Integrin-mediated Cell-matrix Interactions Research Articles

A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution

Frozen shoulder is a spontaneously self-resolving chronic inflammatory fibrotic human disease, which distinguishes the condition from most fibrotic diseases that are progressive and irreversible. Using single-cell analysis, we identify pro-inflammatory MERTKlowCD48+ macrophages and MERTK + LYVE1 + MRC1+ macrophages enriched for negative regulators of inflammation which co-exist in frozen shoulder capsule tissues. Micro-cultures of patient-derived cells identify integrin-mediated cell-matrix interactions between MERTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Cross-tissue analysis reveals a shared gene expression cassette between shoulder capsule MERTK+ macrophages and a respective population enriched in synovial tissues of rheumatoid arthritis patients in disease remission, supporting the concept that MERTK+ macrophages mediate resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identify MERTK + LYVE1 + MRC1+ macrophages and DKK3+ and POSTN+ fibroblast populations analogous to those in frozen shoulder, suggesting that the template to resolve fibrosis is established during shoulder development. Crosstalk between MerTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts could facilitate resolution of frozen shoulder, providing a basis for potential therapeutic resolution of persistent fibrotic diseases.

Bone sialoprotein promotes lung cancer osteolytic bone metastasis via MMP14-dependent mechanisms

Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.

Aligned Collagen-CNT Nanofibrils and the Modulation Effect on Ovarian Cancer Cells.

Fibrillar collagen is a one-dimensional biopolymer and is the most abundant structural protein in the extracellular matrix (ECM) of connective tissues. Due to the unique properties of carbon nanotubes (CNTs), considerable attention has been given to the application of CNTs in developing biocomposite materials for tissue engineering and drug delivery. When introduced to tissues, CNTs inevitably interact and integrate with collagen and impose a discernible effect on cells in the vicinity. The positive effect of the collagen-CNT (COL-CNT) matrix in tissue regeneration and the cytotoxicity of free CNTs have been investigated extensively. In this study, we aimed to examine the effect of COL-CNT on mediating the interaction between the matrix and SKOV3 ovarian cancer cells. We generated unidirectionally aligned collagen and COL-CNT nanofibrils, mimicking the structure and dimension of collagen fibrils in native tissues. AFM analysis revealed that the one-dimensional structure, high stiffness, and low adhesion of COL-CNT greatly facilitated the polarization of SKOV3 cells by regulating the β−1 integrin-mediated cell–matrix interaction, cytoskeleton rearrangement, and cell migration. Protein and gene level analyses implied that both collagen and COL-CNT matrices induced the epithelial–mesenchymal transition (EMT), and the COL-CNT matrix prompted a higher level of cell transformation. However, the induced cells expressed CD44 at a reduced level and MMP2 at an increased level, and they were responsive to the chemotherapy drug gemcitabine. The results suggested that the COL-CNT matrix induced the transdifferentiation of the epithelial cancer cells to mature, less aggressive, and less potent cells, which are inapt for tumor metastasis and chemoresistance. Thus, the presence of CNT in a collagen matrix is unlikely to cause an adverse effect on cancer patients if a controlled dose of CNT is used for drug delivery or tissue regeneration.

Mesenchymal-epithelial transition regulates initiation of pluripotency exit before gastrulation.

The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast cellular morphology and its pluripotency is not well understood. Here, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit, whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.

Decreased Expression of Integrin Subunits α3, α6, and α8 in the Branching Airway Mesenchyme of Nitrofen-Induced Hypoplastic Lungs.

