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Mechanical stress-activated PKC regulates smooth muscle cell migration¹

CHAOHONG LI^*, FLORIAN WERNIG, MICHAEL LEITGES, YANHUA HU and QINGBO XU^*,,2

^* Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria;
Department of Cardiological Sciences, St. George's Hospital Medical School, London, UK; and
Max-Planck-Institute for Experimental Endocrinology, Hannover, Germany

²Correspondence: Department of Cardiological Sciences, St. George's Hospital Medical School, Cranmer Terrance, London SW17 0RE, UK. E-mail: q.xu{at}sghms.ac.uk

SPECIFIC AIMS

Vascular smooth muscle cell (SMC) migration is a key event in the development of vascular diseases, including postangioplasty restenosis and spontaneous atherosclerosis. It has been shown that growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß) induce SMC migration, but the direct effects of mechanical stress on this process of SMC migration have not been studied. The aim of the present study is to evaluate potential effects of mechanical stress-activated protein kinase C delta (PKC) on SMC migration.

PRINCIPAL FINDINGS

1. Cyclic strain stress stimulates PKC and translocation and activation
Traditionally, it has been believed that PKC translocates from the cytoplasm to the membrane in response to specific agonists or other stimuli. To investigate whether this translocation of PKC is altered after mechanical stress, PKC in stressed SMCs was determined separately by Western blot analysis. Surprisingly, strain stress treatment (60 cycles/min, 15% elongation) resulted in significant translocation of PKC to the cytosol, and PKC protein levels were increased in the Triton-insoluble fraction that represents cytoskeleton-related proteins (Fig. 1 a). Kinetic analysis indicates that this response occurred as early as 2 min, with maximum translocation achieved after 2 min in the Triton-insoluble fraction and 30 min in the cytosol after treatment, then declining thereafter (Fig. 1a ). In contrast, PKC translocated from the cytosol to the membrane whereas no PKC proteins were detectable in Triton-insoluble fractions, indicating a "classic" translocation for PKC.

View larger version (55K): [in this window] [in a new window]   Figure 1. PKC translocation in SMCs exposed to mechanical stress. Serum-starved SMCs were treated with cyclic strain stress and harvested. Protein extracts were prepared from the cell membrane, Triton-insoluble fraction, and cytosol. Protein extracts were separated on 10% SDS-polyacrylamide gel, transferred to membranes, and probed using an antibody to PKC and PKC, respectively. a) SMCs were stressed with 15% elongation and 60 cycles/min. b) SMCs were treated for 2 min with 60 cycles/min. c) PKC kinase assays. SMCs were stressed with 15% elongation for 2 min. PKC and PKC were immunoprecipitated from the protein extracts of Triton-insoluble fractions using specific antibodies. Their kinase activities were measured based on the phosphorylation of a myelin basic protein (MBP) substrate. The data represent similar results from 3 independent experiments. SS indicates stretch stress.

To further establish the relationship between mechanical strain stress and PKC translocation to Triton-insoluble fractions, a tensile strength-response analysis of mechanical stress-induced PKC translocations was performed. As shown in Fig. 1b , SMCs were stretched with elongations of 5, 15, and 20% of original size, respectively. The increase of PKC translocation to Triton-insoluble fractions corresponded to the increase in magnitude of stretch stress from 5 to 20%. A decline of PKC proteins in the membrane fraction coincided with increased amounts of PKC proteins in the cytosol in SMCs stimulated by increasing intensities of stretch (Fig. 1b ). Again, no PKC proteins were detectable in Triton-insoluble fractions, although membrane translocation could be observed (Fig. 1b , lower panel). Because of the novel response when PKC proteins translocated to Triton-insoluble fractions, we performed kinase assays for both PKC and PKC using specific antibodies. A marked increase in kinase activities of PKC immunoprecipitated from Triton-insoluble fractions was observed, but no kinase activity for PKC was detected after cyclic strain stress (Fig. 1c ).

To directly confirm PKC translocation, double staining for PKC and the cytoskeleton was used for SMCs stimulated by mechanical stress. There was no significant change in the distribution patterns of PKC (localization in the cytoplasm before and after mechanical stress). However, a double positive staining for PKC proteins and cytoskeleton was observed in stressed SMCs, indicating translocation to the cytoskeleton.

2. Alterations in cytoskeleton rearrangement in PKC-/- SMCs
To further investigate whether PKC influences cytoskeleton rearrangement, SMCs were isolated from the aortic media of PKC-deficient mice generated in our laboratories. During cell spreading, wild-type SMCs showed that actin fibers mainly distributed on the edge of the cell 1 h after seeding and rearranged in the cytoplasm by 6 h. PKC-/- SMCs had a different pattern of actin filament distribution, indicating a loss of normal actin reorganization.

