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Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1*

Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.

For almost 100 years heparin has been used as an anticoagulant. Specific heparin interactions with proteins important in the anti-clotting system are now well understood. Many heparin binding proteins, including quite a few involved in modulating vascular function, inflammation, and angiogenesis, have been identified (reviewed in Ref. 1). The large number of reports indicating evidence of decreased endogenous heparin and heparan sulfates (HS) 3 in atherosclerosis (in model animals and human disease) led to a proposal that decreases in endogenous heparins might be important in the development of atherosclerosis (2). More recent evidence in support of that hypothesis includes increased heparanase expression in atherosclerosis (reviewed in Ref. 3) and increased levels of glycocalyx heparan sulfate in regions of the vasculature where laminar flow decreases the likelihood of atherosclerosis development (4).
In addition to heparin, heparin binding proteins typically also bind HS chains on HS proteoglycans (HSPGs). Although the carbohydrate backbones of heparin and HS are identical, modifications and sulfation patterns vary. HS chains have fewer sulfate residues per disaccharide and a lower overall charge, but their widespread expression suggests that HS may provide many in vivo functions identified originally as heparin functions (reviewed in Ref. 5). Heparin binding to growth factors modulates their activity and appears to protect them from degradation, whereas HSPGs in the extracellular matrix serve as a reservoir of protected growth factors believed to facilitate wound repair after cellular damage (1,5). Interactions of matrix glycoproteins with cell HSPGs (syndecans and glypicans) in cell membranes contribute to appropriate cellular interactions with the matrix (6,7). Heparin interactions with any of these molecules might contribute to the overall responses to heparin treatment.
Heparin also binds specifically to both VSMCs and ECs, suggesting the presence of a receptor for heparin (8 -10). A number of studies have identified physiological changes in heparintreated ECs, including the production and secretion of proteins involved in coagulation (11,12) and changes in inflammatory responses (13)(14)(15)(16)(17). In fact, a study by Li et al. (17) provides evidence that heparin effects involve modulation of p38 activity. Numerous reports have identified VSMC responses to heparin. These responses include decreases in cell proliferation (18,19), ERK pathway activity (19 -21), and activation of specific transcription factors (21)(22)(23). Heparin binding results in increased levels of DUSP1 protein that are required for decreases in ERK activity (24). The heparin-induced increases in VSMC DUSP1 suggest that heparin-induced decreases in EC inflammatory responses might also involve DUSP1 expression. In support of this idea, DUSP1 induction by anti-inflammatory glucocorticoid hormones does increase DUSP1 expression (25,26), and low molecular weight heparin has been reported to decrease peroxide-induced JNK and p38 activity (27). Heparin uptake and many heparin functions likely depend on a heparin/HS receptor.
Monoclonal antibodies that block heparin binding to endothelial cells (HRmAbs) are able to mimic heparin responses in VSMCs (10,19,22,24), providing evidence that the protein to which they bind functions as a heparin receptor. The accompanying report identifies TMEM184A as the heparin-interacting protein to which the HRmAbs bind (28). Knockdown of TMEM184A in VSMCs eliminates heparin responses (28).
Here we report evidence that unfractionated heparin treatment of ECs results in decreased JNK and p38 activity and that HRmAbs mimic heparin effects on JNK and p38 activity. The heparin effects on JNK and p38 depend on increased DUSP1 expression. Heparin effects on TNF␣-induced stress fiber formation also depend on the induction of DUSP1. Furthermore, knockdown of TMEM184A blocks EC heparin responses and indicates that TMEM184A also serves as a receptor for heparin in ECs.
Cell Culture-Bovine aortic endothelial cells (BAOECs) and rat aortic endothelial cells (RAOECs), obtained from Cell Applications (San Diego, CA), were cultured using Cell Applications media according to their recommendations on 0.2% porcine gelatin and exchanged into minimum Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen or Atlanta Biologicals, Atlanta, GA), 5% L-glutamine, 1% sodium pyruvate, 1% minimum non-essential amino acids, and 1% penicillin/streptomycin antibiotics (Sigma). Human brain microvascular endothelial cells (HBMECs) were obtained from Cell Systems (Kirkland, WA) and cultured using Cell Systems complete medium according to their recommendations or minimum Eagle's medium after exchange into minimum Eagle's medium supplemented identically to the BAOEC culture. Typically, primary cells between passages 4 and 25 were used for these experiments. For microscopy, cells were cultured to between 70% and 90% confluence on glass coverslips coated with 0.2% porcine gelatin (Sigma) in 6-well culture dishes.
SDS-PAGE and Western Blotting-Cells were grown to the desired density on 100-mm tissue culture dishes and treated with stressing agents, heparin, and/or HRmAbs, as noted in the text. Cell samples were harvested directly into 2ϫ sample buffer (11,19) for SDS-PAGE. Primary antibody dilutions were 1:5000 except for ␣-tubulin (1:8000) and TMEM184A antibodies (1:1000). Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, or TRITC (diluted 1:5000) were incubated with the blots, and visualization of the bands was accomplished by fluorescence detection using the Bio-Rad ChemiDoc MP system (catalog no. 170-8280). Bands of interest were identified by comparison with lanes developed using only secondary antibodies and by molecular weight on the basis of migration of prestained Rainbow TM molecular weight markers (GE Healthcare LifeSciences). The blots are representative of at least three experiments.
Immunofluorescence Staining-BAOECs, RAOECs, and HBMECs were fixed with 4.0% paraformaldehyde (Pierce) for 20 min at room temperature with gentle shaking and permeabilized with 0.2% Triton-X-100 (Sigma) for 5 min at room temperature with gentle shaking. Coverslips were incubated with 1% BSA for 30 min at room temperature with gentle shaking. Coverslips were incubated with appropriate primary antibodies overnight at 4°C, washed with 1ϫ PBS, and incubated with appropriate secondary antibodies for 1 h at 37°C. Primary and secondary antibodies were used at the recommended optimized dilutions (1:200 or 1:100 for bovine anti-goat conjugated with Alexa Fluor 488, TRITC, and Alexa Fluor 594 or 1:50 for bovine anti-goat conjugated to Alexa Fluor 647). Phalloidin staining used a dilution 1:200. Coverslips were washed with 1ϫ PBS and mounted on Mowiol (Calbiochem, Billerica, MA) to minimize photobleaching. Secondary antibody-only controls were performed to rule out nonspecific staining (data not shown).
Confocal Microscopy-Fluorescent labels were visualized using a Zeiss laser-scanning microscope (510 Meta confocal microscope with a ϫ63 oil immersion lens) at room temperature. Stress fiber images in TMEM184A knockdown cells were taken with a Nikon C2ϩ confocal microscope (ϫ60 oil immer-sion lens). The gain intensity and amplifier settings for each channel were set at a level below saturation and appropriate to provide a dynamic range of intensity for pp38, pJNK, pcJun, pMK2, pHSP27, and DUSP1 using control slides from initial experiments. The gain intensity and amplifier settings for Alexa Fluor 488-phalloidin were set by scanning to approximately the same Z plane in control cells where both the intensity and resolution of stress fibers were optimal. All settings were saved and used to image each sample slide within an experiment and for slides in all subsequent repeats of experiments. Z stacks were taken for all slides. All images of DUSP1 and actin stress fibers represent approximately the same Z plane of each cell.
DUSP1 Knockdown-BAOECs were cultured to confluence in 100-mm plates. Cells were trypsinized, washed with 1ϫ PBS, washed with HEPES-buffered saline, and resuspended in HEPES-buffered saline. Cells were electroporated with 20 g/ml of scrambled control or bovine-specific DUSP1 siRNA (Santa Cruz Biotechnology) in the Bio-Rad Gene Pulser X-Cell system using a modified, preset HeLa protocol (170 V for 7.0 ms). Cells were seeded onto 0.2% porcine gelatin-coated glass coverslips in a 6-well culture dish with Cell Systems BAOEC medium for immunofluorescence staining and quantitative microscopy of pJNK, pp38, DUSP1, and actin stress fibers at 12-24 h post-electroporation.
TMEM184A Knockdown-RAOECs were prepared for knockdown as above for DUSP1 siRNA treatment. Cells were transfected using 20 g/ml of shRNA designed for rat TMEM184A in the pGFP-V-RS plasmid or control shRNA in the same plasmid (Origene, Rockville, MD) using the Bio-Rad Gene Pulser X-Cell system. Cells were seeded and processed as above for the DUSP1 knockdown system. Experimental treatments were accomplished 72 h after electroporation. Imaging of nuclear staining in TMEM184A knockdown cells was accomplished using a Nikon Eclipse TE 2000-U fluorescence microscope. Stress fibers in TMEM184A knockdown cells were visualized as above.
Quantitative Immunofluorescence Image Analysis-To quantify pJNK, pp38, pcJun, and DUSP1, National Institutes of Health ImageJ software was used to determine the mean nuclear fluorescence intensity of at least 75 cells/treatment from each of at least three independent experiments. The nucleus of each cell was outlined accurately with the Oval tool by using the differential interference contrast images taken for each slide on the laser-scanning confocal or Nikon Eclipse microscope, and the nuclear fluorescence intensity for each cell was recorded. The nuclear fluorescence intensity measurements were corrected against minimal background fluorescence for each slide, and untreated controls were set to 100 to standardize between experiments. The averaged nuclear fluorescence intensity for each treatment was compared with untreated samples. To quantitate pMK2 and pHSP27, ImageJ software was used to determine the mean whole-cell fluorescence intensity of at least 75 cells/treatment/experiment and at least three independent experiments. The outline of each cell was determined and outlined with the Freehand Selections tool by referencing the differential interference contrast images, and the whole-cell fluorescence intensity for each cell was recorded. All measurements were corrected against minimal background fluorescence for each slide. The whole-cell fluorescence intensity was averaged for each treatment, and untreated control values were set to 100.
ImageJ software was used to quantitate stress fibers in at least 100 cells/treatment/experiment from at least three independent experiments. The Threshold tool was used to eliminate background fluorescence. Perpendicular line profiling through the cytoplasm of individual cells was used to generate average fluorescence intensity profiles at regular intervals along the freehand line selection, where the y axis represented the level of fluorescence and the x axis represented the distance across each cell. Sharp, distinct peaks above the threshold were counted for each profile and averaged. Cortical actin peaks were excluded from the counts because their dynamics responded differently than the fibers that spanned the cells.
Statistical Analysis-All experiments are representative of at least three independent trials. Graphical data are shown as mean Ϯ S.E. Comparisons between groups were analyzed using analysis of variance followed by a Tukey post hoc test. Comparisons between two groups were analyzed using a Student's t test. Differences with p Ͻ 0.05 were considered significant ( †, p Ͻ 0.05; *, p Ͻ 0.01; ** p Ͻ 0.001).

