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Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

The coordinated action of the actin and microtubule cytoskeletal networks is required for Golgi ribbon assembly. The novel formin FHDC1 accumulates on the Golgi-derived microtubule network, where it acts to regulate Golgi ribbon assembly in an actin- and microtubule-dependent manner.

INTRODUCTION
The Golgi apparatus is the central controller of subcellular trafficking found in all eukaryotes. In most cells, the Golgi occurs as a series of cisternae tethered together as stacks organized from cis to trans. Incoming traffic arrives at the cis face of the Golgi and moves through the medial and trans cisternae before exiting through the trans-Golgi network. At the same time, retrograde trafficking returns cargo proteins to the endoplasmic reticulum (ER) and maintains the localization of Golgi-resident enzymes as the cisternae mature (Rizzo et al., 2013). In higher organisms, these stacks are tethered laterally to form a Golgi ribbon typically found in tight association with the centrosome. The cis-to-trans stacking of the Golgi is found in almost all eukaryotes and is believed to facilitate cargo processing and modification. The additional requirement for the ribbon is unclear, but it has been suggested that ribbon formation may aid in the trafficking of very large cargo and reflects the more complex role of the Golgi in higher organisms (Lavieu et al., 2014). Current models suggest that the assembly of the Golgi ribbon is an actin-and microtubule (MT)-dependent process and that proper positioning and maintenance of the Golgi is required for polarized cellular trafficking and normal cell motility (Dippold et al., 2009;Miller et al., 2009;Yadav et al., 2009;Zhu and Kaverina, 2013;Deakin and Turner, 2014;Guet et al., 2014;Gurel et al., 2014).
Assembly of the perinuclear Golgi ribbon relies on two cytoplasmic MT networks, one originating at the centrosome and one originating at the Golgi itself (Rogalski and Singer, 1984;Miller et al., 2009;Rivero et al., 2009). Golgi-derived MTs are nucleated at the cis-Golgi through the GM130-mediated recruitment of AKAP450, myomegalin, and the γ-TuRC MT-nucleating complex (Chabin-Brion et al., 2001;Rivero et al., 2009;Roubin et al., 2013;Wang et al., 2014). This complex is absent from the trans-Golgi, and it has been proposed that, as the nascent MTs emerge at the cis-Golgi, they are likely captured by CLASP proteins, which are themselves recruited to the trans-cisternae by the Golgin GCC185 (Efimov et al., 2007;Sutterlin and Colanzi, 2010;Vinogradova et al., 2012; MT acetylation, suggesting that the effects of FHDC1 on Golgi assembly are separate consequences of FHDC1 expression. Consistent with these observations, small interfering RNA (siRNA)-mediated knockdown of FHDC1 expression also disrupts Golgi ribbon formation. Together our results suggest that FHDC1 acts at the Golgi-derived MT network to coordinate actin and MT dynamics and facilitate formation of the Golgi ribbon.

A pool of FHDC1 associates with the Golgi ribbon
We first noted a connection between FHDC1 and the Golgi ribbon while investigating a potential role for FHDC1 in cell polarity. In these experiments, the subcellular localization of endogenous FHDC1 protein was determined in NIH 3T3 fibroblasts migrating in a scratch-wound model of directional cell migration ( Figure 1A). The position of the Golgi ribbon was used as a marker of cell polarity and visualized with the cis-Golgi marker GM130; endogenous FHDC1 protein was detected with anti-FHDC1 antibody (Young et al., 2008). Two hours after wounding, cells at the wound's edge reoriented and began to migrate into the wound. Both at the wound's edge and away from the wound, endogenous FHDC1 is associated with the microtubule (MT) network and is concentrated in a perinuclear region corresponding to the position of the Golgi ribbon. A similar distribution of FHDC1 protein was also observed in randomly migrating cells ( Figure 1B). Colocalization of FHDC1 with GM130 was further confirmed by a pixel intensity plot, showing the extensive overlap of the FHDC1 and GM130 signals ( Figure 1B′). We also observed preferential association of endogenous FHDC1 with the Golgi ribbon in spreading cells (Supplemental Figure S1). In newly attached cells, the Golgi and centrosome are not connected (Kisurina-Evgen'eva and Onishchenko, 2004). Under these conditions, FHDC1 accumulates at and around the Golgi and not with the distinct population of cytoplasmic MTs at the cell periphery. These observations prompted us to further investigate the connections between FHDC1, the Golgi ribbon, and Golgi-derived MTs.

FHDC1 accumulates at Golgi-derived microtubules
Golgi ribbon assembly and integrity is MT dependent (Rogalski and Singer, 1984;Chabin-Brion et al., 2001;Vinogradova et al., 2012). Because FHDC1 is a MT-binding protein, we wanted to determine whether the accumulation of FHDC1 at the Golgi is MT dependent. We treated NIH 3T3 cells with nocodazole to dissolve MTs and induce Golgi dispersion . In untreated cells, FHDC1 is bound to MTs and concentrated at the Golgi as before ( Figure  2A). After nocodazole treatment, FHDC1 punctae are distributed diffusely throughout the cytoplasm, and the Golgi is dispersed into ministacks. We did not observe any obvious association of FHDC1 with the Golgi remnants in nocodazole-treated cells ( Figure 2B). After nocodazole washout, FHDC1 can be seen to take on a more filamentous distribution as the Golgi and MTs recover (Figure 2, C-F). We also found that in recovering cells, FHDC1 protein concentrates at cytoplasmic Golgi clusters (Figure 2, C′-E′) that form as the Golgiderived MT network is reestablished and are likely to correspond to the late G phase of Golgi assembly (Efimov et al., 2007;Miller et al., 2009;Vinogradova et al., 2012). Two hours after removal of nocodazole, both Golgi and FHDC1 distributions have fully recovered, and a clear association of FHDC1 with the perinuclear Golgi ribbon is observed (Figure 2,F and F′). This suggests that FHDC1 accumulates at the Golgi in an MT-dependent manner.
Cytoplasmic MTs are nucleated at both the centrosome and the Golgi (Petry and Vale, 2015). The centrosome can be specifically depleted from NIH 3T3 fibroblasts by extended treatment with the Zhu and Kaverina, 2013). Treatment with nocodazole or other MTdisrupting drugs causes the Golgi to disperse into ministacks that redistribute to ER-exit sites (Thyberg and Moskalewski, 1985;Cole et al., 1996). After removal of the drug, reassembly of the ribbon proceeds in two stages . In the G stage, the Golgi-derived MTs collect the dispersed ministacks into larger assemblies, which provide a bigger target for capture by MTs originating from the centrosome. In the C stage of assembly, the captured ministacks are transported along centrosome-derived MTs toward the centrosome, where they are tethered into the perinuclear Golgi ribbon (Corthesy-Theulaz et al., 1992;Burkhardt, 1998;Miller et al., 2009;Vinogradova et al., 2012;Maia et al., 2013). Disruption of the Golgi-derived MT network causes the G stage of reassembly to fail, resulting in Golgi dispersion or formation of compact Golgi circles located proximal to the centrosome Maia et al., 2013).
The asymmetric subcellular positioning of the Golgi ribbon results in the polarized orientation of the Golgi-derived MT network. This asymmetry is required for directional cell migration and is believed to facilitate polarized secretion to the leading edge (Preisinger et al., 2004;Bisel et al., 2008;Vinogradova et al., 2009;Yadav et al., 2009;Hurtado et al., 2011). Golgi-derived MTs are enriched for acetylated α-tubulin (Jasmin et al., 1990;Thyberg and Moskalewski, 1993;Chabin-Brion et al., 2001), which likely affects both MT dynamics and selective recruitment of motor proteins (Reed et al., 2006;Janke, 2014). We previously found a close association between MT acetylation and the function of the novel MT-binding formin FHDC1 (Young et al., 2008;Thurston et al., 2012), raising the possibility that FHDC1 activity might be connected to the Golgi-derived MT network. FHDC1 (previously known as INF1), like other formins, contains formin homology (FH) 1 and FH2 actin regulatory domains (Higgs, 2005;Young et al., 2008). It is distinguished from other formin family members by the presence of a unique C-terminal MT-binding domain (MTBD) and the absence of any of the regulatory domains present in other formins (Young et al., 2008). The full-length protein is constitutively active, and expression of full-length FHDC1 is sufficient to induce actin stress fiber formation, MT stabilization, and MT acetylation. Thus it is distinct from other formins in that its activity is not regulated by autoinhibition (Chesarone et al., 2010). Conversely, knockdown of FHDC1 expression inhibits the accumulation of acetylated MTs (Young et al., 2008), suggesting that FHDC1 participates in the normal regulation of this process. The effects of FHDC1 on actin dynamics are FH2 dependent, as expected. In contrast, expression of either the isolated FH1-FH2 or isolated MTBD is sufficient to induce MT stabilization and acetylation (Young et al., 2008;Thurston et al., 2012). Indeed, the ability to regulate MT modification and stabilization is a common feature of FH2 domains (Bartolini et al., 2008;Bartolini and Gundersen, 2010;Thurston et al., 2012).
As with the Golgi ribbon, FHDC1 is an evolutionary innovation in vertebrates (Young et al., 2008), and there is a growing appreciation that normal Golgi assembly and maintenance is both actin and MT dependent Campellone et al., 2008;Dippold et al., 2009;Farber-Katz et al., 2014). Given the association between FHDC1 and MT acetylation, as well as its ability to regulate both actin and MT dynamics, we investigated the role of FHDC1 in Golgi ribbon formation. We show here that a pool of endogenous FHDC1 protein accumulates at the Golgi and likely has a preferential affinity for the Golgi-derived MT network. FHDC1 overexpression disperses the Golgi into functional ministacks, and FHDC1-induced Golgi dispersion is both FH2 and MTBD dependent. The ability of FHDC1 to induce Golgi dispersion did not correlate with its ability to induce To test the idea that FHDC1 associates with Golgi-derived MTs, we treated cells with brefeldin A (BFA) to disrupt the Golgi ribbon and Golgi-derived MTs while leaving centrosome-derived MTs intact. In vehicle-treated cells, FHDC1 is predominantly associated with the MT network and Golgi ribbon as before ( Figure 4A). BFA treatment quickly induces Golgi ribbon disassembly, leaving behind a remnant Golgi matrix as revealed by GM130 staining (Nakamura et al., 1995). We found that with prolonged BFA treatment (45 min), the accumulation of FHDC1 at the Golgi was lost, and the filamentous distribution of FHDC1 was replaced with a more punctate staining pattern in the majority of cells (Figure 4, B and E). On washout of BFA, the filamentous distribution of FHDC1 was quickly regained, concomitant with the reassembly of the Golgi ribbon (Figure 4, C and D). These results suggest a preferential association of FHDC1 with the Golgi-derived MT network. This hypothesis was further tested by examining the subcellular distribution of endogenous FHDC1 in cells allowed to recover PLK4 inhibitor centrinone (Wong et al., 2015). Surprisingly, this does not block division of the treated cells or affect normal Golgi assembly. We cultured cells in the presence of centrinone for 14 d to completely eliminate centrosome formation and determined the effect on Golgi assembly and endogenous FHDC1 subcellular localization ( Figure 3). In control cells, FHDC1 was present on the MT network, and the centrosome was readily detected with an anti-γ-tubulin antibody ( Figure 3A). In centrinone-treated cells, the centrosome could no longer be detected, the Golgi remained intact, and FHDC1 maintained its association with cytoplasmic MTs (Figure 3, B and C).
The centrinone results prompted us to consider that FHDC1 may have a preferential affinity for the Golgi-derived microtubule network. Golgi-derived MTs are enriched for acetylated MTs (Thyberg and Moskalewski, 1993;Chabin-Brion et al., 2001), and we previously observed a close association between FHDC1 activity and MT acetylation (Young et al., 2008;Thurston et al., 2012).

