A Role for Polo-Like Kinase 4 in Vascular Fibroblast Cell-Type Transition

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SUMMARY
Polo-like kinase 4 (PLK4) is canonically known for its cytoplasmic function in centriole duplication. Here we show a noncanonical PLK4 function of regulating the transcription factor SRF's nuclear activity and associated myofibroblast-like cell-type transition. In this context, we have further found that PLK4's phosphorylation and transcription are respectively regulated by PDGF receptor and epigenetic factor BRD4. Furthermore, in vivo experiments suggest PLK4 inhibition as a potential approach to mitigating vascular fibrosis.   (3,4). Recent reports implicate PLKs in fibrogenic processes. For example, PLK1 is found to be a target gene of FoxM1, a transcription factor that promotes lung fibrosis (5,6). This raises the question of whether fibroblast cell-type transitions involved in fibrosis are regulated by one or more PLKs.
A well known fibroblast cell-type change is its transition into myofibroblast, a fibrogenic process involved in numerous disease conditions (7).
Although the definition of myofibroblasts is still debated, these cells are generally characterized by smooth muscle-like morphologies and proliferative/ migratory behaviors (8). Moreover, they often exhibit high levels of a-smooth muscle actin (aSMA), vimentin, platelet-derived growth factor receptor a (PDGFRa), and extracellular matrix proteins (e.g., collagen). Whereas a variety of cell types can differentiate into myofibroblasts (7), resident fibroblasts have been confirmed as the main source in recent in vivo lineage tracing studies, at least in some vital organs such as the heart (8,9). It is therefore important to identify the regulatory mechanisms in fibroblast cell-type transition. This complex process involves extracellular and cell membrane signaling, cytosolic pathways, epigenetic and transcriptional remodeling, and interactions among these networks (8). The best known fibrogenic signaling pathway is transforming growth factor (TGFb1). In contrast, the PDGF pathways are less well understood (8). In particular, the PDGF-AA homodimer (hereafter denoted as AA), which selectively activates PDGFRa, is inadequately explored relative to PDGF-BB, which activates both PDGFRa and PDGFRb (10).
In the present study, we investigated a possible PLK regulation of vascular adventitial fibroblast celltype transition in the setting of AA-stimulated PDGFRa activation. We focused primarily on the divergent PLK member (PLK4) (3) and included PLK1, the representative member of the PLK family (11). We found that PLK4 inhibition constrained the rat aortic fibroblast proliferative/migratory behaviors and the nuclear activity of serum response factor (SRF), a master transcription factor (8). The latter finding was somewhat surprising, given that PLK4 is deemed to be centriole specific and cytosol localized. We also  or before passage 5 were used for experiments (Supplemental Figure S1). For induction of fibroblast  Eliminator columns provided in the kit. RNA was quantified with a Nanodrop NP-1000 spectrometer, and 1 mg was used for the first-strand cDNA synthesis.  Abbreviations as in Figure 1.
Li et al.  To induce adventitial fibrosis, balloon angioplasty injury was performed in rat common carotid arteries as we previously described (12).
Briefly, rats were anesthetized with isoflurane (5% for inducing and 2.5% for maintaining anesthesia).   Figure 1D). PLK4 inhibition with CenB also effectively attenuated TGFb1-stimulated vimentin upregulation (Supplemental Figure S2). Because this study was focused on PDGFRa pathways, we used AA as a stimulant throughout.
Importantly, we confirmed the PLK4 functional specificity. We did this by silencing PLK4 and showing that aSMA and vimentin protein levels were reduced ( Figure 2A). While it is intuitive that PLK4 as a centriole factor promoted cell proliferation (1), a role for PLK4 in elevating aSMA expression was somewhat unexpected, given PLK4's canonical association with centrioles in the cytoplasm.
We then investigated the mechanism underlying PLK4-stimulated aSMA expression with aSMA transcription is known to be driven by SRF, a master transcription factor. SRF is itself activated by myocardin-related transcription factor A (MRTF-A), a powerful transcription regulator that shuttles between the cytoplasm and nucleus (13). We therefore investigated the influence of PLK4 on MRTF-A protein and SRF transcriptional activity. We found that while treatment with AA elevated total MRTF-A protein levels, either PLK4 silencing (Figure 2A) or inhibition with CenB ( Figure 2B) prevented this elevation.
In the presence of CenB, aSMA was substantially reduced in both cytosol and nucleus, and AA could not increase nuclear MRTF-A protein (Supplemental Figure S3). Furthermore, the SRF transcriptional (luciferase) activity was diminished by CenB ( Figure 2C). These results revealed that, in rat aortic adventitial fibroblasts, PLK4 plays an important role in SRF activation and aSMA production, at least in part by elevating MRTF-A protein levels.