Results
Heparin Decreases TNF␣-induced p38 Activity-To test the hypothesis that unfractionated heparin alters p38 activation in ECs, whole-cell p38 activity was first evaluated in TNF␣treated BAOECs with or without heparin pretreatment. TNF␣ treatment (25 ng/ml) resulted in increased p38 activation, measured as pp38 by Western blotting. In cells pretreated with heparin, the level of pp38 was consistently lower than in TNF␣treated cells without heparin (Fig. 1A). There was little decrease in pp38 at low heparin concentrations. In fact, as shown in Fig.  1A, it sometimes appeared that, at low concentrations of heparin, there was a slight increase in whole-cell pp38. However, by 50 g/ml heparin, it always appeared that there was less pp38 than in cells treated with TNF␣ alone. Neither heparin nor TNF␣ affected the levels of total p38. Activated p38 typically moves to the nucleus, where it phosphorylates numerous substrates (reviewed in Ref. 29). In the nucleus, pp38 can be inactivated by DUSP1 (reviewed in Ref. 30). Therefore, it was important to understand whether there were heparin-induced changes in nuclear pp38. Quantitative analysis of nuclear pp38 using immunofluorescence was determined for a total of more than 200 cells at each time point from at least three experiments per condition. TNF␣ alone resulted in increased active p38 in the nucleus. Heparin treatment for 20 min without TNF␣ caused no change in pp38 levels (Fig. 1B). Heparin pretreatment decreased the TNF␣-induced nuclear pp38 levels over at least 2 h (Fig. 1B). Longer incubations with heparin also resulted in no change in active p38. There were no changes in nuclear total p38 (data not shown). To be certain that the levels of active p38 in human ECs were also sensitive to heparin treatment, we evaluated nuclear pp38 in HBMECs (Fig. 1C). As with the BAOECs, TNF␣ treatment resulted in significant activation of p38, and heparin pretreatment caused a significant decrease in pp38 relative to TNF␣ treatment alone. To investigate the idea that heparin effects on ECs were mediated through a hep-arin receptor similar to heparin effects in VSMCs, HRmAbs effects on TNF␣-induced responses in ECs were investigated. HRmAbs were able to mimic heparin in BAOECs, resulting in decreased pp38 compared with TNF␣ treatment without heparin or HRmAbs (Fig. 1D). Together, these data indicated that heparin treatment caused a decrease of pp38 in the nucleus and that this response depended on the heparin receptor to which the HRmAbs bind.
Heparin Decreases TNF␣-induced JNK Activation-To evaluate whether the decreases in activated p38 would also be observed with JNK, BAOECs were treated as for the p38 studies above and analyzed for JNK activity. Western blotting analysis indicated that BAOECs pretreated with a range of heparin concentrations up to 200 g/ml heparin before TNF␣ showed a decrease in pJNK at higher heparin concentrations compared with cells treated with TNF␣ alone (Fig. 2A). As with heparin effects on pp38, the pJNK response was concentration-dependent. Also, as observed in Western blotting analyses for total pp38, total cell pJNK appeared to be slightly higher at low heparin concentrations, as shown in Fig. 2A. Neither heparin nor TNF␣ treatment caused a change in total JNK levels. As expected from the literature (e.g. Ref. 31), TNF␣ treatment of FIGURE 1. Active p38 is decreased in heparin-treated endothelial cells. BAOECs were treated with 25 ng/ml TNF␣ for varying times. Identical samples were pretreated with 1-200 g/ml heparin 20 min before TNF␣ addition. A, cells treated with varying heparin concentrations were harvested into sample buffer and analyzed by Western blotting. Images are representative of more than three experiments. B, to evaluate pp38 in the nuclei, BAOECs were cultured on coverslips, treated as cells in A, and processed for immunofluorescence as described under "Experimental Procedures." Representative images for one time point are shown. Mean nuclear staining of more than 200 cells/point (from three separate experiments) is shown, with error bars as mean Ϯ S.E. C, HBMECs were treated and analyzed, as were BAOECs. Differences were significant at all times after TNF␣ addition. **, p Ͻ 0.001. D, BAOECs were treated as for A, but some samples contained HRmAb 12B1 at 1.0 g/ml or 200 g/ml heparin. The graph is an analysis of four identical experiments. **, p Ͻ 0.001.
BAOECs significantly increased nuclear pJNK (Fig. 2B). Compared with TNF␣ only, nuclear pJNK was decreased significantly in BAOECs treated with heparin prior to TNF␣. As with pp38, heparin alone caused no change in pJNK (Fig. 2B) at 20 min (or longer, data not shown). We again used HBMECs to confirm that heparin sensitivity was applicable to both human ECs as well as microvessel ECs (Fig. 2C) and found that TNF␣induced pJNK levels were decreased significantly by pretreat-ment with heparin. Pretreatment with HRmAbs also decreased pJNK, comparable with the effect of heparin (Fig. 2D). The results indicate that heparin pretreatment resulted in decreased active JNK in the nuclei and that these effects likely occurred through heparin interactions with its receptor.
Heparin Decreases Activation of Downstream p38 and JNK Substrates-To investigate whether heparin-induced decreases in JNK and p38 activation also decreased downstream signal- ing, the activation status of several JNK and p38 substrates was examined using Western blotting and immunofluorescence microscopy (Fig. 3). Heparin effects on the JNK transcription factor target, cJun, were analyzed by Western blotting (data not shown) and immunofluorescence. Fig. 3A illustrates typical pcJun from at least three independent experiments. As expected, TNF␣ increased nuclear pcJun after 30 min (Fig. 3A). Heparin pretreatment significantly decreased nuclear pcJun, consistent with the heparin-induced decrease in pJNK. Similar results were observed in HBMECs (data not shown). Substrates for p38 include transcription factors and other kinase enzymes, including MK2. Activated MK2 has been shown to be involved in signaling responses to growth factors and inflammation (32). MK2 activity plays an important role in EC actin remodeling (e.g. Ref. 33), making it an important p38 target to evaluate. Therefore, MK2 was tested using quantitative immunofluorescence (Fig. 3B). pMK2 increased rapidly in response to TNF␣ treatment. In cells pretreated with heparin, there were significant decreases in MK2 phosphorylation compared with cells treated with TNF␣ only (Fig. 3B). HSP27, a downstream target of MK2 involved in modulating actin remodeling in ECs (34) and other cell types, was similarly responsive to heparin. Fig. 3C illustrates significant heparin-induced decreases in whole-cell pHSP27 relative to cells treated with TNF␣ alone. These results indicate that both transcription factors and other downstream targets are phosphorylated at lower levels in heparin-treated cells.
DUSP1 Is Critical for Heparin-induced Decreases in JNK and p38 Activity-In VSMCs, heparin has been shown to induce concentration-and time-dependent expression of DUSP1, a dual-specificity phosphatase that localizes to the nucleus, where it regulates MAPK signaling (24). To test the hypothesis that increased DUSP1 is required for heparin-induced decreases in JNK and p38 activation, we first evaluated whether increased DUSP1 is observed in heparin-treated ECs (Fig. 4). BAOECs were treated with heparin, and cells were harvested for Western blotting analysis to determine the relative levels of DUSP1 (Fig. 4A). DUSP1 levels increased by 10 min, and maximal DUSP1 expression was achieved by 30 min in cells treated with 200 g/ml heparin. Quantitative immunofluorescence showed that HBMECs (Fig. 4B) also exhibited a rapid increase in DUSP1.
We then questioned whether the observed decreases in JNK and p38 activation required this heparin-induced DUSP1. BAOECs were electroporated with DUSP1 siRNA as described under "Experimental Procedures," treated, and evaluated using quantitative immunofluorescence assays. Treatment with species-specific DUSP1 siRNA consistently resulted in a significant (p Ͻ 0.05) decrease in nuclear DUSP1 (e.g. Fig. 4C). Wholecell knockdown averaged 54% of control siRNA-treated cells, whereas nuclear DUSP1 levels decreased to 65% of control siRNA-treated cells. Calculated levels of cytoplasmic knockdown indicate that DUSP1 siRNA-treated cells had 45% as much DUSP1 in their cytoplasm as controls, consistent with the fact that this nuclear enzyme is likely in the cytoplasm primarily shortly after synthesis. Knockdown cells treated with heparin did not significantly increase DUSP1 expression (Fig. 4D). TNF␣ treatments activated JNK and p38 in both control and DUSP1 siRNA-treated BAOECs (Fig. 4, E and F). Interestingly, knockdown of DUSP1 resulted in higher levels of nuclear pJNK without any other treatment (Fig. 4E). As expected, heparin pretreatment in control siRNA-treated cells resulted in significantly decreased TNF␣-induced JNK and p38 activity. However, in DUSP1 knockdown cells pretreated with heparin, this heparin-induced reduction in JNK and p38 activation was not observed (Fig. 4, E and F). Therefore, heparin effects on JNK and p38 activation require the induction of DUSP1 in a manner similar to VSMCs (24).
Heparin Decreases TNF␣-induced Stress Fiber Formation and Organization-Activated JNK and p38 have been shown to play roles in actin remodeling and subsequent EC morphology changes (reviewed in Ref. 35). JNK activation is linked with newly organized cortical actin and EC adaptation, whereas p38 appears to be associated with stress fibers and actin events at focal adhesions (35). After observing significant heparin-induced decreases in JNK and p38 activation, it became important to investigate whether these heparin-induced JNK and p38 activity decreases could reduce TNF␣-induced stress fiber formation. BAOECs treated with TNF␣ for at least 30 min exhibited increased stress fibers (e.g. compare Fig. 5, B, c-f, with A). In cells pretreated with heparin (Fig. 5B, g-j), there was an overall decrease in the number of stress fibers per cell. There was individual variability in stress fiber formation between cells, as suggested by the two control actin images (Fig. 5A, a and b). Therefore, stress fibers were counted using ImageJ software as described under "Experimental Procedures" (illustrated in Fig.  5C). Peripheral cortical actin was not included in the line profiling because changes in cortical actin do not correspond to changes in stress fibers traversing the whole cell (36,37). An example of the line profile is shown in Fig. 5C. As illustrated in Fig. 5A and quantified in Fig. 5D (time 0), few stress fibers were found in untreated BAOECs stained with Alexa Fluor 488-phalloidin.
The average number of fluorescence peaks was increased significantly with 30 -120 min of TNF␣ treatment in both BAOECs (Fig. 5, B and D) and HBMECs (Fig. 5E). However, in cells treated with heparin before TNF␣, the average number of fluorescence peaks was significantly lower than in cells treated with TNF␣ alone. In BAOECs, this heparin effect was observed as early as 30 min of TNF␣ treatment and persisted through 120 min (Fig. 5D). In HBMECs, heparin effects on stress fiber levels were also evident by 30 min of TNF␣ treatment and were maintained through 120 min of TNF␣ treatment (Fig. 5E). In cells pretreated with HRmAbs in place of heparin (Fig. 5F), there was also a decrease in the number of stress fibers compared with cells treated with TNF␣ alone. In these experiments, the measured increase in stress fibers induced by TNF␣ was not as great as in the other experiments, possibly because of differences in the batch of TNF␣ or the visualization system (rhodamine-phalloidin).
To test whether the effects of heparin on stress fiber formation were also dependent on DUSP1, stress fibers were quanti- fied in BAOECs transfected with either control or DUSP1 siRNA (Fig. 5G). A 30-min TNF␣ treatment increased stress fibers in both control and DUSP1 siRNA-treated cells. In control siRNA cells, heparin pretreatment resulted in a significant decrease in stress fibers. In cells with DUSP1 knocked down, heparin pretreatment did not decrease stress fibers; rather, an increase was observed. These data indicate that heparin treatment decreases stress fiber responses to TNF␣, which depends on heparin-induced DUSP1 increases that lead to decreased JNK and p38 activity.
Knockdown of TMEM184A Interferes with Heparin Responses-We have recently identified TMEM184A to be a heparin-responsive transmembrane protein required for heparin effects in VSMCs (28). The identification of TMEM184A also involved the use of HRmAbs that mimic effects of heparin in ECs (Figs. 1, 2, and 5). Knockdown of TMEM184A in rat Note that neither cortical actin at two edges nor peaks below a threshold are counted (refer to "Experimental Procedures"). D and E, more than 100 BAOECs (D) or HBMECs (E) for each condition were analyzed for the number of peaks above basal intensity. Error bars represent the mean number of peaks with S.E. *, p Ͻ 0.01. F, BAOECs in k and l were treated as in A and B. Cells in m and n were treated with 1 g/ml HRmAbs H6 or B1 instead of heparin. Cells in k-n were stained with rhodamine-phalloidin. The images are representative of three independent experiments and were analyzed as in D and E. †, p Ͻ 0.05. G, BAOECs were treated with DUSP1 siRNA (light columns) or control siRNA (dark columns) before 30 min of TNF␣ and heparin, and stress fibers were analyzed. **, p Ͻ 0.001. Scale bars ϭ 20 m.
VSMCs eliminated heparin responses (28). To determine whether this heparin-responsive protein identified with our HRmAbs and confirmed in VSMC is also responsible for the heparin effects in ECs reported here, we first confirmed that TMEM184A is present in RAOECs across the range of cell density we studied (Fig. 6A). We then studied RAOEC responses in knockdown compared with control shRNA-treated cells. Knockdown of TMEM184A was typically around 50% on the basis of staining of cells from each knockdown experiment and quantification of the TMEM184A staining. Representative images and data from more than 200 cells/condition are shown in Fig. 6B. Both cell surface TMEM184A and total cell TMEM184A are decreased to a similar extent. We employed an immunofluorescence assay for nuclear JNK and p38 (Figs. 1 and 2) to characterize TMEM184A knockdown effects on heparininduced JNK and p38 activity decreases. Heparin treatment of TMEM184A knockdown cells did not decrease nuclear pp38 levels (Fig. 6C), although control shRNA-transfected cells responded to heparin normally, with a significant decrease at 15 min (Fig. 6C). At 30 min, the level of TNF␣-induced nuclear pp38 was already much lower than at 15 min, and the heparininduced decrease in control cells was not significant. Again, there was no heparin-induced change in knockdown cells. Heparin-induced decreases in nuclear pJNK levels were significant at both 15 and 30 min in TNF␣-activated RAOECs. Knockdown cell nuclear pJNK remained high at both time points despite heparin treatment (Fig. 6D). Knockdown of TMEM184A also blocked the ability of heparin treatment to induce DUSP1 expression (Fig. 6E), consistent with the requirement of heparin-induced DUSP1 expression for resultant decreases in JNK and p38 phosphorylation. Stress fiber formation was significant after 60 min of TNF␣ treatment in control shRNA-treated cells and TMEM184A knockdown cells. In cells pretreated with heparin, control cells exhibited significantly fewer stress fibers. However, there was no stress fiber decrease in heparin-treated knockdown cells (Fig. 6F). The data from the TMEM184A knockdown experiments support the hypothesis that TMEM184A is required for heparin responses in ECs and VSMCs and indicate its involvement in anti-inflammatory EC responses to heparin.

Discussion
Inflammatory mediators such as TNF␣ and other cytokines have been implicated in the chronic pathogenesis of vascular disease. They induce EC p38 and JNK activation, increase immune cell adhesion molecule expression, promote VSMC migration, and cause EC barrier dysfunction, all of which further accelerate local inflammation (38,39). A number of studies have determined that heparin exhibits anti-inflammatory properties and modulates angiogenesis (1). Low molecular weight heparin has been shown to reduce JNK and p38 activation, downstream transcription factor activation, and induction of cell adhesion molecule expression (27). Here we present evidence that unfractionated heparin decreases nuclear JNK and p38 activity and also alters TNF␣ effects on actin remodeling that depend on JNK and p38 activity. Interestingly, at low heparin levels, there appears to be a slight increase in JNK and p38 activation. Because heparin-induced inactivation of these enzymes depends on DUSP1 synthesis and movement of the pJNK and pp38 to the nucleus for dephosphorylation, differences in movement to the nucleus or other cytoplasmic signaling initiated in response to heparin might explain these results and are interesting possibilities to investigate further. The heparin effects in ECs are mimicked by HRmAbs that bind to a heparin receptor. The observations that pretreatment with antibodies to a putative heparin receptor decreases TNF␣-induced JNK and p38 activation and decreases stress fiber formation are consistent with previous reports showing that these antibodies mimic the effects of heparin in VSMCs (19,22,24). Here we provide functional evidence that the anti-inflammatory effects of heparin in ECs are mediated through the heparin receptor TMEM184A, as seen in VSMCs (28). TMEM184A knockdown interferes with heparin responses, confirming our HRmAb evidence. Our data strongly suggest that the TNF␣ and heparin pathways, when activated simultaneously in ECs, engage in cross-talk dependent on the heparin receptor TMEM184A. A schematic for this cross-talk between the TNF␣ and heparin signals is shown in Fig. 7.
EC dysfunction in response to inflammation results in the formation of pericellular gaps, altered interactions between ECs and matrix proteins, decreased calcium-dependent signaling at EC barriers, and changes in mechanosensitivity (39 -41). Reorganization of F-actin into stress fibers through the MAPK signaling cascades in response to TNF␣ and low or disturbed fluid shear stress has also been shown to increase EC permeability (39,41,42). JNK associates with stress fibers in ECs and plays a role in actin remodeling (43,44). Regions of the vasculature with disturbed flow have higher levels of active JNK (45,46). Similarly, p38 plays a role in actin remodeling (41,47,48) and facilitates actin remodeling in response to changes in shear stress (49). This likely requires the p38 substrate MK2 and phosphorylation of its substrate HSP27 (34,47). Our results indicating heparin-induced decreases in pMK2 and pHSP27 are consistent with the evidence that heparin-treated ECs show lower levels of stress fibers when stressed with TNF␣.
Natural inhibitors of inflammation and stress signaling include glucocorticoid hormones, which can decrease signaling through stress kinase pathways by inducing the synthesis of DUSP1 (25,26). JNK and p38, as targets of DUSP1, have been well characterized in vascular cell types, and our data agree with previous reports showing that, after p38 and JNK activation, DUSP1 is effective in decreasing activation and protecting cells from actin stress fiber formation, which often leads to EC dysfunction (39,48,50). In this regard, our finding that heparin treatment of ECs after DUSP1 knockdown resulted in increased TNF␣-induced pJNK and stress fibers indicates that DUSP1 may be critical for routine modulation of stress fiber levels and their remodeling in ECs. ECs express higher levels of DUSP1 in atheroprotective regions of the vasculature, resulting in the lower levels of active JNK and p38 found in those regions (50). The lower levels of DUSP1 in the areas of disturbed flow therefore contribute to increased inflammation in these regions. Although heparin is convenient for cell studies, HSPGs likely interact with most heparin binding proteins in vivo (5). It is interesting that EC HSPGs are involved in EC remodeling in response to shear stress (51). In fact, knocking down synde- FIGURE 6. TMEM184A knockdown blocks heparin responses. A, RAOECs (not transfected) were evaluated for TMEM184A staining (NTD antibody) over a range (a-d) of cell densities. Scale bars ϭ 10 m. RAOECs were transfected with control or TMEM184A shRNA constructs and seeded on coverslips as described under "Experimental Procedures." TMEM knockdown was confirmed by staining some cells from each experiment for TMEM184A using the NTD antibody (e.g. B, images from permeabilized cells). Results from three experiments with more than 200 cells for each condition are shown in the graph. After 72 h, cells were treated with TNF␣ (25 ng/ml) with or without a 20 min pretreatment of 200 g/ml heparin, fixed, and stained for pp38 (C) or pJNK (D). Nuclear levels of the activated enzymes were determined. E, TMEM184A or control shRNA-transfected cells were treated with 200 g/ml heparin for up to 30 min, fixed, and stained for DUSP1, and then nuclear levels of DUSP1 were determined. F, TMEM184A or control shRNA-transfected cells were treated for 60 min with TNF␣ (25 ng/ml) with or without a 20 min pretreatment of 200 g/ml heparin and stained with Alexa Fluor 488-phalloidin. Fluorescence peaks were determined as in Fig. 5. All graphical results are from at least three repeat experiments per condition with at least 50 cells analyzed for each experiment. †, p Ͻ 0.05.
We have recently identified TMEM184A colocalization with caveolin-1 in vascular cells (28). Caveolae organize numerous players in signaling pathways (54). The role of caveolae in endothelial nitrous oxide synthase activation, which results in increased NO production and resultant increases in cGMP (55), and the colocalization of the heparin receptor with caveolin-1 (28) suggest that these players could be linked in modulating healthy endothelial function. The HSPG syndecan-1 requires caveolae for uptake (56), indicating that it might interact with TMEM184A in caveolae. TNF␣ has been shown to inhibit endothelial nitrous oxide synthase activity and, as a result, decrease NO production, which would lead to lower levels of active cGMP-dependent protein kinase. This causes ECs to become vasoconstrictive and, eventually, dysfunctional (38,57). Recent studies have shown that the effects of heparin in VSMCs involve cGMP-dependent protein kinase (22). We hypothesize that the effects of heparin in ECs might also be dependent on cGMP-dependent protein kinase and that the heparin-induced increased cGMP prior to exposure to TNF␣ could prevent the effects resulting from changes in NO production. Elucidating the signaling dynamics between these pathways will be important to understand the protective effects of heparin on the endothelium.
In addition, the significant effects of HSPGs and heparin/HS binding proteins in angiogenesis (1) suggest that the heparin receptor might be a player in that complex process. Endogenous heparin and HS are crucial for vascular health. Decreased glycocalyx HSPGs and increased heparanase, which degrades endogenous HS chains in atherosclerosis (3,4), indicate critical roles for heparin and HS in decreasing atherosclerosis. We provide evidence for cross-talk between heparin through the TMEM184A heparin receptor and TNF␣ through inflammatory signaling. Our results demonstrate that heparin-induced DUSP1 expression in multiple EC types is similar to what has been reported previously in VSMCs and that this induction is necessary to decrease TNF␣-induced JNK and p38 activation to prevent significant stress fiber formation. In vivo, HSPGs are likely natural ligands for TMEM184A, a possibility that requires further study. Additional investigation of the anti-inflammatory potential of heparin in vascular disease is important for improved exploitation of the effects of heparin on vascular cell physiology in a clinical setting.


