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(Circulation Research. 2006;98:306.)
© 2006 American Heart Association, Inc.

Editorials

Organizing Motility

LIM Domains, LPP, and Smooth Muscle Migration

Mark W. Majesky

From the Carolina Cardiovascular Biology Center, and Departments of Medicine & Genetics, University of North Carolina at Chapel Hill.

Correspondence to Mark W. Majesky, PhD, Carolina Cardiovascular Biology Center, 8200 MBRB, Campus Box 7126, University of North Carolina, Chapel Hill, NC 27599-7126. E-mail mmajesky{at}med.unc.edu

See related article, pages 378–385

Key Words: smooth muscle cells • LIM domain • migration • focal adhesions

	Introduction

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
Vascular smooth muscle cells (SMCs) acquire a highly specialized cytoskeleton during development that is organized for efficient transmission of contractile force. This SMC cytoskeleton must be extensively reorganized to support directed cell migrations that are required for repair of arterial injury. Particularly important in this reorganization process are adaptor proteins that mediate the assembly of multiprotein complexes involved in cell adhesion, lamellipodial extensions, signal transduction, and transcriptional activation. One such adaptor protein is called lipoma-preferred partner (LPP), a member of the LIM domain–containing protein family, most closely related to zxyin, ajuba, LIM domain–containing protein-1 (LIMD1), and thyroid receptor-interacting protein-6 (TRIP6).¹ Evidence to suggest that LPP is a smooth muscle–restricted LIM protein that plays an important role in SMC migration after arterial injury is reported by Gorenne et al in this issue of Circulation Research.²

	Discovery of LPP as a Translocation Partner in Human Lipomas

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
LPP was discovered as a component of t(3:12) chromosome translocations found in benign tumors of human adipose tissue (lipomas).³ The chromosome 3 breakpoint occurred in a 400-kb genomic locus that encoded a protein with proline-rich sequences and leucine zipper motifs at its N terminus and three LIM domains at its C terminus. The t(3:12) translocation produced a fusion protein containing the N-terminal DNA binding domain of HMGIC fused to the C-terminal LIM domains of a protein encoded by the chromosome 3 locus. The latter was given the name LIM domain–containing lipoma-preferred partner (LPP).³ The lipoma-associated fusion protein is localized to the nucleus⁴ and can function as a transcription activator.⁵ Translocations involving the LIM domains of LPP are the most common chromosomal translocations found in human tumors. In addition to lipomas, they have been found in various benign and malignant tumors of mesenchymal origin, including leukemias (fused with the mixed lineage leukemia protein (MLL),⁶ and pulmonary chondroid hamartomas (fused with HMGA2).⁷

	The LIM Domain, a Versatile Motif for Protein–Protein Interaction

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
Modular docking domains that mediate protein–protein interactions are critical components of cellular regulatory and signaling networks. The LIM domain, in particular, is a highly versatile peptide cassette found in many adaptor proteins that mediates the assembly of multiprotein complexes involved in cell adhesion, cytoskeletal remodeling, cell motility, and gene transcription. LIM domains were first identified as cysteine and histidine-rich motifs present in Lin-11, a C elegans gene required for asymmetric division of vulval precursors, Isl1, a rat insulin 1 gene enhancer binding protein, and Mec3, a C elegans gene required for mechanosensory neuron development.^1,8 The utility of the LIM domain is reflected by the frequency with which it is found in the human genome, with at least 58 genes containing one or more LIM domains,¹ a number that is similar in frequency to SH2 domain–containing proteins.⁹ The structures of several LIM domains have been determined by NMR spectroscopy and by X-ray crystallography. They fold in the conformation of two zinc fingers stabilized by tetrahedryl zinc coordination.¹⁰ Because most LIM proteins have multiple, often tandem, LIM repeats, it is of interest that the two LIM domains of cysteine-rich LIM protein-1 (CRP1) can fold independently of one another, display distinct protein binding surfaces, and exhibit little or no restraint on rotation or conformation as a result of their proximity.^10,11 These features suggest that a single protein containing multiple LIM domains can probably bind several partners simultaneously, a characteristic feature of a protein with scaffold or adaptor function.

Many LIM proteins, including LPP, can shuttle between the cytoplasm and the nucleus, suggesting they may transfer information directly from the cytoskeleton or focal adhesions to the transcription machinery. However, there is no compelling evidence for sequence-specific DNA binding by a LIM domain. The LIM-homeodomain class of proteins, including Isl1, can bind to DNA via their homeodomains, but not their LIM domains. When implicated in transcriptional control, LIM proteins are usually found to mediate the assembly of interacting partners to form transcriptionally active complexes. For example, LIM only protein-2 (LMO2) is a double LIM domain protein that is required for erythropoiesis in the yolk sac and early embryo.¹² The tandem LIM domains of LMO2 were found to provide docking sites for GATA-1 and TAL-1/E47, thus producing a multiprotein complex with much greater transcriptional activity for key erythrocyte differentiation genes than any of the individual components alone.¹³ Similarly, the LIM and glycine-rich repeat-containing proteins CRP1 and CRP2 were found to form a high affinity complex between serum response factor (SRF) and GATA proteins, that greatly increased the affinity of SRF for its DNA recognition CArG box element in various SMC-specific differentiation marker genes.¹⁴

	LIM Domains Target LPP to Focal Adhesions in Smooth Muscle Cells

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
Through their binding partners, LIM proteins participate in a diverse array of cellular processes.¹ The C-terminal LIM domains of LPP are required for targeting the protein to points of cell–cell and cell–matrix contacts.⁴ LPP also contains N-terminal polyproline-rich motifs and ENA/VASP-homology regions that mediate association with actin filaments and focal adhesions via the vasodilator stimulated phosphoprotein (VASP) and related proteins (Figure).⁴ In intact smooth muscle tissues, Gorenne et al reported finding LPP in punctate foci at the cell surface where it was colocalized with vinculin in peripheral membrane dense bodies involved in actin filament attachment sites.¹⁵ In adult tissues, LPP was present at 100-fold higher levels in smooth muscle-rich tissues including bladder, uterus, ileum, and aorta than in non-smooth muscle organs such as liver, heart, or brain. Overexpression of LPP in cultured human iliac vein SMCs (HIVS) increased EGF-stimulated cell migration by 2.5-fold in a transwell chemotaxis assay. Moreover, treatment of HIVS cells with leptomycin-B, an inhibitor of CRM1-mediated nuclear export, caused accumulation of LPP in the nucleus.¹⁵ Cytoplasmic-nuclear shuttling has also been demonstrated for zyxin¹⁶ and TRIP6,¹⁷ suggesting it is a common property for this class of LIM proteins. Assuming these results for cytoplasmic-nuclear shuttling in vitro have counterparts in vivo, it is intriguing to consider a possible signaling role for LPP in SMCs, possibly in the feedback control of transcription of cytoskeletal proteins.

View larger version (16K): [in this window] [in a new window]   In mature smooth muscle tissues, LPP is localized to membrane-associated peripheral dense bodies (PDBs), sites of actin filament insertion in contractile SMCs. In migrating SMCs, LPP is found colocalized with vinculin at focal adhesions. LPP also contains a nuclear export sequence (NES), and the protein can accumulate in the nucleus through a shuttling mechanism that is sensitive to inhibition of rho kinase activity.15 LPP contains three C-terminal LIM domains that mediate protein–protein interactions, an ENA/VASP homology motif (E/V), and polyproline (PP) domains that can associate with actin binding proteins. Through these diverse structural domains, LPP can function as an adaptor protein to assemble multiprotein complexes at focal adhesions, PDBs, and possibly in the nucleus.

	LPP Is a Smooth Muscle-Restricted Protein That Promotes Cell Migration

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
Extending their previous work, Gorenne et al now report that the LPP gene is responsive to RhoA-mediated signaling pathways that activate serum response factor (SRF)-dependent transcription in SMC differentiation.^2,18,19 Moreover, similar to other SMC specific genes, overexpression of the SRF coactivator myocardin increased LPP gene expression by 3-fold after 48 hours.² Whereas previous results showed that overexpression of LPP enhanced SMC migration in vitro, the present work showed that loss of function using siRNAs against LPP produced a strong inhibition of SMC migration in response to an EGF chemotactic gradient. Of particular interest was the observation that in embryonic fibroblasts lacking focal adhesion kinase (FAK-null), LPP expression was reduced by 10-fold, whereas the contents of other focal adhesion proteins including vinculin, talin, or paxillin were not altered. Moreover, when FAK levels were restored in FAK-null cells by use of a tetracycline-based expression system, LPP levels returned to normal. Furthermore, expression of LPP in migration-defective FAK null cells led to a significant increase in cell spreading on a fibronectin matrix. These results suggest that levels of LPP in SMCs are controlled by FAK signaling and raise intriguing questions about the role of FAK and LPP as determinants of SMC phenotype.

	LPP Is Expressed in Migrating SMCs in Stented Pig Coronary Arteries

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
In adult pig hearts, uninjured coronary arteries exhibit strong immunostaining for LPP that colocalizes with smooth muscle -actin (SMA) and smoothelin (SMO) in the tunica media. No expression of LPP was found in the adventitial layer. In injured vessels 28 days after stent implantation, neointimal cells migrating around the stent wound were positive for LPP and SMA, but not SMO.² Adventitial cells remained negative, but LPP expression could be detected in the walls of microvessels located in the adventitial layer. These dynamic patterns of expression in injured coronary arteries suggest that LPP may partner with multiple players to facillitate cytoskeletal remodeling events needed to accomplish the transition from stationary to migrating SMCs in vivo.

	Summary

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 
LIM domain–containing proteins are versatile adaptors that provide docking sites for assembly of multiprotein complexes that mediate a wide variety of processes in cells. For vascular SMCs, extensive reorganization of cell–matrix contacts and cytoskeletal structure required for conversion from a stationary contractile phenotype to a migratory repair phenotype requires the activity of such adaptor proteins. The findings reported by Gorenne et al suggest that LPP be included in the list of important players in the SMC phenotype transition response that accompanies wound healing.² They raise a number of new questions including: What is the role of LPP in mature adult quiescent SMCs, what proteins associate with LPP at focal adhesions to promote SMC migration, does LPP shuttle to the nucleus in SMCs in vivo, and if so what nuclear proteins does it partner with and what gene targets does it control?

	Acknowledgments

 
Work in the authors laboratory was supported by the National Institutes of Health grant HL-19242. Helpful discussions with Robert J. Schwartz concerning the multiple roles of LIM proteins in cardiovascular development are gratefully acknowledged.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top Introduction Discovery of LPP as... The LIM Domain, a... LIM Domains Target LPP... LPP Is a Smooth... LPP Is Expressed in... Summary References

 

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