Focal Adhesion Sites Research Articles

LRP-1 Promotes Cancer Cell Invasion by Supporting ERK and Inhibiting JNK Signaling Pathways

BackgroundThe low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor mediating the clearance of various extracellular molecules involved in the dissemination of cancer cells. LRP-1 thus appeared as an attractive receptor for targeting the invasive behavior of malignant cells. However, recent results suggest that LRP-1 may facilitate the development and growth of cancer metastases in vivo, but the precise contribution of the receptor during cancer progression remains to be elucidated. The lack of mechanistic insights into the intracellular signaling networks downstream of LRP-1 has prevented the understanding of its contribution towards cancer.Methodology/Principal FindingsThrough a short-hairpin RNA-mediated silencing approach, we identified LRP-1 as a main regulator of ERK and JNK signaling in a tumor cell context. Co-immunoprecipitation experiments revealed that LRP-1 constitutes an intracellular docking site for MAPK containing complexes. By using pharmacological agents, constitutively active and dominant-negative kinases, we demonstrated that LRP-1 maintains malignant cells in an adhesive state that is favorable for invasion by activating ERK and inhibiting JNK. We further demonstrated that the LRP-1-dependent regulation of MAPK signaling organizes the cytoskeletal architecture and mediates adhesive complex turnover in cancer cells. Moreover, we found that LRP-1 is tethered to the actin network and to focal adhesion sites and controls ERK and JNK targeting to talin-rich structures.ConclusionsWe identified ERK and JNK as the main molecular relays by which LRP-1 regulates focal adhesion disassembly of malignant cells to support invasion.

Spreading by snail (Lymnaea stagnalis) defence cells is regulated through integrated PKC, FAK and Src signalling

Cell adhesion and spreading are vital to immune function. In molluscs, haemocytes (circulating phagocytes) are sentinels and effectors of the internal defence system; however, molecular mechanisms that regulate integrin-mediated spreading by haemocytes have not been characterised in detail. Visualisation of Lymnaea stagnalis haemocytes by scanning electron microscopy revealed membrane ruffling, formation of lamellipodia and extensive filopodia during early stages of cell adhesion and spreading. These events correlated with increased phosphorylation (activation) of protein kinase C (PKC) and focal adhesion kinase (FAK), sustained for 60 min. Treatment of haemocytes with the PKC inhibitors GF109203X or Gö 6976, or the Src/tyrosine kinase inhibitors SrcI or herbimycin A, attenuated haemocyte spread by 64, 46, 32 and 35%, respectively (P <or= 0.001); PKC or Src inhibition also prevented focal adhesion formation. Western blotting demonstrated that during spreading and adhesion these inhibitors also impaired PKC and FAK activation, with Gö 6976 or SrcI inhibiting FAK phosphorylation by at least 70% (P <or= 0.001), and herbimycin A or SrcI inhibiting PKC phosphorylation by at least 46% (P <or= 0.01). Confocal microscopy revealed phosphorylated PKC colocalised with focal adhesion sites, particularly during early phases of adhesion and spreading. Finally, fibronectin promoted PKC and FAK phosphorylation in suspended haemocytes demonstrating that activation can occur independent of cell adhesion. These novel data are consistent with PKC and FAK/Src playing an integrated role in integrin activation and integrin-mediated spreading by L. stagnalis haemocytes. We propose a model in which integrin engagement mediates association of PKC with FAK/Src complexes to promote focal adhesion assembly during immune recognition by these cells.

Inhibition of c-Met with the Specific Small Molecule Tyrosine Kinase Inhibitor SU11274 Decreases Growth and Metastasis Formation of Experimental Human Melanoma

The hepatocyte growth factor/scatter factor (HGF/SF) tyrosine kinase (TK) receptor c-Met plays a crucial role in the development of the invasive phenotype of tumors and thus represents an attractive candidate for targeted therapies in a variety of malignancies, including human malignant melanoma (MM). In contrast to what has been shown previously, we were not able to detect any genetic alterations, either in the juxtamembrane- or in the TK-domain of c-Met, in the studied MM cell lines. Nevertheless, c-Met was constitutively active in these cell lines without exogenous HGF/SF stimulation. The active receptor was localized to the adhesion sites of the cells. Addition of the c-Met TK inhibitor SU11274 specifically decreased the phosphotyrosine signal at the focal adhesions sites, which was accompanied by a decrease in cell proliferation as well as an increase in apoptotic cells. In addition, non-apoptotic concentrations of SU11274 significantly reduced the in vitro migratory capacity of MM cells in the modified Boyden-chamber assay. Administration of SU11274 significantly decreased primary tumor growth as well as the capacity for liver colony formation of MM cells in SCID mice. Our study provides the first evidence for an in vivo antitumor activity of SU11274 in a human melanoma xenograft model, and suggests c-Met as a valid target for the therapy of MM. Consequently, SU11274 treatment might represent a useful strategy for controlling melanoma progression and metastasis in patients with MM.