FHDC1 induces Golgi dispersion
We next sought to investigate what role FHDC1 might play in the assembly and positioning of the Golgi. We first chose to address this question by testing the effects of FHDC1 overexpression on Golgi morphology. We expressed a full-length FHDC1 derivative with an N-terminal mCherry tag (FHDC1.mCherry) in NIH 3T3 cells by transient transfection. mCherry-transfected cells served as a negative control. The cells were fixed 24 h posttransfection and the effects of FHDC1 expression on Golgi assembly and localization were assessed by immunofluorescence. In mCherry-expressing controls, the Golgi was organized as a perinuclear ribbon in nearly 90% of transfected cells (Figure 7, A and C). In contrast, in >90% of FHDC1-expressing cells, the Golgi ribbon was completely dispersed ( Figure 7, B and C). The Golgi fragments, while sometimes found in a perinuclear halo, had no consistent, specific subcellular localization (see later discussion of Figures 8B, 9, B, E, and H, 10C, 11A, and 12C for additional examples). This striking phenotype is remarkably robust and is reminiscent of the effects caused by loss or overexpression of other regulators of Golgi assembly (Campellone et al., 2008;Dippold et al., 2009;Yadav et al., 2009Yadav et al., , 2012Farber-Katz et al., 2014). It also suggests that FHDC1 might play an important role in this process. from nocodazole-induced MT disruption in the presence or absence of BFA. As before, FHDC1 was primarily associated with the MT network and enriched at the Golgi in untreated cells ( Figure 5A). In cells treated with nocodazole and BFA, the Golgi is dispersed, and FHDC1 was redistributed as a diffuse array of cytoplasmic punctae ( Figure 5B). Two hours after nocodazole washout, in the absence of BFA, the perinuclear Golgi ribbon had reassembled and FHDC1 had completely regained its MT association ( Figure 5D). In cells recovered from nocodazole treatment in the presence of BFA, however, the Golgi remained dispersed, and FHDC1 failed to regain its filamentous distribution and remained primarily in cytoplasmic punctae ( Figure 5C). We also compared the distribution of endogenous FHDC1 with that of CLASP2, an MT-binding protein and marker for the Golgi-derived MT network. Using antibodies against endogenous FHDC1 and endogenous CLASP2, we found that a pool of both proteins accumulated on MTs at the Golgi ribbon and colocalized on a subset of cytoplasmic MTs ( Figure 6). Accordingly, in cells recovering from nocodazole treatment in the presence of BFA, CLASP2, like FHDC1, no longer accumulates on MTs at the Golgi ribbon (Supplemental Figure S2). Together these data point to a preferential association of FHDC1 with the Golgi-derived MT network. FHDC1-induced Golgi dispersion is FH2 and MTBD dependent FHDC1 binds both actin filaments and MTs (Young et al., 2008), and Golgi ribbon assembly and perinuclear positioning are both actin and MT dependent (Rogalski and Singer, 1984;Magdalena et al., 2003;Lazaro-Dieguez et al., 2006;Dippold et al., 2009;Gurel et al., 2014). We therefore sought to determine which aspect of FHDC1 function might be required for its effects on Golgi dispersion. To this end, we generated the FHDC1.I180A derivative to selectively inactivate FH2. In this mutant, residue 180 in the FHDC1 FH2 is mutated from isoleucine to alanine to abolish the ability of FH2 to bind to the barbed end of F-actin (Xu et al., 2004). We confirmed that this mutant no longer affects actin dynamics but is still able to bind MTs and induce MT acetylation in cell-based assays (Supplemental Figure S3). The I180A mutant and a series of FHDC1 deletion derivatives defective in either FH2 or MTBD activity were tested for their effects on Golgi morphology. As before, expression of FHDC1 is sufficient to induce Golgi dispersion in >90% of transfected cells, and the tagged protein is bound to the MT network. The FHDC1. I180A derivative also associated with MTs and was enriched at the Golgi, but expression of FHDC1.I180A failed to induce Golgi dispersion (Figure 8, C and F). This suggests that FHDC1 effects on Golgi are likely FH2 and actin dependent. Similarly, expression of the isolated FHDC1 MT-binding domain (958C) also had no effect on perinuclear Golgi ribbon assembly (Figure 8, E and F), despite its marked effects on MT bundling and network organization (Young et al., 2008). In the same way, expression of an MTBD-deleted FHDC1 derivative (958N) also failed to induce Golgi dispersion to the same extent as the full-length protein, with a normal perinuclear ribbon present in >60% of transfected cells (Figure 8, D and F). Thus the effects of FHDC1 on Golgi dispersion are both FH2 and MTBD dependent. We also compared the effects of FHDC1 expression to the effects of expressing constitutively active derivatives of other formins believed to participate in the regulation of Golgi dynamics (Colon-Franco et al., 2011;Ramabhadran et al., 2011;Zilberman et al., 2011). In cell-based assays, these proteins (FMNL1, INF2, mDia1) are all potent inducers of F-actin accumulation and MT acetylation (Thurston et al., 2012). As previously observed, expression of FIGURE 4: Brefeldin A treatment decreases FHDC1 microtubule localization. (A) In untreated cells, FHDC1 is found primarily on the microtubule network and in close association with the Golgi. (B) After 45 min of treatment with 5.0 μg/ml brefeldin A, FHDC1 subcellular localization in many cells is less filamentous and has a more punctuate and diffuse cytoplasmic distribution, as typified by the indicated cell (arrow). (C) At15 min after removal of brefeldin A, the Golgi ribbon has begun to reassemble; FHDC1 is regaining a more filamentous distribution and is concentrated at the Golgi. (D) In fully recovered cells, FHDC1 is primarily filamentous and preferentially associated with the Golgi. Scale bar, 10 μm. (E) Quantification of data shown in A-D. In untreated cells, FHDC1 is primarily filamentous (blue bars). In contrast, after 45 min of treatment with brefeldin A, FHDC1 exhibits a more punctuate distribution. N = 3, >100 cells counted per experiment. Error bars indicate SEM. a shift to the permissive temperature of 32°C. Before this shift, cycloheximide is added to inhibit protein synthesis to facilitate the tracking of the VSVG reporter once it is released from the ER. Once shifted to the permissive temperature, VSVG proceeds from the ER to the Golgi and from there to the plasma membrane. We coexpressed VSVG ts045-GFP in NIH 3T3 cells with FHDC1, FHDC1. I180A, or mCherry and followed trafficking of the reporter at nonpermissive and permissive temperatures. After 6 h at 40°C, the VSVG-GFP protein has accumulated in the ER of mCherry-, FHDC1. I180A-, and FHDC1-expressing cells (Figure 9, A-C). Thirty minutes after the shift to 32°C, in the presence of cycloheximide, VSVG-GFP had left the ER and accumulated in the perinuclear Golgi ribbon of mCherry-and FHDC1.I180A-expressing cells (Figure 9, D and F); it could also be clearly seen to have trafficked to the Golgi fragments present in FHDC1-expressing cells ( Figure 9E). After 90 min at 32°C, constitutively active FH1 + FH2-containing derivatives of FMNL1, INF2, and mDia1 had some effect on Golgi morphology but failed to induce dispersion in the majority of cells (Supplemental Figure  S4). These results demonstrate the specificity and striking robustness of the effects of full-length FHDC1 on Golgi assembly.