Taken together, our results indicate that PLK4 regulates fibroblast cell-type transition and, intriguingly, SRF nuclear activity, a function that apparently departs from centriole duplication. To the best of our knowledge, this noncanonical PLK4 function in SRF activation has not been previously reported. It is also noteworthy that pretreatment with CenB largely preserved normal fibroblastic phenotypes ( Figure 1) and did not cause obvious cell death even at high (e.g., 10 mmol/l) concentrations, suggesting a low cytotoxicity of this drug.
We also determined the effect of PLK1 inhibition on fibroblast phenotypes using the PLK1-selective inhibitor GSK461364 (Ki ¼ 2.2 nmol/l) (16). The result ( Figure 3) was similar to that of PLK4 inhibition ( Figure 1). However, the PLK1 inhibitor at high concentrations (e.g., 0.5 mmol/l) reduced cell viability to much below the nonstimulated basal level ( Figure 3C), consistent with its cytotoxicity (16). Nevertheless, the PLK4 loss-of-function and gain-offunction results (Figures 2 and 6) together indicate One-way ANOVA/Bonferroni test: #p < 0.05; ##p < 0.01; ###p < 0.001. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the vehicle control without AA stimulation (the first bar in each plot). Abbreviations as in Figures 1 and 4.   ( Figures 12E and 12F). aSMA; silencing BRD2 slightly reduced the protein level of PLK4 but not that of PLK1, PDGFRa, or aSMA; and silencing BRD3 did not reduce these proteins ( Figure 13A). The robust inhibitory effect of BRD4 silencing on PLK4 and the other 3 proteins is indicated by the quantified data in Figure 13B. Silencing either BRD2 or BRD4 reduced the mRNA levels of PLK4, PLK1, and PDGFRa (though to lesser extents with siBRD2) (Figures 13C and 13D). Given these results, BRD4 appeared to be the predominant BET governing the transcription of the 2 PLKs and PDGFRa.
We next explored a possibility as to whether BRD4 acted downstream of PLK4 in regulating fibroblast phenotypic transition. We first saw that inhibiting BRD4 with JQ1 abolished the up-regulation of MRTF-A protein as well as downstream effectors vimentin and aSMA ( Figure 14A), consistent with the result presented above (Figure 12). We then elevated PLK4 WT protein levels, and a remarkable increase of MRTF-A and aSMA resulted ( Figure 14B). This increase due to PLK4 gain-of-function was completely  arteries compared with vehicle control, as also confirmed by the immunohistochemistry of collagen III ( Figures 15D and 15E). Consistently, the adventitia thickness also significantly decreased after CenB treatment ( Figure 15C). Aside from collagen content measurement, immunofluorescence staining was performed to determine the levels of vimentin and aSMA, important markers of fibroblast activation.
Although vimentin expression mostly occurred in the artery adventitial layer where fibroblast (or its activated form) primarily resides, periadventitial treatment with CenB reduced vimentin protein levels in the artery wall by >70% (Figure 16). Marked decrease of aSMA staining in the adventitial layer was also observed as a result of CenB treatment ( Figure 17).

DISCUSSION
The major findings of this study are: 1) Though previously known as centriole-specific, PLK4 positively regulated MRTF-A protein levels, SRF nuclear activity, and the target gene aSMA's expression; 2) upon AA stimulation, PLK4 phosphorylation was activated by PDGFR and downstream kinase P38; and 3) the   Rat adventitial fibroblasts were cultured and starved as described in Figure 1. identified (33). Thus, PLK4 is much less understood than PLK1, the best studied PLK member, which mediates multiple mitotic processes (1,32).
Given that PLK4 is a centriole-associated factor, it was not surprising to learn that PLK4 is proproliferative (1) in vascular fibroblasts, as observed here, and in cancer progression, as previously reported (34,35).
PLK4 was also recently linked to cancer cell migration and invasion (33), consistent with our result that PLK4 inhibition mitigates vascular fibroblast migration. On the other hand, cell-type transition is a process beyond cell proliferation and migration; it       Figure 14C).  The means from all animals in each group were then averaged to produce the final mean AE SEM (n ¼ 3 animals per group). Student t-test: *p < 0.05. (42). The bromodomains of BETs "usher" this transcription assembly to target genes by binding to bookmarked (acetylated) chromatin loci. This BRD4directed mechanism has been recently recognized as critical in orchestrating cell-state transitions in various pathobiological contexts (12,(26)(27)(28)(29). As indicated by our data, BRD2 also participated in regulating the PLK4 pathway but played a lesser role (compared with BRD4). The mechanistic difference between the effects of BRD4 and BRD2 on PLK4 awaits further investigation.