Published online before print May 22, 2008, doi: 10.1161/CIRCRESAHA.107.170357
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© 2008 American Heart Association, Inc.
Molecular Medicine |
The LIM Protein Leupaxin Is Enriched in Smooth Muscle and Functions As an Serum Response Factor Cofactor to Induce Smooth Muscle Cell Gene Transcription
From the Departments of Pathology (L.J.S.-S., L.A.D., C.P.M., J.M.T.) and Physiology (R.L.S.) and the Carolina Cardiovascular Biology Center (C.P.M., J.M.T.), University of North Carolina, Chapel Hill.
Correspondence to Joan M. Taylor, Department of Pathology and Laboratory Medicine, 501 Brinkhous-Bullitt Bld CB 7525, University of North Carolina, Chapel Hill, NC 27599-7525. E-mail jmt3x{at}med.unc.edu
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Abstract |
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Leupaxin is a LIM domain–containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor–dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix–dependent signals may regulate phenotypic switching of smooth muscle cells.
Key Words: smooth muscle • differentiation • LIM proteins • adhesion • signal transduction
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Introduction |
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Mature medial smooth muscle cells (SMCs) express high levels of the SMC differentiation marker genes (ie, smooth muscle [SM] myosin heavy chain [SM-MHC], SM
-actin, SM22 among others) that contribute to the regulation of SMC contractility.1 Unlike cardiac and skeletal muscle, SMCs never terminally differentiate and can transition to a synthetic phenotype characterized by decreased SMC marker gene expression, increased matrix production, and responsiveness to progrowth and migratory signals. This unique plasticity is critical for proper vessel development, blood pressure homeostasis, and injury repair but can also contribute to the development of various vascular pathologies.1 Thus, defining the signaling mechanisms that regulate SMC growth and differentiation will be important for understanding the processes that modulate vascular development and is critical for the design of agents that might regulate aberrant SMC responses in diseased vessels. The transcription mechanisms that regulate SMC differentiation are starting to become clear. Serum response factor (SRF) binding to conserved CArG [CC(A/T)6GG] promoter elements is required for the expression of most SMC differentiation marker genes. It is well known that SRF activity is regulated through interactions with additional ubiquitous and cell type–selective transcription factors or cofactors including the ternary complex factors (Elk-1, Sap-1, SAP-2/NET/ERP), the GATA factors, Nkx2.5, and the LIM domain proteins CRP1, CRP2, and FHL2.1 The myocardin factors (myocardin, MRTF-A, and MRTF-B) are particularly potent activators of SRF-dependent transcription.2–4 Their importance in the regulation of SMC differentiation is underscored by the lethal defects in SMC differentiation observed in myocardin–/– and MRTF-B–/– mice.5,6 The presence of multiple cofactors likely provides the opportunity for precise transcriptional control of the numerous SRF target genes that are known to regulate SMC growth, migration, and differentiation.
Transforming growth factor (TGF)-β, which promotes SMC differentiation,7,8 and platelet-derived growth factor-BB, which promotes phenotypic modulation, are important extrinsic regulators of SMC phenotype, and genetic ablation of these genes resulted in defective vasculogenesis.9,10 In addition to these soluble factors, extracellular matrix molecules also regulate SMC phenotype. Deletion of either fibronectin (FN), the 5 integrin FN receptor, or focal adhesion kinase (FAK) (the kinase that mediates 5-dependent signaling) result in extraembryonic and embryonic vessel defects leading to lethality in the mouse from embryonic day 8.5 to 10.11–13 In vitro, FN (which is enriched in the developing vasculature) supports SMC proliferation and limits SMC differentiation, whereas the basement membrane components collagen type IV and laminin (which are more prominent in the mature vessel) promote SMC differentiation.14,15 However, it is currently unclear how integrin-dependent signals interface with the transcriptional machinery to regulate SMC phenotype.
Herein, we identified leupaxin, an understudied LIM protein in the paxillin family, as a FAK binding partner in SMCs. We found that leupaxin is particularly abundant in SMCs, where it localizes both to focal adhesions and the nucleus, indicating that it may be a bifunctional adapter protein. Indeed, we found that nuclear localized leupaxin acts as an SRF cofactor to enhance SMC differentiation and that leupaxin undergoes regulated cytoplasmic–nuclear shuttling that is dependent on FAK activity. These data highlight the possibility that extrinsic signals can regulate the SMC gene profile by modulating the activation state of FAK and the localization of LIM-containing SRF cofactors.
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Materials and Methods |
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See the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org, for a complete description of the reagents, tissue samples, DNA constructs, and general methods used for these studies.
Statistical Analysis
All promoter measurements were performed using the indicated reporter construct in parallel with a minimal thymidine kinase (TK-Luc) promoter. Data are presented as fold levels of SM promoter activity over TK promoter activity (to rule out generic increases in gene transcription), and all data represent at least 3 separate experiments presented as means±SEM. Means were compared by 2-tailed Student’s t test or ANOVA (where indicated), and P<0.05 was considered statistically significant, as indicated by an asterisk. All other data, including Western blot analysis and chromatin immunoprecipitation assays, are representative of at least 3 individual experiments.
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Results |
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We identified leupaxin as a putative FAK binding partner in a yeast 2-hybrid screen of an aortic smooth muscle cell library. This finding was somewhat surprising, because leupaxin was previously reported to be selectively expressed in lymphoid tissues, as well as several cultured hematopoietic cell lines, osteoclasts, and a bone-derived cancer cell line.16,17 However, as our RT-PCR results demonstrate (Figure 1A), high levels of leupaxin message were detected in several mouse tissues including spleen, aorta, lung, and uterus, whereas lower levels were observed in stomach, bladder, heart, and brain. Western blot analysis using a monoclonal antibody that is completely specific for human leupaxin yielded a similar expression pattern and also that leupaxin runs as a doublet of approximately 45/47 kDa in some tissues (Figure 1B). Because a single mRNA species was identified by Northern blot analysis (data not shown), the slower migrating form is likely caused by posttranslational modification. Indeed, the leupaxin mobility shift was reversed by treatment of human aortic lysates with calf intestinal alkaline phosphatase (CIP) (Figure 1B, bottom left). Leupaxin expression was also strong in cultured human coronary SMCs (huCSMCs) (Figure 1B, bottom right) and treatment of these cells with calyculin A induced a mobility shift (data not shown), in further support of a phosphorylation-induced change in motility on SDS-PAGE. Importantly, the leupaxin antibody used does not recognize its closely related family members paxillin (68 kDa) or Hic-5 (50 kDa), which are also expressed in huCSMCs (Figure IA in the online data supplement).
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In accordance with the expression of leupaxin in huCSMCs, immunohistochemical analysis of human aorta revealed strong leupaxin expression throughout the media, as well as in the smooth muscle layers (but not endothelium) in the microvessels within the adventitia (Figure 1C and supplemental Figure IB). Because several extracellular matrix/integrin signaling components that regulate SMC function have been shown to be developmentally regulated, we also examined leupaxin expression at several stages of human aortic development. Figure 1D demonstrates that leupaxin expression was relatively low in fetal human thoracic aorta (37 weeks postconception) but was much higher at P14 and in adult (55 years), which contain more highly differentiated SMCs as revealed by higher levels of SM-actin. Interestingly, leupaxin was expressed in a reciprocal fashion relative to its family members, paxillin, and Hic-5. Quantitative RT-PCR analysis for leupaxin message in thoracic aorta isolated from postnatal day 4 to adult (8 weeks) mice revealed a similar striking increase in leupaxin expression in the more mature vessels (supplemental Figure IC). Notably, the 8-week samples contained 6-fold higher levels of SM22 message when compared to day 4 vessels (data not shown). Collectively, these data indicate that leupaxin is expressed strongly in SMCs and may play a previously unrecognized role during SMC maturation.
Given the high level of leupaxin expression in huCSMCs, we used these cells to confirm an interaction between FAK and leupaxin. Leupaxin localizes within focal adhesions and colocalizes with FAK in these cells (Figure 2A and supplemental Figure IIA). In addition, a glutathione S-transferase (GST) fusion protein containing the entire FAK C terminus (GST-CTII) but not GST alone strongly precipitated endogenous leupaxin from huCSMC lysates (Figure 2B). This interaction was subsequently confirmed in vivo by coimmunoprecipitation of a FAK–leupaxin complex from Cos7 cells (Figure 2C). Somewhat surprisingly, a GST fusion protein containing the more C-terminal FAT domain of FAK (GST-CTI) efficiently precipitated paxillin but not leupaxin (Figure 2B). These results indicate that leupaxin association with FAK requires sequences N-terminal to the FAT domain and, perhaps, that leupaxin may not directly compete with paxillin and/or Hic-5 for FAK binding. Reciprocal mapping studies revealed that the N-terminal LD motifs in leupaxin were sufficient to precipitate FAK from cell lysates (Figure 2D) and subsequent deletion studies revealed that LD3 is the major site on leupaxin that directs binding to FAK (Figure 2E and supplemental Figure IIB).
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Interestingly, we also observed a subset of huCSMCs (approximately 15% of cells maintained in serum) that exhibited both focal adhesion and nuclear-localized leupaxin, suggesting that like several other LIM proteins, leupaxin shuttles between focal adhesion and nuclear compartments (Figure 2A). To test this more directly, we treated huCSMCs with the CRM1-dependent nuclear export inhibitor leptomycin B (LMB). LMB treatment for 20 minutes to 1 hour induced a dramatic redistribution of endogenous leupaxin to the nuclear compartment (Figure 3A and 3B), whereas treatment for 3 hours resulted in nearly complete nuclear localization (data not shown). Importantly, immunohistochemical localization of leupaxin in the media of aortic tissue sections also revealed both nuclear and cytoplasmic staining (Figure 3B).
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As noted above, several LIM domain proteins that exhibit nuclear accumulation have been implicated in the regulation of gene transcription.18 In fact, the smooth muscle-specific LIM proteins, CRP1 and CRP2, and the cardiac/SMC-selective LIM protein FHL2 have been shown to interact with SRF and to regulate SRF-dependent gene expression.19,20 To test whether leupaxin can contribute to the regulation of transcription in SMCs, we cotransfected Flag-tagged leupaxin along with SM22 or SM -actin promoter/luciferase constructs into multipotential mouse 10T1/2 cells. As shown in Figure 4, expression of leupaxin strongly increased SM22 and SM -actin promoter activity in a dose-dependent fashion and enhanced the effects of TGF-β in this model. Expression of leupaxin also significantly upregulated SM22 (Figure 4C) and SM -actin promoter activity (data not shown) in primary rat aortic SMCs (rASMCs). The more modest effects of leupaxin in SMCs are most likely attributable to the already high levels of SMC-specific transcriptional activity in primary SMC cultures. Importantly, ectopic expression of leupaxin in 10T1/2 cells induced the expression of endogenous SM gene transcription as assessed by quantitative RT-PCR for SM22 (Figure 4D) and by Western blot analysis for SM-MHC (the canonical smooth muscle-specific marker) and SM -actin (Figure 4E).
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We hypothesized that nuclear leupaxin, like CRP1/2, may regulate SMC-specific gene expression by interacting with SRF. In support of this idea, leupaxin failed to activate a SM -actin promoter that contained mutations to all 3 SRF-binding CArG boxes (ABI) (Figure 5A). In addition, we found a coassociation of green fluorescent protein (GFP)-leupaxin and flag-SRF in reciprocal coimmunoprecipitation assays performed in Cos7 cells (Figure 5B) and that GST–leupaxin interacted directly with 35S-labeled SRF translated in vitro (Figure 5C). We also used chromatin immunoprecipitation assays to demonstrate that endogenous leupaxin associated with the CArG-containing regions of the SM -actin and SM-MHC promoters (but not with the c-fos promoter) in huCSMCs grown in serum (Figure 5D). When combined with results from gel shift assays, demonstrating that leupaxin did not associate directly with the SM -actin CArGs (data not shown), these results strongly suggest that leupaxin interacts with SRF in vivo. Interestingly, leupaxin was not found in association with CArG elements in serum-starved huCSMCs; however, under these conditions, TGF-β significantly promoted leupaxin association with CArG-containing region of the SM-actin promoter, without effecting leupaxin expression levels (Figure 5E). These data corroborate the finding that leupaxin and TGF-β exhibit functional synergy in the promotion of SM marker gene expression. To directly test whether the effects of leupaxin on SMC differentiation marker gene required SRF, we expressed leupaxin in SRF–/– embryonic stem cells. As expected, expression of leupaxin did not enhance SM22 reporter gene expression in SRF–/– embryonic stem cells, but this response could be rescued by coexpression of SRF (supplemental Figure III). Collectively, these data strongly support our hypothesis that leupaxin is recruited to the SMC-specific promoters through a direct interaction with SRF and that this leads to increased SMC differentiation marker gene expression.
