No abstract

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKC␦ is the most strongly phosphorylated isoform, and the preferential PKC␦ inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKC␦ is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKC␦ activation, whereas lack of PLC␥ signaling resulted in delayed PKC␦ activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKC␦ is required, a dominant-negative PKC␦ construct or RNAi-mediated PKC␦ depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKC␦ activation. All of the interventions that blocked PKC␦ activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKC␦ activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKC␦ to promote the acute phase of EGF-induced MLC activation.Cell motility induced by activation of epidermal growth factor receptor (EGFR), 1 and related receptor tyrosine kinases, can be deconstructed into a series of orchestrated events: lamellipodial extension, formation of forward adhesions, exertion of contractile forces to pull the cell body forward, and detachment of the rear (1). While each process is required for net cell locomotion, it is not necessarily the case that signals downstream of receptor activation must concomitantly be involved in triggering all of the processes. Despite longstanding anecdotal indications, only recently have formal demonstrations emerged that signaling via EGFR actually elicits cell contractile force generation (2, 3) along with the other biophysical processes (4 -6). Because of the central importance of growth factor-induced cell motility in physiological and pathological applications, such as organogenesis, wound repair, and tumor invasion, determination of key pathways involved in connecting EGFR activity to contractile force generation, as well as the other processes underlying motility, is a crucial undertaking. Myosin motors operating on cytoskeletal actin filaments are presumed to be involved in growth factor-induced cell motility in manne...

A combined effect of protein coating and plasma modification on the quality of the osteoblast-scaffold interaction was investigated. Three-dimensional polycaprolactone (PCL) scaffolds were manufactured by the precision extrusion deposition (PED) system. The structural, physical, chemical and biological cues were introduced to the surface through providing 3D structure, coating with adhesive protein fibronectin and modifying the surface with oxygen-based plasma. The changes in the surface properties of PCL after those modifications were examined by contact angle goniometry, surface energy calculation, surface chemistry analysis (XPS) and surface topography measurements (AFM). The effects of modification techniques on osteoblast short-term and long-term functions were examined by cell adhesion, proliferation assays and differentiation markers, namely alkaline phosphatase activity (ALP) and osteocalcin secretion. The results suggested that the physical and chemical cues introduced by plasma modification might be sufficient for improved cell adhesion, but for accelerated osteoblast differentiation the synergetic effects of structural, physical, chemical and biological cues should be introduced to the PCL surface.

The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 m in width at a constant depth of 1 m. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm 2 ), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm 2 ) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow. endothelial cell alignment; shear stress; focal adhesion ENDOTHELIAL CELL MIGRATION plays a critical role in vascular remodeling processes such as angiogenesis, vasculogenesis, and wound healing (24,25,27,30,35). The fluid shear stress experienced by vascular endothelial cells in the in vivo hemodynamic milieu provides an important mechanical cue that can direct cell migration and induce the activation of biochemical processes (8,15,35). Integrins (16, 38), focal adhesion (FA) proteins (24, 26, 27, 37), cytoskeletal components (6, 16, 18, 19, 23, 27, 28, 34, 37), regulatory proteins (13, 35, 41), and intracellular ion concentrations (12, 28) are amongst the molecular components that regulate the morphological changes and the physiological responses of endothelial cell migration upon exposure to shear stress. Therefore, the modulation of endothelial cell migration in such vascular remodeling processes requires an understanding of how the cells interact and respond to the shear stress environment.In physical terms, cell migration proceeds in three coordinated steps: 1) membrane extension and formation of FAs at the leading edge, 2) forward movement of the cell body through contraction of the actin cytoskeleton by myosin-based motors, and 3) detachment of the trailing edge of the cell, which completes the cycle of the migration process (25,30,35). When cultured cells are exposed to a steady, laminar flow, lamellipodial protrusions develop within minutes from the cell periphery in the direction of flow (27, 29), followed by the recrui...

Nerve growth factor (NGF) is a well known neurotropic and neurotrophic agonist in the nervous system, which recently was shown to also induce angiogenic effects in endothelial cells (ECs). To measure NGF effects on the migration of cultured ECs, an important step in neoangiogenesis, we optimized an omnidirectional migration assay using human aortic endothelial cells (HAECs) and validated the assay with human recombinant basic fibroblast growth factor (rhbFGF) and human recombinant vascular endothelial growth factor (rhVEGF). The potencies of nerve growth factor purified from various species (viper, mouse, and recombinant human) to stimulate HAEC migration was similar to that of VEGF and basic fibroblast growth factor (bFGF) (EC 50 of ϳ0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by ϳ30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(ı)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist ) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system.