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Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2α stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide. The same decoy peptide also significantly inhibits PGF-2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par-4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par-4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par-4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.
Smooth muscle contraction in the vasculature is a key determinant of blood pressure and blood flow. A major mechanism of initiation of smooth muscle contraction is phosphorylation of the regulatory myosin light chain (LC20), which is largely determined by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. The myosin phosphatase holoenzyme is a heterotrimer composed of the 38 kD protein phosphatase 1 (PP1) catalytic subunit, a 110–133 kD myosin phosphatase target subunit (MYPT1), also termed myosin binding subunit (MBS), and a 20 kD subunit of unknown function . While initially assumed to be constitutively active, myosin phosphatase is now considered as regulated by various pathways, most of which lead to inhibition of myosin phosphatase by phosphorylation of MYPT1 at threonine-696 and/or threonine-850. The kinases that have been implicated in inhibitory phosphorylation of MYPT1 are ROCK [2, 3], ILK  and ZIPK [5, 6]. ZIPK is expressed in various smooth muscle tissues such as intestine, vascular tissues and bladder, where it is associated with the regulatory subunit of myosin phosphatase, MYPT1 [5, 6].
In the process of investigating signalling pathways involved in regulation of smooth muscle myosin phosphatase, the pro-apoptotic protein Par-4 (prostate apoptosis response-4) attracted our interest, since Par-4 has been shown to interact with ZIPK in non-contractile cells [7, 8]. Par-4 was originally identified as the product of a gene that is up-regulated upon ionomycin-induced apoptosis in prostate cancer cells . Subsequent reports also characterized Par-4 chiefly as a proapoptotic protein. Loss of Par-4 function was found to play a role in tumourigenesis, identifying the protein as a tumour suppressor. On the other hand, up-regulation of Par-4 has been linked to neuronal apoptosis in neurodegenerative diseases like amyoloid lateral sklerosis (ALS) and Alzheimer's disease, and in animal models of Parkinson's disease and Huntington's disease .
In the setting of apoptosis in non-muscle cells, the Preuss group  has shown that Par-4 targets ZIPK to the cytoskeleton, leading to apoptosis. In the same study, an increased LC20 phosphorylation was also observed, but a cause-and-effect relationship was not shown. Here, we have suggested (1) that Par-4 modulates the ZIPK-mediated regulation of myosin phosphatase in general; and, (2) that Par-4 specifically regulates the contractility of fully differentiated vascular smooth muscle. We show here, that Par-4 is expressed at high protein levels in contractile, differentiated vascular smooth muscle (dVSM). The data presented in this manuscript show that Par-4 facilitates smooth muscle contraction, and acts as a cytoskeletal scaffold for ZIPK, dynamically targeting the kinase to the vicinity of its substrates, LC20 and MYPT1. Our results identify Par-4 as a novel regulator of LC20 phosphorylation and hence, contractility, in smooth muscle.
All procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee. Male ferrets (Mustela putorius furo, Marshall Farms, North Rose, NY) were killed by an overdose of chloroform, and tissues were prepared as previously described [12, 13]. Ferret tissues were used in this study because of the increased similarity of ferret proteins and physiological functions to the human relative to rodents [14–16].
A7r5 rat aorta cells were cultured in DMEM high glucose (Invitrogen) with 10% FCS, 1% glutamine, 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were transiently transfected with a GFP-tagged ZIPK construct  performed with the jetPEI transfection reagent (PolyPlus, Illkirch, France). Transfected cells were subjected to a 24 hrs serum starvation prior to preparation of cell extracts.
Cells were fixed, stained and analyzed as described previously . As primary antibodies, the rabbit polyclonal anti-ZIPK (1:1000, Biomol) and anti-Par-4 (1:1000, Sigma) antibodies were used. As secondary antibodies, goat anti-rabbit Alexa Fluor® 488 and Alexa Fluor® 568 (1:1000, Molecular Probes) were used. Filamentous actin was stained with Alexa Fluor® 568 phalloidin (1:500, Molecular Probes).