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Our next goal was to test whether leupaxin nuclear/cytoplasmic shuttling was an important mechanism that regulated the effects of leupaxin on SMC-specific transcription. The effects of LMB indicated that leupaxin likely contains a leucine-rich CRM-1–dependent nuclear export sequence (NES), and we identified 2 regions (amino acids 4 to 11 and 134 to 144) that conformed to consensus NES sequences identified in the LIM proteins Trip6, zyxin, LPP, and Hic-5 (Figure 6A).21 Four leucine to alanine mutations were made at each site (mNES1 [L4,7,8,11A] and mNES2 [L134,137,141,144A]) separately and in combination (dbl-mNES) in the context of GFP–leupaxin. We used 10T1/2 cells and rat primary aortic SMCs (rASMCs) for these and subsequent localization experiments because the huCSMCs proved to be remarkably resistant to transfection. Importantly, significant leupaxin mRNA is expressed in both 10T1/2 and rASMCs (not shown), and the subcellular localization of GFP–leupaxin in these cells was virtually identical to that of flag–leupaxin and to the localization of endogenous leupaxin in huCSMCs (Figure 6A). Similar to our previous results, 15% of 10T1/2 cells exhibited nuclear accumulation of GFP–leupaxin, whereas treatment with LMB for 1 hour resulted in nuclear accumulation in nearly all cells (Figure 6A). Whereas individual mutation of either NES1 or NES2 increased the percentage of cells with nuclear GFP–leupaxin (to approximately 45%), mutation of both sites resulted in nuclear localization in approximately 90% of transfected cells (Figure 6A and supplemental Figure IVA). Similar results were observed in rASMCs (not shown). Importantly, transfection of these constructs did not significantly affect cell morphology (supplemental Figure IVB). These data provide strong evidence to support that leupaxin undergoes CRM-1–dependent export and that the NES sequences identified are important for the rapid shuttling of leupaxin from the nucleus to the cytoplasm.
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Although the dbl mNES variant did exhibit nuclear-restricted expression, this construct contains disrupted LD1 and LD4 binding motifs that could affect normal leupaxin function. Thus, to directly test the effects of nuclear localization on transcriptional activity we generated a leupaxin variant that was targeted to the nucleus by fusion of a triple NLS tag to the N terminus of wild-type leupaxin. This construct exhibited nearly complete nuclear localization in 100% of transfected 10T1/2 cells (Figure 6A) and SMCs (data not shown) and was functional, as shown by its ability to activate the SM22 promoter even more strongly than wild-type leupaxin (Figure 6B). These data strongly support the idea that the ability of leupaxin to induce gene expression is dependent on nuclear not focal adhesion localization. Furthermore, these data suggest that the function of leupaxin as a transcriptional regulator can be modified by altering its subcellular localization.
Our demonstration of a FAK–leupaxin interaction suggested that FAK may regulate leupaxin localization and/or transcriptional activity. We reasoned that enhanced FAK activity may lead to sequestration of leupaxin in focal adhesions, thus limiting its effects in the nucleus. To test this hypothesis, we examined endogenous leupaxin localization in huCSMCs under different conditions that modulate FAK activity. huCSMCs plated on tissue culture plastic under serum-starved conditions exhibit low FAK activity (as assessed by autophosphorylation of FAK on Y397) and near-complete restriction of leupaxin within the nucleus (Figure 7A an 7B). Interestingly, in serum-starved cells plated on FN for 90 minutes (that exhibit high FAK activity), leupaxin was exclusively associated with focal adhesions (Figure 7A). We next used a pharmacological approach to determine whether endogenous leupaxin shuttling was regulated by FAK/Src activity. We found that treatment with the FAK/Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine) attenuated FAK activity and led to remarkable nuclear accumulation of leupaxin in huCSMCs plated on FN (Figure 7A and 7B).
