Cells communicate with their microenvironment and neighbors by several specialized cell membrane-associated structures, which transduce diverse mechanical and biochemical signals across cell membrane and play essential roles in regulating morphogenesis and maintaining tissue structural and functional integrity and homeostasis. Cell and extracellular matrix (ECM) interaction is mediated mainly by integrin and its associated protein complexes, including integrin-linked kinase (ILK), parvin, and particularly interesting cysteine- and histidine-rich (PINCH) proteins.1 Engagement of integrins with the components of the ECM leads to recruitment and formation of a cytoplasmic focal adhesion complex, referred to as IPP (ILK, parvin, and PINCH) complex.1,2 Formation of the IPP complex is essential for targeting of IPP to focal adhesion sites and for stabilization of each component of the complex, preventing them from proteosomal degradation.3-6 Signaling through integrins is bidirectional. Changes in intracellular signaling pathways and cytoskeletal organization modulate the binding of intracellular molecules to the integrin cytoplasmic tail, which in turn modifies integrin binding affinity to the ECM, and deposition and remodeling of the ECM.1,2,7 Recent studies have provided an insight into the molecular interactions and functions of these proteins in cell adhesion, migration, proliferation, differentiation, and survival and have revealed a central role for ILK and PINCH in mediating bidirectional integrin signaling. The focus of this review will be on the molecular interactions and in vivo functions of PINCH. We will discuss unresolved questions and future directions in dissecting the molecular mechanism of PINCH and highlight any potential clinical implications.
MOLECULAR INTERACTIONS AND FUNCTIONS OF PINCH
Integrin-linked kinase contains N-terminal ankyrin repeat, pleckstrin homology (PH), and C-terminal Ser/Thr kinase domains.8,9 Integrin-linked kinase binds directly to the cytoplasmic tail of β1 and β3 integrins through its C-terminal kinase domain. Integrin-linked kinase also binds to the Lin11-Isl-1-Mec-3 (LIM) domain-only protein PINCH10-12 and a number of actin cytoskeleton-associated proteins, such as parvin and paxillin, thus linking ECM-integrin to the actin cytoskeleton and other intracellular pathways.1,13-17 These interactions of ILK are fundamental to the establishment of the integrin-actin cytoskeleton network and for the accurate control of basic cellular functions such as cell migration, spreading, growth, and survival. Disruption of these interactions by various experimental approaches targeting PINCH, ILK, or parvin; such as dominant-negative overexpression; or small interfering RNA gene knockdown or gene knockout lead to defects in cell migration, spreading, survival, and ECM assembly.3,14,15,17-27
The kinase activity of ILK is regulated by cell-matrix adhesion and growth factors in a phosphoinositol-3 kinase (PI3K)-dependent manner (Fig. 1).8,9 Cell adhesion and growth factor stimulation activate PI3K, which in turn increases the production of phosphatidylinositol 3,4,5-trisphosphate.8 The 3-phosphoinoside lipid binds to the PH motif of ILK and activates its kinase activity, which in turn activates multiple signaling pathways involved in cell adhesion, migration, growth and survival.1,2,22,27-32 Overexpression of the phosphatidylinositol 3,4,5-trisphosphate phosphatase, phosphatase and tensin homolog (PTEN), or treatment of cells with the PI3K inhibitors Wortmannin or Ly294002 inhibits ILK activation.8 Activated ILK phosphorylates and activates protein kinase B (PKB)/Akt at Ser473, an event critical for cell growth and survival.8 Integrin-linked kinase can also phosphorylate and inhibit GSK3β, leading to the stabilization and translocation of β-catenin to the nucleus and activation of gene expression (Fig. 1).33
The N-terminal ankyrin domain of ILK binds directly to the LIM domain-only adaptor protein PINCH. Two PINCH proteins have been characterized in mammals, including PINCH1 (LIM and senescent cell antigen-like-containing domain protein 1 [LIMS1]) and PINCH2 (LIMS2). Particularly interesting cysteine- and histidine-rich protein 1 was originally identified in an antibody screen of a human complementary DNA library as a marker for senescent erythrocytes.34 A yeast 2-hybrid screen using the N-terminal ankyrin domain of ILK as bait identified PINCH as a direct binding partner of ILK.11 Particularly interesting cysteine- and histidine-rich protein 2, a close homolog of PINCH1, was identified by complementary DNA sequence database mining. Proteins PINCH1 and PINCH2 share high sequence and structural homology, and both are localized to focal adhesions and the nucleus.12,35 Particularly interesting cysteine- and histidine-rich protein, through its LIM domain-mediated protein interactions, functions as a molecular scaffold that supports the assembly of a multi-protein complex at sites of integrin enrichment.
