Measurement of local strain on cell membrane at initiation point of calcium signaling response to applied mechanical stimulus in osteoblastic cells

In adaptive bone remodeling, it is believed that bone cells such as osteoblasts, osteocytes and osteoclasts can sense mechanical stimuli and modulate their remodeling activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into intracellular biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical conditions under which the cellular mechanosensing mechanism is activated. The aims of this study were to evaluate a mechanical condition, that is, the local strain on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, intracellular calcium ions and the cell membrane were labeled using fluorescent dyes and simultaneously observed by confocal laser scanning microscopy. The strain distribution on the cell membrane attributable to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling responses were analyzed using obtained fluorescence images. From two-dimensionally projected images, it was found that there is a local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with a higher sensitivity to a higher deformation velocity. From the viewpoint of cell biomechanics, these results provide us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells.     

Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes

Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Ca_v3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.     

Load Regulates Bone Formation and Sclerostin Expression through a TGFβ-Dependent Mechanism

Bone continually adapts to meet changing physical and biological demands. Osteoblasts, osteoclasts, and osteocytes cooperate to integrate these physical and biochemical cues to maintain bone homeostasis. Although TGFβ acts on all three of these cell types to maintain bone homeostasis, the extent to which it participates in the adaptation of bone to mechanical load is unknown. Here, we investigated the role of the TGFβ pathway in load-induced bone formation and the regulation of Sclerostin, a mechanosensitive antagonist of bone anabolism. We found that mechanical load rapidly represses the net activity of the TGFβ pathway in osteocytes, resulting in reduced phosphorylation and activity of key downstream effectors, Smad2 and Smad3. Loss of TGFβ sensitivity compromises the anabolic response of bone to mechanical load, demonstrating that the mechanosensitive regulation of TGFβ signaling is essential for load-induced bone formation. Furthermore, sensitivity to TGFβ is required for the mechanosensitive regulation of Sclerostin, which is induced by TGFβ in a Smad3-dependent manner. Together, our results show that physical cues maintain bone homeostasis through the TGFβ pathway to regulate Sclerostin expression and the deposition of new bone.     

Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. During embryonic development, myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ influx in myocytes. Evidence of the involvement of Ca2+ -dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea of its implication during the initial events of muscle development.     

Functional classification of scaffold proteins and related molecules

In this series of four minireviews the field of scaffold proteins and proteins of similar molecular/cellular functions is overviewed. By binding and bringing into proximity two or more signaling proteins, these proteins direct the flow of information in the cell by activating, coordinating and regulating signaling events in regulatory networks. Here we discuss the categories of scaffolds, anchors, docking proteins and adaptors in some detail, and using many examples we demonstrate that they cover a wide range of functional modes that appear to segregate into three practical categories, simple proteins binding two partners together (adaptors), larger multidomain proteins targeting and regulating more proteins in complex ways (scaffold/anchoring proteins) and proteins specialized to initiate signaling cascades by localizing partners at the cell membrane (docking proteins). It will also be shown, however, that the categories partially overlap and often their names are used interchangeably in the literature. In addition, although not usually considered as scaffolds, several other proteins, such as regulatory proteins with catalytic activity, phosphatase targeting subunits, E3 ubiquitin ligases, ESCRT proteins in endosomal sorting and DNA damage sensors also function by bona fide scaffolding mechanisms. Thus, the field is in a state of continuous advance and expansion, which demands that the classification scheme be regularly updated and, if needed, revised.     

Mechanical stimulation increases proliferation, differentiation and protein expression in culture: stimulation effects are substrate dependent

Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects various signal transduction pathways, gene sequences and protein synthesis. One indication of which cells are competent to undergo the fusion process is their expression of two proteins, Myo-D and myogenin. The mechanism by which the cells are able to to regulate Myo-D and myogenin is poorly understood. In the present work, we investigate the role of mechanical loading, through specific receptors to intracellular matrix proteins such as laminin and fibronectin, in both Myo-D and myogenin expression in C(2)C(12) cells. We propose to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin recepotors plays a role in myoblast differentiation and fusion.     

Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography

Cellular response to scaffold materials is of great importance in cellular and tissue engineering, and it is perhaps the initial cell contact with the scaffold that determines development of new tissue. Material surface morphology has strong effects on cell cytoskeleton and morphology, and it is thought that cells may react to the topography of collagen and surrounding cells during tissue embryology. A poorly understood area is, however, gene-level responses to topography. Thus, this paper used microarray to probe for consistent gene changes in response to lithographically produced topography (12.5 x 2-microm grooves) with time. The results showed many initial gene changes and also down-regulation of gene response with time. Cell and nucleus morphology were also considered, with nuclear deformation linked to cell signaling.     

