P0-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin.
ABSTRACT: P0-related protein (PZR), a Noonan and Leopard syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and Leopard syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with ?1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(?5?1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.
Project description:Protein-zero related (PZR) is an immunoglobulin V (IgV)-type immunoreceptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PZR interacts with Src homology 2 domain-containing tyrosine phosphatase (SHP-2) via its tyrosine-phosphorylated ITIMs, for which c-Src is a putative kinase. Towards elucidating PZR function in endothelial cells (ECs), the authors cloned PZR from bovine aortic endothelial cells (BAECs) and characterized it. Mature bovine PZR had 94.8% and 92.7% sequence identity with canine and human proteins, respectively, and the two ITIM sequences were conserved among higher vertebrates. PZR was expressed in many cell types and was localized to cell contacts and intracellular granules in BAECs and mesothelioma (REN) cells. Coimmunoprecipitation revealed that PZR, Grb-2-associated binder-1 (Gab1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) were three major SHP-2-binding proteins in BAECs. H(2)O(2) enhanced PZR tyrosine phosphorylation and PZR/SHP-2 interaction in ECs in a dose-and time-dependent manner. To see if tyrosine kinases other than Src are also capable of phosphorylating PZR, the authors cotransfected HEK293 cells with PZR and one of several tyrosine kinases and found that c-Src, c-Fyn, c-Lyn, Csk, and c-Abl, but not c-Fes, phosphorylated PZR and increased PZR/SHP-2 interaction. These results suggest that PZR is a cell adhesion protein that may be involved in SHP-2-dependent signaling at interendothelial cell contacts.
Project description:The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of ??-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of ??-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.
Project description:Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision-making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin-associated adhesion complex composition.Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature-based reference set of cell adhesion-related components and using ontological and protein-protein interaction network analyses.Adhesion complex-specific proteins in MSCs were identified that comprised predominantly cell adhesion-related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain-containing proteins in MSC adhesion complexes were highlighted, which may act as force-sensing components.These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention.
Project description:Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.
Project description:Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an "open" conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation-defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-?B pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice, and PZRY242F mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.
Project description:Noonan syndrome (NS) is a common autosomal dominant disorder that presents with short stature, craniofacial dysmorphism, and cardiac abnormalities. Activating mutations in the PTPN11 gene encoding for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) causes approximately 50% of NS cases. In contrast, NS with multiple lentigines (NSML) is caused by mutations that inactivate SHP2, but it exhibits some overlapping abnormalities with NS. Protein zero-related (PZR) is a SHP2-binding protein that is hyper-tyrosyl phosphorylated in the hearts of mice from NS and NSML, suggesting that PZR and the tyrosine kinase that catalyzes its phosphorylation represent common targets for these diseases. We show that the tyrosine kinase inhibitor, dasatinib, at doses orders of magnitude lower than that used for its anticancer activities inhibited PZR tyrosyl phosphorylation in the hearts of NS mice. Low-dose dasatinib treatment of NS mice markedly improved cardiomyocyte contractility and functionality. Remarkably, a low dose of dasatinib reversed the expression levels of molecular markers of cardiomyopathy and reduced cardiac fibrosis in NS and NSML mice. These results suggest that PZR/SHP2 signaling is a common target of both NS and NSML and that low-dose dasatinib may represent a unifying therapy for the treatment of PTPN11-related cardiomyopathies.
Project description:Local signals from tissue-specific extracellular matrix (ECM) microenvironments, including matrix adhesive ligand, mechanical elasticity and micro-scale geometry, are known to instruct a variety of stem cell differentiation processes. Likewise, these signals converge to provide multifaceted, mechanochemical cues for highly-specific tissue morphogenesis or regeneration. Despite accumulated knowledge about the individual and combined roles of various mechanochemical ECM signals in stem cell activities on 2-dimensional matrices, the understandings of morphogenetic or regenerative 3-dimenstional tissue microenvironments remain very limited. To that end, we established high-throughput platforms based on soft, fibrous matrices with various combinatorial ECM proteins meanwhile highly-tunable in elasticity and 3-dimensional geometry. To demonstrate the utility of our platform, we evaluated 64 unique combinations of 6 ECM proteins (collagen I, collagen III, collagen IV, laminin, fibronectin, and elastin) on the adhesion, spreading and fate commitment of mesenchymal stem cell (MSCs) under two substrate stiffness (4.6 kPa, 20 kPa). Using this technique, we identified several neotissue microenvironments supporting MSC adhesion, spreading and differentiation toward early vascular lineages. Manipulation of the matrix properties, such as elasticity and geometry, in concert with ECM proteins will permit the investigation of multiple and distinct MSC environments. This paper demonstrates the practical application of high through-put technology to facilitate the screening of a variety of engineered microenvironments with the aim to instruct stem cell differentiation.
Project description:SHP2 is an allosteric phosphatase essential for growth factor-mediated Ras activation. Germ-line mutations in SHP2 cause clinically similar LEOPARD and Noonan syndromes, two of several autosomal-dominant conditions characterized by gain-of-function mutations in the Ras pathway. Interestingly, Noonan syndrome SHP2 mutants are constitutively active, whereas LEOPARD syndrome SHP2 mutants exhibit reduced phosphatase activity. How do catalytically impaired LEOPARD syndrome mutants engender gain-of-function phenotypes? Our study reveals that LEOPARD syndrome mutations weaken the intramolecular interaction between the N-SH2 and phosphatase domains, leading to a change in SHP2 molecular switching mechanism. Consequently, LEOPARD syndrome SHP2 mutants bind upstream activators preferentially and are hypersensitive to growth factor stimulation. They also stay longer with scaffolding adapters, thus prolonging substrate turnover, which compensates for the reduced phosphatase activity. The study provides a solid framework for understanding how individual SHP2 mutations cause diseases.
Project description:SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY(246)AQL and VVY(263)ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34(+)CD38(+) and early clonogenic progenitors, and at lower levels on CD34(+)CD38(-) cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133(+) precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Delta 46-110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility.
Project description:The tyrosine phosphatase SHP2 (PTPN11) regulates cellular proliferation, survival, migration, and differentiation during development. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, which have overlapping clinical features. Paradoxically, Noonan syndrome mutations increase SHP2 phosphatase activity, while LEOPARD syndrome mutants are catalytically impaired, raising the possibility that SHP2 has phosphatase-independent roles. By comparing shp2-deficient zebrafish embryos with those injected with mRNA encoding LEOPARD syndrome point mutations, we identify a phosphatase- and Erk-dependent role for Shp2 in neural crest specification and migration. We also identify an unexpected phosphatase- and Erk-independent function, mediated through its SH2 domains, which is evolutionarily conserved and prevents p53-mediated apoptosis in the brain and neural crest. Our results indicate that previously enigmatic aspects of LEOPARD syndrome pathogenesis can be explained by the combined effects of loss of Shp2 catalytic function and retention of an SH2 domain-mediated role that is essential for neural crest cell survival.