Focal Adhesion Complexes Research Articles

Abstract P1-05-22: Breast cancer cells that undergo an Epithelial-to-Mesenchymal transition co-opt LPP, a regulator of mesenchymal cell migration and invasion.

Abstract Transforming Growth Factor β (TGFβ) promotes breast cancer cell metastasis to multiple sites, including the bone and lungs. TGFβ is a strong inducer of Epithelial-to-Mesenchymal transitions (EMT) and breast cancers that exhibit features of an EMT acquire stem cell-like characteristics, are highly aggressive, are resistant to therapy and are refractory to tumor suppressive processes. While TGFβ itself is non-oncogenic, it is a potent modifier of the malignant phenotype in breast cancer and is capable of enhancing the migration, invasion and metastasis of ErbB2 expressing breast cancer cells. We have identified Lipoma Preferred Partner (LPP) as an indispensable regulator of TGFβ-induced migration and invasion of ErbB2 expressing breast cancer cells. LPP is ubiquitously expressed in smooth muscle cells where it mediates cell adhesion, migration and the formation of lamellipodial extensions. We hypothesize that breast cancer cells capable of undergoing an EMT can utilize novel mediators that are engaged in promoting the migration and invasion of mesenchymal cells. We propose that LPP is one such example, which promotes the migration and invasion of ErbB2 expressing breast cancer cells that have undergone a TGFβ-induced EMT. We demonstrate that ErbB2 expressing breast cancer cells display significant increases in cell migration and invasion upon TGFβ stimulation, and such responses are dependent on LPP expression. We show that LPP re-localizes to focal adhesion complexes following TGFβ-induced EMT and it is a critical determinant in focal adhesion turnover. Furthermore, we determined that LPP targeting to focal adhesions through its LIM1 domain requires the cooperation of ErbB2 and TGFβ signaling pathways. Finally, we demonstrate that LPP promotes TGFβ-induced migration and invasion of ErbB2 expressing breast cancer cells through recruitment of α-Actinin, an actin cross-linking protein. Overall, we have identified LPP as a novel mediator that integrates TGFβ and ErbB2 signaling to promote the migration and invasion of breast cancer cells that undergo an EMT. Our data reveal that breast cancer cells, which can transition from an epithelial to mesenchymal phenotype, can engage a regulator of mesenchymal cell migration and invasion. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-22.

Functional Proteomic Analysis of Long-term Growth Factor Stimulation and Receptor Tyrosine Kinase Coactivation in Swiss 3T3 Fibroblasts

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome.

MECHANICS IN MECHANOSENSITIVITY OF CELL ADHESION AND ITS ROLES IN CELL MIGRATION

Cells sense and respond to external stimuli and properties of their environment through focal adhesion complexes (FACs) to regulate a broad range of physiological and pathological processes, including cell migration. Currently, the basic principles in mechanics of the mechanosensitivity of cell adhesion and migration have not been fully understood. In this paper, an FEM-based mechano-chemical coupling model is proposed for studying the cell migration behaviors in which the dynamics of stability of FACs and the effect of cell shape on cell traction force distribution are considered. We find that the driving force of cell migration is produced by the competition of stability of cell adhesion between the cell front and cell rear, which consequently controls the speed of cell migration. We show that the rigidity gradient of matrix can bias this competition which allows cell to exhibit a durotaxis behavior, i.e. the larger the gradient, the higher the cell speed.