FHDC1-dispersed Golgi fragments support subcellular trafficking
To gain insight into the nature of the FHDC1-dispersed Golgi remnants, we sought to determine whether the Golgi fragments are still capable of supporting intracellular trafficking. To do this, we took advantage of the temperature-sensitive vesicular stomatitis virus G (VSVG) ts045-green fluorescent protein (GFP) trafficking reporter (Presley et al., 1997). At the nonpermissive temperature (40°C), the mutant VSVG protein is trapped in the ER and is released only upon FIGURE 5: Brefeldin A blocks FHDC1 recruitment to microtubules during recovery from nocodazole treatment. (A) In untreated cells, FHDC1 (red) is concentrated on microtubules associated with the Golgi (GM130; green). (B) Cells were treated for 2 h with 2.5 μg/ml nocodazole and 5.0 μg/ml brefeldin A. The Golgi is dispersed throughout the cytoplasm; FHDC1 has a punctate cytoplasmic distribution. (C) Cells were allowed to recover after nocodazole washout for 120 min in the presence of brefeldin A. FHDC1 does not recover its filamentous staining pattern and maintains its punctate cytoplasmic distribution. (D) Cells were allowed to recover for 120 min in the absence of BFA, with FHDC1 regaining its association with the microtubule network. Scale bar, 10 μm. expressing cells, LatB treatment altered ribbon morphology and induced the formation of perinuclear Golgi clusters ( Figure 10, A and B), as observed previously Zilberman et al., 2011). Similar effects were observed in FHDC1-expressing cells, for which LatB treatment reversed FHDC1-induced Golgi dispersion and resulted in the reassembly of perinuclear Golgi clusters (Figure 10, D and E). This suggests that the effects of FHDC1 are actin dependent, consistent with the results obtained with the I180A mutant.
The data in Figures 2-6 suggest that FHDC1 may preferentially associate with Golgi-derived MTs; however, the FHDC1 overexpression phenotype could also be caused by disrupting the connection between the Golgi ribbon and the centrosome-derived MT network Yadav et al., 2009). We thus wished to determine whether or not either of these two networks is required for the effects of FHDC1 on Golgi assembly. To determine whether FHDC1induced dispersion was dependent on the centrosome-derived MT network, we expressed FHDC1 in centrinone-treated cells and assessed the effects on Golgi assembly. In both control and centrosome-depleted cells, FHDC1 induced Golgi dispersion in >90% of transfected cells (Figure 11), suggesting that the effects of FHDC1 are not centrosome dependent.
VSVG-GFP had moved from the Golgi to the plasma membrane in all cells, including those with FHDC1-dispersed Golgi (Figure 9, G-I). We also examined distribution of the lysosome marker LAMP1-RFP and the ER marker calreticulin-GFP in untransfected and FHDC1-expressing cells and found no obvious differences in their distribution (Supplemental Figure S5). Thus the FHDC1-induced Golgi fragments were still able to support trafficking from the Golgi to the plasma membrane, the lysosome, and the ER.

FHDC1-induced Golgi dispersion is F-actin and MT dependent
Golgi assembly and positioning relies on the coordinated action of both the actin and MT networks, and we observed that FHDC1induced dispersion is both FH2 and MTBD dependent. The FHDC1 FH2 domain, however, is able to regulate both actin and MT dynamics (Young et al., 2008;Thurston et al., 2012). To determine whether the effects of FHDC1 expression on Golgi assembly are actin dependent, we tested the effects of the actin-disrupting drug latrunculin B (LatB) on FHDC1-induced Golgi dispersion. FHDC1-expressing cells were fixed after 120 min of treatment with 2 μM LatB, and the effects on actin assembly and Golgi morphology were determined by immunofluorescence. In control mCherry-FIGURE 6: FHDC1 and CLASP2 localize to an overlapping microtubule network. (A) NIH 3T3 cells were stained with rabbit anti-FHDC1 antibody to detect endogenous FHDC1 (red) and rat anti-CLASP2 antibody to detect endogenous CLASP2 (green), and mouse anti-GM130 (white) was used as a Golgi marker. FHDC1 and CLASP2 colocalize (yellow) to an overlapping set of cytoplasmic microtubules and concentrate at the Golgi ribbon. Boxes 1 and 2 highlight areas of FHDC1 and CLASP2 colocalization. The white arrows indicate MT-associated FHDC1 and CLASP2 codistribution. Cells were fixed in 4% paraformaldehyde in PHEM buffer for optimal CLASP2 staining. Scale bar, 10 μm. (B) A pixel intensity plot used to assess the colocalization of FHDC1 and CLASP2 in the region indicated by the white line in B′. The pixel intensity histogram reveals extensive colocalization of the FHDC1 and CLASP2 signals.
The depletion of GM130, however, caused a distinct reduction in filamentous FHDC1 staining despite the presence of abundant cytoplasmic MTs ( Figure 12B and Supplemental Figure S6, A and B). Indeed, the FHDC1 distribution was very similar to that seen in cells recovering from nocodazole treatment in the presence of BFA (Figure 4 and Supplemental Figure S2). GM130 depletion did not affect FHDC1 expression levels as assessed by immunoblotting ( Figure 12F and Supplemental Figure S6G).
Depletion of GM130 also inhibited the ability of FHDC1 expression to induce Golgi dispersion. In control cells, as before, FHDC1 expression induced Golgi dispersion, and intact perinuclear Golgi ribbons were observed in <10% of FHDC1-expressing cells ( Figure  12, C and E). In GM130-depleted cells, giantin staining was used to visualize the Golgi. In these cells, the Golgi appeared somewhat Assembly of the Golgi-derived MT network requires the GM130dependent recruitment of AKAP450 to the cis-Golgi, which in turn recruits the γ-TuRC MT-nucleating complex to allow assembly of Golgi-derived MTs (Rivero et al., 2009;Roubin et al., 2013;Rios, 2014;Petry and Vale, 2015). Knockdown of GM130 expression should therefore selectively inhibit MT nucleation at the cis-Golgi while leaving the Golgi ribbon and the centrosome-derived MT network largely intact. We therefore examined the effects of GM130 depletion on FHDC1 function and subcellular localization. We transfected cells with GM130-targeted siRNA or control duplex and assessed the efficiency of GM130 depletion by immunoblotting and immunofluorescence (Figure 12, D and F, and Supplemental Figure  S6). In control cells, endogenous FHDC1 was associated with cytoplasmic MTs and concentrated at the Golgi as before ( Figure 12A).  cells expressing FHDC1. GM130 knockdown had no effect on the ability of FHDC1 to induce MT acetylation (Supplemental Figure  S6, C-E), consistent with previous reports that loss of the Golgi-derived MT network does not affect acetylation of non-Golgi MTs (Rivero et al., 2009). This suggests that FHDC1 activity was not generally inhibited in GM130-depleted cells and that the centrosome-derived MT network remains intact ( Figure 12B). We also found that GM130 depletion neither enhanced nor suppressed the effects on Golgi dispersion of an FHDC1 derivative lacking the MTBD (FHDC1.958N; Figure 12E). This is consistent with the protective effects of GM130 depletion being dependent on the loss of Golgi-derived MTs. Together these data point to a model in which the ability of FHDC1 to affect Golgi morphology depends on its ability to bind to the Golgi-derived MT network and to actin filaments. In support of this model, we find that the full-length FHDC1 protein is able to bind directly to both F-actin and MTs in vitro (Supplemental Figure S7).

