LPP Expression During In Vitro Smooth Muscle Differentiation and Stent-Induced Vascular Injury
http://www.100md.com
《循环研究杂志》
the Department of Molecular Physiology and Biological Physics (I.G., L.J., T.Y., G.K.O., A.P.S., A.V.S.), Department of Medicine (J.M.S., I.J.S.)
Cardiovascular Research Center (T.Y., G.K.O., A.V.S.), University of Virginia, Charlottesville, Va.
Abstract
Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM -actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acideCtreated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducible expression of FAK. LPP expression in FAK-null fibroblasts enhanced cell spreading. In stented pig coronary vessels, LPP was expressed in the neointima of cells lacking smoothelin and showed expression patterns identical to those of SM -actin. In conclusion, LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.
Key Words: lipoma preferred partner smooth muscle vascular injury cell migration focal adhesion kinase
Introduction
The lipoma-preferred partner (LPP)1 belongs to a subclass of related LIM proteins which includes zyxin, Trip6, Ajuba, LIMD1, WTIP, and Cal.2 LPP structure, like other members of this gene family, possesses multiple proteineCprotein interaction domains including C terminally located LIM domains, proline-rich motifs, and ENA/VASP-homology regions.1 Through these domains, LPP can bind focal adhesions1 and, like some of its homologues,2 may function as an adaptor protein to anchor structural or regulatory proteins to focal adhesion complexes. Recently it has been shown that LPP3 and its closest homologue, Trip6,4 can enhance cell migration in vitro. In addition, LPP has the ability to shuttle from the cell periphery to the nucleus.3,5 LPP showed capability to activate a reporter gene expression in a GAL4-transactivation assay in nonmuscle cells, and it has been suggested that LPP belongs to a new family of proteins which communicate between the cell surface and the nucleus.5 This potential property is highly relevant to smooth muscle (SM) where the SM differentiation marker genes are sensitive to cytoskeletal dynamics.6eC8
Our group recently showed that LPP expression is highly selective for SM cells (SMCs) in adult animals.3 The strict specificity of LPP for SMCs has been questioned because it has been detected in several cultured cell lines including fibroblasts cell lines, epithelial cell lines, and several cancer cell lines,5 albeit at approximately 100-fold lower expression levels. However, in vivo, LPP was exclusively found in SMCs in sections of ileum, kidney, heart, and bladder.3 LPP has also been identified among a core of computationally identified SM-specific genes from expressed sequence tag data.9 In differentiated quiescent SMCs, LPP is associated with membrane dense body (MDB) complexes involved in actin filament attachment sites.10 MDBs can act as mechanosensors transducing integrin-mediated stretch signals of muscle cells.11 MDBs cluster specific proteins (metavinculin, SM -actin [SMA], SM -actinin), as well as regulatory proteins such as focal adhesion kinase (FAK) and in that respect resemble the focal adhesions of migrating cells in culture, suggesting a role for this complex of proteins in migration and phenotypic modulation during vascular injury.12
Analysis of transcription factors and regulatory elements that convey the specificity of expression in SMCs, have led to the demonstration that a subset of SM gene promoters, including those of SMA, myosin heavy chain (MHC), and SM22 genes, require the presence of CC(A/T)6GG motifs which are targets for the concerted action of SRF and the very potent SRF coactivator myocardin,12,13 which is expressed exclusively in SM and cardiac cells.13eC15 SRF-dependent transcriptional regulation of SM specific genes is dependant on RhoA/Rho kinase (ROK) activation.6
Importantly, although LPP appears to be highly selective for SMC, as yet no studies have examined mechanisms that regulate its expression. The aim of the present study was to determine mechanisms that regulate expression of LPP and to ascertain whether LPP expression is controlled by myocardin- and/or RhoA/ROK-dependent pathways. Based on our previous3 and present findings on LPP contributions to SMC migration,3 we also addressed whether LPP is expressed in neointimal SMCs within a model of stent-induced vascular injury.
Materials and Methods
Cultured Cells
A404 cells are derived from mouse embryonic carcinoma P19 cells stably cotransfected with SMA promoter (eC2560, 2784)/puromycin N-acetyltransferase, to select differentiated cells, and a cytomegalovirus promoter/hygromycin gene to maintain the selection16 and described in detail in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Human iliac vein SMCs (HIVS-125) were purchased from the American Type Culture Collection (Rockville, Md). Protocols for treatment with ROK inhibitor and C3 and mRNA extraction are described in the expanded Materials and Methods section in the online data supplement. FAK-null embryonic cells (FAKeC/eC) and wild-type (WT) cells (FAK+/+), a gift from Dr Thomas Parson (University of Virginia), were cultured as previously described.17 The FAK tetracycline repression system for inducible expression of FAK in FAKeC/eC fibroblasts were a gift from Dr Steven Hanks (Vanderbilt University) and are detailed in the online expanded Materials and Method section.
Western blotting was performed as previously described.3
Adenovirus Constructs
Undifferentiated A404 cells were infected, 24 hours after plating with purified viruses for 48 hours at a multiplicity of infection (MOI) of 50, which infected greater than 95% of SMCs, as previously defined by using green fluorescent protein (GFP)-expressing adenovirus. Adenoviral production is described in the online expanded Materials and Methods section.
Plasmid Constructs and Transfections
RNA silencing was performed by transfection of a plasmid which expresses short interfering RNA (siRNA). The plasmid-based pMighty system15 for production of the silencing RNA specific for LPP is described in online expanded Materials and Methods section. The cells were assayed 48 hours posttransfection for Western blot analysis and cell motility assays.
The fusion construct of LPP and enhanced GFP (EGFP) was prepared as previously reported,3 with transfection and cell growth protocols described online expanded Materials and Methods section.