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We next used the GFP-tagged leupaxin construct to track leupaxin shuttling in real time in transfected SMCs to determine whether FAK/Src activity regulates the rate of leupaxin translocation to the nucleus. To this end, we identified cells in which GFP–leupaxin was predominantly cytoplasmic, pretreated the cells with PP2 or vehicle for 10 minutes, and then performed time-lapse imaging immediately following LMB treatment. LMB-induced leupaxin nuclear accumulation was evident much earlier in PP2-treated SMCs compared to vehicle-treated cells (Figure 7C and supplemental Movies 1 and 2). Indeed, 90% of PP2 pretreated cells exhibited marked leupaxin nuclear localization within 5 minutes following LMB treatment, whereas vehicle-treated cells required at least 20 minutes to exhibit significant nuclear accumulation.
In support of a functional significance of FAK-dependent leupaxin shuttling, we found that overexpression of a constitutively active FAK variant (termed SuperFAK;22 promoted focal adhesion-associated leupaxin localization (similar to plating on FN) and partially reversed the marked leupaxin-induced SMC marker gene transcription in 10T1/2 cells (Figure 7D). It should be noted that although both ectopic expression of SuperFAK (data not shown) and plating cells on FN resulted in pronounced focal adhesion localization in cells maintained in 10% serum, treatment of both populations of cells with LMB for 1 hour did result in substantial nuclear accumulation of leupaxin. Thus, even under conditions of enhanced FAK activity some nuclear shuttling of leupaxin does occur, which may (in part) account for the incomplete rescue of promoter activity observed following SuperFAK expression. Taken together, these results indicate that activation of FAK inhibits leupaxin-induced transcription by sequestering leupaxin to focal adhesions.
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Discussion |
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Leupaxin is an understudied 43-kDa protein that was originally reported as having a lymphoid-restricted expression pattern.16 We show for the first time that leupaxin is also highly expressed in aorta and in cultured SMCs. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an SRF cofactor in the nucleus. We found that leupaxin forms a complex with SRF, associates with CArG-containing regions of the SMC-specific promoters, and that ectopic expression of leupaxin induces SMC differentiation marker gene expression. Subsequent studies indicated that enhanced FAK activity attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance SRF-dependent gene transcription. Thus, these studies indicate that sequestration of leupaxin to focal adhesion plaques may be 1 mechanism by which extracellular matrix–dependent signals might regulate phenotypic switching of SMCs.
Leupaxin belongs to a family of proteins including paxillin and Hic-5 that may share some overlapping cellular functions. Each of these proteins are comprised of 4 N-terminal LD motifs and 4 C-terminal LIM domains,21 and they share approximately 40% identity at the amino acid level. Leupaxin has also been reported to share several binding partners with paxillin such as Pyk2, FAK, Src, PTP-PEST, and p95 paxillin kinase linker.16,17,23,24 Leupaxin was previously shown to colocalize with Pyk2 and FAK in the cortical F-actin domain in JY8 lymphoblasts and within podosomes of osteoclasts.16,23 Thus far, known functions for leupaxin include suppression of B-cell antigen receptor signaling in lymphoblasts,25 induction of bone resorption in osteoclasts,24 and induction of cell motility in bone-derived prostate cancer cells.17
Treatment with the nuclear export inhibitor leptomycin B causes retention of paxillin, Hic-5, and leupaxin in the nucleus, providing evidence that each of these family members may share a common function to coordinate cell adhesion status with specific changes in gene expression.21 Although numerous LIM proteins have been shown to undergo nucleocytoplasmic shuttling, it is becoming clear that distinct mechanisms regulate their trafficking. Hic-5 demonstrates oxidant-sensitive nuclear export that was shown to be dependent on 2 cysteine residues located proximal to the canonical NES identified within the Hic-5 LD3 motif.26 Notably, Hic-5 appears to be unique in this regulation because neither paxillin nor leupaxin harbors similarly located cysteine residues. Recently, elegant studies by Tsujita et al indicate that nuclear shuttling of zyxin in cardiomyocytes is regulated in a cGMP and AKT-dependent fashion and that agonists that stimulate protein kinase G (including estradiol and insulin-like growth factor-1) induce dramatic nuclear accumulation of zyxin and paxillin in these cells.27 On the other hand, the LIM only protein FHL2 has been shown to translocate to the nucleus in response to Rho A–dependent signals.28 Although we have not observed Rho- or cGMP-dependent nuclear accumulation of leupaxin in huCSMCs in these studies (data not shown), we did find that leupaxin localization was regulated by the activation state of FAK in these cells. Interestingly, leupaxin was recently shown to be a substrate for Src in A20 B lymphoma cells,25 and we have shown that leupaxin is a direct substrate for FAK in vitro (L.J. Sundberg-Smith, et al., unpublished data, 2007). Thus, we favor a model whereby activation of FAK and/or Src likely promotes phosphotyrosine-dependent leupaxin protein interactions within focal adhesions, thus limiting the amount of leupaxin available to shuttle to the nucleus. It remains to be determined whether FAK activity may also regulate SM gene expression through cytoplasmic retention of other LIM-containing SRF cofactors.