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231

BackgroundGonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion.MethodsActin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay.ResultsAgonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers.ConclusionsWe here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

Enhanced Adhesion, Migration and Pyk2 Expression in K562 Cells Following Imatinib Treatment

AbstractAbstract 4426 Background/Aims:Despite improved prognosis of CML patients with the use of imatinib (IM), its administration is associated with extramedullary disease (EMD) occurrence. We postulate that, like in the metastatic processes, changes in migration and adherence potential may enable leukemic cells to inhabit extramedullary sites. Focal adhesion complexes linking between extracellular matrix and the cell cytoskeleton are likely to play an important role in these processes.Pyk2 is a tyrosine kinase highly expressed in hematopoietic cells, localized to focal adhesion complexes, and known to participate in adhesion and migration processes. We have previously shown that Pyk2 participates in NB4 (an acute promyelocytic leukemia [APL] cell line) cells' adhesion and migration following exposure to the APL targeted therapy ATRA.We postulate that similar to the effect of ATRA on NB4 cells, IM being also a targeted therapy, might also be associated with enhanced adhesion and migration abilities of the CML cell line K562.Our objectives were to identify the effect of IM administration on pyk2 expression and on K562 cell adhesion and migration ability and to establish the role of these changes in treatment-associated EMD. Results:We found a 2.6-fold increase in pyk2 mRNA expression in K562 cells following exposure to IM. We also found that 30% of IM-treated K562 cells adhered to fibronectin (FN) compared to untreated cells having no adhesion ability. In addition, a 3-fold induction in migration was seen in K562 cells following treatment. Furthermore, K562 cells treated with IM demonstrated a 2-fold increase in invasion potential as compared to untreated cells. In order to assess whether Pyk2 is essential for IM-dependent adhesion and migration of K562 cells, these cells were infected with pyk2 specific shRNAs. While 30% of the non-infected NB4 cells adhered to FN following IM treatment, only 12% of the pyk2-shRNA–infected K562 cells exhibited adhesion potential (Pvalue<0.002). In addition, we witnessed over a 3-fold reduction in the ability of pyk2-shRNA–infected K562 cells to migrate following exposure to IM when compared to parental K562 cells. These data support the role of Pyk2 in IM-mediated adhesion and migration.Finally, we found that IM treatment induced an in-vivo increase in pyk2 mRNA expression level in leukocytes derived from 3 out of 5 CML patients studied. Conclusions:IM induces K562 cell adhesion, migration and invasion accompanied by increased pyk2 expression. Pyk2 is one of the key proteins regulating IM-induced cell migration and adhesion. Collectively our data suggest a critical role of Pyk2 in adhesion and migration initiated by the targeted therapy IM and a possible role in EMD development. These data support a common mechanism for the development of EMD in hematological malignancies treated by targeted therapies via pyk2 expression. Disclosures:No relevant conflicts of interest to declare.

Integrin bi‐directional signaling across the plasma membrane

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi-directional signals across the plasma membrane. Inside-out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside-in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi-directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside-out and outside-in signaling with specific focus on the mechanism how integrins transmit bi-directional signals through transmembrane/cytoplasmic domains.

Mass‐producible Nano‐featured Polystyrene Surfaces for Regulating the Differentiation of Human Adipose‐derived Stem Cells

In this study, we report an efficient and cost-effective method of fabricating polystyrene (PS) nano-featured substrates containing nanopore (NPo) and nanopillar (NPi) arrays based on hot embossing using nickel nano-stamps. We investigate the behavior of adipose-derived stem cells (ASCs), including adhesion, morphology, proliferation and differentiation, on the replicated PS surfaces. Compared to a flat substrate, NPo- and NPi-featured substrates do not alter the morphology of stem cells. However, both NPo- and NPi-featured substrates induce different integrin expression and lower formation of focal adhesion complexes. In addition, ASCs on the NPo-featured substrate exhibit greater adipogenic differentiation, while the NPi-featured substrate induces higher osteogenic differentiation.

MiR-127 Protects Proximal Tubule Cells against Ischemia/Reperfusion: Identification of Kinesin Family Member 3B as miR-127 Target

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.