Immunostaining
Immunostaining of cultured cells is described in the online expanded Materials and Methods section.
Twenty-eight days after the stenting procedure, animals were anesthetized, euthanized by exsanguinations, and pressure-perfused with 10% buffered formalin phosphate. Tissues were processed and embedded in paraffin as outlined in the online expanded Materials and Methods section.
Real-Time RT-PCR
Total RNA extraction, reverse-transcription, and amplification steps are detailed in the online expanded Materials and Methods section. BioRad iCycler detection system (Bio-Rad Laboratories Inc, Hercules, Calif) was used with the QuantiTect RT-PCR kit (Qiagen) for reverse transcription and real-time amplification of RNA samples. Primers and probes (see supplemental Table I) were designed using the Beacon Designer software (Bio-Rad) and synthesized by MWG Biotech AG.
Cell Motility Assays
Conditions for cell migration assays are detailed in the online expanded Materials and Methods section. Cell migration was monitored by time-lapse video imaging using a Zeiss Axiovert 100TV inverted microscope with a x10 objective collecting images with a Hamamatsu ORCA digital charge-coupled device camera at 5-minute intervals over a 2-hour period of time. Dynamic motion of individual cells was analyzed using Simple PCI imaging software (Compix Inc, Cranberry Township, Pa). Transwell assays of cell migration have been described previously.3 Cell spreading assays on fibronectin coated coverslips are described in the online expanded Materials and Methods section.
Animal Surgery for Stent Placement
Cardiac catheterization and coronary stenting procedures were performed on 15- to 20-kg male Yorkshire swine that were 8 to 12 weeks old. All procedures were in compliance with the Animal Care and Use Committee at the University of Virginia Health System and are described in detail in the online expanded Materials and Methods section.
Results
LPP Expression Was Increased During All Trans-Retinoic AcideCInduced A404 Cell Differentiation
A404 cells represent a clonal line of SMC progenitor cells and have been shown to activate expression of all known SMC differentiation marker genes when treated with all trans-retinoic acid (RA).16 RA induced profound changes in cell morphology and large increases in LPP and SMA staining at focal adhesions and actin stress fibers (Figure 1A), as well as increases in LPP mRNA expression (Figure 1B). These results suggest that expression of the LPP gene and other SM marker genes may be regulated through common mechanisms.
LPP Expression Was RhoA/ROK Dependent
As a number of SMC genes are dependent on RhoA/ROK-mediated SRF activation,6 we determined whether RA-induced LPP expression was ROK dependent. A404 cells and SM HIVS-125 cells were treated with the ROK inhibitor H1152.18 LPP expression in A404 cells or HIVS-125 cells was downregulated by approximately 50% by H1152 (0.5 eol/L) or Y-27632 (10 eol/L data not shown) either when added before (Figure 2A) or following RA (Figure 2B) treatment. Smoothelin (SMO) gene expression was completely inhibited by H1152 treatment (Figure 2A). Similarly, H1152 treatment inhibited protein expression of LPP in HIVS-125 cells (Figure 2B).
Myocardin Increased LPP Expression
Although myocardin has been described as a master SMC differentiation control gene, it appears to only activate a subset of CArG-SRFeCdependent SMC differentiation marker genes but not FRNK, or SMO in embryonic stems cells or A404 cells.19 As such, it was of interest to determine whether LPP is a myocardin target gene. LPP mRNA expression in A404 cells was increased by approximately 300% in 48 hours postinfection with an adenovirus expressing myocardin15 (Figure 3). SMA and MHC mRNA levels were enhanced by 2000% and 400%, respectively, whereas SMO and GAPDH gene expression were not significantly modified. Thus, myocardin can activate LPP expression.
Myocardin Gene Expression Was Decreased by Inhibition of RhoA or ROK
Consistent with previous studies,19 treatment of A404 cells with RA was associated with an approximately 2.5-fold increase in myocardin expression (Figure 4A), which was suppressed by the ROK inhibitor (Figure 4A). H1152 induced a 60% reduction in myocardin in HIVS-125 cells. ADP ribosylation of RhoA by C3 exoenzyme inhibited actin stress fiber formation and expression of myocardin by 90% (Figure 4B). Electroporation per se had no effect (not shown). Thus, ROK signaling pathways modulate CArG SRF responsiveness of SMC genes such as SMA, at least in part, by modulating myocardin expression.
SMC Migration Was Modulated by LPP Expression
Expression of exogenous GFP-LPP in HIVS cells was associated with development of increased focal adhesions but no visible modification of cell shape. However, tracking analysis by time-lapse video imaging showed a significant increase in cell motility in control and epidermal growth factor (EGF)-stimulated cells (Figure 5A) when compared with GFP-transfected cells. Inhibition of endogenous LPP, using expression plasmids coding for an LPP siRNA but not a scrambled sequence, caused inhibition of LPP protein expression after 3 days (Figure 5B). Two different LPP siRNAs, but not a scrambled construct, caused a significant decrease in LPP protein expression and Transwell cell migration (Figure 5C). SMA, myosin light chain and GAPDH protein expression was not altered by treatment with LPP siRNA (only GAPDH shown). LPP siRNA had no effect on cell growth.