Pulmonary hypoplasia (PH), characterized by smaller lung size and reduced airway branching, remains a major cause of neonatal mortality in newborns with congenital diaphragmatic hernia (CDH). Integrin-mediated cell-matrix interactions play an essential role in the fetal lung mesenchyme by stimulating branching morphogenesis. Mice lacking integrin subunits α3 (Itga3) and α6 (Itga6) exhibit severe PH. Furthermore, Itga8-knockout mice show defective airway branching, suggesting that Itga3, Itga6, and Itga8 are crucial for fetal lung development. We hypothesized that expression of Itga3, Itga6, and Itga8 is decreased in the branching airway mesenchyme of hypoplastic rat lungs in the nitrofen-induced CDH model. Time-mated rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D15, D18, and D21, and dissected lungs were divided into control and nitrofen-exposed specimens (n = 12 per time-point and group, respectively). Pulmonary gene expression of Itga3, Itga6, and Itga8 was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double-staining for Itga3, Itga6, and Itga8 was combined with the mesenchymal marker Fgf10 to evaluate protein expression and localization in branching airway tissue. Relative mRNA expression of Itga3, Itga6, and Itga8 was significantly decreased in lungs of nitrofen-exposed fetuses on D15, D18, and D21 compared with controls. Confocal laser scanning microscopy showed markedly diminished immunofluorescence of Itga3, Itga6, and Itga8 mainly in mesenchymal cells surrounding branching airways of nitrofen-exposed fetuses on D15, D18, and D21 compared with controls. Decreased expression of Itga3, Itga6, and Itga8 in the pulmonary mesenchyme may lead to disruptions in airway branching morphogenesis, thus contributing to PH in the nitrofen-induced CDH model.

Integrin-Linked Kinase Regulates Bone Formation by Controlling Cytoskeletal Organization and Modulating BMP and Wnt Signaling in Osteoprogenitors.

Cell-matrix interactions constitute a fundamental aspect of skeletal cell biology and play essential roles in bone homeostasis. These interactions are primarily mediated by transmembrane integrin receptors, which mediate cell adhesion and transduce signals from the extracellular matrix to intracellular responses via various downstream effectors, including integrin-linked kinase (ILK). ILK functions as adaptor protein at focal adhesion sites, linking integrins to the actin cytoskeleton, and has been reported to act as a kinase phosphorylating signaling molecules such as GSK-3β and Akt. Thereby, ILK plays important roles in cellular attachment, motility, proliferation and survival. To assess the in vivo role of ILK signaling in osteoprogenitors and the osteoblast lineage cells descending thereof, we generated conditional knockout mice using the Osx-Cre:GFP driver strain. Mice lacking functional ILK in osterix-expressing cells and their derivatives showed no apparent developmental or growth phenotype, but by 5 weeks of age they displayed a significantly reduced trabecular bone mass, which persisted into adulthood in male mice. Histomorphometry and serum analysis indicated no alterations in osteoclast formation and activity, but provided evidence that osteoblast function was impaired, resulting in reduced bone mineralization and increased accumulation of unmineralized osteoid. In vitro analyses further substantiated that absence of ILK in osteogenic cells was associated with compromised collagen matrix production and mineralization. Mechanistically, we found evidence for both impaired cytoskeletal functioning and reduced signal transduction in osteoblasts lacking ILK. Indeed, loss of ILK in primary osteogenic cells impaired F-actin organization, cellular adhesion, spreading, and migration, indicative of defective coupling of cell-matrix interactions to the cytoskeleton. In addition, BMP/Smad and Wnt/β-catenin signaling was reduced in the absence of ILK. Taken together, these data demonstrate the importance of integrin-mediated cell-matrix interactions and ILK signaling in osteoprogenitors in the control of osteoblast functioning during juvenile bone mass acquisition and adult bone remodeling and homeostasis. © 2017 American Society for Bone and Mineral Research.

Design of an osteoinductive extracellular fibronectin matrix protein for bone tissue engineering.

Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05).

Integrin β1 controls VE-cadherin localization and blood vessel stability.

Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin β1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that β1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin β1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin β1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

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

Integrin-Linked Kinase Deletion in the Developing Lens Leads to Capsule Rupture, Impaired Fiber Migration and Non-Apoptotic Epithelial Cell Death

The lens is a powerful model system to study integrin-mediated cell-matrix interaction in an in vivo context, as it is surrounded by a true basement membrane, the lens capsule. To characterize better the function of integrin-linked kinase (ILK), we examined the phenotypic consequences of its deletion in the developing mouse lens. ILK was deleted from the embryonic lens either at the time of placode invagination using the Le-Cre line or after initial lens formation using the Nestin-Cre line. Early deletion of ILK leads to defects in extracellular matrix deposition that result in lens capsule rupture at the lens vesicle stage (E13.5). If ILK was deleted at a later time-point after initial establishment of the lens capsule, rupture was prevented. Instead, ILK deletion resulted in secondary fiber migration defects and, most notably, in cell death of the anterior epithelium (E18.5-P0). Remarkably, dying cells did not stain positively for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or activated-caspase 3, suggesting that they were dying from a non-apoptotic mechanism. Moreover, cross to a Bax(fl/fl)/Bak⁻/⁻ mouse line that is resistant to most forms of apoptosis failed to promote cell survival in the ILK-deleted lens epithelium. Electron microscopy revealed the presence of numerous membranous vacuoles containing degrading cellular material. CONCLUSIONS. Our study reveals a role for ILK in extracellular matrix organization, fiber migration, and cell survival. Furthermore, to our knowledge we show for the first time that ILK disruption results in non-apoptotic cell death in vivo.

Integrin-Mediated Cell-Matrix Interaction in Physiological and Pathological Blood Vessel Formation

Physiological as well as pathological blood vessel formation are fundamentally dependent on cell-matrix interaction. Integrins, a family of major cell adhesion receptors, play a pivotal role in development, maintenance, and remodeling of the vasculature. Cell migration, invasion, and remodeling of the extracellular matrix (ECM) are integrin-regulated processes, and the expression of certain integrins also correlates with tumor progression. Recent advances in the understanding of how integrins are involved in the regulation of blood vessel formation and remodeling during tumor progression are highlighted. The increasing knowledge of integrin function at the molecular level, together with the growing repertoire of integrin inhibitors which allow their selective pharmacological manipulation, makes integrins suited as potential diagnostic markers and therapeutic targets.

Lack of α8-integrin aggravates podocyte injury in experimental diabetic nephropathy

Development of diabetic nephropathy is accompanied by changes in integrin-mediated cell-matrix interactions. The α8-integrin chain is specifically expressed in mesangial cells of the glomerulus. During experimental hypertension, α8-integrin plays a protective role in the glomerulus. We hypothesized that α8-integrin is involved in maintaining the integrity of the glomerulus in diabetic nephropathy. Experimental streptozotocin (STZ) diabetes led to an increased expression and glomerular deposition of α8-integrin. To test the functional role of α8-integrin, STZ diabetes was induced in mice with a homozygous (α8-/-) or heterozygous (α8+/-) deletion of the α8-integrin gene and in wild-type litters (α8+/+). Blood glucose and mean arterial blood pressure were not different in α8-/- and α8+/+ mice after 6 wk of diabetes. However, diabetic α8-/- mice developed significantly higher albuminuria and more glomerulosclerosis than diabetic α8+/+ mice. Moreover, in diabetic α8-/- mice, the number of glomerular cells staining positive for the podocyte markers WT-1 and vimentin were reduced more prominently than in diabetic α8+/+. The filtration barrier protein nephrin was downregulated in diabetic glomeruli with the strongest reduction observed in α8-/- mice. Taken together, α8-/- mice developed more severe glomerular lesions and podocyte damage after onset of STZ diabetes than α8+/+ mice, indicating that α8-integrin is protective for the structure and function of the glomerulus and maintains podocyte integrity during the development of diabetic nephropathy.

β1 Integrin Deficiency Results in Multiple Abnormalities of the Knee Joint

The lack of beta1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of beta1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the beta1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to beta1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. beta1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of beta1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.

Functional integrin subunits regulating cell–matrix interactions in the intervertebral disc

Cellular interactions with the extracellular matrix are key factors regulating cell survival, differentiation, and response to environmental stimuli in cartilagenous tissues. Much is known about the extracellular matrix proteins in the intervertebral disc (IVD) and their variations with region, age, or degenerative state of the tissue. In contrast, little is known of the integrin cell surface receptors that directly bind to and interact with these matrix proteins in the IVD. In almost all tissues, these integrin-mediated cell-matrix interactions are important for transducing environmental cues arising from mechanical stimuli, matrix degradation fragments, and cytokines into intracellular signals. In this study, cells from the nucleus pulposus and anulus fibrosus regions of porcine IVDs were analyzed via flow cytometry to quantify integrin expression levels upon isolation and after monolayer culture. Assays of cell attachment to collagens, fibronectin, and laminin were performed after functional blocking of select integrin subunits to evaluate the role of specific integrins in cell attachment. In situ distribution and co-localization of integrins and laminin were also characterized. Results identify integrin receptors critical for IVD cell interactions with collagens (alpha1beta1) and fibronectin (alpha5beta1). Additionally, dramatic differences in cell-laminin interactions were observed between cells of the nucleus and anulus regions, including differences in alpha6 integrin expression, cell adhesion to laminin, and in situ pericellular environments. These findings suggest laminin-cell interactions may be important and unique to the nucleus pulposus region of the IVD. The results of this study provide new information on functional cell-matrix interactions in tissues of the IVD.

Molecular phenotypes of notochordal cells purified from immature nucleus pulposus

The immature nucleus pulposus (NP) is populated by cells of notochordal-origin that are larger and contain an extensive cytoskeletal network and numerous vacuoles. The disappearance of these cells with age is believed important in regulating metabolic shifts that may contribute to age-related disc degeneration. The precise biological function of these notochordal cells in the immature NP remains unclear, however, because of challenges in studying the mixed cell population in the NP. In this study, notochordal-like cells were purified from immature NP cells using a new fluorescence-activated cell sorting (FACS) protocol with auto-fluorescence and size analysis. The unique molecular phenotypes of sorted notochordal-like cells were characterized by the mRNA expression pattern for key matrix proteins and modulators, and by the expression of cell-matrix receptor integrin subunits. An FACS analysis showed that the immature NP contained a majority of cells that were larger than anulus fibrosus (AF) cells and with fluorescence higher than AF cells. In comparison with the small NP cells separated by the FACS protocol, sorted notochordal-like cells expressed lower mRNA levels of type I collagen, biglycan, TIMP1, HSP70 and c-fos, and did not express detectable mRNA levels of decorin, lumican, multiple MMPs or IL-1beta via real-time quantitative RT-PCR. A greater number of these notochordal-like cells also expressed the higher levels of alpha6, alpha1 and beta1 integrin subunits as compared to small NP cells. Together, our results point towards a unique molecular phenotype for these notochordal-like cells of NP, characterized by the absence of gene expression for specific small proteoglycans and higher protein expression of integrin subunits that regulate interactions with collagens and laminin. Future studies will be important for revealing if this unique molecular profile is coordinated with functional differences in pericellular matrix regions and/or integrin-mediated cell-matrix interactions for these notochordal-like cells within the NP.

Integrin-mediated cell–matrix interactions for prosurvival and antiapoptotic signaling after genotoxic injury

Interactions of cells with their microenvironment modify the cellular sensitivity of normal and tumor cells for radiation- and drug-induced genotoxic injury. The preexistent or acquired cellular resistance against such agents aggravates anticancer therapies and, therefore, complicates the recovery of patients. Recently, integrin-mediated adhesion was shown to improve cell survival of both normal and cancer cells following DNA damage. Here, I will discuss the role of integrins and integrin-mediated signaling cascades in the survival or death response upon genotoxic stress. Detailed knowledge of the responsible molecular processes might provide implications for putative therapies targeting integrins or integrin-associated molecules to achieve an optimization of anticancer treatments.