Once in the nucleus, LIM proteins can participate in transcriptional control, a function likely dependent on the ability of these proteins to provide scaffolds for transcription factors and/or chromatin-remodeling factors. Both paxillin and Hic-5 bind to steroid receptors and have been shown to coactivate androgen, glucocorticoid, and progesterone response genes by bridging an association with the nuclear matrix.29,30 In addition, Hic-5 has been shown to influence Sp1-dependent c-fos and p21 expression by inducing a complex between Sp1, SMAD3, and the histone acetyltransferase p300.31 The finding that Hic-5 and leupaxin appear to be reciprocally expressed during development (with higher levels of Hic-5 observed in less-differentiated SMCs) coupled with evidence that Hic-5 regulates Sp1-dependent transcription, whereas leupaxin regulates SRF-dependent transcription, is particularly interesting with respect to SMC phenotypic switching. Although Sp1 can act as either a transcription enhancer in certain cell types, in SMCs, Sp1 acts as a repressor of SMC marker gene transcription by interacting with G/C-rich repressor elements contained in SM22 and SM-MHC promoters.32 Thus, it is possible that these 2 FAK binding partners have opposing functions with respect to SM gene transcription.
Although the paxillin family members may induce divergent gene regulation in SMCs, several other LIM domain proteins besides leupaxin including CRP1/2, LPP, and FHL2 (which also cycle between focal adhesions and the nucleus) share the ability to influence SRF-mediated gene transcription.19,20,33 Although some of these LIM proteins may have partially overlapping functions, it is likely that additional regulatory pathways that alter the expression levels, localization, or protein binding interactions ultimately impart specific functions for each of these molecules.
In conclusion, we propose a model that leupaxin may play a dual role in SM function, aiding to coordinate multiprotein complexes in focal adhesions and in the nucleus. Because leupaxin localization is clearly modulated by FAK/Src activity in SMCs, we postulate that extracellular matrix– and agonist-dependent regulation of FAK activity in vessels could impart precise control of leupaxin localization to balance the migratory and contractile capacities necessary for proper vasculogenesis during development and following vascular injury. Interestingly, recent studies in our laboratory indicate that FAK inactivation in SMCs by homologous recombination promotes TGF-β–induced SMC differentiation but attenuates platelet-derived growth factor–stimulated SMC motility (L.J. Sundberg-Smith, et al., unpublished observations, 2007). Thus, it will be of future interest to determine to what extent these phenotypes are attributable to mislocalization of leupaxin. Although our data provide clear evidence of a role for nuclear leupaxin in promoting SRF-dependent gene transcription, future gene targeting studies will be necessary to determine under which circumstances leupaxin activity may be necessary for specific SMC functions.
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Acknowledgments |
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We thank Marisa Deburkarte (laboratory assistant, Department of Pathology, University of North Carolina, Chapel Hill) for excellent technical assistance and Vincent Moylan (clinical investigator, Department of Pathology, University of North Carolina, Chapel Hill) for human tissue procurement.
Sources of Funding
This work was supported in part by NIH/National Heart, Lung, and Blood Institute grants HL-081844 and HL-071054 (to J.M.T.) and HL070953 (to C.P.M.) and American Heart Association grants 0355776U (to J.M.T.), 0555476U (to C.P.M.), and 0515329U (to L.J.S.-S.).
Disclosures
None.
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Footnotes |
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Original received December 29, 2007; revision received May 2, 2008; accepted May 8, 2008.
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References |
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