Nonmotile FAK-Null Embryonic Fibroblasts Showed Marked Reductions in LPP Expression
FAK plays a critical role in regulation of cell motility.17 Interestingly, FAK-null embryonic cells contain approximately 10-fold lower levels of LPP mRNA and protein than their WT controls. Among the focal adhesion proteins tested, such as vinculin, talin, or paxillin, only LPP was inhibited (Figure 6B and data not shown). The analysis of motility of the FAKeC/eC cells versus FAK+/+ cells plated on hFN was 2.5±0.4 and 36.0±3.0 e/h, respectively (n=3). Expression of GFP-LPP in FAKeC/eC cells led to a significant increase (P<0.05) in cell spreading on hFN compared with control transfected cells (Figure 6D). Furthermore, when FAK expression is induced in FAKeC/eC fibroblasts using a tetracycline repression system, FAK and LPP proteins are expressed at levels comparable to WT DU17 FAK+/+ cells when normalized to GAPDH content (Figure 6C). Although LPP may not be the only protein with modified expression in FAKeC/eC cells, these results support the idea that LPP belongs to a class of structural and/or regulatory proteins involved in controlling SMC migration and that FAK is necessary for LPP expression at least in fibroblasts.
Expression of LPP Appeared To Be Colocalized With SMA in Normal and Injured Pig Coronary Vessels
To assess the potential role of LPP in control of SMC migration during in-stent restenosis, we performed immunohistochemical analysis of expression of LPP relative to SMA and SMO (reviewed previously12) in normal and stented pig coronary artery segments 28 days postsurgery. LPP, SMA, or SMO antibody labeling was strongly associated with SMCs of the media in normal vessels (Figure 7A through 7C) but not detectable in adventitial fibroblasts. In mild lesions (Figure 7D through 7F), showing compression of the media but no rupture of the external elastic lamina, LPP staining was associated with regions expressing SMA. Staining of vessels with more pronounced injury and a thicker neointima (Figure 7G through 7I) also showed the presence of LPP in areas where cells express SMA, particularly in neovessels, but not in regions infiltrated by macrophages identified by the anti-Mac2 antibody (Figure 8). Overall, the percentage of cells expressing SM markers in the neointima was lower in segments where neointima was more developed and accompanied by a disorganization of the SMCs circular pattern in the medial layer, possibly attributable to a disruption of the external elastic lamina (Figure 7). Some cells expressing LPP and SMA, and presenting the typical elongated shape of differentiated cells, could be detected in cells migrating around the stent wound (Figure 8). LPP, SMA, or SMO labeling was not found in the adventitia of any injured artery, apart from microvessels supporting the notion that SMCs are the main contributor to neointima formation (Figure 7A, 7D, and 7G). Interestingly, SMO antibody showed lower staining of the neointima when compared with staining of adjacent sections stained with SMA and LPP antibodies (Figure 7). Immunofluorescence labeling also showed greater downregulation of SMO in the neointima (see supplemental Figure I), suggesting a differential regulation of SMO and LPP in either less differentiated and/or migrating cells.
Discussion
The major new findings of the present studies are that (1) the LIM domain protein, LPP, contributes significantly to SMC migration; (2) its expression is activated by the SMC restricted SRF cofactor myocardin; (3) ROK appears to modulate expression of myocardin in addition to its known role in regulating SRF transcriptional activity; (4) inhibition of ROK lead to appropriately 50% inhibition of LPP gene expression; (5) LPP is expressed in SMCs which migrate into the neointima of injured vessels and is coincident with expression of SMA rather than SMO; and (6) FAK-null fibroblasts exhibit a marked reduction in LPP expression, which is rescued by expression of FAK and accompanied by a significant increase in cell adhesion.
LPP expression was found to be induced by myocardin in an in vitro model of early SMC differentiation. As previously shown, RA induced expression of myocardin in A404 cells, as well as the expression of a large panel of differentiated SMC markers, SMA, SM-MHC, SM22, and SM calponin.16 Recently the LPP gene was also shown to be induced following RA-induced differentiation of A404 cells using oligonucleotide microarray transcriptional profiling.20 Thus, these findings, plus the high selectivity of LPP expression in SM, establish that LPP is a novel additional marker of differentiated SMCs. Myocardin is an extremely potent SRF coactivator that is exclusively expressed in cardiac and differentiated SMC.14,15,21,22 It activates transcription of a large subset of SMC marker genes by binding of SRF to CArG elements (CC (A/T) 6-GG motif) found in the promoters of these genes.12,23 However, a recent report from 1 of our groups19 showed that myocardin did not directly activate expression of all SMC marker genes including ACLP or SMO, whose promoters do not appear to contain functional CArG elements.19 A homology search for SRF binding sites in promoter-enhancer regions of the LPP gene reveals at least one possible CArG motif. However, as yet, the function of this CArG element has not been directly assessed nor has a functional LPP promoter-enhancer yet been identified that can recapitulate expression of this gene in transgenic mice. Nevertheless, the results of the present studies showing marked activation of LPP expression by myocardin (Figure 3) provide strong evidence that LPP falls into the CarG\SRF\myocardin dependent class of SM marker genes. Interestingly, our results and those of previous studies6,24 indicate that all of these SMC promoters are also regulated by the RhoA/ROK pathway.
In the present study, the RhoA/ROK pathway was found to be an upstream regulator of myocardin as inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors inhibited myocardin mRNA expression in RA-treated A404 cells as well as in human SMCs (HIVS-125). It is not clear whether RhoA/ROK acts directly on myocardin or through modulation of SRF activity or both. Myocardin, unlike SRF7,8 and other myocardin family members, does not translocate to the nucleus in a RhoA·GTP-dependent manner. As such, it is unlikely to be able to directly "sense" the cytosolic G:F actin dynamics as has been shown to be the case for regulation of ets-independent, CArG-dependent SMC marker genes such as SMA.6,8 We have previously shown that inhibition of the RhoA pathway significantly decreases the activity of SM specific promoters6 and this activity was sensitive to the extent of actin polymerization. SRF-mediated transcription has also been shown to be regulated by RhoA with the G-actin level controlling SRF activity.25 Thus, inhibition or activation of the RhoA pathway may impact on the transcription of SMC marker genes in multiple ways including through the regulation of cytoskeletal dynamics, as well as through alterations in the expression and/or activity of myocardin or the myocardin related factors A and B (MKL1/1, MRTF-A, -B).
Although LPP is localized to membrane associated dense bodies in tissues and their equivalent, focal adhesions, in cultured cells, its function and regulation remain to be determined. LPP is associated with membrane but not cytosolic dense bodies, both attachment sites of actin filaments in differentiated SM. However, LPP, which has a nuclear export signal and in vitro transcriptional activation capacity, has been detected in the nucleus under specific conditions,3,5 such as in the presence of a ROK inhibitor. This, plus its high homology with other LIM domain transcriptional regulators,2 suggests that the function of LPP may vary depending on the phenotype of the cell or indeed may play a role in establishing the phenotype. A special feature of LPP is the polyproline-rich regions FPPPPP and FLPPPPPP at the amino-terminal half of the molecule.1 By analogy with other actin accessory proteins, thought to be important regulators of actin assembly, such as zyxin, palladin, and vinculin where FPPPP serves as a minimal docking site for the vasodilator-stimulated phosphoprotein Ena/VASP,26eC28 a similar role for LPP is likely. The second proline sequence is also found in other actin regulatory proteins and is implicated in actin-based motility. We show using time-lapse video imaging and Transwell migrations assays that siRNA induced down regulation of LPP significantly reduced cell migration, whereas overexpression of LPP increased cell motility, which was further enhanced in the presence of EGF. The ability of the ROK inhibitor to significantly depress LPP mRNA expression in both HIVS-125 cells and in A404 cells induced to differentiate by RA correlates with its ability as well as that of C3-exoenzyme to decrease stress fibers and focal adhesions, structures that undergo complex dynamics and remodeling in the establishment of cell polarity and cell migration.29,30 A role for LPP in cell migration was further explored in FAK-null cells and in the neointima of injured pig coronary vessels.
FAK plays an important role in promoting cell invasion, and embryonic cells from FAK-null mice display reduced cell motility with enhanced focal adhesion contact formation.17 Interestingly, both LPP protein and mRNA levels were decreased in FAK-null cells and inducible expression of FAK in fibroblasts derived from FAK-null embryos lead to marked expression of LPP and enhanced cell spreading. Expression of other focal adhesion proteins such as vinculin, talin, or paxillin were enhanced in FAKeC/eC cells.17 A decrease in RhoA/ROK signaling does not account for the reduced LPP expression (data not shown) as GTP·RhoA content was actually increased in FAKeC/eC cells, as has been reported by others.31 Surprisingly, induced expression of FAK using a tetracycline repression system in FAK-null fibroblasts led to a marked expression of LPP consistent with FAK regulating the expression of LPP, as it has been reported to induce expression of the homeodomain protein Prx1,32 and Prx1 has been shown to increase SRF binding to CArG elements before the recruitment of myocardin in SMCs.33 The role of FAK and LPP in nonmotile cells is less clear. Focal adhesion proteins including FAK and LPP are present at the surface dense bodies in differentiated SMCs in adult tissues, and FAK is phosphorylated/activated in stretch-induced responses in SM tissue of the trachea34 but, in this case, is not promoting cell movement. Thus, not surprisingly, the involvement of a protein in cell motility does not mean that its function will be similar in differentiated cells.
The above observations implicating LPP in SMC migration led to the exploration of LPP expression in the migration and proliferation of SMCs in the neointima of pig injured coronary vessels. In pig stented vessels, LPP was expressed in the neointima and showed expression patterns identical to those of SMA independently of the severity of the lesion. With vascular injury and the ensuing release of growth factors and other signaling molecules, SMCs undergo phenotypic switching to a primarily synthetic proliferative phenotype that migrate to the neointima. Indeed, cells stained for LPP and SMA, but not SMO, appear to be migrating around the stent lesions (rich in macrophages), and these elongated cells reorient at the luminal edge of the vessel matching the orientation of the medial SM. SMO is considered a marker of highly differentiated SMCs35,36 and can be found in neointimal hyperplasia in stented pig coronary vessels, its expression increasing with time postinjury.36 Our findings suggest that LPP, unlike SMO, is not a marker of the contractile phenotype and may be a particularly useful tool for monitoring the phenotypic switching of SMCs in vascular lesions. Thus although much remains to be explored, it is not surprising that the expression of LPP is coincident with SMA expression in the neointima of injured coronary arteries where LPP and its partners may be important for the organization of the actin cytoskeleton in these migrating cells.
In conclusion, our results indicate that LPP is a SMC differentiation marker that is regulated through CArG-SRF-myocardin, and ROK-dependent pathways also involved in regulating many other SMC differentiation marker genes. Of major interest, results of the present studies also demonstrated that LPP plays a key role in regulation of SMC migration in cultured SMCs and is expressed within neointimal SMC. However, further direct studies will be needed to ascertain the primary functional role of LPP in vivo under both normal conditions and in response to vascular injury. The ability of ROK inhibitors to suppress the expression of myocardin a key regulator of SMC genes and their products, and their ability to inhibit SMC migration make them excellent potential candidates for drug-coated stent therapy for reduction of coronary artery neointimal growth.37
Acknowledgments
This work was supported by NIH grants PO1 HL48807, PO1 HL19242, and PO1 HL 55798 (to I.J.S. and Jerry Nadler). We thank Dr Isa M. Hussaini for advice and for the use of video imaging instrumentation and Mark Hoofnagle for advice and help in the design and construction of siRNA constructs. We are grateful to Dr Jerry Nadler for generosity in providing the pig coronary specimens.
Both authors contributed equally to the study.
Deceased January 14, 2004.
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Cardiovascular Research Center (T.Y., G.K.O., A.V.S.), University of Virginia, Charlottesville, Va.
Abstract
Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM -actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acideCtreated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducible expression of FAK. LPP expression in FAK-null fibroblasts enhanced cell spreading. In stented pig coronary vessels, LPP was expressed in the neointima of cells lacking smoothelin and showed expression patterns identical to those of SM -actin. In conclusion, LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.
Key Words: lipoma preferred partner smooth muscle vascular injury cell migration focal adhesion kinase
Introduction
The lipoma-preferred partner (LPP)1 belongs to a subclass of related LIM proteins which includes zyxin, Trip6, Ajuba, LIMD1, WTIP, and Cal.2 LPP structure, like other members of this gene family, possesses multiple proteineCprotein interaction domains including C terminally located LIM domains, proline-rich motifs, and ENA/VASP-homology regions.1 Through these domains, LPP can bind focal adhesions1 and, like some of its homologues,2 may function as an adaptor protein to anchor structural or regulatory proteins to focal adhesion complexes. Recently it has been shown that LPP3 and its closest homologue, Trip6,4 can enhance cell migration in vitro. In addition, LPP has the ability to shuttle from the cell periphery to the nucleus.3,5 LPP showed capability to activate a reporter gene expression in a GAL4-transactivation assay in nonmuscle cells, and it has been suggested that LPP belongs to a new family of proteins which communicate between the cell surface and the nucleus.5 This potential property is highly relevant to smooth muscle (SM) where the SM differentiation marker genes are sensitive to cytoskeletal dynamics.6eC8
Our group recently showed that LPP expression is highly selective for SM cells (SMCs) in adult animals.3 The strict specificity of LPP for SMCs has been questioned because it has been detected in several cultured cell lines including fibroblasts cell lines, epithelial cell lines, and several cancer cell lines,5 albeit at approximately 100-fold lower expression levels. However, in vivo, LPP was exclusively found in SMCs in sections of ileum, kidney, heart, and bladder.3 LPP has also been identified among a core of computationally identified SM-specific genes from expressed sequence tag data.9 In differentiated quiescent SMCs, LPP is associated with membrane dense body (MDB) complexes involved in actin filament attachment sites.10 MDBs can act as mechanosensors transducing integrin-mediated stretch signals of muscle cells.11 MDBs cluster specific proteins (metavinculin, SM -actin [SMA], SM -actinin), as well as regulatory proteins such as focal adhesion kinase (FAK) and in that respect resemble the focal adhesions of migrating cells in culture, suggesting a role for this complex of proteins in migration and phenotypic modulation during vascular injury.12
Analysis of transcription factors and regulatory elements that convey the specificity of expression in SMCs, have led to the demonstration that a subset of SM gene promoters, including those of SMA, myosin heavy chain (MHC), and SM22 genes, require the presence of CC(A/T)6GG motifs which are targets for the concerted action of SRF and the very potent SRF coactivator myocardin,12,13 which is expressed exclusively in SM and cardiac cells.13eC15 SRF-dependent transcriptional regulation of SM specific genes is dependant on RhoA/Rho kinase (ROK) activation.6
Importantly, although LPP appears to be highly selective for SMC, as yet no studies have examined mechanisms that regulate its expression. The aim of the present study was to determine mechanisms that regulate expression of LPP and to ascertain whether LPP expression is controlled by myocardin- and/or RhoA/ROK-dependent pathways. Based on our previous3 and present findings on LPP contributions to SMC migration,3 we also addressed whether LPP is expressed in neointimal SMCs within a model of stent-induced vascular injury.
Materials and Methods
Cultured Cells
A404 cells are derived from mouse embryonic carcinoma P19 cells stably cotransfected with SMA promoter (eC2560, 2784)/puromycin N-acetyltransferase, to select differentiated cells, and a cytomegalovirus promoter/hygromycin gene to maintain the selection16 and described in detail in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Human iliac vein SMCs (HIVS-125) were purchased from the American Type Culture Collection (Rockville, Md). Protocols for treatment with ROK inhibitor and C3 and mRNA extraction are described in the expanded Materials and Methods section in the online data supplement. FAK-null embryonic cells (FAKeC/eC) and wild-type (WT) cells (FAK+/+), a gift from Dr Thomas Parson (University of Virginia), were cultured as previously described.17 The FAK tetracycline repression system for inducible expression of FAK in FAKeC/eC fibroblasts were a gift from Dr Steven Hanks (Vanderbilt University) and are detailed in the online expanded Materials and Method section.
Western blotting was performed as previously described.3
Adenovirus Constructs
Undifferentiated A404 cells were infected, 24 hours after plating with purified viruses for 48 hours at a multiplicity of infection (MOI) of 50, which infected greater than 95% of SMCs, as previously defined by using green fluorescent protein (GFP)-expressing adenovirus. Adenoviral production is described in the online expanded Materials and Methods section.
Plasmid Constructs and Transfections
RNA silencing was performed by transfection of a plasmid which expresses short interfering RNA (siRNA). The plasmid-based pMighty system15 for production of the silencing RNA specific for LPP is described in online expanded Materials and Methods section. The cells were assayed 48 hours posttransfection for Western blot analysis and cell motility assays.
The fusion construct of LPP and enhanced GFP (EGFP) was prepared as previously reported,3 with transfection and cell growth protocols described online expanded Materials and Methods section.
Immunostaining
Immunostaining of cultured cells is described in the online expanded Materials and Methods section.
Twenty-eight days after the stenting procedure, animals were anesthetized, euthanized by exsanguinations, and pressure-perfused with 10% buffered formalin phosphate. Tissues were processed and embedded in paraffin as outlined in the online expanded Materials and Methods section.
Real-Time RT-PCR
Total RNA extraction, reverse-transcription, and amplification steps are detailed in the online expanded Materials and Methods section. BioRad iCycler detection system (Bio-Rad Laboratories Inc, Hercules, Calif) was used with the QuantiTect RT-PCR kit (Qiagen) for reverse transcription and real-time amplification of RNA samples. Primers and probes (see supplemental Table I) were designed using the Beacon Designer software (Bio-Rad) and synthesized by MWG Biotech AG.
Cell Motility Assays
Conditions for cell migration assays are detailed in the online expanded Materials and Methods section. Cell migration was monitored by time-lapse video imaging using a Zeiss Axiovert 100TV inverted microscope with a x10 objective collecting images with a Hamamatsu ORCA digital charge-coupled device camera at 5-minute intervals over a 2-hour period of time. Dynamic motion of individual cells was analyzed using Simple PCI imaging software (Compix Inc, Cranberry Township, Pa). Transwell assays of cell migration have been described previously.3 Cell spreading assays on fibronectin coated coverslips are described in the online expanded Materials and Methods section.
Animal Surgery for Stent Placement
Cardiac catheterization and coronary stenting procedures were performed on 15- to 20-kg male Yorkshire swine that were 8 to 12 weeks old. All procedures were in compliance with the Animal Care and Use Committee at the University of Virginia Health System and are described in detail in the online expanded Materials and Methods section.
Results
LPP Expression Was Increased During All Trans-Retinoic AcideCInduced A404 Cell Differentiation
A404 cells represent a clonal line of SMC progenitor cells and have been shown to activate expression of all known SMC differentiation marker genes when treated with all trans-retinoic acid (RA).16 RA induced profound changes in cell morphology and large increases in LPP and SMA staining at focal adhesions and actin stress fibers (Figure 1A), as well as increases in LPP mRNA expression (Figure 1B). These results suggest that expression of the LPP gene and other SM marker genes may be regulated through common mechanisms.
LPP Expression Was RhoA/ROK Dependent
As a number of SMC genes are dependent on RhoA/ROK-mediated SRF activation,6 we determined whether RA-induced LPP expression was ROK dependent. A404 cells and SM HIVS-125 cells were treated with the ROK inhibitor H1152.18 LPP expression in A404 cells or HIVS-125 cells was downregulated by approximately 50% by H1152 (0.5 eol/L) or Y-27632 (10 eol/L data not shown) either when added before (Figure 2A) or following RA (Figure 2B) treatment. Smoothelin (SMO) gene expression was completely inhibited by H1152 treatment (Figure 2A). Similarly, H1152 treatment inhibited protein expression of LPP in HIVS-125 cells (Figure 2B).
Myocardin Increased LPP Expression
Although myocardin has been described as a master SMC differentiation control gene, it appears to only activate a subset of CArG-SRFeCdependent SMC differentiation marker genes but not FRNK, or SMO in embryonic stems cells or A404 cells.19 As such, it was of interest to determine whether LPP is a myocardin target gene. LPP mRNA expression in A404 cells was increased by approximately 300% in 48 hours postinfection with an adenovirus expressing myocardin15 (Figure 3). SMA and MHC mRNA levels were enhanced by 2000% and 400%, respectively, whereas SMO and GAPDH gene expression were not significantly modified. Thus, myocardin can activate LPP expression.
Myocardin Gene Expression Was Decreased by Inhibition of RhoA or ROK
Consistent with previous studies,19 treatment of A404 cells with RA was associated with an approximately 2.5-fold increase in myocardin expression (Figure 4A), which was suppressed by the ROK inhibitor (Figure 4A). H1152 induced a 60% reduction in myocardin in HIVS-125 cells. ADP ribosylation of RhoA by C3 exoenzyme inhibited actin stress fiber formation and expression of myocardin by 90% (Figure 4B). Electroporation per se had no effect (not shown). Thus, ROK signaling pathways modulate CArG SRF responsiveness of SMC genes such as SMA, at least in part, by modulating myocardin expression.
SMC Migration Was Modulated by LPP Expression
Expression of exogenous GFP-LPP in HIVS cells was associated with development of increased focal adhesions but no visible modification of cell shape. However, tracking analysis by time-lapse video imaging showed a significant increase in cell motility in control and epidermal growth factor (EGF)-stimulated cells (Figure 5A) when compared with GFP-transfected cells. Inhibition of endogenous LPP, using expression plasmids coding for an LPP siRNA but not a scrambled sequence, caused inhibition of LPP protein expression after 3 days (Figure 5B). Two different LPP siRNAs, but not a scrambled construct, caused a significant decrease in LPP protein expression and Transwell cell migration (Figure 5C). SMA, myosin light chain and GAPDH protein expression was not altered by treatment with LPP siRNA (only GAPDH shown). LPP siRNA had no effect on cell growth.