PECAM‐1 isoform‐specific activation of MAPK/ERKs and small GTPases: Implications in inflammation and angiogenesis

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) and leukocytes. PECAM-1 plays an important role in endothelial-leukocyte and endothelial-endothelial cell-cell interactions. The anti-PECAM-1 antibody-mediated blockade of these interactions inhibits transendothelial migration (TEM) of leukocytes and angiogenesis. PECAM-1 may accommodate these processes through the regulation of cell adhesive and migratory mechanisms. How PECAM-1 regulates these dynamic processes remain unknown. Here we show that PECAM-1 transduces outside-in signals, which activate MAPK/ERKs and small GTPases. This occurs through PECAM-1-mediated formation of intracellular-signaling complexes, Shc/Grb2/SOS1 and/or Crkl/C3G, which is initiated by PECAM-1 engagement on the surface of leukocytes and/or EC. Src, SHP2, and alternative PECAM-1 pre-mRNA splicing play a regulatory role in these signaling events. Our findings reveal that PECAM-1 engagement on the cell surface can transduce "outside-in" signals and activate MAPK/ERKs and small GTPases, impacting both cadherin-mediated cell-cell and integrin-mediated cell-matrix interactions. Thus, we propose PECAM-1 is an important mediator of vascular barrier and regulator of leukocyte and EC adhesion and migration.

Mechanism of Perturbation of Integrin-Mediated Cell-Matrix Interactions by Reactive Carbonyl Compounds and Its Implication for Pathogenesis of Diabetic Nephropathy

Perturbation of interactions between cells and the extracellular matrix (ECM) of renal glomeruli may contribute to characteristic histopathological lesions found in the kidneys of patients with diabetic nephropathy. However, the mechanism by which the diabetic conditions may affect cell-ECM interactions is unknown. Existing hypotheses suggest a role of glucose in direct modification of ECM. Here, we have demonstrated that carbonyl compound methylglyoxal (MGO) completely inhibited endothelial cell adhesion to recombinant alpha3 noncollagenous 1 domain of type IV collagen mediated via a short collagenous region containing RGD (Arg-Gly-Asp) sequence as well as binding of purified alpha(v)beta(3) integrin to this protein. Specific MGO adducts of the arginine residue were detected within RGD sequence using mass spectrometry. Modification by carbonyl compounds glyoxal or glycolaldehyde had similar but smaller effects. MGO strongly inhibited adhesion of renal glomerular cells, podocytes, and mesangial cells to native collagen IV and laminin-1 as well as binding of collagen IV to its major receptor in glomerular cells, alpha(1)beta(1) integrin. In contrast, modification of these proteins by glucose had no effect on cell adhesion. Pyridoxamine, a promising drug for treatment of diabetic nephropathy, protected cell adhesion and integrin binding from inhibition by MGO. We suggest that in diabetes, perturbation of integrin-mediated cell-matrix interactions occurs via the modification of critical arginine residues in renal ECM by reactive carbonyl compounds. This mechanism may contribute to the development of diabetic nephropathy.

Gp38k (CHI3L1) is a novel adhesion and migration factor for vascular cells

gp38k (CHI3L1) is a secreted heparin-binding glycoprotein whose expression, in vitro, is associated with vascular smooth muscle cell (VSMC) migration and invasion into the underlying gelatinous matrix. gp38k is expressed at high levels in postconfluent “nodular” VSMC cultures and at low levels in subconfluent proliferating cultures. In vivo, expression of gp38k homologs is high in regions of tissue remodeling and now has been detected in atherosclerotic plaques and in the developing heart. We tested the hypothesis that gp38k functions to modulate VSMC adhesion and migration. By use of modified Boyden chambers, gp38k at a concentration as low as 1 ng/ml has profound effects on VSMC migration but little or no effect on fibroblast migration. In addition, gp38k adsorbed to polystyrene surfaces directly promotes VSMC attachment and spreading. Attachment is inhibited in the presence of affinity-purified anti-gp38k or 10 mM EDTA. These results establish that gp38k is a new vascular cell adhesion and migration factor that may have a role in processes leading to vascular occlusion and heart development. gp38k may interact with VSMC via an EDTA-sensitive mechanism consistent with integrin mediated cell–matrix interaction.