SOX 7 promotes the maintenance and proliferation of B cell precursor acute lymphoblastic cells

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent type of cancer in children. Despite progresses in curative treatment, intensive chemotherapy regimens still cause life threatening complications. A better understanding of the molecular mechanisms underlying the emergence and maintenance of BCP-ALL is fundamental for the development of novel therapies. Here, we establish that SOX7 is frequently and specifically expressed in BCP-ALL and that the expression of this transcription factor does not correlate with any specific cytogenetic abnormalities. Using human leukemia model systems, we establish that the down-regulation of SOX7 in BCP-ALL causes a significant decrease in proliferation and clonogenicity in vitro that correlates with a delay in leukemia initiation and burden in vivo. Overall, these results identify a novel and important functional role for the transcription factor SOX7 in promoting the maintenance of BCP-ALL.

INTRODUCTION
Acute lymphoblastic leukemia (ALL) is the most frequent type of cancer in children and comprises neoplastic precursor cells committed to the B cell (BCP-ALL) or the T cell (T-ALL) lineages. BCP-ALL represents the majority of ALL, accounting for up to 85% of childhood ALL and 75% of adult ALL [1]. Genes involved in B cell development such as EBF1, IKZF1, PAX5 or PBX1 are frequently found mutated in BCP-ALL [2,3]. Additionally, cytogenetic abnormalities are frequently detected and often define specific subtypes of the disease with unique prognostic features. While childhood BCP-ALL is curable in most cases with a survival rate approaching 80%, the rate of cure for adult patients presenting with BCP-ALL is only 40% [4,5]. Despite progresses in the treatment of BCP-ALL, intensive chemotherapy regimens cause life threatening complication and around 20% of the treated patients relapse presenting then a very poor outcome [6]. There is a great need for the development of less toxic compounds and novel therapies for the treatment of relapsed and specific subgroups of BCP-ALL patients with poor prognosis.
The SOX family of transcription factor comprises 20 members divided into subgroups based on sequence similarities [7]. SOX factors are essential for the maintenance of various stem cell compartments during embryogenesis and adult life, such as for example SOX2 in neuronal stem progenitor cells [8] or SOX17 in fetal hematopoietic stem cells [9]. In addition, evidences for the involvement of SOX factors in cancers, either as tumour suppressors or as proto-oncogenes, have emerged recently. Deregulation of SOX gene expressions have been widely documented in cancer [10], and a few studies have identified a direct involvement of SOX factors in tumorigenesis [11][12][13][14]. SOX7 and its close homologues, Research Paper www.impactjournals.com/oncotarget SOX17 and SOX18, belong to the SOX F subgroup and play important roles in cardiovascular development [15][16][17]. The SOX F factors are also involved in the development of the hematopoietic system with SOX17 driving the expansion of fetal hematopoietic stem cells [9] while SOX7 and SOX18 are expressed at the earliest stages of blood development [18,19]. The expression of SOX7 is frequently down-regulated in solid tumours such as prostate, colon and endometrial cancers, but the functional relevance of these findings remain unknown [20,21]. A recent study has also revealed a tumor suppressor role for SOX7 in acute myeloid leukemia (AML) [22]. In the present study, we establish that SOX7 is frequently expressed in BCP-ALL and that the expression of this transcription factor is critical for BCP-ALL proliferation and maintenance.