A7r5 cells were lysed in isotonic lysis buffer and subjected to immunoprecipitation with the mouse monoclonal anti-Par-4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The antigen–antibody complexes were adsorbed to Protein G-Agarose (Sigma-Aldrich Co., St. Louis, MO, USA). For testing of the Par-4 decoy peptide, recombinant purified Par-4 protein  immobilized on StrepTactin beads (IBA, Göttingen, Germany) was incubated with extracts from GFP-ZIPK transfected A7r5 cells in the absence or presence of 10 μmol/L Par-4 decoy peptide or scrambled control peptide. Samples were washed three times with lysis buffer and analyzed by western blotting.
Samples were separated on 10% polyacrylamide gels according to standard procedures or on urea gels as described previously , and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were stained with anti-ZIPK or anti-Par-4 in the same dilutions as described above, or with the following antibodies: mouse monoclonal, anti-Tubulin α (1:3000, Santa Cruz), anti-LC20 (1:1500, Sigma), anti-Vimentin (1:1000, Sigma); rabbit polyclonal, anti-MYPT1 (1:2000, Covance), anti-phospho-MYPT1 T850 (1:1000, Upstate), anti-actin (1:2000, Cytoskeleton). Goat Oregon Green® 488 or Alexa Fluor® 568 labelled anti-rabbit or antimouse IgG were used as secondary antibodies (1:1000, Calbiochem). Bands were visualized on a Bio-Rad PhosphorImager. Densitometry analysis was performed with the MultiAnalyst software. Ponceau staining was used to verify equal protein loading and transfer.
Experiments were carried out with peptides consisting of the HIV TAT sequence (GYGRKKRRQRRR) to facilitate cellular peptide uptake as previously described , followed by either a sequence derived from the Par-4 leucine zipper motif (LKQENKTLLKVVGQLTR, decoy peptide), or a corresponding scrambled sequence (TVLKGVLNTRQLQKKEL, control peptide). Peptides were N-terminally tagged with fluorescein isothio-cyanate (FITC). For immunofluorescence experiments, cells were incubated on ice for 1 hr with 10 μmol/L peptide solution in Ca2+/Mg2+ free HBSS. For force transduction experiments, portal vein strips were incubated at 37°C with 50 μmol/L peptide in oxygenated PSS for 3 hrs.
Morpholino oligonucleotides were purchased from Gene Tools, LLC (Philomath, OR). Par-4 protein expression was suppressed using the anti-sense morpholino oligonucleotide 5′-CGGTAGCCACCGGTCGCCATATTCC-3′ complementary to the 5′ part of the ferret Par-4 gene including the initiation codon (underlined). The scrambled morpholino nucleotide 5′-CGCCGTGTCTGATCTTCGTGCATCC-3′ served as control. Ferret portal vein strips were chemically loaded with the morpholino oligonucleotides at 50 μmol/L performed with a previously described protocol . On the fifth day of organ culture after loading, the muscle strips were quick-frozen following contractility assessment.
Cells were analyzed by confocal fluorescence microscopy using the anti-ZIPK antibody. In three individual experiments, cells were categorized as showing a filamentous or non-filamentous ZIPK staining pattern. The staining pattern was considered filamentous when at least two filaments of at least 5 μm length were visible. All cells on each coverslip were analyzed without bias. The percentage of cells showing a filamentous staining pattern was calculated from the total number of analyzed cells.
PSS contained (in mmol/L) 120 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2 25 NaHCO3, 1.2 NaH2PO4 and 11.5 dextrose at pH 7.4 when bubbled with 95% O2 5% CO2. HBSS contained (in mmol/L) 137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 NaH2PO4, 4.17 NaHCO3, 5.55 glucose and 10 HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.4. PBS-Tween solution contained (in mmol/L) 80 Na2HPO4, 20 NaH2PO4, 100 NaCl and 0.05% Tween. Isotonic lysis buffer contained (in mmol/L) 10 NaPO4, pH 8.0, 140 NaCl, 3 MgCl2, 1 dithiothreitol and 0.5% Nonidet-P40.PGF-2α was purchased from Sigma. General laboratory reagents were of analytical grade or better and were purchased from Sigma and Fisher Scientific.
All values given in the text are mean ± SE. Differences between means were evaluated using a two-tailed Student's t-test. Significant differences were taken at the P < 0.05 level. For MYPT1 and LC20 phosphorylation levels, a one-tailed t-test was used, since based on contraction data from the same experiments, only reduction of phosphorylation was predicted for the antisense treated samples.
After testing for antibody specificity (Fig. 1A), western blot analysis (Fig. 1B) demonstrates a strong expression of Par-4 in aorta, portal vein and trachea tissue, and weaker expression in brain, kidney, liver and lung. In kidney, a small band of 30 kD, probably representing a smaller splice isoform of Par-4 that has been described recently , is also present. ZIPK protein expression is strongest in lung, liver, heart and portal vein, followed by brain, trachea and aorta The data clearly show that Par-4 is expressed at high levels in dVSM tissues, and open the possibility of a functional role for the Par-4/ZIPK interaction in dVSM. Since ZIPK has been implicated in regulation of myosin phosphatase [5, 6, 20, 21], portal vein was chosen for the following experiments, as in this tissue, inhibition of myosin phosphatase contributes significantly to contractility  and cells are readily enzymatically dispersed from this tissue.
We next analyzed the subcellular distribution of ZIPK and Par-4 in fully differentiated vascular smooth muscle cells (dVSMCs). For this purpose, we fixed freshly isolated cells in the absence of any stimulus and stained them with the anti-Par-4 antibody and the anti-ZIPK antibody, respectively. Actin filaments, as a marker of the contractile filaments, were co-stained with phal-loidin. As shown in (Fig. 1C), Par-4 is localized along the actin filament bundles, while ZIPK (Fig. 1D) shows a diffuse staining of the cytoplasm.
To test for interaction of endogenous Par-4 and ZIPK in smooth muscle cells, we performed co-immunoprecipitation experiments. Since the antibody used for western blot and immuno-fluorescence staining of Par-4 is not suitable for immunoprecipitation experiments, we used the mouse monoclonal anti-Par-4 antibody. This antibody, however, failed to immunoprecipitate endogenous Par-4 from ferret tissue lysates. Since the antibody was raised against Par-4 of rat origin, we performed immunoprecipitation experiments using the rat aorta smooth muscle cell line A7r5. Figure 1E shows that endogenous ZIPK is coprecipitated upon immunoprecipitation of endogenous Par-4 with the anti-Par-4 antibody, demonstrating the in vivo interaction of the two proteins in smooth muscle cells.
Since in resting dVSM cells, ZIPK and Par-4 stainings show only partial overlap, the question arises as to whether the colocaliza-tion increases upon stimulation. We therefore performed a detailed time course analysis of the Par-4 and ZIPK subcellular distributions in cells fixed after stimulation with PGF-2α. Par-4 is associated with actin filaments at all time-points analyzed (1 to 15 min, data not shown). ZIPK, however, consistently redistributes to the actin filaments 1 min after stimulation with PGF-2α, while at later time-points the staining pattern gradually changes to a morepronounced cell surface staining and reduced nuclear staining (Fig. 2A). Quantitation reveals that at 1-min PGF-2α stimulation, the percentage of cells with filamentous staining pattern is significantly increased compared to resting cells (70.08 ± 3.39% compared to 34.03 ± 4.03%, Fig. 2A, graph).
Next, we investigated whether Par-4 is involved in ZIPK redistribution in dVSMCs in a cause-and-effect manner. For this purpose, we designed and synthesized a cell permeant version of a Par-4 decoy peptide that contains amino acids 326–342 of Par-4 (see schematic drawing in Fig. 2B). The selected sequence is part of the C-terminal leucine zipper motif that has been shown to mediate the interaction between Par-4 and ZIPK as well as other Par-4 interaction partners [7, 22, 23]. By choosing this sequence, the decoy peptide was designed to compete with endogenous Par-4 in binding to ZIPK. Pull-down experiments were performed to test the efficacy of the decoy peptide. As shown in Figure 2C, the amount of ZIPK that bound to immobilized purified recombinant Par-4 protein was decreased in the presence of the Par-4 decoy peptide compared to that in the presence of the scrambled control peptide, or in the absence of peptides, demonstrating that the Par-4 decoy peptide interferes with the Par-4/ZIPK interaction and confirming that the decoy peptide can be used as a tool to disrupt the Par-4/ZIPK interaction.
Pre-treatment of cells with the decoy peptide before stimulation with PGF2α for 1 min inhibits the targeting of ZIPK to the actin filaments (Fig. 2D). Quantitative analysis shows that the percentage of cells with filamentous ZIPK staining pattern significantly decreases in the decoy peptide treated cells compared to the scrambled peptide treated cells.
Our results predict that Par-4 targets ZIPK to the contractile filament bundles and to its substrates MYPT1 and LC20, thus presumably supporting contraction. To prove whether Par-4 indeed modulates contractility, we employed the previously described cell permeant Par-4 decoy peptide (see Fig. 2B) in force transduction experiments. Tissues were pre-treated with either the scrambled control peptide or with the decoy peptide prior to contractility assessment. Loading efficiency was monitored by immunofluorescence analyses of tissue pieces (data not shown). Compared to the control strips, contraction of the tissues treated with the decoy peptide is significantly reduced after 10-min PGF-2α stimulation (Fig. 3A). Although the effect is not large, consistent with the known redundancy of multiple signalling pathways in dVSM , it is highly statistically significant. Furthermore, the effect of the decoy peptide was specific for PGF-2α and also for KCl-induced contraction, since it had no effect on phenylephrine-induced contractility (data not shown).
As a second approach to analyze the contribution of Par-4 in smooth muscle contractility, we used antisense knockdown of Par-4 with antisense morpholino nucleotides. On day five after morpholino loading in serum-free organ culture, the contractility of the treated tissues was significantly reduced (Fig. 3B). Thus, very similar results were obtained with two very different techniques.
The same tissue strips as used in the contractility assay following morpholino treatment were quick-frozen at the steady-state contraction and analyzed for protein expression. Western blot analysis after five days of organ culture shows that Par-4 protein levels were significantly reduced to 78.50 ± 3.91% (Fig. 3C, light grey bar) in the Par-4 antisense morpholino treated strips, while the level of MYPT1, vimentin and actin, which served as control proteins that were analyzed in parallel, remained at around 100% (Fig. 3C, black bars). To test our hypothesis that Par-4 promotes ZIPK translocation, thereby facilitating ZIPK-mediated phosphorylation of MYPT1 and LC20, we analyzed the phosphorylation of these ZIPK substrates in the morpholino-treated tissues For MYPT1, the threonine-850 phosphorylation site was chosen since its phosphorylation has been connected with detachment of MYPT1 from myosin , which may cause the agonist-induced redistribution of MYPT1 that has been previously observed in this same cell type . Interestingly, a significant reduction of both MYPT1 and LC20 phosphorylation could be demonstrated: At steady-state contraction, MYPT1 phosphorylation at threonine-850 was reduced to 87.07 ± 5.22% in the antisense-treated strips compared to the control strips, and LC20 phosphorylation was reduced to 87.67 ± 4.67% (Fig. 3C, dark grey bars).
Here, we demonstrate for the first time that Par-4 plays a role in the signalling pathways that regulate dVSM contractility. In summary, we provide evidence that (1) Par-4 is expressed at high protein levels in dVSM, (2) that it regulates targeting of zipper-interacting protein kinase, ZIPK, to its substrates MYPT1 and LC20 and (3) that phosphorylation levels of LC20 and MYPT1 as well as tissue contractility are decreased upon downregulation of Par-4 or decoy peptide interference, indicating a role for Par-4 in the regulation of contractility.
A recent publication has suggested that the Par-4/ZIPK interaction is restricted to rodent cells . However, our finding that Par-4 regulates the targeting and function of ZIPK and co-localizes with ZIPK argues against this concept. Clearly, the data presented here with contractile ferret smooth muscle cells show that ZIPK targeting by Par-4 is not restricted to rodent cells and are consistent with those of Kawai et al. , who have shown interaction of endogenous human Par-4 and ZIPK in HeLa cells.
The cause-and-effect relationship between Par-4 and regulation of contractility was shown by two different experimental approaches: (1) interference with Par-4 interactions by using a Par-4 decoy peptide, and (2) downregulation of Par-4 protein expression. We chose to use antisense morpholino oligomers in this study, even though this method has not previously been reported for dVSM, because morpholinos are reported to be more stable, more specific and more effective than phosphorothioate nucleotides , which we have used in our previously published studies [12, 18, 28, 29]. It is worth noting that even with this approach, endogenous Par-4 protein was reduced by only 23%. Since sequences corresponding to the same Par-4 mRNA region have been successfully used in antisense experiments by various labs [30–32], we can exclude the possibility of ineffective targeting. The corresponding region of the ferret Par-4 mRNA was cloned and sequenced (data not shown) and proved to be 100% identical with the human Par-4 mRNA. The loading efficiency of the morpholinos was confirmed by visual and confocal inspection of the FITC signal (data not shown). The most likely factor affecting the degree of knock down is therefore the stability of the Par-4 protein. Protein stability is a general problem with antisense approaches in dVSM, since the rate of protein turnover is extremely low , so that even when mRNA translation is completely blocked, the efficiency of the knockdown approach still depends on the degradation of preexisting target protein. However, despite the challenge of using the antisense method in dVSM tissue, we were able to achieve significant downregulation of the Par-4 protein, and furthermore could show significant effects of the Par-4 downregulation on LC20 and MYPT1 phosphorylation levels and on tissue contractility.
It is noteworthy that another protein with a similar cytoskeletal scaffold function has been identified recently: the protein p116RIP/M-RIP binds both RhoA and MYPT1, thus directing myosin phosphatase to the cytoskeleton [34–36]. The protein has been shown to participate in regulation of myosin phosphatase in various cell lines including aorta and coronary smooth muscle cells [36–38]. Both Par-4 and M-RIP may represent scaffold proteins that target myosin phosphatase to the contractile filament bundles and, upon stimulation, assemble it with one of its upstream kinases. Since the substrates of ROCK and ZIPK are overlapping and both kinases have similar consensus phosphorylation targets, specific binding of the Rho/ROCK complex to M-RIP and specific binding of ZIPK to Par-4 may mediate substrate specificity in vivo or lead to distinct subcellular localizations of Rho/ROCK and ZIPK, which might also result in different downstream effects. Alternatively, Par-4 and M-RIP might channel separate upstream pathways to the same downstream effect, thus potentiating MYPT1 phosphorylation and myosin phosphatase inhibition by the super-assembly of the Rho/ROCK pathway and the ZIPK pathway.
To the best of our knowledge, besides the data presented here, only one other non-apoptotic function for Par-4 has been described so far: the regulation of dopamine signalling in neuronal cells by interaction with the dopamine D2 receptor . Apart from dopamine signalling, Par-4 has been associated with neuronal apoptosis and appears to be involved in the pathology of several neurodegenerative diseases . We have analyzed the protein expression in different ferret tissues including brain, and found that, despite of the important role Par-4 seems to play in the nervous system, the protein expression in brain was low compared to vascular smooth muscle from aorta and portal vein. A possible explanation is that apoptotic stimuli trigger Par-4 protein expression in neuronal cells [31, 41, 42], while basal expression levels are kept low to protect the cells from accidental activation of the apoptotic response. However, it is intriguing that the high Par-4 protein expression in aorta and portal vein does not lead to induction of apoptosis. Our data show that the anti-apoptotic 30 kD splice isoform of Par-4  is barely detectable in vascular smooth muscle and is therefore unlikely to account for this phenomenon. Thus, it is currently unclear what cellular signals ultimately determine whether Par-4 induces apoptosis or not. However, this will be important to resolve in future studies, should Par-4 have potential as a therapeutic target.
In summary, we have shown here that Par-4, although previously characterized mainly as a pro-apoptotic protein, supports contractility in dVSM. This novel role for Par-4 is associated with its capacity to target ZIPK to the cytoskeleton.
We thank Paul Leavis and his Peptide Core Facility for synthesizing the peptides. This study was supported by NIH grants HL31704 and HL80003 to K.G.M., and an Endre A. Balazs scholarship and an American Heart Association fellowship to S.V.