Nonmotile FAK-Null Embryonic Fibroblasts Showed Marked Reductions in LPP Expression
FAK plays a critical role in regulation of cell motility.17 Interestingly, FAK-null embryonic cells contain approximately 10-fold lower levels of LPP mRNA and protein than their WT controls. Among the focal adhesion proteins tested, such as vinculin, talin, or paxillin, only LPP was inhibited (Figure 6B and data not shown). The analysis of motility of the FAKeC/eC cells versus FAK+/+ cells plated on hFN was 2.5±0.4 and 36.0±3.0 e/h, respectively (n=3). Expression of GFP-LPP in FAKeC/eC cells led to a significant increase (P<0.05) in cell spreading on hFN compared with control transfected cells (Figure 6D). Furthermore, when FAK expression is induced in FAKeC/eC fibroblasts using a tetracycline repression system, FAK and LPP proteins are expressed at levels comparable to WT DU17 FAK+/+ cells when normalized to GAPDH content (Figure 6C). Although LPP may not be the only protein with modified expression in FAKeC/eC cells, these results support the idea that LPP belongs to a class of structural and/or regulatory proteins involved in controlling SMC migration and that FAK is necessary for LPP expression at least in fibroblasts.
Expression of LPP Appeared To Be Colocalized With SMA in Normal and Injured Pig Coronary Vessels
To assess the potential role of LPP in control of SMC migration during in-stent restenosis, we performed immunohistochemical analysis of expression of LPP relative to SMA and SMO (reviewed previously12) in normal and stented pig coronary artery segments 28 days postsurgery. LPP, SMA, or SMO antibody labeling was strongly associated with SMCs of the media in normal vessels (Figure 7A through 7C) but not detectable in adventitial fibroblasts. In mild lesions (Figure 7D through 7F), showing compression of the media but no rupture of the external elastic lamina, LPP staining was associated with regions expressing SMA. Staining of vessels with more pronounced injury and a thicker neointima (Figure 7G through 7I) also showed the presence of LPP in areas where cells express SMA, particularly in neovessels, but not in regions infiltrated by macrophages identified by the anti-Mac2 antibody (Figure 8). Overall, the percentage of cells expressing SM markers in the neointima was lower in segments where neointima was more developed and accompanied by a disorganization of the SMCs circular pattern in the medial layer, possibly attributable to a disruption of the external elastic lamina (Figure 7). Some cells expressing LPP and SMA, and presenting the typical elongated shape of differentiated cells, could be detected in cells migrating around the stent wound (Figure 8). LPP, SMA, or SMO labeling was not found in the adventitia of any injured artery, apart from microvessels supporting the notion that SMCs are the main contributor to neointima formation (Figure 7A, 7D, and 7G). Interestingly, SMO antibody showed lower staining of the neointima when compared with staining of adjacent sections stained with SMA and LPP antibodies (Figure 7). Immunofluorescence labeling also showed greater downregulation of SMO in the neointima (see supplemental Figure I), suggesting a differential regulation of SMO and LPP in either less differentiated and/or migrating cells.
Discussion
The major new findings of the present studies are that (1) the LIM domain protein, LPP, contributes significantly to SMC migration; (2) its expression is activated by the SMC restricted SRF cofactor myocardin; (3) ROK appears to modulate expression of myocardin in addition to its known role in regulating SRF transcriptional activity; (4) inhibition of ROK lead to appropriately 50% inhibition of LPP gene expression; (5) LPP is expressed in SMCs which migrate into the neointima of injured vessels and is coincident with expression of SMA rather than SMO; and (6) FAK-null fibroblasts exhibit a marked reduction in LPP expression, which is rescued by expression of FAK and accompanied by a significant increase in cell adhesion.
LPP expression was found to be induced by myocardin in an in vitro model of early SMC differentiation. As previously shown, RA induced expression of myocardin in A404 cells, as well as the expression of a large panel of differentiated SMC markers, SMA, SM-MHC, SM22, and SM calponin.16 Recently the LPP gene was also shown to be induced following RA-induced differentiation of A404 cells using oligonucleotide microarray transcriptional profiling.20 Thus, these findings, plus the high selectivity of LPP expression in SM, establish that LPP is a novel additional marker of differentiated SMCs. Myocardin is an extremely potent SRF coactivator that is exclusively expressed in cardiac and differentiated SMC.14,15,21,22 It activates transcription of a large subset of SMC marker genes by binding of SRF to CArG elements (CC (A/T) 6-GG motif) found in the promoters of these genes.12,23 However, a recent report from 1 of our groups19 showed that myocardin did not directly activate expression of all SMC marker genes including ACLP or SMO, whose promoters do not appear to contain functional CArG elements.19 A homology search for SRF binding sites in promoter-enhancer regions of the LPP gene reveals at least one possible CArG motif. However, as yet, the function of this CArG element has not been directly assessed nor has a functional LPP promoter-enhancer yet been identified that can recapitulate expression of this gene in transgenic mice. Nevertheless, the results of the present studies showing marked activation of LPP expression by myocardin (Figure 3) provide strong evidence that LPP falls into the CarG\SRF\myocardin dependent class of SM marker genes. Interestingly, our results and those of previous studies6,24 indicate that all of these SMC promoters are also regulated by the RhoA/ROK pathway.
In the present study, the RhoA/ROK pathway was found to be an upstream regulator of myocardin as inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors inhibited myocardin mRNA expression in RA-treated A404 cells as well as in human SMCs (HIVS-125). It is not clear whether RhoA/ROK acts directly on myocardin or through modulation of SRF activity or both. Myocardin, unlike SRF7,8 and other myocardin family members, does not translocate to the nucleus in a RhoA·GTP-dependent manner. As such, it is unlikely to be able to directly "sense" the cytosolic G:F actin dynamics as has been shown to be the case for regulation of ets-independent, CArG-dependent SMC marker genes such as SMA.6,8 We have previously shown that inhibition of the RhoA pathway significantly decreases the activity of SM specific promoters6 and this activity was sensitive to the extent of actin polymerization. SRF-mediated transcription has also been shown to be regulated by RhoA with the G-actin level controlling SRF activity.25 Thus, inhibition or activation of the RhoA pathway may impact on the transcription of SMC marker genes in multiple ways including through the regulation of cytoskeletal dynamics, as well as through alterations in the expression and/or activity of myocardin or the myocardin related factors A and B (MKL1/1, MRTF-A, -B).