CTEN (C-terminal tensin-like), a novel oncogene overexpressed in invasive breast carcinoma of poor prognosis

C-terminal tensin-like (CTEN) gene is a member of the TENSIN gene family, involved in cell migration and localised at focal adhesion sites. This study was designed to explore the prevalence and clinical significance of CTEN expression in a large and well-characterised series (n = 1,409) invasive breast cancer (BC) cases with long term follow-up (median 11 years), using immuno-histochemistry and tissue microarray. Moderate to strong cytoplasmic immunoreactivity for CTEN was observed in 90% of the studied cases. CTEN expression was significantly associated with poor prognostic variables including larger tumour size (P = 0.044), higher histological grade (P = 0.019), axillary nodal involvement (P = 0.035) and poor Nottingham Prognostic Index (P = 0.016). Significant associations were observed between increased CTEN expression and up-regulation of phosphorylated-Akt (P-Akt), PIK3 and N-cadherin proteins (P\0.001). Kaplan-Meier survival analysis demonstrated that patients with high CTEN expression had significantly shorter Breast Cancer Specific Survival (P = 0.004) and Metastasis-Free Survival (P = 0.041) than those with low-CTEN expression. Multivariate analysis showed that CTEN was not an independent prognostic marker in BC. In conclusion, our results demonstrated the oncogenetic role of increased CTEN expression and its association with poor prognostic parameters. These data could help in prognostic assessment in BC patients.

The Role of Syndecan‐1 in Arterial Mechanotransduction

Hemodynamic forces induced by blood flow are potent regulators of vascular homeostasis and arterial structure. While previous work has focused on the role of integrin receptors in mechanotransduction, little is known about the role of cell surface proteoglycans in these processes. This work focuses specifically on the role of the cell surface heparan sulfate proteoglycan syndecan‐1 in vascular cell mechanotransduction. We isolated vascular smooth muscle cells from syndecan‐1 knockout mice and tranfected them with wild‐type and mutated forms of syndecan‐1 using lentiviral vectors. Wild‐type, syndecan‐1 knockout and mutated syndecan‐1 overexpressing mouse vascular smooth muscle cells were cultured in plates with collagen coated silicone membranes. The cells were then subjected to 10% strain at 1 Hz biaxial or uniaxial loads for up to two hours. We found that syndecan‐1 knockout or deletion of the cytoplasmic region of syndecan‐1 caused an increase in actin stress fibers and focal adhesion sites in response to mechanical strain. Further, knockout of syndecan‐1 led to enhanced ERK and Src phosphorylation in response to mechanical strain. These findings support that syndecan‐1 is an important mediator of mechanotransduction in vascular cells.

Behavior of dermal fibroblasts on microdot arrays yields insight into wound healing mechanisms

The behavior of fibroblasts on patterned substrates was examined in order to elucidate the role of dermal structure in wound healing. Dermal fibroblasts were cultured on micro-patterned silicone elastomer substrates designed to enforce cell adhesion only to fibronectin microdots. The morphology, expression of α-smooth muscle actin (α-SMA), proliferation, apoptotic cells, and soluble collagen production of cells were measured. Cells grown on patterned substrates showed some signs of a scar-fibroblast phenotype such as: elongated pseudopodia, enhanced expression of alpha smooth muscle actin (α-SMA), and increased collagen/pre-collagen, in comparison to unpatterned controls. Cells also showed low proliferation rates and high apoptotic index. The results showed that the microdot arrays, acting as a grid of limited focal adhesion sites, could force cells to adopt constrained morphologies and limited adhesion areas, which affect the cytoskeleton, ultimately leading to expression of a scar-tissue fibroblast phenotype. This study provides insight into the regulatory mechanisms of micro-topology on cell behavior in wound healing.