FHDC1 depletion inhibits Golgi assembly
Given the dramatic effect that FHDC1 expression has on Golgi morphology, we sought to determine whether FHDC1 activity is required for Golgi ribbon formation. We transfected NIH 3T3 cells with control siRNA duplexes or duplexes targeting FHDC1. The efficiency of FHDC1 knockdown was assessed by immunoblotting ( Figure 13F), and the effects on Golgi morphology were determined by immunofluorescence, using GM130 as a Golgi marker. As expected, a normal perinuclear Golgi ribbon was observed in the majority of control cells (Figure 13, A and E). In contrast, major defects in Golgi assembly were observed in >80% of cells transfected with the FHDC1 siRNA duplex. The defects fell into three categories: cytoplasmic Golgi dispersion ( Figure 13B), perinuclear compact Golgi circles (Figure 13C), and a diverse group of fragmented Golgi structures ( Figure 13D). Knockdown of FHDC1 with a second siRNA duplex yielded similar results ( Figure 13E). Each of these phenotypes is consistent with defects in the function of the Golgi-derived MT network during Golgi assembly Vinogradova et al., 2012), which suggests that FHDC1 plays an important role in this process.

DISCUSSION
Our findings indicate that FHDC1 likely acts at the Golgi-derived MT network to regulate Golgi assembly. Golgi-derived MTs are highly acetylated (Thyberg and Moskalewski, 1993), and, as noted fragmented but retained its perinuclear position ( Figure 12D). Despite this fragmentation, knockdown of GM130 expression actually protected the Golgi from the effects of FHDC1; in GM-130 depleted cells, normal Golgi ribbons were observed in >30% of transfected from the abundant cytoplasmic microtubules present in newly attached cells. Conversely, in BFA-treated cells, the association of FHDC1 with MTs is reduced, and BFA treatment largely prevents the reassociation of FHDC1 with MTs during recovery from nocodazole treatment. Consistent with the effects of BFA on MT binding by FHDC1, we find that endogenous FHDC1 is efficiently recruited to MTs in centrosome-depleted cells, but MT binding is reduced in GM130-knockdown cells, where Golgi-derived MT formation should be inhibited (Rivero et al., 2009;Rios, 2014). Two major phenotypes were observed in FHDC1-depleted cells: Golgi dispersion and formation of compact Golgi circles. Both of these are consistent with defects in the G phase of Golgi assembly as observed either during recovery from nocodazole treatment  or during postmitotic Golgi reassembly (Vinogradova et al., 2012;Maia et al., 2013).
As with FHDC1 depletion, FHDC1 overexpression also has major effects on Golgi structure. Expression of the full-length FHDC1 protein induces Golgi dispersion. The dispersed Golgi fragments most likely represent Golgi ministacks and are still able to support most aspects of Golgi trafficking. It is unlikely that FHDC1-induced dispersion arises as a secondary effect of FHDC1-induced changes in MT dynamics. First, there is no correlation between the ability of FHDC1 to induce MT acetylation and the ability to induce Golgi dispersion. All of the tested FHDC1 derivatives induce MT acetylation to a similar extent (Young et al., 2008;Thurston et al., 2012), but only the full-length protein induces dispersion. Second, dispersion is unrelated to effects on MT stability: the isolated FHDC1 MTBD is a much more effective inducer of MT stabilization than the fulllength protein (Young et al., 2008), yet expression of the isolated MTBD does not induce Golgi dispersion.
We also found that FH2 activity is required for the effects of FHDC1 on Golgi assembly and that these effects are actin dependent. It is not clear how FHDC1-induced changes in actin dynamics induce dispersion directly. Previous studies suggested that F-actin formation would be expected to induce Golgi compaction  rather than the dispersion we observe with FHDC1 expression. In addition, expression of either full-length FHDC1 or the 958N deletion derivative is sufficient to induce extensive thin stress fiber formation (Young et al., 2008), but 958N expression has only minimal effects on Golgi dispersion.
We propose that FHDC1-induced Golgi dispersion is not a simple consequence of disrupting normal actin or MT dynamics. Instead, FHDC1 expression disrupts a specific connection between Golgi, actin filaments, and the Golgi-derived MT network. This is consistent with the protective effect that GM130 knockdown has on FHDC1-induced Golgi dispersion. GM130 depletion should inhibit the nucleation of Golgi-derived MTs and would therefore remove the proposed site of FHDC1 action. In agreement with this model, we find that loss of GM130 does not modify the effects of FHDC1.958N expression on Golgi morphology. The 958N derivative lacks the MTBD, and therefore its activity should be unaffected by loss of Golgi-derived MTs, precisely as observed. It is likely that the overexpressed FHDC1 protein is able to bind both Golgi-and centrosome-derived MTs (Young et al., 2008), and our data do not exclude additional FHDC1 functions at centrosome-derived MTs, which may also be affected by GM130 knockdown (Kodani and Sutterlin, 2008;Kodani et al., 2009). GM130 depletion does not affect the ability of FHDC1 to induce MT acetylation, and thus not all aspects of FHDC1 activity are affected by the loss of GM130. Given that the loss of Golgi-derived MTs does not affect the acetylation of centrosome-derived MTs (Rivero et al., 2009) this suggests that the centrosome-derived MT network is still intact in GM130-depleted previously, FHDC1 preferentially associates with acetylated MTs (Young et al., 2008). We find that the endogenous FHDC1 protein accumulates on MTs in close association with the Golgi ribbon. This association is also observed during recovery from nocodazole treatment at cytoplasmic Golgi clusters. These clusters likely represent late G phase of Golgi assembly as Golgi-derived MTs are reformed , suggesting that FHDC1 is recruited to nascent Golgi-derived MTs. Similar results were obtained in spreading cells, where FHDC1 first accumulates at the Golgi and is largely absent tance for maintaining Golgi structure in these cells so as to assist in their primary function of generating and secreting ECM proteins. Our results identify a new role for FHDC1 in the assembly of the Golgi ribbon and suggest that FHDC1 plays an important role in the function of the Golgi-derived MT network during this process. How endogenous FHDC1 is preferentially recruited to Golgi-derived MTs is being investigated.
Cell culture, transfections, and treatments NIH 3T3 fibroblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the cells but does not contribute significantly to the effects of FHDC1 on Golgi assembly. This is entirely consistent with the observation that centrosome depletion does not affect FHDC1-induced Golgi dispersion.
Our results therefore point to a specific function for FHDC1 at the Golgi-derived MT network during Golgi assembly. We favor a model in which FHDC1 acts as a cytoskeletal bridging factor required to promote the stability of Golgi-derived MTs by coordinating actin and MT dynamics (Rodriguez et al., 2003;Gundersen et al., 2004;Dippold et al., 2009). In this scenario, FHDC1 would be recruited to the Golgi-derived MT network at the cis-Golgi, where it acts in an actin-dependent manner to stabilize the nascent MTs as they are handed over from the GM130/AKAP450/γ-TuRC complex at the cis-Golgi to GCC185/CLASP proteins at the trans-Golgi (Millarte and Farhan, 2012;Zhu and Kaverina, 2013). Our results suggest that FHDC1 would then remain bound to the Golgi-derived MTs as they extend into the cytoplasm and track along actin filaments. This model accounts for the accumulation of FHDC1 with cis-Golgi markers, the association of FHDC1 with the Golgi in spreading cells, and the ability of brefeldin A treatment or GM130 depletion to inhibit MT binding by FHDC1. It also accounts for the FH2/F-actin and MTBD/MT-dependent effects of FHDC1 expression on Golgi assembly and is consistent with the Golgi phenotypes observed in FHDC1-depleted cells.
In conclusion, the assembly of a perinuclear Golgi ribbon is a vertebrate evolutionary innovation whose function has still not been clearly defined (Nakamura et al., 2012). FHDC1, like the Golgi ribbon, is also unique to the vertebrate lineage. It was proposed recently that ribbon assembly is required to facilitate the efficient transport of large cargo such as extracellular matrix (ECM) proteins (Lavieu et al., 2014). We reported previously that FHDC1 is highly expressed in fibroblasts (Young et al., 2008). It is therefore interesting to speculate that FHDC1 activity may be of particular impor-  gies and Dharmafect1 transfection reagent according to the supplier's protocol. We used FHDC1 siRNA duplex 1, 5′-GCUAUAG-CACCAAAGAGAAAUUCCT-3′ and 5′-AGGAAUUUCUCUUUG-GUGCUAUAGCAU-3′ (Young et al., 2008); duplex 2, 5′-CCAUC-GUAGAGGAUAUCUATT-3′ and 5′-UAGAUAUCCUCUACGAUG-GAC-3′; and GM130 siRNA duplex, 5′-GCAUGUCAAGAAA-GAGCUAGCC CGG-3′ and 5′-CCGGGCUAGCUCUUUCUUGACA-UGCUG-3′. Efficient knockdown of FHDC1 was obtained by performing the initial transfection, followed by a repeat transfection 48 h later. Cells were fixed 48 h after the second transfection. For FHDC1 expression in GM130 knockdown cells, the cells were transfected with siRNA duplex, replated after 48 h, and transfected supplied guidelines in DMEM (Wisent, St. Jean Baptiste, Canada) supplemented with 10% donor bovine serum (DBS; ATCC) in 5% CO 2 . Transient transfections were performed using polyethylenimine (PEI) as described previously (Young et al., 2008). Briefly, 1.5 μg of total plasmid DNA was diluted in 50 μl of OptiMEM, 5 μl of 1 mg/ml PEI was added, and the mixture was incubated for 25-30 min at room temperature. The DNA/PEI mix was added to cells in 1 ml of OptiMEM and left for 5 h under normal culture conditions. At the end of 5 h, the medium was replaced with 2 ml of the appropriate culture medium. siRNA-mediated knockdown was performed as previously described (Young et al., 2008) with Dicer substrate siRNA duplexes from Integrated DNA Technolo- (F) Whole-cell lysates from FHDC1-knockdown, control transfected, and untransfected cells were immunoblotted for FHDC1 and α-tubulin. In the gels shown, duplex 1 resulted in a 63% knockdown of FHDC1 protein levels relative to untransfected cells, and duplex 2 resulted in a 51% knockdown.
with the appropriate primary antibody in 0.03% Triton X-100 and 5% DBS in 1× PBS for 1 h at room temperature. The coverslips were washed three times in 1× PBS and then incubated with secondary antibody in the same solution for 1 h at room temperature. After being washed in 1× PBS, the coverslips were mounted in Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) and sealed with nail polish.

Microscopy
All microscopy was performed on a Zeiss AXIO Imager.Z1 with a Zeiss Apotome.2 structured illumination system for optical sectioning and using a 63× (numerical aperture [NA] 1.4) oil immersion lens or a 40× (NA 0.75) dry objective and a Zeiss AxioCam HRm camera (60N-C 1′′ 1.0X 426114) controlled with AxioVision (release 4.8.2; Zeiss). Coverslips were mounted in Vectashield (Vector Labs) with or without DAPI. Cy5 and Alexa Fluor 488 and 594 secondary antibodies were from Jackson ImmunoResearch. Figures were prepared in Adobe Photoshop and Adobe Illustrator.
with the appropriate plasmid the next day. Cells were fixed 96 h after the initial siRNA transfection.
Cells were treated with nocodazole for 2 h at 37°C at a final concentration of 2.5 μg/ml. After treatment, cells were washed five times in ice-cold complete medium and 37°C medium and allowed to recover for 20, 40, 60, and 120 min before fixation in methanol. Cells were treated with brefeldin A for 5, 10, 15, 10, and 45 min at a final concentration of 5.0 μg/ml, recovered by washing twice in complete medium, and fixed 5, 15, 30, 60, 90, and 120 min after inhibitor removal. In combined nocodazole/brefeldin A treatments, cells were incubated with nocodazole (2.5 μg/ml) and brefeldin A (5.0 μg/ml) for 2 h and then washed with ice-cold medium with or without brefeldin A, allowed to recover at 37°C, and then fixed in methanol at 20, 40, 60, and 120 min after removal of nocodazole. For latrunculin B treatment, cells were treated with the drug (2 μM) for 120 min before fixation. Cells were treated with 300 nM centrinone for 14 d to induce centrosome depletion, as previously described (Wong et al., 2015).

Cell-based assays
Scratch wounds were created in confluent cells using a sterile P200 pipette tip. Three wounds were created per coverslip, and cells were fixed in methanol at 0, 1, 2, 4, and 6 h after wounding. For spreading-cell experiments, cells were trypsinized, reseeded at 19,000 cells/cm 2 , and fixed in methanol 1, 2, and 4 h after plating.
The Golgi was scored as "dispersed" when the ribbon was fragmented into >30 units with an average fragment size of ≤1 μm and there was loss of polarized distribution of the fragments. The Golgi was scored as "intermediate" if either it was fragmented but still had an asymmetrical perinuclear position or the ribbon was intact (≤25 subunits, ≥2 μm in size) but perinuclear polarity was lost. An intact ribbon obviously located to one side of the nucleus was scored as "normal." To control for cell-cycle effects on Golgi morphology, we included a separate mCherry-transfected control sample as a comparator. The initial quantification of the effects of FHDC1 overexpression on Golgi morphology were confirmed by a second observer working blind with respect to the identity of the sample.
SRF reporter gene assays were performed as in Copeland et al. (2007). Briefly, 50 ng of the SRF reporter p3D.ALuc and 0.25 μg of the transfection control reporter pMLV-LacZ were transfected for each sample. Cells transfected with 50 ng of pEF-SRF.VP16 were included as a positive control standard and set to 100% in each reporter gene experiment. After transfection, cells were incubated overnight in DMEM plus 0.5% DBS and harvested in reporter gene lysis buffer (Promega).
To monitor VSV-G trafficking, cells were transfected with 0.25 μg of pCDM8.1-VSVG.ts045-GFP (Presley et al., 1997) as described. After 16 h, transfected cells were shifted to 42°C for 6 h. Cycloheximide (20 μg/ml final concentration) was added 5 min before the shift to 32°C. The temperature shift was performed by changing to 32°C medium also containing cycloheximide. Cells were fixed in methanol at 30, 60, and 90 min after the 32°C shift.

Immunofluorescence
Cells were prepared for immunofluorescence as in Young et al. (2008). Briefly, cells cultured on acid-washed glass coverslips were fixed for 10 min directly in 2 ml of ice-cold methanol or 4% paraformaldehyde freshly prepared in PHEM (60 mM PIPES, 21 mM HEPES, 10 mM EGTA, 2 mM MgCl 2 , pH 7.0) buffer (Schliwa and van Blerkom, 1981). After fixation, the cells were permeabilized and blocked for 20 min in 0.3% Triton X-100 and 5% DBS in 1× phosphate-buffered saline (PBS). The coverslips were washed in 1× PBS and incubated


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
A Novel Protective Function of 5-Methoxytryptophan in Vascular Injury

5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of tryptophan metabolism, was recently shown to suppress inflammatory mediator-induced cancer cell proliferation and migration. However, the role of 5-MTP in vascular disease is unknown. In this study, we investigated whether 5-MTP protects against vascular remodeling following arterial injury. Measurements of serum 5-MTP levels in healthy subjects and patients with coronary artery disease (CAD) showed that serum 5-MTP concentrations were inversely correlated with CAD. To test the role of 5-MTP in occlusive vascular disease, we subjected mice to a carotid artery ligation model of neointima formation and treated mice with vehicle or 5-MTP. Compared with vehicle-treated mice, 5-MTP significantly reduced intimal thickening by 40% 4 weeks after ligation. BrdU incorporation assays revealed that 5-MTP significantly reduced VSMC proliferation both in vivo and in vitro. Furthermore, 5-MTP reduced endothelial loss and detachment, ICAM-1 and VCAM-1 expressions, and inflammatory cell infiltration in the ligated arterial wall, suggesting attenuation of endothelial dysfunction. Signaling pathway analysis indicated that 5-MTP mediated its effects predominantly via suppressing p38 MAPK signaling in endothelial and VSMCs. Our data demonstrate a novel vascular protective function of 5-MTP against arterial injury-induced intimal hyperplasia. 5-MTP might be a therapeutic target for preventing and/or treating vascular remodeling.

Scientific RepoRts | 6:25374 | DOI: 10.1038/srep25374 5-Methoxytryptophan (5-MTP) is a 5-methoxyindole metabolite of tryptophan metabolism 9,10 . 5-Methoxyindoles such as melatonin, 5-methoxytryptamine, and 5-methoxytryptophol participate in the control of circadian rhythm in the pineal gland and retina [11][12][13][14][15] and are regarded as neuromodulators 16,17 . However, unlike these 5-methoxyindoles, the physiological function of 5-MTP is unclear, although it has been implicated a role in the pineal gland and retina 11,18 . Interestingly, we recently identified a function of 5-MTP in tissues other than pineal gland and retina. We found that 5-MTP could suppress inflammatory mediator-induced cancer cell proliferation and migration, thereby reducing tumor growth and metastasis 9,10 . Another study showed that 5-MTP is able to protect cardiomyocytes from H 2 O 2 -induced oxidative injury 19 . However, the role of 5-MTP in vascular disease is unknown. The pathogenic VSMC migration and proliferation are somewhat similar to that of cancer cell growth and invasion. We hypothesized that 5-MTP might protect against vascular injury-induced medial VSMC proliferation and migration and subsequent neointima formation and occlusive vascular remodeling.
The goal of the present study was to investigate whether 5-MTP has a role in vascular disease. Measurements of serum 5-MTP levels in healthy subjects and patients with coronary artery disease (CAD) showed that serum 5-MTP concentrations are inversely correlated with CAD. In a mouse carotid artery ligation model, we showed that 5-MTP significantly reduced neointima area after ligation, likely through attenuating VSMC proliferation and migration. In addition, 5-MTP decreased ligation-induced endothelial adhesion molecule expressions and inflammatory cell infiltration. Our results indicate that in response to arterial injury, 5-MTP protects against endothelial dysfunction and VSMC proliferation and migration, thereby reducing intimal hyperplasia. Taken together, 5-MTP might be a therapeutic target for preventing and/or treating vascular remodeling.

Results
Serum 5-MTP concentrations are inversely correlated with coronary artery disease. To investigate the clinical relevance of 5-MTP in human CAD, we measured serum 5-MTP levels in control subjects and CAD patients. Control subjects did not have any coronary vessel and known systemic disease. The presence of CAD was confirmed by coronary angiography and CAD was defined as more than 50% angiographic diameter stenosis in one or more coronary arteries. Patients who had diabetes, hypertension, or stroke were excluded from the study. Twenty control subjects (10 males and 10 females, mean age 59 ± 7 years) and 23 CAD patients (17 males and 6 females, mean age 61 ± 10 years) were included in the study. The body mass index and blood pressure were not different between control and CAD groups (Table 1). Serum was obtained from blood drawn from controls or patients prior to angiography for biochemical analysis (Table 1). Although serum creatinine levels were higher in CAD group but it did not reach a statistical significance. Patients in CAD group had higher serum glucose levels than those in the control group but they were not diabetic. Cholesterol and triglyceride levels were within normal ranges in both groups. Importantly, serum IL-6 levels, an indicator of inflammation, were significantly higher in CAD group ( Table 1). Measurements of 5-MTP with enzyme-immunoassays revealed that the mean serum 5-MTP levels from controls were 0.78 ± 0.15 μ mol/L (Fig. 1a). In contrast, serum 5-MTP concentrations of CAD patients were significantly reduced to 0.22 ± 0.13 μ mol/L (Fig. 1a, P < 0.0001 vs. control). These results indicate that serum 5-MTP concentrations are inversely correlated with CAD, implicating a potential role of 5-MTP in occlusive vascular disease.

5-MTP inhibits smooth muscle cell proliferation after arterial injury.
We next investigated the mechanisms by which 5-MTP protects against neointima formaton. Proliferation and migration of VSMCs from the media into intima contributes to arterial intimal thickening and subsequent arteriosclerosis 2,4 . In the carotid artery ligation model, VSMC proliferation is a prominent feature of neointima formation and the index is high in the media and intima one week after ligation 20,21 . Thus, we examined neointima formation at this time point. As reported previously 22 , one week after ligation neointima was evident, although small, in vehicle-treated mice (Fig. 3a,c). In comparison, 5-MTP substantially reduced neointimal area (Fig. 3b,d) and significantly decreased the intima/media ratio from 0.50 ± 0.16 of vehicle group (n = 6) to 0.16 ± 0.04 (n = 9; P < 0.05; Fig. 3e).
To examine whether the reduced neointima formation by 5-MTP was due to decreased proliferation, mice were injected with BrdU at 16-18 h and 1-2 h before harvest. We then assessed BrdU incorporations by immunostaining vessel sections with BrdU antibodies. Many BrdU-positive cells were detected in the neointima and media from vehicle-treated ligated carotids (Fig. 3f,h) whereas 5-MTP treatment decreased BrdU-positive cell number in the neointima and media (Fig. 3g,i). Quantitative analysis showed that proliferation index was much higher in vehicle-treated vessels with 21.4 ± 3.0% BrdU incorporation in the neointima and 9.2 ± 2.9% in the  (Fig. 3j, white bars). In 5-MTP-treated vessels, we observed 7.3 ± 2.1% of BrdU incorporation in the neointima (P < 0.05 vs. vehicle group) and 3.1 ± 1.0% in the media (P < 0.05 vs. vehicle group; Fig. 3j, black bars), indicating reduced cellular proliferation in 5-MTP-treated mice in both neointima and media.

5-MTP inhibits IL-1β-induced smooth muscle cell proliferation and migration.
To further confirm that the reduced intimal hyperplasia was due to decreased VSMC proliferation but not increased cell death by 5-MTP, we treated VSMCs with 5-MTP and assessed cell viability. Increasing concentrations of 5-MTP, even up to 500 μ mol/L, did not exert any cytotoxic effect (Fig. 4a), ruling out the possibility that 5-MTP affected cell viability. Proinflammatory cytokine IL-1β has been shown to play a critical role in neointima formation following vessel injury, such as balloon angioplasty 23 , vein graft remodeling 24 , or carotid artery ligation 25,26 . Indeed, we found that carotid ligation induced IL-1β expression in the vessel wall (Fig. 4b,c). Furthermore, IL-1β stimulated VSMC proliferation in a concentration-dependent manner (Fig. 4d). Given the critical role of IL-1β in vessel injury and VSMC proliferation, we pretreated VSMCs with different concentrations of 5-MTP, stimulated with or without IL-1β , and then assessed proliferation. In the absence of IL-1β , 5-MTP did not affect VSMC proliferation (Fig. 4e). In contrast, 5-MTP significantly decreased IL-1β -induced proliferation (Fig. 4e). Intriguingly, 5-MTP did not affect growth factor PDGF-BB-induced VSMC proliferation (Fig. 4f), suggesting 5-MTP preferentially affects IL-1β -mediated effects. In addition to proliferation, VSMC migration contributes to intimal hyperplasia. We then performed migration assays to test whether 5-MTP affects VSMC migration in response   to chemoattractant stimulation. Interestingly, IL-1β treatment increased VSMC migration while 5-MTP abrogated the enhanced migration of IL-1β (Fig. 4g). Taken together, these results indicate that 5-MTP attenuates IL-1β -induced VSMC proliferation and migration and reduces neointima formation following arterial injury.

5-MTP reduces carotid artery endothelial damage after ligation. Endothelial damage/dysfunction
is one of the initial events and an underlying cause of vascular remodeling/disease. Therefore, we hypothesized that in addition to VSMCs, 5-MTP might also protect endothelium against injury and subsequent neointima formation. To test our hypothesis, we examined carotid arteries at an early time point (4 d) after ligation. In the carotid artery ligation model, although endothelium is not removed it is frequently found to be detached from the underlying internal elastic lamina at an early time point 20 . Elastin staining showed intact unligated arteries ( Fig. 5a,b). However, 4 d after ligation we observed endothelium detachment in vehicle-but not 5-MTP-treated ligated arteries (Fig. 5c,d). Endothelial marker CD31 staining demonstrated intact endothelium in the unligated arteries (Fig. 5e,f). In contrast, detachment and loss of endothelium in vehicle-treated ligated arteries (Fig. 5g,i) were obvious while endothelium was relatively intact or with minor damage from mice treated with 5-MTP (Fig. 5h,j). Quantitative analysis revealed that ligation caused 20.1 ± 7.4% endothelial detachment and 58.9 ± 9.3% denudation whereas 5-MTP reduced endothelial detachment and denudation to 4.7 ± 3.1% and 24.7 ± 8.1%, respectively (P < 0.05 vs. vehicle group, n = 5 each group) (Fig. 5k), indicating 5-MTP protects against endothelial injury.

5-MTP decreases endothelial ICAM-1 and VCAM-1 expressions in the ligated arteries.
In light of the apparent morphological improvement of ligated carotid endothelium in 5-MTP-treated mice, we next determined whether 5-MTP treatment protects against endothelial activation/dysfunction. Immunohistochemistry revealed that adhesion molecule ICAM-1 was normally expressed in the endothelium of unligated control arteries (Fig. 6a,b). Despite that ligation resulted in certain degree of endothelial loss (Fig. 5i,j), ligation enhanced ICAM-1 expression by 3.0 ± 0.6 fold in the remaining endothelium (Fig. 6c,e). In comparison, 5-MTP treatment significantly reduced ICAM-1 expression to 1.6 ± 0.2 fold of control (P < 0.05 vs. vehicle group, n = 5 each group) (Fig. 6d,e). Another adhesion molecule VCAM-1 was not expressed in the control unligated arteries (Fig. 6f,g), but markedly induced in the vehicle-treated ligated arteries, not only in endothelium but also in medial layer (7.1 ± 1.9% area of endothelium and media; Fig. 6h,j). The upregulation of VCAM-1 in medial layer after injury is consistent with the notion that VCAM-1 induction in VSMCs has a role in vascular disease 3,34 . Importantly, 5-MTP decreased ligation-induced VCAM-1 expression in the endothelium and medial layer to 2.5 ± 1.1% (P < 0.05 vs. vehicle group, n = 5 each group) (Fig. 6i,j).
We next investigated potential signaling mechanisms that mediate the inhibitory effect of 5-MTP on IL-1β -induced adhesion molecule expressions. As with VSMCs, IL-1β substantially induced phosphorylation of p38 MAPK, JNK, and NFκ B-p65, but rather moderate of ERK1/2, in endothelial cells (Fig. 7e). Interestingly, 5-MTP predominantly reduced phosphorylation of p38 MAPK, but did not have much effect on ERK1/2, JNK, or NFκ B-p65 phosphorylations (Fig. 7e). These results suggest that the decreases of endothelial adhesion molecule expressions by 5-MTP might be mediated through attenuated p38 MAPK activation.

5-MTP reduces inflammatory cell adhesion and infiltration to the vessel wall after carotid artery ligation.
ICAM-1 and VCAM-1 are important for leukocyte adhesion and trans-endothelial migration 35 . Given that 5-MTP attenuated the induction of ICAM-1 and VCAM-1 after arterial injury, 5-MTP might attenuate inflammatory cell adhesion to the luminal surface and infiltration into the vessel wall. We therefore stained vessel sections with CD45 antibodies to identify inflammatory cells. CD45-positive cells were not detectable in unligated arteries (Fig. 8a,b). In vehicle-treated ligated arteries, we detected many CD45-positive cells adhered to the endothelium and in the enlarged subendothelial space, and some infiltrated into medial layer (Fig. 8c,f). We also observed CD45-positive cells adhered to the denuded luminal surface (Fig. 8d,g). In contrast,   (Fig. 8e,h). Quantitative analysis revealed that 5-MTP decreased CD45-positive cell number from 17 ± 4 to 4 ± 2 per vessel section (P < 0.05 vs. vehicle group, n = 5 each group; Fig. 8i), indicating 5-MTP protects vascular endothelium from injury-induced activation/dysfunction and subsequent inflammatory cell adhesion and infiltration.

Discussion
We demonstrated in this study a novel protective function of 5-MTP in vascular disease. We showed that CAD patients exhibit significantly lower levels of serum 5-MTP than control subjects. In a mouse model, 5-MTP protects against arterial injury-induced neointima formation through attenuating vascular injury-induced VSMC proliferation and migration and endothelial dysfunction. Furthermore, the protective effects of 5-MTP on vascular cells are predominantly mediated through p38 MAPK signaling pathway.
Various metabolites and their metabolic intermediates and byproducts participate in cellular processes. Metabolic aberrations and their downstream effects contribute to the pathogenesis of many disorders 36 . For example, succinate accumulation from ischemic insults contributes to reperfusion injury in mouse models of heart attack and stroke 37 . The tryptophan metabolite serotonin is a well-known neurotransmitter; however, a recent report showed that peripheral serotonin contributes to obesity and metabolic dysfunction 38 . On the other hand, organisms also exhibit intrinsic defense mechanisms to protect cells and tissues against pathophysiological stress and/or to repair damage. Tryptophan metabolites are involved in diverse physiological functions, including regulating circadian rhythms and reproduction 12,39 . In contrast to investigations in the immune and nervous systems of tryptophan metabolites 40,41 , study of tryptophan metabolism in vascular disease has been limited [42][43][44][45] . Nonetheless, it has been reported that coronary heart disease patients have lower serum tryptophan concentrations while the ratio of its metabolite kynurenine to tryptophan ratio is increased 43 . Another study showed that kynurenine pathway metabolites are associated with endothelial dysfunction markers and carotid artery intima media thickness values in patients with chronic kidney disease 46 . These studies suggest that activation of tryptophan metabolic pathways might be involved in the pathogenesis of cardiovascular disease. In line with this notion and an intrinsic defense mechanism of organisms, we found that in contrast to kynurenine, another tryptophan metabolite 5-MTP has a protective function in vascular disease. Supporting this concept, serum 5-MTP levels in humans are inversely correlated with CAD (Fig. 1a). It is tempting to speculate that reduced 5-MTP levels might contribute in part to the development of occlusive vascular disease. 5-MTP reduces intimal hyperplasia after carotid ligation (Figs 2 and 3). Consistent with the notion that development of neointima is mainly characterized by migration and proliferation of VSMCs, 5-MTP not only decreases ligation-mediated neointimal and medial VSMC proliferation in vivo (Fig. 3f-j) but also inflammatory cytokine IL-1β -induced VSMC proliferation and migration in vitro (Fig. 4e,g), suggesting a direct effect of 5-MTP on VSMCs. Intriguingly, 5-MTP did not inhibit PDGF-BB-induced VSMC proliferation (Fig. 4f), indicating 5-MTP preferentially influences IL-1β -mediated cellular effects. One early phase in the development of vascular disease involves adhesion of inflammatory cells to the vascular endothelium and subsequent infiltration into the subendothelial space and vessel wall, which are mediated through cellular adhesion molecules 47  concept, we found that 5-MTP reduces ligation-induced ICAM-1 and VCAM-1 expressions (Figs 6 and 7) and the consequent inflammatory cell recruitment and infiltration (Fig. 8). Thus, 5-MTP reduces ligation-induced inflammatory responses, including endothelium detachment and denudation at an early time point (Fig. 5). These findings are in accordance with the notion that 5-MTP attenuates inflammation and the consequent reduction of proinflammatory gene expressions 9,10 .
Signaling pathway analyses reveal that 5-MTP attenuates IL-1β -induced VSMC proliferation via suppressing p38 MAPK and ERK1/2 activation, particularly p38 MAPK (Fig. 4h). Supporting our findings, it has been shown that p38 MAPK is activated after injury and promotes neointimal formation 32 . Interestingly, in endothelial cells 5-MTP mainly decreases IL-1β -induced p38 MAPK phosphorylation (Fig. 7e). It is of interest that in endothelial cells p38 MAPK inhibitor RWJ-67657 blocked IL-1β -induced expression of ICAM-1 and VCAM-1 at 24 h 48 . Together, these results indicate that the anti-inflammatory effects 5-MTP might be mediated predominantly through p38 MAPK pathway in vascular cells.
In conclusion, we have identified in the present study a novel protective function of 5-MTP in vascular disease. Serum 5-MTP levels are inversely correlated with CAD in humans. 5-MTP prevents VSMC proliferation and migration, endothelial damage, and the consequent intimal hyperplasia and vascular remodeling. Based on these results, 5-MTP might be a valuable lead compound for developing new strategy for preventing and/or treating vascular injury and intimal hyperplasia.

Methods
Patient enrollment and measurement of serum 5-MTP levels. Patients with coronary artery disease (CAD) and control subjects were enrolled in the study between September 2013 and July 2015. The Ethics Committee on Human Studies at Tri-Service General Hospital, National Defense Medical Center in Taiwan approved the study protocol (TSGHIRB # 2-102-05-104 and 2-102-05-105) and written informed consent was obtained from all participants. The study was conducted in accordance with the Declaration of Helsinki. The presence of CAD was confirmed by coronary angiography and CAD was defined as more than 50% angiographic diameter stenosis in one or more coronary arteries. CAD patients who had diabetes, hypertension, or stroke were excluded from the study. Twenty-three patients with CAD (17 males and 6 females) and 20 control subjects (10 males and 10 females) without CAD and known systemic disease were included in the study. Blood was drawn from patients prior to angiography and serum collected and stored at − 80 °C until analysis. Clinical Characteristics of study participants were measured and are shown in Table 1. 5-MTP was measured by a competitive ELISA in a 96-well microtiter plate coated with polyclonal rabbit anti-5-MTP antibodies (Abcam, ab6476) using a coating buffer of 0.05 mol/L carbonate-bicarbonate (pH 9.6) at 4 °C overnight. After PBST washing and treatment with blocking buffer, mixture of 5-MTP-conjugated HRP generated by using a NH 2 peroxidase labeling kit (Abnova) and 5-MTP standards, serum samples were added to the wells and incubated at 4 °C overnight. The wells were washed and treated with a substrate tetramethylbeuzidine at room temperature for 30 min. Reaction was stopped with 0.1N H 2 SO 4 and the product was measured at 450 nm. The calibration curve was established by using pure 5-MTP at concentrations from 0.01 to 50 μ mol/L.
Measurement of blood pressure in mice. C57BL/6 wild-type mice (National Laboratory Animal Center, Taiwan) were used for experiments. A noninvasive tail-cuff method was used to measure systolic blood pressure (SBP) using a non-preheating MK-2000ST system (Muromachi Kikai Corp.). Conscious mice (~12-week-old male mice) were placed in special mouse holders and acclimated to the device for 10 min before measurement. A minimum of 3 serial measurements was made and the average value calculated. The SBP of each mouse was measured at baseline before IP injection of vehicle (PBS) or 5-MTP (Sigma, M4001) (100 mg/kg, 3 times a week 9 ) and at different time points following initial injection. 5-MTP was dissolved in 0.05 N HCl (made in PBS) first, pH adjusted to 7.4 by NaOH, and then a stock solution of 6.5 mg/mL was prepared with PBS. week-old male C57BL/6 wild-type mice were subjected to a neointima formation model by ligating the left common carotid artery (LCCA) as described 22 . All experimental procedures were performed in accordance with NIH guidelines (Guide for the care and use of laboratory animals) and approved by the Institutional Animal Care and Use Committee of National Health Research Institutes, Taiwan (#NHRI-IACUC-101144-A). Mice were anesthetized with tribromoethanol solution at a dose of 250 mg/kg by IP injection. After assessing the level of anesthesia by checking the pedal reflex, the LCCA was exposed through a midline incision in the neck. The LCCA was then ligated near the carotid bifurcation with a suture, skin closed, and the animals were allowed to recover from anesthesia and showed no symptoms of stroke. Following surgery, mice were treated with vehicle (PBS) or 5-MTP (100 mg/kg) by IP injection 3 times a week 9 . At indicated time points, mice were sacrificed by an overdose of tribromoethanol solution (500-750 mg/kg) by IP injection, perfused with saline, followed by 10% neutral-buffered-formalin. The contralateral right common carotid artery (RCCA, as control) and ligated LCCA were then carefully dissected, excised, and immersed in 10% formalin before processing and embedding in paraffin.