Although LPP is localized to membrane associated dense bodies in tissues and their equivalent, focal adhesions, in cultured cells, its function and regulation remain to be determined. LPP is associated with membrane but not cytosolic dense bodies, both attachment sites of actin filaments in differentiated SM. However, LPP, which has a nuclear export signal and in vitro transcriptional activation capacity, has been detected in the nucleus under specific conditions,3,5 such as in the presence of a ROK inhibitor. This, plus its high homology with other LIM domain transcriptional regulators,2 suggests that the function of LPP may vary depending on the phenotype of the cell or indeed may play a role in establishing the phenotype. A special feature of LPP is the polyproline-rich regions FPPPPP and FLPPPPPP at the amino-terminal half of the molecule.1 By analogy with other actin accessory proteins, thought to be important regulators of actin assembly, such as zyxin, palladin, and vinculin where FPPPP serves as a minimal docking site for the vasodilator-stimulated phosphoprotein Ena/VASP,26eC28 a similar role for LPP is likely. The second proline sequence is also found in other actin regulatory proteins and is implicated in actin-based motility. We show using time-lapse video imaging and Transwell migrations assays that siRNA induced down regulation of LPP significantly reduced cell migration, whereas overexpression of LPP increased cell motility, which was further enhanced in the presence of EGF. The ability of the ROK inhibitor to significantly depress LPP mRNA expression in both HIVS-125 cells and in A404 cells induced to differentiate by RA correlates with its ability as well as that of C3-exoenzyme to decrease stress fibers and focal adhesions, structures that undergo complex dynamics and remodeling in the establishment of cell polarity and cell migration.29,30 A role for LPP in cell migration was further explored in FAK-null cells and in the neointima of injured pig coronary vessels.
FAK plays an important role in promoting cell invasion, and embryonic cells from FAK-null mice display reduced cell motility with enhanced focal adhesion contact formation.17 Interestingly, both LPP protein and mRNA levels were decreased in FAK-null cells and inducible expression of FAK in fibroblasts derived from FAK-null embryos lead to marked expression of LPP and enhanced cell spreading. Expression of other focal adhesion proteins such as vinculin, talin, or paxillin were enhanced in FAKeC/eC cells.17 A decrease in RhoA/ROK signaling does not account for the reduced LPP expression (data not shown) as GTP·RhoA content was actually increased in FAKeC/eC cells, as has been reported by others.31 Surprisingly, induced expression of FAK using a tetracycline repression system in FAK-null fibroblasts led to a marked expression of LPP consistent with FAK regulating the expression of LPP, as it has been reported to induce expression of the homeodomain protein Prx1,32 and Prx1 has been shown to increase SRF binding to CArG elements before the recruitment of myocardin in SMCs.33 The role of FAK and LPP in nonmotile cells is less clear. Focal adhesion proteins including FAK and LPP are present at the surface dense bodies in differentiated SMCs in adult tissues, and FAK is phosphorylated/activated in stretch-induced responses in SM tissue of the trachea34 but, in this case, is not promoting cell movement. Thus, not surprisingly, the involvement of a protein in cell motility does not mean that its function will be similar in differentiated cells.
The above observations implicating LPP in SMC migration led to the exploration of LPP expression in the migration and proliferation of SMCs in the neointima of pig injured coronary vessels. In pig stented vessels, LPP was expressed in the neointima and showed expression patterns identical to those of SMA independently of the severity of the lesion. With vascular injury and the ensuing release of growth factors and other signaling molecules, SMCs undergo phenotypic switching to a primarily synthetic proliferative phenotype that migrate to the neointima. Indeed, cells stained for LPP and SMA, but not SMO, appear to be migrating around the stent lesions (rich in macrophages), and these elongated cells reorient at the luminal edge of the vessel matching the orientation of the medial SM. SMO is considered a marker of highly differentiated SMCs35,36 and can be found in neointimal hyperplasia in stented pig coronary vessels, its expression increasing with time postinjury.36 Our findings suggest that LPP, unlike SMO, is not a marker of the contractile phenotype and may be a particularly useful tool for monitoring the phenotypic switching of SMCs in vascular lesions. Thus although much remains to be explored, it is not surprising that the expression of LPP is coincident with SMA expression in the neointima of injured coronary arteries where LPP and its partners may be important for the organization of the actin cytoskeleton in these migrating cells.
In conclusion, our results indicate that LPP is a SMC differentiation marker that is regulated through CArG-SRF-myocardin, and ROK-dependent pathways also involved in regulating many other SMC differentiation marker genes. Of major interest, results of the present studies also demonstrated that LPP plays a key role in regulation of SMC migration in cultured SMCs and is expressed within neointimal SMC. However, further direct studies will be needed to ascertain the primary functional role of LPP in vivo under both normal conditions and in response to vascular injury. The ability of ROK inhibitors to suppress the expression of myocardin a key regulator of SMC genes and their products, and their ability to inhibit SMC migration make them excellent potential candidates for drug-coated stent therapy for reduction of coronary artery neointimal growth.37
Acknowledgments
This work was supported by NIH grants PO1 HL48807, PO1 HL19242, and PO1 HL 55798 (to I.J.S. and Jerry Nadler). We thank Dr Isa M. Hussaini for advice and for the use of video imaging instrumentation and Mark Hoofnagle for advice and help in the design and construction of siRNA constructs. We are grateful to Dr Jerry Nadler for generosity in providing the pig coronary specimens.
Both authors contributed equally to the study.
Deceased January 14, 2004.
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