IGF-1 increases macrophage motility via PKC/p38-dependent αvβ3-integrin inside-out signaling

Macrophage migration is a key aspect in the initiation and progression of atherosclerosis. Insulin-like growth factor (IGF)-1 is highly expressed in macrophages in human atheroma. Its function in macrophage motility, however, remains to be elucidated. The aim of this study was to investigate the impact of IGF-1 on macrophage migration, its signaling pathways and the involvement of integrins and/or matrix metalloproteinases (MMPs). Results Migration checker-box experiments demonstrated that IGF-1 induced chemotaxis in human THP-1/macrophages. IGF-1 induced migration was inhibited by RGD-containing peptides and the αvβ3-blocking antibody LM609, but was unaffected by the MMP-inhibitor GM6001. Immunoblotting demonstrated that IGF-1 did not affect the activation of MMPs or TIMPs, nor did it increase αv-integrin protein levels. However, IGF-1 induced recruitment of αvβ3, as well as trans-location of the integrin adaptor protein phospho-paxillin to focal adhesion sites. Pharmacological blocking experiments with specific inhibitors of Akt, PKC and p38 MAP-kinase revealed that IGF-1-dependent activation of focal adhesion kinase (FAK) and paxillin, and consecutively IGF-1 facilitated migration, required IGF-1/IGF-1R-mediated PI3-kinase/PKC/p38-dependent integrin inside-out signaling. Conclusion IGF-1 plays a vital role in macrophage migration critically implicated in tissue inflammation. This involves activation of integrins and focal adhesion formation via inside-out PI3-kinase/PKC/p38-dependent signaling, but does not require MMP activation.

Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4

In addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importin-independent manner and is exported by the chromosome region maintenance-1 (CRM1)-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF)-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Basis for the Isoform-specific Interaction of Myosin Phosphatase Subunits Protein Phosphatase 1c β and Myosin Phosphatase Targeting Subunit 1

Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c beta and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c beta, and, using chimeric analysis and site-directed mutations, that the central region of PP1c beta confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c beta as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function.

117. THE ROLE OF FOCAL ADHESION PROTEINS AND THEIR HORMONAL REGULATION IN RAT UTERINE EPITHELIAL CELLS DURING EARLY PREGNANCY

Uterine epithelial cells (UECs) undergo extensive alteration during early pregnancy followed by their removal in order for the implanting blastocyst to penetrate into the underlying endometrium. Focal adhesions (FAs) may play a role during this process as FAs provide adhesion between the cell and its underlying basal lamina. The present study investigated the distribution and expression of FA proteins in rat UECs at the time of implantation and their hormonal control as well as their expression in blastocyst stage embryos. Immunofluorescence microscopy showed that the principal focal adhesion proteins, talin and paxillin, were localised along the basal cell surface of UECs on day 1 of pregnancy, however they were markedly reduced from the site of FAs at the time of implantation. This is thought to be a critical process in the removal of UECs, which allows the invasion of the blastocyst into the underlying endometrium. Hormone treatments in ovariectomised rats showed that disassembly of talin and paxillin from the site of FAs were predominantly under the control of progesterone. Formation of FAs is initiated by the clustering of specific integrin subunits and both integrin beta1 and beta3 colocalised and interacted with talin at the site of FAs on day 1 of pregnancy. Integrin beta1 and beta3 disassembled from the site of FAs at the time of implantation, however integrin beta3 significantly increased along the apical membrane at this time suggesting a role in embryo attachment. In the rat blastocyst, integrin beta3 was concentrated around the nuclei of the trophoblast cells and once the blastocyst was placed onto a receptive endometrial cell line, Ishikawa cell line, integrin beta3 relocated to the apical membrane of the trophoblast cells. Taken together, our results show that disassembly of FA proteins plays a pivotal role in the removal of UECs in order to establish successful implantation and is tightly regulated by ovarian hormones.

Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.

Integrin cleavage regulates bidirectional signalling in vascular smooth muscle cells

Integrins link the cytoskeleton to the extracellular matrix, providing outside-in/inside-out signalling essential for vascular smooth muscle cell (VSMC) migration in atherosclerosis. The integrin av subunit is synthesised from its precursor via furin-dependent endoproteolytic cleavage. Furin is a proprotein convertase (PC) highly expressed in VSMCs and in human atherosclerotic lesions. Inhibition of av processing inhibits binding to vitronectin and migration. However, the precise role of furin-dependent av cleavage on integrin bidirectional signalling and subsequent VSMC functions is unknown. Our present study demonstrates that the furin-like PC inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited av cleavage. This reduced vitronectin-induced (outside-in) focal adhesion kinase (FAK)- and paxillin-phosphorylation, and VSMC motility. Inside-out-stimulated, integrin- mediated VSMC adhesion/migration relied on integrin-adaptor protein activation following protein kinase C (PKC) and ERK1/2 phosphorylation. In contrast to outside-in signalling, PKC-dependent phosphorylation of FAK and paxillin was unaffected by the status of integrin cleavage. Still, cytoskeleton and focal adhesion site rearrangements were modulated by the inhibition of furin-dependent integrin cleavage, thereby lessening inside-out dependent migration. Hence, we find that integrin bidirectional signalling is critically controlled by furin. Furin- dependent integrin processing modulates rapid adaptive integrin/cytoskeleton changes, essential to VSMC motility, which represents a crucial component in atherosclerosis and restenosis.