SOX7 is frequently over-expressed in human BCP-ALL
To determine whether SOX7 expression was associated with hematological malignancies, we interrogated the Oncomine database for differential SOX7 expression in leukemias. This analysis revealed that SOX7 expression levels were frequently and significantly higher in childhood and adult BCP-ALL when compared to normal bone marrow and other types of leukemia, including T-ALL, acute myeloid leukemia (AML), chronic leukemias and myelodysplastic syndrome (MDS) ( Figure 1A and S1A-S1B). In contrast, the expression of SOX17 and SOX18 did not show any specific or restricted pattern of expression in any hematological malignancies ( Figure 1A). To validate this observation, the expression of SOX7 and SOX18 was evaluated in a small cohort of human ALL samples ( Figure 1B-1C). These analyses confirmed that whilst SOX7 expression was high in multiple cases of BCP-ALL, SOX18 expression remained undetectable. Overall, SOX7 expression did not correlate with any specific type of chromosomal translocation or mutation known to be involved in BCP-ALL as confirmed by further analysis of the Oncomine datasets (Supplementary Figure  S1C-S1D). Altogether, these results establish that the transcription factor SOX7 is specifically and frequently expressed in human BCP-ALL, without an obvious association with specific chromosomal abnormalities or cytogenetic subtypes.

SOX7 knock-down impairs the proliferation and clonogenicity of BCP-ALL cells
To investigate how SOX7 expression might contribute to the leukemic process, we first analyzed its expression in multiple human BCP-ALL cell lines with different cytogenetic characteristics (Figure 2A). The six lines evaluated expressed varying level of SOX7 transcript ( Figure 2B) and protein ( Figure 2C); MV4;11 an AML line used as a negative control did not expressed SOX7. To modulate SOX7 expression, we tested several shRNAs against SOX7 transcripts ( Figure  2D), all of which induced greater than 95% decrease in SOX7 transcript levels in the endothelial HUVEC cells expressing high level of the SOX7 transcripts ( Figure  2E). However, in BCP-ALL cells, these shRNAs induced much lower levels of knock-down. To evaluate the role of SOX7 in BCP-ALL, we therefore used two shRNAs conferring around 50% decrease in SOX7 transcript levels in RS4;11 and NALM6 expressing intermediate and high SOX7 level respectively ( Figure 3A-3B). The two SOX7 shRNAs tested induced a significant decrease in proliferation in both leukemic cell lines when compared to the control shRNA ( Figure 3C-3D). The cotransduction of two different SOX7 shRNAs in RS4;11 cells did not further decrease SOX7 transcript levels or proliferation when compared to cells transduced with a single shRNA (Supplementary Figure S2A-S2B). Cell cycle status via EdU incorporation revealed a reduction in the frequency of cells in the S-phase of the cell cycle for both leukemic cell lines transduced with either shRNAs ( Figure 3E). Apoptosis assay showed that BCP-ALL cells were viable without significant increased apoptosis upon SOX7 knock-down ( Figure 3F, positive control of staining Supplementary Figure S3). Next, to test whether SOX7 down-regulation impacted the clonogenic capacity of the BCP-ALL cells, we cultured the transduced cells in semisolid colony assay, which represents a more stringent test of clonal proliferative capacity. The down-regulation of SOX7 by either shRNAs resulted in a significant decrease in colony formation in both leukemia lines ( Figure 4A-4B), with much smaller colonies formed upon SOX7 knock-down ( Figure 4C-4D). Altogether, these data demonstrate that the down-regulation of SOX7 in BCP-ALL human cell lines induced a significant decrease in proliferation, even though SOX7 transcript levels were only decreased by 50%.

SOX7 knock-down delays leukemogenesis in vivo
To investigate the role of SOX7 in the progression of leukemia in vivo, recipient mice were engrafted either with RS4;11 leukemia cells transduced with a SOX7 or control shRNA or with NALM6 leukemia cells transduced with an inducible lentiviral vector encoding a SOX7 shRNA. An inducible system was tested to determine if acute deletion of SOX7 transcripts might be more efficient but similar results to the constitutive system were observed (Supplementary Figure S4A-S4B). Upon engraftment of the BCP-ALL control cells, recipient mice progressively developed symptoms indicative of leukemia, such as pale extremities, piloerection, hunched posture and continuous weight loss, ultimately leading to death. The knock-down of SOX7 expression in both cell lines induced a significant delay in the onset of these symptoms hence resulting in prolonged survival ( Figure 5A-5B). A decrease in the leukemic burden was also observed post-mortem in the spleen of recipient mice engrafted with RS4;11 cells in which SOX7 expression was knocked-down ( Figure  5C-5D). To further document leukemia development, in vivo imaging was performed for luciferase activity carried along the GFP reporter by the shRNA constructs ( Figure 6A). Cells transduced with both shSOX7-2 and shSOX7-3 showed an overall similar level of GFP expression (hence luciferase) than the scramble control cells ( Figure 6B), whereas cells transduced with a single SOX7 shRNA had lower GFP levels (hence luciferase) and therefore were not used for comparative imaging. Non-invasive in vivo luciferase imaging at the early stage of the disease clearly revealed a delay in the proliferation of BCP-ALL cells and in the spread of the leukemia through organ infiltration ( Figure 6C). Taken together, these results demonstrate that the downregulation of SOX7 delays the onset of BCP-ALL and decreases the leukemic burden, establishing the critical contribution of SOX7 expression in maintaining the high proliferative status of the leukemic cells. Quantitative RT-PCR analysis of SOX7 and SOX18 transcript levels relative to β2-microglobulin in childhood human BCP-ALL samples (bone marrow). C. Quantitative RT-PCR analysis of SOX7 and SOX18 transcript levels relative to β2microglobulin in adult human BCP-ALL samples (blood). www.impactjournals.com/oncotarget DISCUSSION While specific recurrent cytogenetic abnormalities and mutations in key transcription factors of B cell lymphopoiesis have been identified in most BCP-ALL cases [1,2], the molecular mechanisms underlying the emergence and maintenance of this type of leukemia still remain poorly understood. In the present study, we identify SOX7 as a novel and important player in BCP-ALL. We show that SOX7 is frequently and specifically expressed in BCP-ALL, independently of any specific chromosomal translocation, and that this transcription factor is implicated in the proliferative potential and clonogenicity of leukemic cells. Our study suggests that SOX7 expression in BCP-ALL is an important factor contributing to leukemia.
The analysis of SOX7 expression revealed the significant and specific expression of this transcription factor in BCP-ALL but not in other types of blood malignancy or in healthy bone marrow. Expression of SOX7 in BCP-ALL had no obvious association with specific chromosomal abnormalities or cytogenetic subtypes. Chromosomal translocations frequently found in BCP-ALL such as t(4;11) or t(12;21) producing the MLL-AF4 and ETV6-RUNX1 fusion proteins respectively, are not able on their own to fully recapitulate the human disease, suggesting that other critical events are required to lead to BCP-ALL [23][24][25][26], the deregulation of SOX7 expression could be one of these events. It remains to be determined however how SOX7 expression becomes deregulated, to date no chromosomal translocations or mutations have been identified within the SOX7 locus. RT-PCR analysis of SOX7 transcript level relative to β2microglobulin in leukemia cell lines. Error bars indicate mean ± standard deviation (n=3). C. Protein expression level for SOX7 and β-actin for the indicated cell lines measured by Western blot. Data are representative of two independent experiments. D. Binding regions of the different shRNAs to the human SOX7 locus; shRNAs are indicated as sense together with a number. Only shRNA 4, 5 and 7 are binding to SOX7 coding sequence, all the other shRNAs are binding to the UTR region. The alignment was performed using BLAST browser. E. Quantitative RT-PCR analysis of SOX7 transcript level relative to β2-microglobulin in HUVEC transduced with shRNA against SOX7 or not. HEK cells were used as negative control. Error bars indicate mean ± standard deviation (n=3). www.impactjournals.com/oncotarget An alternative possibility is that SOX7 is part of the transcriptional machinery regulating B cell development and as a result of mutational events leukemic cells become trapped in a SOX7-expressing stage of B lymphopoiesis in which SOX7 is a critical player of proliferation.
To investigate the role for SOX7 in leukemogenesis, we knocked-down the expression of SOX7 in human BCP-ALL cell lines using shRNAs. Eight different shRNA constructs were tested in HUVEC (endothelial cells) and all successfully and efficiently decreased SOX7 transcripts to less than 5% of the control levels. In contrast, in BCP-ALL cell lines, none of the shRNA constructs was able to decrease SOX7 transcript levels to less than 50% of the control levels. It is possible that   this difference in knockdown efficiency is related to differences in the 3'UTR region of SOX7 mRNA between HUVEC and BCP-ALL cells as 3'UTR sequences are known to affect mRNA stability and post-transcriptional regulation [27]. Alternatively, it is possible that HUVECs do not rely on SOX7 expression for survival as much as BCP-ALL cell lines as HUVECs also express SOX17 and SOX18 that can control similar gene expression to SOX7 in endothelial cells [16]. Given that BCP-ALL cells do not express the two other SOXF factors, their survival might be compromised by a complete absence of SOX7 expression. However, even with this partial knock-down of SOX7, we observed a marked effect on proliferation and clonogenicity. It is likely that a more complete reduction in SOX7 level might result in more severe consequences in term of proliferation, survival or differentiation. Attempts to further decrease SOX7 transcript levels were investigated through transduction with two independent shRNAs or using an inducible shRNA system but none of these attempts was effective at further knocking-down SOX7 expression. These observations suggest that SOX7 expression is dispensable for the maintenance of HUVEC cells but that it is most likely critically required for BCP-ALL cell survival and proliferation. Few SOX7 transcriptional targets have been identified to date and among them, we recently described VE-cadherin as one of SOX7 prime targets during the establishment of the cardiovascular system [28]. Interestingly, the expression of VE-cadherin has been reported in BCP-ALL cell lines and primary samples [29]. Furthermore, down-regulation of VE-Cadherin expression with siRNA or antagonist was shown to induce increase sensitivity to chemotherapy while VE-cadherin over-expression resulted in increased survival upon chemotherapy treatment [30]. Furthermore, the expression of endothelial adhesion molecules such as VE-cadherin has been linked to enhanced migration across the brain-blood endothelial barrier, promoting infiltration of the central nervous system by leukemic cells, an event contributing to relapse and poor outcome [31]. It is possible therefore that part of the leukemogenic potential of SOX7 might be achieved through its direct transcriptional activation of VE-cadherin. It will be important in future work to define the downstream program regulated by SOX7 in BCP-ALL and to identify the key pathways regulating the proliferative potential controlled by SOX7. They might represent potential novel druggable targets for the treatment of poor prognosis and relapsed BCP-ALL. Alternatively, the identification of small molecules that can interfere with SOX7 transcriptional activity either through SOX7-DNA interaction or through protein complex formation will represent interesting avenues of investigation with potential therapeutic benefits.