β-Actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration

Directed cell motility is at the basis of biological phenomena such as development, wound healing, and metastasis. It has been shown that substrate attachments mediate motility by coupling the cell's cytoskeleton with force generation. However, it has been unclear how the persistence of cell directionality is facilitated. We show that mRNA localization plays an important role in this process, but the mechanism of action is still unknown. In this study, we show that the zipcode-binding protein 1 transports β-actin mRNA to the focal adhesion compartment, where it dwells for minutes, suggesting a means for associating its localization with motility through the formation of stable connections between adhesions and newly synthesized actin filaments. In order to demonstrate this, we developed an approach for assessing the functional consequences of β-actin mRNA and protein localization by tethering the mRNA to a specific location-in this case, the focal adhesion complex. This approach will have a significant impact on cell biology because it is now possible to forcibly direct any mRNA and its cognate protein to specific locations in the cell. This will reveal the importance of localized protein translation on various cellular processes.

Insights into the role of focal adhesion modulation in myogenic differentiation of human mesenchymal stem cells.

We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the β(3) integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and α-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering.

Laser-engineered topography: correlation between structure dimensions and cell control

Topographical cues have a significant impact on cell responses and by this means, on the fabrication of innovative implant materials. However, analysis of cell-topography interactions in dependence of the surface feature dimensions is still challenging due to limitations in the fabrication technology. Here, we introduce surface structuring via picosecond laser systems, which enable a fast production of micro-sized topologies. Changes in the processing parameters further control the feature sizes of so-called spikes. Using surfaces with big and small spike-to-spike-distances for comparisons, we focussed on cell adhesion via extracellular matrix adsorption and focal adhesion complexes, morphology, localisation and proliferation of fibroblasts. The observed cell control was dependent on a turnover point related to the structure dimensions: only big spike-to-spike-distances reduced cell behaviour. Therefore, this technology offers a platform to study cell and tissue interactions with a defined microenvironment.

Paxillin phosphorylation by JNK and p38 is required for NFAT activation

Paxillin is an adaptor protein associated with focal adhesion complex, and is activated by tyrosine phosphorylation through focal adhesion kinase (FAK) and Src kinase. Recent studies reveal that serine phosphorylation of paxillin by JNK and p38 MAPK is essential for cell migration or neurite extension, but their cellular targets remain unclear. In this study, we examined the requirement of paxillin phosphorylation by p38 MAPK or JNK in T-cell motility and activation using paxillin mutants at the respective phosphorylation sites, Ser85, and Ser178. (S85A)-paxillin, (S178A)-paxillin, or (S85A/S178A)-paxillin inhibited the motility of NIH/3T3 fibroblasts, but did not interfere with T-cell migration and integrin-mediated T-cell adhesion. In contrast, activation of T cells was effectively suppressed by (S85A/S178A)-paxillin. Transgenic (S85A/S178A)-paxillin expression inhibited T-cell proliferation and reduced the production of IL-2, IFN-γ, and IL-4. In searching for signals modulated by (S85A/S178A)-paxillin, we found that NFAT activation was specifically blocked by (S85A/S178A)-paxillin. This could be partly attributed to diminished stromal interaction molecule 1 (STIM1) expression and attenuated TCR-induced Ca(2+) influx. Our results demonstrate that dual phosphorylation of paxillin by JNK and p38 MAPK is essential for T-cell activation and suggest that NFAT is a functional target of the JNK/p38 phosphorylated paxillin.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.

CalpB modulates border cell migration in Drosophila egg chambers

BackgroundCalpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility.ResultsWe demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), β-PS integrin ( mys) and talin ( rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-headR367A, a mutant form which is not able to bind β-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts.ConclusionsThe physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.

