and 10% acetonitrile. Trypsin was added at a 1:40 enzyme/substrate ratio in two additions once at the start of the digestion and once more, 2 h later. Digestion was completed at 37°C overnight.

Following digestion the sample was diluted into 25% acetonitrile, pH 3.0 in order to reduce the buffer concentration below 10 mM. The resulting peptide mixture was injected to a PolyLC (4.6 × 100 mm Polysulfoethyl A) SCX column on Vision workstation (Applied Biosystems, Framingham, MA, USA) at a flow rate of 1 mL/min using a binding buffer (buffer A) 10 mM KH₂PO₄, 25% acetonitrile, pH 3 and an elution buffer (buffer B) of 350 mM KCL, 10 mM KH₂PO₄, 25% acetonitrile, pH 3. The gradient was 0% B to 10% B in 2 min, 20% B in 15 min, 45% B in 3 min, 100% B in 10 min, and an additional 8 min hold at 100% B. Twenty-three SCX fractions of 1.5 mL volume were collected.

The pH was adjusted for each SCX fraction to 7.2 with 10× PBS buffer (pH 10) and injected to the avidin affinity column as prescribed by the manufacturer (Applied Biosystems, Framingham, MA, USA). Cysteine-containing peptides bound to the avidin column were washed three times: 1 mL of Wash 1, 1 mL of Wash 2, and 1 mL of de-ionized water. Elution of the cysteine-containing peptides was performed with 800 μL of avidin elution buffer. The samples were then concentrated by a speed-vac to dry, cleaved with the ICAT ^tm cleaving reagent containing 95% TFA and concentrated again to remove the cleaving reagent. The samples were then taken up in 2% acetonitrile, 0.1% TFA for re-injection for a nanoHPLC MS and MS/MS analysis.

Avidin purified SCX fractions were subjected to MALDI and ESI HPLC-MS and MS/MS analysis in a 50:50 split. For the MALDI based workflow, these fractions were injected in the HPLC loading buffer onto a 100 μm × 15 mm C₁₈ Magic column (Auburn, Michrom Bioresources, CA, USA) using a CapTrap pre-column (Auburn, Michrom Bioresources, CA, USA). Mobile phase A was 2% acetonitrile, 0.1% TFA, mobile phase B was 85% acetonitrile, 5% n-PrOH, 10% water, 0.1% TFA. HPLC elution was carried out at a 1-μL/min flow rate on an Ultimate nanoHPLC workstation (Dionex-LC Packings, Hercules, CA, USA). The HPLC elution from the column was collected at 20-s intervals on the MALDI plate using a Probot fraction collector (Dionex-LC Packings). Forty-eight-min HPLC elution was collected to the MALDI plates as an array of 12 × 12 sample spots. The HPLC eluent was mixed with the MALDI matrix (7.5 mg/mL α-cyano-4-hydroxycinnamic acid dissolved in 60:40 acetonitrile-water containing 0.15 mg/mL dibasic ammonium-citrate) through a mixing tee (Upchurch, WA, USA) at a flow rate of 2-μL/min. The most abundant, middle-fractions of the SCX separation were spotted onto two plates using a 90-min HPLC gradient.

MALDI plates were analyzed in automated mode on the AB4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). First the MS spectra were collected from the entire HPLC run. Then, using an in-house developed program, MS/MS precursors were selected by applying an exclusion algorithm to eliminate: (a) redundant precursors carrying over multiple HPLC fractions; and (b) using only the more abundant members of peptide pairs for MS/MS analysis. MS/MS spectra were acquired using up to 2,500 laser shots/precursor unless the predefined signal-to-noise level in the MS/MS acquisitions was achieved sooner. The MS/MS data were submitted for database searching as a batch to Mascot (http://​www.​matrixscience.​com) through its automation interface of Mascot (Mascot Daemon). The non-redundant NCBI protein database was used. A detailed description of the acceptance criteria for the database searching results will be described elsewhere. In brief, tryptic peptides containing arginine residues were accepted at a Mascot score >20, peptides not containing arginine at a Mascot score >25. Peptides with less-than-significant Mascot score were thoroughly inspected, considering the correlation of peptide basicity with SCX fraction numbers, presence of characteristic high-energy CID fragments, and accurate mass measurements through internal calibration of the MS spectra using the masses of confidently identified peptides as internal mass references.

ESI based LC-MS/MS analyses were carried out using an Ultimate nanoHPLC system (Dionex-LC Packings, CA, USA) on a 75 μM × 150 mm Picofrit C18 column at a 300-nl/min flow rate with a gradient of 5% B to 30% B over 60 min. Mobile phase A was 2% acetonitrile, 0.1% formic acid, mobile phase was 85% acetonitrile, 5% n-PrOH, 10% water, 0.1% formic. A CapTrap (Michrom Bioresources, CA, USA) precolumn was used to preconcentrate the sample.

A platinum electrode was placed behind the HPLC column using a microtee (Upchurch, WA, USA) in order to apply the electrospray voltage (3 kV) for LC-MS/MS analysis. LC-MS/MS was performed using a QSTAR quadrupole time-of-flight instrument (Applied Biosystems, CA, USA). The instrument was set up to perform a 1-s MS scan (300–1500 Da) followed by three most abundant components (two to four charges were allowed) exceeding an intensity threshold of 35 counts. To improve the protein coverage the samples were halved and the analysis was replicated. LC-MS and MS/MS analyses were processed by the ProICAT software (Applied Biosystems, Foster City, CA, USA). Database search results were accepted at >95% confidence interval. Database searching and quantification results from multiple MS platforms were consolidated by parsing all the qualitative and quantitative peptide results into an Oracle database. The set of all the identified peptides were assigned to a minimum set of proteins that could explain all the confidently identified peptide sequences. The abundance ratios of peptide pairs labeled with the heavy (9 × ¹³ C) and light (0 × ¹³ C) ICAT^TM reagents were normalized to the median of all peptide ratios in order to generate normalized expression ratios. Expression ratios of peptides matching to the same protein were averaged to generate expression ratios at the protein level.

Data are expressed as mean ± SEM. Student’s t-test was used to evaluate the differences of the means between groups. Differences were considered significant at P < 0.05. All analyses were performed using GraphPad Prism software version 4.00 (GraphPad Software, San Diego, CA, USA).