Particularly interesting cysteine- and histidine-rich protein 1 is composed of 5 LIM domains (LIM1-5) and a short C-terminal sequence. It shares high homology to that of PINCH2, expect in the LIM5 domain and the C-terminal tail.10,12,35,36 The binding of the ankyrin repeat domain of ILK has been mapped to the first LIM domain (LIM1) of PINCH, which is required for localization and function of the ILK-PINCH complex (Fig. 1).36 In addition to promoting ILK-mediated phosphorylation and activation of PKB/Akt at Ser473, PINCH1 is also required for phosphorylation of PKB/Akt at Thr308 and survival even in cells with a constitutively active form of PKB/Akt.3 These data suggest that PINCH1 activates PKB/Akt in ILK-dependent and -independent manners and functions both upstream and downstream of PKB/Akt.3 Interestingly, the 2 PINCHs compete for binding to ILK, and the PINCH1-ILK and PINCH2-ILK interactions are mutually exclusive.12 Overexpression of PINCH2 inhibits the PINCH1-ILK interaction and reduces cell spreading and migration, suggesting an intriguing role for PINCH2 in fine tuning the PINCH1-ILK interaction in cell adhesion and migration.3,12,36 In addition, the expression of a chimeric PINCH with PINCH1 LIM domains and PINCH2 C-terminal tail cannot rescue the spreading defect in PINCH1-knockdown HeLa cells,37 suggesting that the C-terminal of PINCH1 is required for its function. Nevertheless, expression of a full-length PINCH2 completely restores the adhesion and spreading defects of PINCH1-null fibroblasts,38 suggesting a redundant role of PINCH1 and PINCH2 in this context. Furthermore, global knockout of PINCH2 and single knockout of PINCH1 in the myocardium did not result in any basal cardiac phonotype, which has been attributed to the redundant role of PINCH1 and PINCH2.39
NCK2 is a Src homology 2/3 (SH2-SH3) adaptor protein implicated in various signaling pathways, including that of growth factors, cell adhesion receptors, and the cytoskeleton.40 It has been shown that integrin-mediated signaling is required for potentiating growth factor signaling.1,2,40 NCK2 has been shown to associate with receptor tyrosine kinases via its SH2 domain. NCK2, via its interactions with Rho effectors and other cytoskeleton-associated proteins such as Wiskott-Aldrich syndrome (WASP) and neural Wiskott-Aldrich syndrome proteins (N-WASP), is implicated in linking receptor tyrosine kinases to actin cytoskeleton remodeling (Fig. 1).1,41,42 The LIM4 domain of PINCH1 has been shown to bind to NCK2 but not its homolog NCK1,40,43 thus linking growth factor signaling pathways to integrin and cytoskeletal signaling. However, the role of PINCH1-NCK2 interaction remains to be determined because the binding affinity of NCK2 to PINCH1 is very weak and NCK2 knockout mice did not present any phenotype.40,44
Ras suppressor protein 1 (Rsu-1) is a highly conserved leucine-rich repeat (LRR) protein, identified as a Ras suppressor based on its ability to inhibit transformation by Ras.45,46 Ras suppressor protein 1 is colocalized with PINCH1 and ILK in focal adhesions.47 Moreover, studies have shown that the LIM5 domain of PINCH1, but not that of PINCH2, binds to Rsu-1, and this interaction plays a role in targeting PINCH1 to focal adhesions, stabilizing the IPP complex and inhibiting migration.10,47,48 Ectopic expression of Rsu-1 inhibits anchorage-independent growth of Ras-transformed cells and human tumor cell lines, in which both expression of ILK and PINCH are increased. Thus, Rsu-1 may represent an important cross talk between Ras and integrin signaling pathways and play an important role in cell growth and tumoriogenesis.47-49
The G-actin-sequestrating peptide thymosin-β4 binds to LIM4 and LIM5 domains of PINCH1 and forms a functional complex with PINCH1 and ILK, which activates PKB/Akt and promotes migration and survival of embryonic and postnatal cardiac cells in culture (Fig. 1).50 In a mouse myocardial infarction model, thymosin-b4 treatment results in up-regulation of ILK and Akt activity in the heart, enhanced cardiomyocyte survival, and improved cardiac function.50 Although ILK-Akt pathway has been implicated in these regeneration processes, the underlying molecular mechanisms remain to be determined, which may involve multiple cell types, alterations in metabolism and energy consumption, and enhanced angiogenesis that promote cell survival. It is unknown whether PINCH2 also binds to thymosin-β4; however, given the redundant role of the 2 PINCHs in the myocardium, it is likely that PINCH2 may also bind to thymosin-β4 and act redundantly with PINCH1 in this context. Thus, thymosin-β4 PINCH-ILK pathway may represent a promising therapeutic target for cardiac disease.51
ROLES OF PINCH1 DURING EARLY DEVELOPMENT STAGE AND EMBRYONIC STEM CELL
Genetic studies in Caenorhabditis elegans and Drosophila have revealed an essential role for PINCH in mediating integrin-ILK-dependent signaling.35,52 The deletion of UNCoordinated-97, an orthologue of PINCH1, in C. elegans results in muscle detachment and an embryonic-lethal phenotype called paralyzed and arrested elongation at the 2-fold stage (PAT)35 resembling that of β1 integrin/PAT-3 or ILK/PAT-4.53,54 In Drosophila muscle, PINCH displays a completely overlapping expression pattern with ILK and βPS integrin, prominently enriched at the muscle attachment sites.52 Flies deficient in PINCH1 (named stck in Drosophila) exhibit muscle detachment, similar to the phenotypes of ILK and PS integrin.52,55-57
During early mouse embryogenesis, the inner cell mass (ICM) of the blastocyst develops into the primitive endoderm and the epiblast.58 The primitive endoderm forms the surface of the ICM of the blastocyst and deposits a basement membrane. The basement membrane is required for adjacent ICM cells to polarize and to establish the columnar epiblast.59 The importance of integrin-ILK-mediated cell-cell and cell-matrix interactions during early embryonic development is highlighted by genetic studies in mouse models.60-64 In β1 integrin null embryos, the primitive endoderm fails to deposit laminin α1 and form the basement membrane.60,62 In ILK null mouse embryos, the primitive endoderm differentiates and produces a basement membrane, but the epiblast fails to polarize or cavitate, and mutants die at the peri-implantation stage.63 In contrast to that of invertebrates, 2 PINCH isoforms, PINCH1 and PINCH2, are expressed in mammals.10,12,34 However, PINCH2 is not expressed until a later developmental stage from E14.5 onward. Particularly interesting cysteine- and histidine-rich protein 1 is readily detectable in blastocysts at approximately E3.5.6,39 Particularly interesting cysteine- and histidine-rich protein null mouse embryos die at E5.5, exhibiting a disorganized egg cylinder, with decreased cell proliferation and excessive cell death, highlighting an important role of PINCH1 during early embryogenesis.5,6 In addition, studies from Fässler laboratory using a PINCH1 null embryoid body model and comparing with that of an ILK null embryoid body highlighted an ILK-independent role of PINCH1 in endoderm survival and cell-cell adhesion.5
ROLE OF PINCH1 IN NEURAL CREST AND OUTFLOW TRACT MORPHOGENESIS
Neural crest cells (NCCs) are a transient embryonic stem cell population that originate from the dorsal neural tube and migrate along well-defined migratory pathways to their final destinations, giving rise to a diversity of cell types and contributing to craniofacial and cardiac outflow tract morphogenesis, and formation of the entire peripheral nervous system.65-67 Cardiac NCCs migrate into outflow tract and contribute to the smooth muscle component of the outflow tract and outflow tract septation and endocardial cushion morphogenesis.66-72 Perturbation of cardiac NCC development causes congenital heart defects in animal models and in humans, affecting the outflow tract and great vessels.70,73-76