Vascular endothelial growth factor C enhances cervical cancer migration and invasion via activation of focal adhesion kinase

Vascular endothelial growth factor C (VEGF-C) is correlated positively with clinical cervical cancer metastasis and survival. Previously we showed that VEGF-C directly activated actin-binding protein moesin, leading to the formation of membrane protrusions. However, whether VEGF-C alters cervical cancer cell adhesion to the extra-cellular matrix is currently unknown. In this study, we investigated the effects of VEGF-C on the formation of focal adhesion complexes, which provide anchoring sites for cell attachment to the extracellular matrix. On cultured cervical carcinoma cell line SiHa cells, VEGF-C enhanced focal adhesion kinase (FAK) phosphorylation. As a result, VEGF-C led to increased formation of focal adhesion complexes and enhanced migration and invasion, which was reversed by siRNA abrogating FAK. VEGF-C resulted in increased interaction of its receptor Flt-4 with non-receptor tyrosine kinase c-Src, leading to c-Src phosphorylation. The specific inhibitor of c-Src kinase, PP2, or the transfection with specific c-Src siRNA largely impaired VEGF-C-enhanced FAK phosphorylation, suggesting that Flt-4/c-Src cascade plays a central role in these processes. These results implied that VEGF-C promotes cervical cancer metastasis by activation of FAK protein through Flt-4/c-Src pathway.

Scanning angle interference microscopy reveals cell dynamics at the nanoscale

Emerging questions in cell biology necessitate nanometer-scale imaging in live cells. Here we present scanning angle interference microscopy, capable of localizing fluorescent objects with nanometer-scale precision along the optical axis in motile cellular structures. We use this approach to resolve nano-topographical features of the cell membrane and cytoskeleton, as well as the temporal evolution, three-dimensional architecture, and nano-scale dynamics of focal adhesion complexes.

Mechanotransduction and focal adhesions

Cellular FAs (focal adhesions) respond to internal and external mechanical stresses which make them prime candidates for mechanotransduction. Recent observations showed that the FA proteins including vinculin, FAK (FA kinase) and p130Cas are crucial for the ability of cells to transmit forces and to generate cytoskeletal tension. When mechanically stimulated, cells respond by modulating the spreading area, remodel the actin cytoskeleton, activate actomyosin interactions, recruit integrins and reinforce FAs and cytoskeletal structures. These complex cellular responses are orchestrated such that mechanical stresses within the FA complex remained within a narrow range.

EGF-Like Factors Induce Expansion of the Cumulus Cell-Oocyte Complexes by Activating Calpain-Mediated Cell Movement

Cumulus cell-oocyte complex (COC) expansion is obligatory for LH-induced ovulation and is initiated by LH induction of the epidermal growth factor (EGF)-like factors that mediate the synthesis of the hyaluronan-rich matrix and hyaluronan-stabilizing factors. COC expansion also involves the movement of cumulus cells within the matrix by mechanisms that have not been characterized. We document herein that two proteases, calpain 2 and to a lesser extent calpain 1, are expressed in cumulus cells and that the proteolytic activity of these enzymes is rapidly and significantly increased in COC isolated from human chorionic gonadotropin-induced ovulatory follicles in vivo. Stimulation of calpain activity was associated with proteolytic degradation of paxillin and talin (two components of focal adhesion complexes), cell detachment, and the formation of cell surface bleb-like protrusions. Injection of a calpain inhibitor in vivo reduced 1) human chorionic gonadotropin-stimulated calpain enzyme activity, 2) cell detachment, 3) membrane protrusion formation, and 4) COC expansion by mechanisms that did not alter Has2 expression. During EGF-like factor induction of COC expansion in culture, calpain activity was increased by ERK1/2 and intracellular Ca(2+) signaling pathways. Inhibition of calpain activity in cultured COC blocked cumulus cell detachment, protrusion formation, and the vigorous movement of cumulus cells. As a consequence, COC expansion was impaired. Collectively, these results show that two highly coordinated processes control COC expansion. One process involves the synthesis of the hyaluronan matrix, and the other mediates cumulus cell detachment and movement. The latter are controlled by calpain activation downstream of the EGF receptor activation of the Ca(2+) pathway and ERK1/2 pathways.

An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

IntroductionIn humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and MethodsHere, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. ResultsMice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. ConclusionMaintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declared

An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

Introduction In humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and Methods Here, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. Results Mice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. Conclusion Maintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared