Oscillatory Flow Accelerates Autocrine Signaling due to Nonlinear Effect of Convection on Receptor-Related Actions

We study effects of oscillatory convective flow in extracellular space on the velocity of chemical signal propagation having a form of a front wave above a cellular layer. We found that the time-averaged propagation velocity under oscillatory flow for a particular Péclet number amplitude is slower than the velocity under steady laminar flow regime for the same value of the Péclet number, but significantly faster than under no-flow conditions. We derive asymptotic values of the propagation velocity and asymptotic characteristics of the corresponding concentration fronts in high- and low-frequency regimes and show that the reason for the observed velocity increase under the oscillatory flow stems from a nonlinear dependence of the propagation velocity on the Péclet number, particularly from the convex character of the dependence. Our findings suggest that the specific responses of cellular cultures to different flow conditions in the extracellular space (for example, expression of atherosclerosis protective genes under steady laminar flow but not under oscillatory flow) is a consequence of a nonlinear coupling between the extracellular transport and complex intracellular reaction cascades forming a positive feedback loop of the autocrine signaling. This mechanism can operate independently of, or in conjunction with, a direct stress-sensing due to mechanotransduction.     

Oscillatory Flow Accelerates Autocrine Signaling due to Nonlinear Effect of Convection on Receptor-Related Actions

We study effects of oscillatory convective flow in extracellular space on the velocity of chemical signal propagation having a form of a front wave above a cellular layer. We found that the time-averaged propagation velocity under oscillatory flow for a particular Péclet number amplitude is slower than the velocity under steady laminar flow regime for the same value of the Péclet number, but significantly faster than under no-flow conditions. We derive asymptotic values of the propagation velocity and asymptotic characteristics of the corresponding concentration fronts in high- and low-frequency regimes and show that the reason for the observed velocity increase under the oscillatory flow stems from a nonlinear dependence of the propagation velocity on the Péclet number, particularly from the convex character of the dependence. Our findings suggest that the specific responses of cellular cultures to different flow conditions in the extracellular space (for example, expression of atherosclerosis protective genes under steady laminar flow but not under oscillatory flow) is a consequence of a nonlinear coupling between the extracellular transport and complex intracellular reaction cascades forming a positive feedback loop of the autocrine signaling. This mechanism can operate independently of, or in conjunction with, a direct stress-sensing due to mechanotransduction.     

Integrin Signaling and Lipid Rafts

Integrin-mediated adhesion regulates the recruitment of the small GTPase Rac to the plasma membrane and subsequent activation of downstream signaling. We recently reported that Rac binds preferentially to cholesterol-rich membranes (“lipid rafts”), and integrins regulate Rac function by preventing the internalization of its binding sites within these domains. Regulation of lipid rafts by integrins may be important for the spatial control of cell migration and signaling pathways involved in anchorage-dependent cell growth.     

Genetic Analyses of Integrin Signaling

The development of multicellular organisms, as well as maintenance of organ architecture and function, requires robust regulation of cell fates. This is in part achieved by conserved signaling pathways through which cells process extracellular information and translate this information into changes in proliferation, differentiation, migration, and cell shape. Gene deletion studies in higher eukaryotes have assigned critical roles for components of the extracellular matrix (ECM) and their cellular receptors in a vast number of developmental processes, indicating that a large proportion of this signaling is regulated by cell-ECM interactions. In addition, genetic alterations in components of this signaling axis play causative roles in several human diseases. This review will discuss what genetic analyses in mice and lower organisms have taught us about adhesion signaling in development and disease.Almost all cells in multicellular organisms are surrounded by a three-dimensional organized meshwork of macromolecules that constitute the extracellular matrix (ECM). The ECM is a dynamic structure that is generated and constantly remodeled by cells that secrete and manipulate its components into a precise configuration. It functions as a structural framework that provides cells with positional and environmental information, but also forms specialized structures such as cartilage, tendons, basement membranes (BM), bone, and teeth. In addition to its structural properties, the ECM acts as a signaling platform that regulates a large number of cellular functions. It is capable of binding growth factors, chemokines, and cytokines thereby modulating their bioavailability and activity. On the other hand, the ECM is recognized by multiple cell surface receptors that transmit information from the extracellular environment by propagating intracellular signals (for a review, see Hynes 2009).The major cell surface receptors that recognize and assemble the ECM are integrins. Integrins are heterodimeric transmembrane proteins composed of α and β subunits. Eighteen α subunits and eight β subunits can assemble in 24 different combinations with overlapping substrate specificity and cell-type-specific expression patterns (Hynes 2002; Humphries et al. 2006). This, together with the ability of different heterodimers to assemble specific intracellular signaling complexes, provides multiple layers of signaling specificity to these receptors. Conversely, the integrin expression profile of a given cell type determines which ECM components it can bind. Signals arising from integrins regulate virtually all aspects of cell behavior, including cell migration, survival, cell cycle progression, and differentiation.Genetics has proven to be a powerful tool to dissect the functions of ECM–cell interactions in complex organisms. To date, all of the integrin subunits and their major ligands have been deleted in mice. Given the large variety of cellular processes regulated by adhesion signaling, it is not surprising that a significant subset of these proteins has proven to be essential for embryonic development and/or tissue maintenance. However, in addition to underlining the importance of cell-ECM interactions in development, genetic studies also revealed critical roles for tissue- and cell-type-specific modes of adhesion signaling and provided important insights into human disease.     