Recent studies from our group have shown that PINCH1 is highly expressed in NCCs and that neural crest-specific ablation of PINCH1 leads to severe cardiovascular defects, including an enlarged common arterial trunk, ventricular septal defects, and defective cushion/valve maturation.77 In addition to cardiovascular defects, mutants exhibited defects in craniofacial structures, such as hypoplastic thymus and craniofacial malformation. Interestingly, PINCH1-deficient NCCs did migrate correctly into the pharyngeal apparatus as demonstrated by fate mapping and by in situ hybridization using the neural crest marker cellular retinol-binding protein type 1 (Crbp1).77 Importantly, we found that from E11.5 onward, cardiac NCCs in the outflow tract continue to proliferate, fail to exit the cell cycle, and undergo smooth muscle cell differentiation. Particularly interesting cysteine- and histidine-rich protein 1-deficient NCCs in the outflow tract cushion underwent markedly increased apoptosis at E11.5 to E13.5, associated with an observed failure of cushion remodeling and valve maturation.77 These observations demonstrate that PINCH1 plays important roles in proliferation, differentiation, and survival of NCCs, but it appears dispensable for NCC migration into the outflow tract.
It has been shown that interaction of PINCH and ILK is essential for their stability, targeting to focal adhesion sites and function.3-6 However, we found no significant change in ILK expression in cultured NCCs and in the outflow tract of PINCH1 mutants, suggesting that inactivation of PINCH1 in NCCs does not affect formation of the focal adhesion complex and cardiac phenotypes may be caused by an ILK independent mechanism. Supporting this, PINCH1 in NCCs was found to be predominantly nuclear, and PINCH1 contains a presumed leucine-rich nuclear export signal and an overlapping basic nuclear localization signal, suggesting that it may act as a shuttling/signaling protein or directly involved in regulation of gene expression.77,78
A unique feature of the neural crest PINCH1 mutant is the aneurysmal arterial trunk. It is important to note that several human syndromes that include aortic and vascular aneurysms have been associated with alterations in transforming growth factor β signaling.79-81 Transforming growth factor β signaling plays an important role in specification, migration, survival, and differentiation of NCCs,65,82,83 and its expression in the PINCH1 mutant outflow tract was dramatically down-regulated. It is tempting to speculate that PINCH1 mutation might be involved in human syndromes characterized by aortic and vascular aneurysms.
PINCH IN MYOCARDIAL GROWTH, MATURATION, AND REMODELING
Myocardial cells interact with each other and the matrix at specialized membranous structures, referred to as the intercalated discs and costameres, respectively,1,31,61,84-86 which provide mechanical and electrical coupling between myocytes and enable the myocardium to function as a syncytium. During late development and early postnatal life, cardiomyocytes undergo physiological hypertrophic growth, realign cytoskeletal components, and acquire a mature cytoarchitecture to meet a dramatically increased hemodynamic load. One feature of these postnatal myocardial remodeling and maturation is the redistribution and segregation of distinct cell-matrix and cell-cell adhesions.84,86-89 During embryonic development, myocytes interact with each other extensively via the cadherin-mediated adherens junction, which seems to play a dominant role in mediating myocyte adhesion and function.90 During perinatal development, the myocytes continue to grow, elongate, and interact with each other only at the bipolar ends by intercalated discs. Intercalated discs are disassembled from lateral cell membranes and reassembled at the bipolar ends of the cells, whereas costameres remain in the lateral cell membranes.91,92 However, molecular mechanisms regulating the segregation and redistribution of cell adhesions during postnatal myocardium remodeling and maturation remain largely unknown.
The integrin signaling pathway has been shown to be important for maintenance of cardiac structure and function.93-95 Cardiac-specific deletion of β1 integrin or ILK in mice resulted in disruption of the focal adhesion complex and development of cardiomyopathy and heart failure.93-95 Deletion of PINCH1 in mouse myocardium did not lead to any basal cardiac phenotype, In addition, global PINCH2 knockout did not result in any phenotype.5,6,39
However, analyses of mice that were doubly homozygous null for PINCH1 and PINCH2 in the myocardium (conditional double knockout [CDKO]) have revealed a redundant yet essential role for PINCH in postnatal myocardial remodeling and maturation and in maintaining myocardial integrity.39 Hearts of CDKO mutants were dilated, ventricular wall thickness was highly irregular with abnormal trabeculation, and mutant mice developed cardiomyopathy and heart failure within 4 weeks.39 Consistent with the observed role of PINCH in mediating cell-matrix interaction, PINCH CDKO myocytes in culture failed to attach and spread and exhibited disrupted focal adhesions. Moreover, expression of ILK, parvin, and β1 integrin and phosphorylation of Akt were significantly reduced, and there was markedly increased cell death in hearts of PINCH CDKO mice.39 In addition, electron microscopic analysis revealed disruption of costameric structures and intercalated discs in the PINCH CDKO myocardium.39
Particularly interesting cysteine- and histidine-rich protein also plays a critical role in modulating the stability and polarized distribution of intercalated disc proteins during postnatal myocardial maturation and remodeling.39 The expression of proteins that function in adherens and gap junctions of the intercalated discs is markedly affected in the PINCH mutant myocardium. In CDKO myocardium, Connexin43, α-E-catenin, and ZO-1 were significantly reduced and retained a disperse expression pattern throughout the lateral membrane comparable with that seen at earlier stages of development, rather than being expressed at the intercalated discs. Similarly, expression of N-cadherin, β-catenin, and vinculin remained largely in the lateral cell membrane of PINCH mutant heart; however, their expression at the intercalated discs were lost despite that the overall amounts of these 3 proteins were unchanged. Taken together, our study demonstrates that PINCH proteins play essential roles in myocardial growth, maturation, remodeling, and function and highlights the importance of studying the role of PINCH proteins in human cardiac injury and cardiomyopathy.
Despite significant progress in our understanding of the molecular regulation of cell adhesions by integrin and its associated complex, many outstanding questions remain. Integrin-linked kinase and PINCH are ubiquitously expressed in all cell types. However, it is not clear how 1 specific function in a given cellular context is achieved over others, such as survival versus growth and differentiation and migration. Particularly interesting cysteine- and histidine-rich protein 1 plays a critical role in the neural crest in a seemingly ILK-independent manner, and ILK expression is not affected in the PINCH1 null neural crest. In addition, PINCH1 in NCCs was found to be predominantly nuclear, and hence, it will be important to determine whether PINCH1 functions as a shuttling/signaling protein or is directly involved in the regulation of gene expression (Fig. 1). It will also be interesting to determine the potential role of ILK in neural crest cells to establish how PINCH and ILK function inside and outside focal adhesion complexes.
Cardiac integrity and function are maintained by dynamic interactions of multiple cell types within the heart, including myocytes, fibroblasts, and endothelial cells, and therefore, it would be interesting to define the role for PINCH in these contexts. Given the important role of thymosin β4 in mediating cardiomyocyte survival, it is important to determine the detailed molecular mechanisms by which the interactions of thymosin β4, ILK, and PINCH promote cardiomyocyte survival. Answers to these questions will increase our understanding of fundamental ECM-integrin signaling and facilitate the development of novel therapies to human heart disease.
The authors thank Drs Indroneal Banerjee and Robert Lyon for critical reading of the manuscript.
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