Western blotting
Protein samples were isolated using the RIPA lysis buffer and quantified followed by electrophoresis on 10% NuPAGE Novex Bis-Tris Mini Gels and blotting onto nitrocellulose membranes using Mini iBlot Gel Transfer Stacks Nitrocellulose (Invitrogen) Proteins were detected using human SOX7 antibody (R&D) and β-actin (Sigma).
Cell cycle analysis 10 6 cells were incubated with 10 μM EdU for 2 hours, harvested, fixed and permeabilized. EdU detection is based on a reaction between the azide, coupled to Alexa Fluor ® 647 dye, and the alkyne in the EdU (Invitrogen). DNA content was measured using Violet406. Acquisition analysis was performed on a LSRII (BD cytometer).

Transplantation
RS4;11 or NALM6 human leukemia cells were transduced with lentiviruses for 30 minutes at 1200 rpm at MOI10-50. Transduced cells were sorted and transplanted i.v. into sub-lethally irradiated (125cGy) NSG mice. Leukemia growth was assessed by blood analysis, weight and general health monitoring. All animal work was performed under regulations governed by the Home Office Legislation under the Animal Scientific Procedures Act of 1986.

Statistical analyses
Data were analyzed using a Student's t Test. Significant differences are indicates with * (P<0.05) and ** (P<0.01). Survival analysis was performed using the Kaplan-Meier method and Log-rank statistical test.

ACKNOWLEDGMENTS
We thank Dr Tim Somervaille for scientific advices and input. We thank the staff at the Flow Cytometry, Advanced Imaging, Biological Research Unit and Molecular Biology Core facilities of CRUK Manchester Institute for technical support. Works in our laboratories are supported by Cancer Research UK (CRUK), Biotechnology and Biological Sciences Research Council (BBSRC) and Leukemia Lymphoma Research / Bloodwise. V.S. is a Wellcome-DBT India Alliance Margdarshi Fellow.

CONFLICTS OF INTEREST
The authors declare no competing financial interests.


extfieldsofstudy: Biology Medicine

text:
Determinants of Amyloid Formation for the Yeast Termination Factor Nab3

Low complexity protein sequences are often intrinsically unstructured and many have the potential to polymerize into amyloid aggregates including filaments and hydrogels. RNA-binding proteins are unusually enriched in such sequences raising the question as to what function these domains serve in RNA metabolism. One such yeast protein, Nab3, is an 802 amino acid termination factor that contains an RNA recognition motif and a glutamine/proline rich domain adjacent to a region with structural similarity to a human hnRNP. A portion of the C-terminal glutamine/proline-rich domain assembles into filaments that organize into a hydrogel. Here we analyze the determinants of filament formation of the isolated low complexity domain as well as examine the polymerization properties of full-length Nab3. We found that the C-terminal region with structural homology to hnRNP-C is not required for assembly, nor is an adjacent stretch of 16 glutamines. However, reducing the overall glutamine composition of this 134-amino acid segment from 32% to 14% destroys its polymerization ability. Importantly, full-length wildtype Nab3 also formed filaments with a characteristic cross-β structure which was dependent upon the glutamine/proline-rich region. When full length Nab3 with reduced glutamine content in its low complexity domain was exchanged for wildtype Nab3, cells were not viable. This suggests that polymerization of Nab3 is normally required for its function. In an extension of this idea, we show that the low complexity domain of another yeast termination factor, Pcf11, polymerizes into amyloid fibers and a hydrogel. These findings suggest that, like many other RNA binding proteins, termination factors share a common biophysical trait that may be important for their function.

Introduction
The study of low complexity protein domains (LCDs), particularly those that are considered intrinsically disordered, has gained focus recently because of the unusually large number of proteins that contain them, and their association with prion-like structural properties [1,2]. RNA binding domains are over-represented in proteins with prion-like regions. While 1% of the proteins in the human genome have an RNA recognition motif, 12% of proteins with a prion-like domain have an RNA recognition motif [2]. LCD sequences commonly lack stable, folded secondary structural elements and have been referred to as intrinsically disordered [1].
Although unstructured, these domains can assume stable folded states through intermolecular aggregation or polymerization; in some cases assembling into regular filaments which can further organize into hydrogels [3]. Some of the proteins bearing these domains form heritable prion-like particles [4]. A bioinformatics analysis of the yeast proteome revealed that many recombinant low complexity prion-like domains formed amyloid polymers in vitro [5]. In vivo, some of the candidates displayed features of aggregation and some were able to stimulate prion formation. This initial survey has been seminal in the study of LCDs. We recently showed that LCDs from the transcription termination factors Nrd1 and Nab3 which were predicted to be prion-like, indeed form amyloid-like filamentous polymers in vitro [6].
One mechanism for terminating transcription by RNA polymerase II (pol II) employs the hnRNP-like proteins Nab3 and Nrd1 and the helicase-like protein, Sen1 [7]. The Nab3-Nrd1--Sen1 (NNS) complex is the major termination machinery for short, non-coding RNAs. Nab3 and Nrd1 bind RNA through a conserved RNA recognition motif [8,9]. Sen1 is thought to unwind the nascent RNA from the transcription bubble [8]. The three proteins interact with each other, with pol II, and with nascent RNA. They also recruit the TRAMP complex and nuclear exosome to process or degrade RNA substrates [10,11]. This complex network of interactions is important for termination activity as pairwise mutations that disrupt these interactions are deleterious and often lethal [12]. Nrd1 has a motif (CTD-interaction domain [13], CID) that has been shown to bind the carboxy-terminal domain (CTD) of the pol II's largest subunit [14,15]. Interestingly, other proteins that associate with pol II, including subunits of the Mediator complex (Med2, Med3, Med15) and termination factors associated with cleavage and polyadenylation of mRNA (Pcf11, Hrp1, Rat1), also contain prion-like domains as predicted by a Hidden Markov Model algorithm [5]. Med3, Med15, and Hrp1 have been shown to form amyloid assemblies [5,16]. The termination factor Pcf11, like Nrd1, contains a CID that binds a specific phosphorylated form of the pol II CTD [17,18]. Curiously, the mammalian RNA-binding protein FUS, which has an LCD and forms protein polymers, has been shown to interact with the pol II CTD in a phosphorylation-dependent manner, suggesting that this may be an additional type of contact that enables the recognition of pol II across the transcription cycle [19].
Recently, it has been shown that Nab3 can employ its LCD to detoxify a derivative of the polyglutamine-containing huntingtin protein in living yeast [20,21]. This is thought to operate through a direct interaction of the two polyglutamine-containing proteins. Prior work showed that a portion of the Nab3 LCD fused to GFP formed punctate aggregates in yeast as observed by fluorescent microscopy [5]. These findings suggest that Nab3's polyglutamine domain can be functionally deployed in vivo to form higher order structural assemblies. Nab3 contains a lengthy prion-like domain that is glutamine and proline rich over 235 amino acids, including a tract of 16 glutamines. The domain ends with a C-terminal region with structural homology to an α-helix found in human hnRNP-C that is known to self-associate into bundles [22]. Mutations in this part of Nab3 were picked up as terminator override mutants in yeast [23]. Loss of the final 134 residues, is lethal [12]. Genetic and biochemical evidence suggest that multiple copies of Nab3-Nrd1 dimers bind to nascent RNA during the termination of transcription [9,12]. Recent work on a number of RNA binding proteins also suggests that higher order LCD interactions are important for compartmentalization of RNA metabolism [24][25][26][27]. Hence, a working hypothesis is that the LCDs of RNA-binding proteins enable non-membrane delimited compartments to form that house RNA metabolism centers [3]. Here we have investigated the determinants of filament formation of Nab3's low complexity domain. Recently, the expression and purification of full-length Nab3 has been reported [28]. We now show that, as observed for its isolated LCD, the entire protein also forms amyloid fibers. As well, the termination factor Pcf11's low complexity domain, forms filaments and a hydrogel. These findings suggest that self-assembly of low complexity domains is an important part of the function of proteins that interact with pol II and in particular, factors involved in terminating transcription.

Plasmid and protein preparation
Full-length Nab3 expressed from a modified pET41a plasmid (pDL469, courtesy of D. Libri) was transformed into BL21(DE3) E. coli and expressed by autoinduction as described [28]. Cells were resuspended in lysis buffer (50mM Tris, pH 7.5, 500mM NaCl, 10mM imidazole) containing protease inhibitors (EasyPack, Roche, Inc.), lysed with 10 μg/ml lysozyme, sonicated, and centrifuged at 27,000 X g for 30 min. Supernatants were applied to a 1 ml HisTrap HP nickel column (GE Healthcare) equilibrated in lysis buffer, washed with 25 ml of lysis buffer, and eluted with lysis buffer containing 250 mM Imidazole. Fractions were pooled and digested with TEV protease, for 16 h at 22°C. Digested proteins were exchanged into lysis buffer by centrifugal filtration in Vivaspin units (GE Healthcare) and re-run on a HisTrap HP column. Nab3 in the flow-through were concentrated with Vivaspin units with intermittent pipetting to promote mixing while exchanging into 0.2M NaCl, 50mM Tris, pH 7.5. Amyloid formation was initiated by incubating concentrated, purified protein, at 4 degrees without agitation. Viscosity and gel formation were monitored visually. At varying times following incubation, extending from days to weeks, samples were taken for amyloid and filament formation assays. While not quantified, filament formation tended to proceed faster at higher protein concentrations and thus varied from preparation to preparation and protein to protein.
The sequence of Nab3 encoding its C-terminal domain was deleted from pDL469 using Phusion DNA polymerase (NEB) and the oligonucleotides 5'-aggtggttgaggaggcggacc-3' and 5'-gaaaacctgtattttcagggacaccacc-3'. This plasmid (pET41-Nab3Δ134) was transformed into BL21(DE3) cells and this truncated version of Nab3 was expressed and purified as described above for full-length Nab3.
Derivatives of the Nab3 low complexity region (Nab3 134 ) protein were expressed from plasmids pET32a-Nab3-134, pET32-Nab3 134ΔQ16 , pET32-Nab3 134Δα , and pET32-134QE. pET32a-Nab3-134 has been described [12], it encodes the C-terminal 134 amino acids of Nab3 fused to thioredoxin by inserting the coding sequence between the BglII and XhoI sites of pET32a. pET32-Nab3 134ΔQ16 , was made by deleting the DNA encoding the stretch of sixteen Qs from pET32a-Nab3-134 using Phusion DNA polymerase and the oligonucleotides 5'-cctgctggcaataatgttcaaagtctatta-3' and 5'-aggtggcggaggttggtgtgac-3' in mutagenic PCR. pET32-Nab3 134Δα was similarly made by deleting the last 18 amino acids of Nab3 from pET32a-Nab3-134 using the oligonucleotides 5'-aggttgctgttgctgttgctgttgctg-3' and 5'-tgactcgagcaccaccaccaccac-3'. pET32-134QE was created by synthesis (Genscript, Inc.) of the portion of Nab3 shown in Fig  1A in which 24 Q codons were changed to glutamate codons inserted into the EcoRV site of pUC57. Synthesis included BglII and XhoI sites at the 5' and 3' ends respectively which were used to excise the fragment for insertion into similarly digested pET32a. All plasmids were transformed into E. coli, which were induced with IPTG, lysed with lysozyme, sonicated, and the lysate was chromatographed over HisTrap media as described above. Pooled fractions were digested with thrombin, washed back into lysis buffer, and re-chromatographed on HisTrap columns as described above. The resulting proteins were concentrated by centrifugal filtration and left undisturbed at 4°C or 22°C.
pET32-Pcf11-LCD was assembled by synthesizing the sequence from codon 228 to codon 287 of Pcf11 with BglII and XhoI sites on its ends for insertion into similarly cut pET32a. The plasmid was transformed into E. coli for expression and purification of the thioredoxin-Pcf11 LCD fusion protein. Cells were lysed and chromatographed on HisTrap as described above. Eluted protein was cleaved with thrombin and re-chromatographed on HisTrap as described for the Nab3 polypeptides.

Yeast Strains
Strains used in this study are listed in Table 1. The strain DY3111, bearing a chromosomal deletion of NAB3 and a wildtype copy of NAB3 on a URA3-marked plasmid, was used as a starting strain for plasmid shuffling. It was transformed with the LEU2-marked plasmids pRS315-Nab3-11QE, pRS315-Nab3-14QE, or pRS315-Nab3 to yield strains with two plasmids. Counterselection against the URA3 plasmid on 5-fluoroortic acid (FOA)-containing medium yielded the strains DY368, DY384, and DY357, respectively. DY382 was made by transforming DY3111 with pRS315-Nab3 24Q>E . For PCF11 shuffles, H-314 (courtesy of Dr. M. Hampsey) was used as a starting strain. pRS315-PCF11 or pRS315-PCF11-ΔPrLD were transformed into H-314 to generate DY361 and DY362, respectively. Counterselection against the URA3marked plasmid following growth in the presence of FOA led to strains DY363 and DY364, respectively.

Semi-denaturing detergent agarose electrophoresis
Purified protein samples were adjusted to 2% SDS, 140 mM β-mercaptoethanol, 10% glycerol, 0.002% bromphenol blue, 80 mM Tris, pH 6.8 and separated by electrophoresis in agarose gels (1.5% w/v) in 40 mM Tris-acetate, pH 7.8; 1 mM EDTA, 0.1% SDS run at 4°C. Bio-Rad Precision Plus Protein Kaleidoscope (Cat. No. 161-0375) molecular weight markers were run as standards. Proteins were blotted to Protran nitrocellulose transfer membrane (Whatman) by capillary action for 18 hrs. Filters were blocked in 5% (w/v) nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 [29] and probed with rabbit anti-S tag antibody (catalog no. PM021; MBL International Corp., Woburn, MA). Electron microscopy Five μl of sample suspension were placed on a 400-mesh carbon coated copper grid that had been made hydrophilic by glow discharge. After 5 minutes, the grid was rinsed by briefly touching the sample side to a drop of distilled water. The residual water was then removed by blotting to filter paper. For negative staining, 5μl 1% aqueous phosphotungstic acid (pH 6.5) was applied to the grid immediately after water removal, and excess liquid was removed by blotting after 30 seconds. The grid was air dried before viewing on a JEOL (Tokyo, Japan) IEM-1400 transmission electron microscope equipped with a Gatan (Pleasanton CA) 2k x 2k US1000 CCD camera. While filaments and less well-defined amorphous material were both apparent, the bulk of the negatively stained material resided in filaments. Representative images were captured digitally as.tif files.

Thioflavin T fluorescence
Assays were carried out as described by LeVine [30]. Thioflavin T (Sigma Chemical, St. Louis, MO) was dissolved in 50mM glycine-NaOH, pH 8.5 and diluted to 10 μM when mixed with proteins. The fluorescence was read in a Shimadzu RF-5301PC spectrofluorophotometer in spectrum mode, with the excitation filter at 450+/-5 nm and the emission filter at 485+/-5 nm or using a Biotek Synergy 4 plate reader (384 well; 50 μl per well; triplicate wells) with excitation at 450 nm and emission at 490 nm.

Results
We previously showed that a recombinant version of the Saccharomyces cerevisiae Nab3 LCD (its C-terminal 134 amino acids; "Nab3 134 ") formed amyloid polymers and an ordered hydrogel [6,12]. This region of the protein is important for its function as a termination factor and for cell viability. To learn more about the sequence determinants of self-assembly, we examined Nab3 134 with a specific set of mutations. Because we identified mutations in Nab3 that impact its termination activity by deleting all or part of 16 consecutive glutamines in the LCD [22,23], we deleted this homopolymer tract (Fig 1A, ΔQ16). Filaments readily formed from this derivative as scored by electron microscopy (Fig 1B, middle). We then introduced a deletion of the terminal 18 amino acids which have structural homology to an α-helix Δα) of human hnRNP-C that can self assemble into a tetrameric bundle, and which is also important for Nab3's termination function [12,23] (Fig 1A, Δα). This purified protein was also competent to form filaments (Fig 1B, right panel). Although both of these segments are important for termination activity, neither the loss of the stretch 16 glutamines nor the terminal 18 residues were lethal in the context of the rest of the Nab3 protein [22]. Nab3 134 is 32% glutamine, and it resembles prion-like domains of yeast that polymerize into filaments by stacking their β-sheet-forming, low complexity domains perpendicular to the direction of filament polymerization [31]. The primary sequence of domains such as these is less important than the overall amino acid composition, and the polymer fails to form if charged residues are introduced by mutation since they occur in-register in the β sheet stack providing unfavorable charge-repulsion [31]. To test if this was true for the Nab3 LCD, we substituted glutamate for glutamine at every-other position of runs of consecutive glutamines, as well as for isolated residues in the domain (Fig 1A). This change of 24 glutamines to glutamates altered the composition from 32 to 14% glutamine. This low-glutamine version of the recombinant Nab3 LCD was purified from E. coli. The introduction of charged glutamic acid residues resulted in an anomalously slow mobility of the polypeptide on SDS-PAGE relative to the wildtype LCD (Fig 1C) [6]. When subjected to filament forming conditions for the same length of time as that described for the wildtype Nab3 134 domain, the low-glutamine Nab3 LCD failed to form filaments or a hydrogel, or to bind the β-sheet-binding dye thioflavin T ( Fig  1D). Continued incubation for many months under filament forming conditions did not result in detectable polymer formation.
Nab3 134 represents the C-terminal 17% of the entire Nab3 protein. To test if it potentiates polymerization for full-length Nab3, we purified the entire 802-residue protein from E. coli with and without its final 134 amino acids and subjected the respective proteins to polymerization conditions. Full-length Nab3 formed filaments as scored by electron microscopy (Fig 2A). Assembly was dependent upon the C-terminal domain that autonomously forms filaments, since Nab3 lacking that region (Nab3Δ134) failed to show filaments (Fig 2A). Since the C-terminus of Nab3 is intrinsically unstructured and sensitive to proteolysis [12], we needed to show that the filaments were composed of full-length Nab3 and not the LCD released from the protein by spurious proteolysis following expression in E. coli. Filaments were pelleted by centrifugation (13,000 x g; 5 min) and analyzed by Western blotting with an anti-Nab3 antibody. We found that the pelletable fibers were indeed formed from full-length protein (Fig 2B; "Nab3" lane "P"). This procedure also confirmed that purified Nab3Δ134 did not assemble into filaments (Fig 2B, "Nab3Δ134" lane "P"). Thioflavin T-binding and fluorescence also revealed amyloid formation by the full-length protein (Fig 2C, ▼). Nab3 lacking the filament- forming C-terminal 134 amino acids bound the dye less well (■); residual fluorescence could be due to the glutamine/proline-rich bias of a small part of the prion-like domain still retained in this derivative [5].
An additional test for amyloid formation is the use of semi-denaturing agarose gel electrophoresis (SDD-AGE) to separate monomeric protein from high molecular weight polymers [32]. As shown previously, Nab3 134 assembled into an SDS-resistant form that was readily separable from the soluble form of the protein (Fig 2D, left panel). Similarly, full-length Nab3 formed high molecular weight, SDS-resistant aggregates while Nab3 lacking the Nab3 134 assembly domain did not (Fig 2D, right panel).
If assembly of Nab3 through its LCD is important for its function, the 24 glutamine to glutamate substitutions in the LCD that impair filament and hydrogel formation should impact cell physiology. To test this, we introduced this low-glutamine version of the LCD into full length Nab3 expressed from its own promoter and tested if it could support cell viability by plasmid shuffling. This test revealed that cells are not viable with only the mutant form of Nab3 ( Fig  3A). To insure that the mutant Nab3 protein was synthesized, we examined cells with a plasmid expressing wildtype Nab3 and with one expressing the low-glutamine version of Nab3. We anticipated that full-length Nab3 with the 24 glutamine to glutamate substitutions (Fig 1A) would also display an unusually slow mobility on SDS-PAGE as observed above for the isolated domain (Fig 1C), and thus we would be able to distinguish the wildtype from the mutant protein in cells expressing both. This was indeed the case (Fig 3B, lane 4), and the mobility anomaly was further confirmed by comparing the 24 glutamine to glutamate version of Nab3 to derivatives of the protein with 11 or 14 glutamine to glutamate substitutions which expressed intermediate mobility on SDS-PAGE and could serve as the sole Nab3 in these strains (Fig 3B). Thus, Nab3 24Q>E was ineffective in supporting cell survival. In fact, when cells with both plasmids (one containing wildtype NAB3 marked with URA3 and another with Nab3 24Q>E marked with LEU2) were grown on media selective only for the latter (leucine-dropout media), both were retained because, even though there was no selection for URA3, wildtype NAB3 had to be retained to cover for the inactive nab3 24Q>E allele. A number of yeast transcription termination factors have a low complexity domain that appears prion-like when analyzed by a Hidden Markov Model algorithm trained on well-characterized prions [5]. Since, we observed that the LCDs of Nab3 and Nrd1 both formed homopolymeric filaments, we tested if that from Pcf11 could as well. The low complexity domain purified from E. coli formed filaments that could be observed by electron microscopy (Fig 4A). Polymers could be formed at 4 or 22°C and they were resistant to boiling in the presence of SDS as seen by SDD-AGE analysis (Fig 4B). The filaments readily organized into a hydrogel ( Fig 4C) and they bound thioflavin T and fluoresced in a manner similar to Nab3 134 (Fig 4D). Deletion of the LCD of Nab3 compromises cell viability [9,12]. To test if this is the case for Pcf11, a version of the protein lacking its LCD (residues 228-287) on a LEU2-marked plasmid was transformed into yeast cells harboring a URA3-marked plasmid encoding wildtype Pcf11. Cells that lost the latter plasmid were selected on FOA-containing medium and were able to grow well when the only copy of PCF11 was the one lacking its LCD (Fig 5). Thus, Pcf11's LCD is not required for cell viability. Discussion A growing body of evidence indicates that RNA binding proteins often possess low complexity, unstructured regions that can form amyloid polymers [2,3,33]. These domains are functionally important and are involved in the assembly of granules or droplets in living cells [3,24,26,33,34]. Here we provide evidence that the determinants of the Nab3 LCD's ability to polymerize reside in the glutamine-rich quality of the domain. This is similar to what has been seen for the LCDs that enable prions to form filaments and render them prone to aggregation [35,36]. When the LCD's glutamine content is drastically reduced by mutation and introduced into the context of the full-length yeast Nab3 protein, it failed to support cell viability, suggesting that polymer-formation is important for its function. It remains possible that the increased charge introduced into Nab3 following the substitution of glutamines with glutamates, renders the protein non-functional for reasons other than its amyloid-forming capacity. In any case, genetic and biochemical evidence indicate that multiple copies of Nab3, with its partner Nrd1, associate with a nascent transcript to provoke transcription termination [9,12,22]. Also apparent, is the evolutionary plasticity of the LCD's primary sequence, and particularly the glutamine positioning, as seen by its polymorphic nature which is apparent when S. cerevisiae Nab3 is aligned with that from five other Saccharomyces species (Fig 6). While the exact positions of the glutamines vary, the fractional glutamine composition has been retained in the range of 26 to 33% (Fig 6, box lower right), reinforcing the notion that the primary sequence is less important than overall composition [35,36]. Interestingly, the very C-terminal region of Nab3 that has structural homology to an α-helical region of hnRNP-C and is not required for filament formation (Fig 1A), is amongst the best conserved parts of the LCD (Fig 6). This portion of Nab3 can self-assemble independently of the LCD, as can the cognate region of hnRNP-C [22,37]. The domain is important for the termination function of Nab3 as revealed by mutations, including some that are structurally subtle such as a substitution of alanine for leucine only two positions from the C-terminus which reduces termination efficiency in vivo [22].
We also show that the Nab3 LCD, which independently forms amyloid polymers, is responsible for filament formation of the full-length protein with its RNA-binding domain. The finding that full length Nab3 forms filaments is an important extension of the initial computational analysis identifying LCDs in the yeast proteome that have assembly potential [5]. In this regard, we also show here that another prion-like domain identified in that study can assemble into filaments and a hydrogel in vitro. This domain is contained in the termination factor Pcf11, thereby extending the trend for termination complexes to contain subunits with this ability. Pcf11 co-exists in the CFI polyadenylation complex with Hrp1, whose prion-like domain scores very strongly in the study of Alberti et al., including the ability to form filaments in vitro [5]. Combined with the potential of LCD-containing Mediator subunits to polymerize [16], Fig 5. Pcf11's LCD is not essential for viability. Yeast bearing a LEU2-marked plasmid with wildtype PCF11 or PCF11 from which the region encoding its LCD was deleted (pcf11ΔLCD), were grown in liquid media, serially diluted, spotted onto solid media, and grown at 30°C. doi:10.1371/journal.pone.0150865.g005 and the finding that fibrous polymers formed from LCDs interact with pol II's CTD [38], LCD polymerization is a recurring feature of complexes that interact with pol II across the transcription cycle. It is also significant to note that we have found conditions in which the low complexity domains from three different proteins, Nab3, Nrd1, and Pcf11, form amyloid polymers. These were initially identified in the yeast proteome computationally but in vitro assembly had not been detected [5]. One possibility is that purification here was done in the absence of denaturants. Another consideration may be our use of thioredoxin as a fusion partner during expression in E. coli. In any case, our findings suggest that under certain conditions there may be additional members of the computationally-derived set of predicted yeast prion-like domains identified by Alberti et al. that indeed polymerize. was aligned using CLUSTAL Omega with the cognate region from S. kluyveri, S. bayanus, S. kudriavzevii, S. mikatae, and S. paradoxus. The numbering of amino acids for each is shown at right. Glutamine residues (Q) were tallied for each species, divided by the total number of residues in its LCD (tot), and described as a percentage thereof in the box at lower right. doi:10.1371/journal.pone.0150865.g006