The Role of Actin Cytoskeletal Tension in Oscillatory Fluid Flow Induced Osteogenesis

This article has no abstract.     

Integrin Signalling and the Cellular Response to Ionizing Radiation

Cell survival and cycling in mammalian cells are both greatly affected by ionizing radiation and are both strictly controlled by integrated integrin-mediated adhesion to extracellular matrix (ECM) proteins and by binding of growth factors to their cognate receptors. Recent emerging findings show a diverse panel of integrin-dependent signals that are channelled into the regulation and modification of the cellular response to ionizing radiation. Cell adhesion-mediated radioresistance and alteration of DNA damage-induced cell cycle arrest in cells attached to the ECM can be linked to focal adhesion protein signalling. This review summarizes the latest radiobiological and radiooncological findings about integrins and their signal transduction pathways.     

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

Notch receptors play a role in skeletal development and homeostasis, and Notch activation in undifferentiated and mature osteoblasts causes osteopenia. In contrast, Notch activation in osteocytes increases bone mass, but the mechanisms involved and exact functions of Notch are not known. In this study, Notch1 and -2 were inactivated preferentially in osteocytes by mating Notch1/2 conditional mice, where Notch alleles are flanked by loxP sequences, with transgenics expressing Cre directed by the Dmp1 (dentin matrix protein 1) promoter. Notch1/2 conditional null male and female mice exhibited an increase in trabecular bone volume due to an increase in osteoblasts and decrease in osteoclasts. In male null mice, this was followed by an increase in osteoclast number and normalization of bone volume. To activate Notch preferentially in osteocytes, Dmp1-Cre transgenics were crossed with Rosa^Notch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences. Dmp1-Cre^+/−;Rosa^Notch mice exhibited an increase in trabecular bone volume due to decreased bone resorption and an increase in cortical bone due to increased bone formation. Biomechanical and chemical properties were not affected. Osteoprotegerin mRNA was increased, sclerostin and dickkopf1 mRNA were decreased, and Wnt signaling was enhanced in Dmp1-Cre^+/−;Rosa^Notch femurs. Botulinum toxin A-induced muscle paralysis caused pronounced osteopenia in control mice, but bone mass was preserved in mice harboring the Notch activation in osteocytes. In conclusion, Notch plays a unique role in osteocytes, up-regulates osteoprotegerin and Wnt signaling, and differentially regulates trabecular and cortical bone homeostasis.     

Rigidity-Dependent Cross Talk between Integrin and Cadherin Signaling

Integrin-cadherin cross talk is an important aspect of cell function. We explored this signaling using substrates micropatterned with islands of fibronectin surrounded by E-cadherin, capturing the segregation of these signals in normal tissue. While MDCK cells were able to concurrently form adhesive structures with these two proteins, engagement of fibronectin by MCF-7 cells, an adenocarcinoma cell line, inhibited response of these cells to E-cadherin. We further demonstrated that this inhibition is rigidity dependent; on soft elastomer substrates with Young's modulus in the range of tens of kiloPascals, MCF-7 cells were able to engage both integrin and cadherin ligands.     

Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH''s anabolic actions on bone.     

Non-Newtonian Behavior of Blood in Oscillatory Flow

Sinusoidal oscillatory flow of blood and of aqueous glycerol solutions was produced in rigid cylindrical tubes. For aqueous glycerol, the amplitude of the measured pressure gradient wave form conformed closely to that predicted by Womersley's theory of oscillatory flow, up to Reynolds numbers approaching 2000. Blood differed significantly from aqueous glycerol solutions of comparable viscosity, especially at low frequencies and high hematocrits. As frequency increased, the hydraulic impedance of blood decreased to a minimum at a frequency of about 1-2 CPS, increasing monotonically at higher frequencies. The dynamic apparent viscosity of blood, calculated from Womersley's theory, decreased with increasing flow amplitude. The reactive component of the hydraulic impedance increased with frequency as predicted by theory; the resistive component decreased with increasing frequency, differing from the resistance of a Newtonian fluid which increased with frequency.     

Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β₁, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr³⁹⁷ as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.     

Viscoelasticity of Bone Cells Exposed to Fluid Flow

This article has no abstract.     

Plasticity of the MAPK Signaling Network in Response to Mechanical Stress

Cells display versatile responses to mechanical inputs and recent studies have identified the mitogen-activated protein kinase (MAPK) cascades mediating the biological effects observed upon mechanical stimulation. Although, MAPK pathways can act insulated from each other, several mechanisms facilitate the crosstalk between the components of these cascades. Yet, the combinatorial complexity of potential molecular interactions between these elements have prevented the understanding of their concerted functions. To analyze the plasticity of the MAPK signaling network in response to mechanical stress we performed a non-saturating epistatic screen in resting and stretched conditions employing as readout a JNK responsive dJun-FRET biosensor. By knocking down MAPKs, and JNK pathway regulators, singly or in pairs in Drosophila S2R+ cells, we have uncovered unexpected regulatory links between JNK cascade kinases, Rho GTPases, MAPKs and the JNK phosphatase Puc. These relationships have been integrated in a system network model at equilibrium accounting for all experimentally validated interactions. This model allows predicting the global reaction of the network to its modulation in response to mechanical stress. It also highlights its context-dependent sensitivity.