Non-overlapping functions for Pyk2 and FAK in osteoblasts during fluid shear stress-induced mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2(+/+) and Pyk2(-/-) primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2(+/+) and Pyk2(-/-) osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK(-/-) osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts.     

Mechanical regulation of signaling pathways in bone

A wide range of cell types depend on mechanically induced signals to enable appropriate physiological responses. The skeleton is particularly dependent on mechanical information to guide the resident cell population towards adaptation, maintenance and repair. Research at the organ, tissue, cell and molecular levels has improved our understanding of how the skeleton can recognize the functional environment, and how these challenges are translated into cellular information that can site-specifically alter phenotype. This review first considers those cells within the skeleton that are responsive to mechanical signals, including osteoblasts, osteoclasts, osteocytes and osteoprogenitors. This is discussed in light of a range of experimental approaches that can vary parameters such as strain, fluid shear stress, and pressure. The identity of mechanoreceptor candidates is approached, with consideration of integrins, pericellular tethers, focal adhesions, ion channels, cadherins, connexins, and the plasma membrane including caveolar and non-caveolar lipid rafts and their influence on integral signaling protein interactions. Several mechanically regulated intracellular signaling cascades are detailed including activation of kinases (Akt, MAPK, FAK), β-catenin, GTPases, and calcium signaling events. While the interaction of bone cells with their mechanical environment is complex, an understanding of mechanical regulation of bone signaling is crucial to understanding bone physiology, the etiology of diseases such as osteoporosis, and to the development of interventions to improve bone strength.     

Three dimensional multi-scale modelling and analysis of cell damage in cell-encapsulated alginate constructs

One of the major challenges in scaffold guided regenerative therapies is identifying the essential cues such as mechanical forces that induce cellular responses to form functional tissue. Developing multi-scale modelling methods would facilitate in predicting responses of encapsulated cells for controlling and maintaining the cell phenotype in an engineered tissue construct, when mechanical loads are applied. The objective of this study is to develop a 3D multi-scale numerical model for analyzing the stresses and deformations of the cell when the tissue construct is subjected to macro-scale mechanical loads and to predict load-induced cell damage. Specifically, this methodology characterizes the macro-scale structural behavior of the scaffold, and quantifies 3D stresses and deformations of the cells at the micro-scale and at a cellular level, wherein individual cell components are incorporated. Assuming that cells have inherent ability to sustain a critical load without damage, a damage criterion is established and a stochastic simulation is employed to predict the percentage cell viability within the tissue constructs. Bio-printed cell-alginate tissue constructs were tested with 1%, 5% and 10% compression strain applied and the cell viability were characterized experimentally as 23.2±16.8%, 9.0±5.4% and 4.6±2.1%. Using the developed method, the corresponding micro-environments of the cells were analyzed, the mean critical compressive strain was determined as 0.5%, and the cell viability was predicted as 26.6±7.0, 13.3±4.5, and 10.1±2.8. The predicted results capture the trend of the damage observed from the experimental study.     

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.     

Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 tat protein

The human immunodeficiency virus type 1 Tat protein is a potent activator of viral gene expression and replication. Tat can also affect the expression of cellular genes including cytokines, extracellular matrix proteins, enzymes degrading the basement membrane and cell cycle-related proteins, and can regulate cellular functions such as growth, migration and angiogenesis. In addition, under certain circumstances, Tat may have tumorigenic effects. These activities of Tat appear to be mediated by different mechanisms such as the transactivation of cellular gene expression or the interaction of extracellular Tat with the cell membrane through both receptor-mediated and nonreceptor-mediated interactions. Deregulation of cellular gene expression and function by Tat cause abnormalities which may participate in AIDS pathogenesis and in the development of AIDS-associated disorders.     

Mechanical aspects of cell shape regulation and signaling

Physical forces play a critical role in cell integrity and development, but little is known how cells convert mechanical signals into biochemical responses. This mini-review examines potential molecular mediators like integrins, focal adhesion proteins, and the cytoskeleton in the context of a complex cell structure. These molecules-when activated by cell binding to the extracellular matrix-associate with the skeletal scaffold via the focal adhesion complex. Vinculin is presented as a mechanical coupling protein that contributes to the integrity of the cytoskeleton and cell shape control, and examples are given of how mechanical signals converge into biochemical responses through force-dependent changes in cell geometry and molecular mechanics.