Neointima formation model of carotid artery ligation in mice.
Histological analysis and immunohistochemistry. Vessel sections (4 μ m) were stained with H&E for morphology. To delineate elastin layers, we stained sections with Verhoeff 's stain (Sigma) for elastin. Three sets of sections at 150-μ m intervals were used for morphometry. The intimal and medial areas were measured using NIH ImageJ software. The average intimal area, medial area, and intima-to-media ratio were calculated. To determine cellular proliferation in the injured vessel wall, mice were injected twice with BrdU (Sigma) at 16-18 h and 1-2 h before harvest. Vessel sections were stained with a monoclonal BrdU antibody (Dako) to detect proliferating cells and counterstained with hematoxylin. Immunostained positive cells were quantified as the number of positive cells divided by total number of nuclei in the neointima or media, and expressed as % of total cells.
Immunostaining was performed on sections with IL-1β antibody (R&D) to detect IL-1β expression. To detect endothelium, sections were incubated with CD31 antibody (Abcam). To examine the extent of endothelial damage, we assessed endothelial detachment and denudation. Three sets of sections at 150-μ m intervals were used for morphometry. For endothelial detachment, CD31-positive endothelium that was detached from internal elastic lamina (IEL) was divided by the circumference of the lumen and multiplied by 100. For endothelial denudation, CD31-negative luminal lining was divided by the circumference of the lumen and multiplied by 100. The expressions of adhesion molecules, ICAM-1 and VCAM-1, were examined by incubating sections with ICAM-1 (R&D) or VCAM-1 antibodies (Santa Cruz), respectively, and measured by colorimetric analysis using NIH ImageJ software. To measure ICAM-1 expressions, we set a threshold just below unligated vessel staining and calculated positively stained pixel number in the endothelium 49 . The number of positively stained pixels was divided by total pixel number of endothelium. The ICAM-1 expression level is then expressed relative to unligated vessels. For VCAM-1, the number of positively stained pixels in the endothelium and media was divided by the total number of pixels and then multiplied by 100. To detect inflammatory cells, sections were incubated with an antibody against the common leukocyte antigen, CD45 (BD Pharmingen). To assess inflammatory cell accumulation, the total number of CD45-positive cells at three sets of sections at 150-μ m intervals was counted and averaged. VSMC culture, viability, proliferation, and migration assays. Primary VSMCs were isolated from mouse aortas and cultured in DMEM as described 50 and cells of passages 5-8 were used for experiments. To determine the effect of 5-MTP on cell viability, VSMCs were plated in 24-well plates (1.6 × 10 6 cells/well), serum starved in quiescent medium (0.4% FBS) containing different concentrations of 5-MTP for 24 h. MTT assays were then performed and cell viability presented as a percentage of control without 5-MTP. To determine the effect of IL-1β on cell proliferation, VSMCs were plated in 24-well plates (8 × 10 3 cells/cm 2 ), serum starved for 24 h, and then treated with different concentrations of IL-1β (PeproTech) along with bromodeoxyuridine (BrdU) for 24 h. Proliferation was then assessed according to manufacturer's instructions (Millipore). To assess the effect of 5-MTP on IL-1β -induced proliferation, VSMCs were plated, serum starved in the presence of vehicle (DMSO) or 5-MTP, and then treated with 10 ng/mL IL-1β along with BrdU for 24 h, and proliferation assessed. To determine the effects of MAP kinases on VSMC proliferation, VSMCs were pretreated with different concentrations of p38 MAPK inhibitor SB203580 (InvivoGen), SB202190 (Sigma), or MEK inhibitor U0126 (Invivogen) 30 min prior to IL-1β stimulation. BrdU incorporation assays were then performed 24 h later to assess proliferation.
To assess migration, VSMCs were serum-starved for 24 h in the absence or presence of 5-MTP (100 μ mol/L) and then treated with or without IL-1β (10 ng/mL) for 24 h. Cells were placed in the upper chamber of 24-well transwell plates (Millipore, 8-μm pore size) in triplicate (10,000 cells/well). The bottom chambers were filled with quiescent medium containing PDGF-BB (Peprotech, 10 ng/mL) as a chemoattractant. After 4 h, the upper layer was scraped free of cells, membrane fixed, and stained with DAPI (Sigma). Cells that had migrated to the underside of the membrane were visualized using a fluorescent microscope. Cell number of the whole membrane was counted using × 40 images and normalized to control without IL-1β and 5-MTP treatment.
Phosphorylation of MAP kinases and NFκB and p38 MAPK activity assays. To evaluate the effect of IL-1β and 5-MTP on MAPK and NFκ B phosphorylation, VSMCs were serum starved for 24 h in the presence of vehicle (PBS) or 5-MTP (100 μ mol/L), and then stimulated with or without IL-1β (10 ng/mL) for 15 min. Total proteins were then extracted for Western blotting to detect phosphorylated p38 MAPK, ERK1/2, JNK, and NFκ B-p65 (Ser536) using phospho-antibodies (Cell Signaling Technology). The blots were subsequently probed with a pan-actin antibody (Millipore) to verify equivalent loading. To measure p38 MAPK activity, a p38 MAPK activity assay kit (Cell Signaling Technology, #9820) was used by immunoprecipitating active p38 with phospho-p38 antibody, followed by measuring phosphorylation of its substrate ATF2. Briefly, VSMCs were serum starved for 24 h in the absence or presence of 5-MTP (100 μ mol/L), and then stimulated with or without IL-1β (10 ng/mL) for 15 min. Cell lysates were prepared and incubated with immobilized phospho-p38 antibody overnight at 4 °C. Immunocomplex kinase assays were performed at 30 °C for 30 min using ATF2 fusion protein and ATP in kinase buffer. The reaction was terminated with SDS sample buffer and the p38 activity was determined by phospho-ATF2 immunoblotting. Aliquots of cell lysates from different treatment groups were subjected to immunoblotting with p38 MAPK antibody (Cell Signaling Technology) to detect total p38 and verify equivalent amount of protein for immunoprecipitation.

Endothelial cell culture, viability, detection of adhesion molecules, and phosphorylation of MAP kinases and NFκB. Primary human umbilical vein endothelial cells (HUVECs) (Bioresource Collection and
Research Center, Hsinchu, Taiwan) were grown in endothelial basal media (Lonza), supplemented with endothelial growth media (EGM)-2 SingleQuot Kit (Lonza) containing defined supplements. Cells of passages 5-8 were used for experiments. To determine the effect of 5-MTP on HUVEC viability, cells were seeded at a density of 1.6 × 10 4 cells per well of 96-well plate. After overnight incubation, HUVECs were treated with indicated concentrations of 5-MTP in culture medium for 24 h. MTT assays were then performed and cell viability was presented as a percentage of control without 5-MTP.
To evaluate the effect of IL-1β and 5-MTP on endothelial adhesion molecule expressions, HUVECs were plated at a density of 5 × 10 4 cells/cm 2 and incubated overnight. Following 24 h vehicle (DMSO) or 5-MTP (100 μ mol/L) pretreatment, cells were stimulated with indicated concentrations of IL-1β for 24 h, and total proteins isolated for Western blot analysis. To detect adhesion molecules, membranes were probed with ICAM-1 (R&D) and VCAM-1 (Santa Cruz) antibodies. The blots were subsequently probed with a pan-actin antibody (Millipore) to verify equivalent loading. To examine the effect of IL-1β and 5-MTP on phosphorylation of MAP kinases and NFκ B, HUVECs were pretreated with vehicle (PBS) or 5-MTP (100 μ mol/L) for 24 h, and then stimulated with different concentrations of IL-1β for 15 min. Total proteins were then prepared for Western blotting. Phosphorylations of p38 MAPK, ERK1/2, JNK, and NFκ B were detected using antibodies as described for VSMCs. The blots were subsequently probed with a α -tubulin antibody (GeneTex) to verify equivalent loading.
Statistical analysis. Data are presented as mean ± S.E. of at least three independent experiments. All results were analyzed statistically by Student's t-test. P values < 0.05 are considered statistically significant.