Factor XIII Associates with the Guanine Exchange Factors Cool-1 and Cool-2 in Human Platelets.

Abstract 4015Poster Board III-951A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42, Rac1 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We previously identified the presence of two homologous GEFs, Cool-1 and Cool-2, in human platelets. In nucleated cells, the activities and substrate specificities of the Cool GEFs are regulated by complex interactions with p21-activated kinases (PAK), Gb/g heterodimers, focal adhesion kinase, and the scaffolding proteins GIT-1 and GIT-2. The Cool GEFs are found at the sites of focal adhesions during spreading and migration in nucleated cells, however, little is known about the regulation or activity of Cool-1 or Cool-2 in platelets.Co-immunoprecipitation experiments were performed with Cool-1 and Cool-2 antibodies using lysates from resting and thrombin stimulated human platelets. We analyzed the precipitated proteins by Mass Spectroscopy and found a number of structural and scaffold proteins bound to the Cool GEFs in the thrombin activated lysates. These cytoskeletal proteins include talin, multimerin, tubulin, filamin A, actin and fibrinogen. Interestingly, a large number of Factor XIII also co-immunoprecipitated with Cool-1 and Cool-2 in the thrombin activated platelet lysates. Western analysis of the co-immunoprecipated platelet proteins from thrombin, TRAP1 and TRAP4 demonstrated that the association of factor XIII with the Cool GEFs is calcium dependent. Inhibition of transglutaminase activity and presence of RGD peptide did not affect the association of Factor XIII with Cool-1 or Cool-2.Factor XIII deficiency is commonly thought to result in bleeding due to the impaired crosslinking of fibrin. However, platelet factor XIII may also play an important role in cross linking platelet cytoskeleletal proteins such as actin and myosin. Consistent with that hypothesis is a recent report showing that platelet factor XIII deficiency results in decreased lamellapodia formation under static adhesive conditions. The association of Factor XIII with the Cool GEFs further supports investigating the potential for the transglutaminase to crosslink platelet cytoskeletal proteins. Disclosures:No relevant conflicts of interest to declare.

Abstract 5472: Nox4 is a Novel Inducible Source of Intracellular Reactive Oxygen Species in Human Monocyte-Derived Macrophages and Mediates Oxidized LDL-induced Macrophage Death

Nox2/gp91 phox is the large subunit of the integral membrane component of the phagocytic NADPH oxidase (Nox) complex and was believed to be the sole Nox isoform present in monocytes and macrophages. Previously, we reported that increased intracellular ROS formation is required for OxLDL-induced cell death in human monocyte-derived macrophage (HMDM), but the source of these intracellular ROS was unknown (Asmis, Circ.Res. 2003). We now identified Nox4, not Nox2, as the source of OxLDL-induced ROS generation. Nox4 mRNA was detected in human blood monocytes and HMDM. Using a novel rabbit monoclonal antibody that detects Nox4 as a single 66 kDa band, we localized Nox4 to the ER, the nucleus and focal adhesion sites of HMDM, suggesting that Nox4 may play a role in macrophage redox signaling. After stimulation of HMDM with OxLDL, but not native LDL, Nox4 mRNA and protein expression were up-regulated in a dose- and time-dependent manner. Nox4 protein expression was maximal after 6 h and coincided with both increased p22 expression and increased intracellular ROS formation, suggesting that the Nox4/p22 complex is responsible for OxLDL-induced ROS formation in HMDM. Inhibition of MEK1 and ERK1/2 phosphorylation blocked both the induction of Nox4 expression and intracellular ROS formation induced by OxLDL, and protected HMDM from OxLDL-mediated cytotoxicity. In contrast, inhibitors of p38 MAPK or JNK affect neither Nox4 induction nor ROS formation, and showed no cytoprotection. Knockdown of Nox4 with adenovirus-delivered siRNA decreased OxLDL-induced ROS formation and protected macrophages from OxLDL-induced cell injury. In summary, we identified Nox4 as a novel, inducible source of intracellular ROS in human monocytes and macrophages. Nox4 is likely to play important roles in redox signaling and macrophage function. However, if overinduced Nox4-derived ROS promote macrophage death, a mechansim which may have important implications for the pathogenesis of atherosclerosis. This research has received full or partial funding support from the American Heart Association, South Central Affiliate (Arkansas, New Mexico, Oklahoma &amp; Texas).

Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

Synemin is a unique cytoplasmic intermediate filament protein for which there is limited understanding of its exact cellular functions. The single human synemin gene encodes at least two splice variants named α-synemin and β-synemin, with the larger α-synemin containing an additional 312 amino acid insert within the C-terminal tail domain. We report herein that, by using the entire tail domain of the smaller β-synemin as the bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interaction partner for human synemin. The synemin binding site in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within β-synemin is within the C-terminal 332 amino acid region (SNβTII) at the end of the long tail domain. Transient expression of SNβTII within mammalian cells markedly reduced zyxin protein level, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesion and increased motility. Knockdown of synemin expression with siRNAs within mammalian cells resulted in significantly compromised cell adhesion and cell motility. Our results suggest that synemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.

Mitochondrial bioenergetics and distribution in living human osteoblasts grown on implant surfaces.

Osseointegration of implants is crucial for the long-term success of oral implants. The periimplant bone formation by osteoblasts is strongly dependent on the local mechanical environment in the interface zone. Robust demands for energy are placed on osteoblasts during the adhesion process to solid surfaces, and mitochondria are capital organelles in the production of most of the ATP needed for the process. We have assessed the relationship between osteoblast differentiation and mitochondrial bioenergetics in living cells grown on two different titanium surfaces, in order to provide valuable information for the design of material surfaces required for the development of the most appropriate osteogenic surface for osteoblastic anchorage. Combined backscattered and fluorescence confocal microscopy showed that in flat cells grown on a machined surface, highly energized mitochondria were distributed along the cell body. In contrast, cells grown on the rough surface emitted long protrusions in search of surface roughness, with actin stress fibers clearly polarized and highly energized mitochondria clustered at focal adhesion sites. This report using normal human osteoblastic cells indicates that these cells are especially sensitive to surface cues through energy production that enhances the necessary adhesion required for a successful osseointegration.

Flightless I Regulates Hemidesmosome Formation and Integrin-Mediated Cellular Adhesion and Migration during Wound Repair

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

AMPA receptors promote perivascular glioma invasion via β1 integrin–dependent adhesion to the extracellular matrix

High-grade gliomas release excitotoxic concentrations of glutamate, which has been shown to enhance tumor proliferation and migration. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors are abundantly expressed at the invading edge of glioblastoma specimens, suggesting they may play an important biologic role in tumor invasion. In this study, we examined potential mechanisms by which AMPA receptor (AMPAR) expression and stimulation promote glioma cell migration and invasion. Overexpression of GluR1, the most abundant AMPAR subunit in gliomas, positively correlated with glioma cell adhesion to type I and type IV collagen, which was decreased in cells with knockdown of GluR1 and with blocking antibodies to beta1 integrin. Furthermore, stimulation of the AMPAR led to detachment of cells from the extracellular matrix (ECM). Immunoprecipitation studies showed that GluR1 associated with the actin cytoskeleton-linked protein band 4.1B (brain type), which may serve as a link between GluR1 and integrins. Overexpression of GluR1 correlated with increased cell-surface expression of beta1 integrin, increased phosphorylation of focal adhesion kinase (FAK-Y397), and enhanced numbers of focal adhesion (FA) complexes. Cells overexpressing GluR1 had increased colocalization of actin and paxillin at FAs and, in several glioma cell lines, significantly increased invasion in an in vitro Matrigel transwell assay. Likewise, in an intracranial xenograft model, overexpression of GluR1 led to perivascular and subependymal glioma cell invasion similar to patterns of tumor dissemination described in human glioblastoma. Together, these results suggest that AMPARs may link signals from the ECM to sites of FA, where signal integration promotes tumor invasion.

Human aortic endothelial cell response to 316L stainless steel material microstructure

The role of metal microstructure (e.g. grain sizes) in modulating cell adherence behavior is not well understood. This study investigates the effect of varying grain sizes of 316L stainless steel (SS) on the attachment and spreading of human aortic endothelial cells (HAECs). Four different grain size samples; from 16 to 66 microm (ASTM 9.0-4.9) were sectioned from sheets. Grain structure was revealed by polishing and etching with glycergia. Contact angle measurement was done to assess the hydrophilicity of the specimens. Atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the roughness and surface chemistry of the specimens. Cells were seeded on mechanically polished and chemically etched specimens followed by identification of activated focal adhesion sites using fluorescently tagged anti-pFAK (phosphorylated focal adhesion kinase). The 16 microm grain size etched specimens had significantly (P < 0.01) higher number of cells attached per cm(2) than other specimens, which may be attributed to the greater grain boundary area and associated higher surface free energy. This study shows that the underlying material microstructure may influence the HAEC behavior and may have important implications in endothelialization.