Because cytoskeleton rearrangement requires actins and related enzymes or proteins, we examined several types of actins and actin-related proteins by Western blot analysis. There was no difference in F-actin, G-actin, and -actin between PKC-/- and PKC+/+ SMCs in response to mechanical stress. FAK, paxillin, and vinculin phosphorylation was markedly induced in PKC+/+ SMCs stimulated by cyclic strain but much less so in PKC-/- SMCs. Paxillin protein levels were significantly lower in PKC-/- SMCs compared with PKC+/+ SMCs. To further scrutinize the distribution of actin-related proteins, double staining for vinculin and actin fibers of PKC+/+ and PKC-/- SMCs was performed. In response to mechanical stress, vinculin proteins were relocalized from even to cluster patterns in wild-type SMCs; such changes in vinculin distribution in PKC-/- SMCs were significantly diminished (Fig. 2 ), confirming the above findings of abnormal functioning in actin-related proteins.

View larger version (79K): [in this window] [in a new window]   Figure 2. Double staining for vinculin and actin fibers. SMCs were serum-starved for 3 days and treated with strain stress (60 cycles/min, 15% elongation). SMCs were fixed, incubated with rabbit anti-vinculin antibodies for 1 h, and visualized with swine anti-rabbit Ig conjugated with FITC. After washing cells were stained with rhodamine phalloidin for 30 min. Photographs were taken with a confocal microscope, original magnification x400.

3. Impact of PKC in SMC migration
Because of the observed PKC-dependent alterations in the cytoskeleton, it would be interesting to investigate the effects of PKC on SMC migration, a key event in the pathogenesis of vascular diseases. After the cell layer was disrupted by scraping, SMC migration was evaluated at 0 and 24 h. PKC deficiency markedly reduced SMC migration, resulting in slower closure of the wound. Pretreatment with cyclic strain stress significantly enhanced SMC migration: complete closure of the wound in wild-type SMCs, but less effect on PKC-deficient SMCs.

CONCLUSIONS AND SIGNIFICANCE

Migration of vascular SMCs plays an important role in the pathogenesis of vascular diseases. In the present study we provide the first evidence that mechanical stretch-enhanced SMC migration is mediated, at least in part, by PKC activation. We demonstrate that PKC translocates to the cytoskeleton, which is abnormal in PKC-/- SMCs. Because cell migration is a coordinated process consisting of signaling and cytoskeleton rearrangement, our data suggest that PKC could be a link between mechanical stress and actin fiber structuring during cell migration. Thus, these findings could be crucial to better understand the molecular mechanisms of SMC migration and find new targets for therapeutic intervention.

Previous work has established that members of the classic PKC family (e.g., PKC) translocate to the cell membrane in response to TPA and other stimuli. Concerning novel PKC, recent data indicate that PKC translocates into the mitochondria and the nucleus in U937 cell lines in response to TBA. Recent studies demonstrate that the mechanical treatment of SMCs is associated with translocation of PKC to the cytoskeleton, while PKC is found in the membrane. These findings have been confirmed by cell fractionation, kinase assays, and immunofluorescence studies. The results indicate a diversity of translocation mechanisms for PKC in response to different stimuli, which may be related to different functions. In fact, Majumder et al. provided evidence that the mitochondrial translocation of PKC is associated with cytochrome c release and apoptosis. We found that a proportion of PKC proteins also translocates to the mitochondria in response to mechanical stress (data not shown), as implicated by an increase of PKC in the cytosol (Fig. 1) . A proportion of PKC proteins appear in the cytoskeleton of SMCs stimulated by mechanical stress, indicating the presence of multiple translocations and functions for PKC in a variety of cell types.

How does mechanical stress lead to SMC migration? Our hypothesis is schematically illustrated in Fig. 3 . Two main signal pathways link mechanical stress to cell migration. One is the PDGF receptor–MAPK–MMP pathway that is responsible for cell detachment from matrix proteins. The other involves a PKC paxillin–cytoskeleton pathway essential for cell movement. Supporting this model is our previous finding that mechanical stress can directly activate tyrosin kinase-coupled receptors, including PDGF receptor, followed by PI₃K and MAPK activation. There is also evidence indicating that mechanical stress activates the transcription factor AP-1 in SMCs in vivo and in vitro, which leads to production of matrix metalloproteinases, e.g., collagenase. Furthermore, previous studies established that mechanical stress influences the structure of the cytoskeleton in endothelial cells and SMCs. In the present study, we demonstrated the crucial role of PKC in mediating the actin fiber rearrangement that influences SMC migration. These findings close the gap between mechanical stress and cytoskeleton alterations; therefore, the model formulated in Fig. 3 could provide a better understanding for the molecular mechanisms of signaling involved in SMC migration.

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic representation of PKC-dependent migration stimulated by mechanical stress. Mechanical stress can directly activate PDGF receptor (PDGFR)–MAPK pathways leading to matrix metalloproteinase (MMP) production, which is responsible for cell detachment from the matrix proteins. In parallel, mechanical stress activates integrins (ITG) resulting in cytoskeleton rearrangement, which is essential for cell movement. Both pathways are coordinately regulated by PKC leading to SMC migration.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0150fje; doi: 10.1096/fj.03-0150fje

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