Mechanical stretching stimulates smooth muscle cell growth, nuclear protein import, and nuclear pore expression through mitogen-activated protein kinase activation

Although it is known that mechanical stretching of cells can induce significant increases in cell growth and shape, the intracellular signaling pathways that induce this response at the level of the cell nucleus is unknown. The transport of molecules from the cell cytoplasm to the nucleoplasm through the nuclear pore is a key pathway through which gene expression can be controlled in some conditions. It is presently unknown if mechanical stimuli can induce changes in nuclear pore expression and/or function. The purpose of the present investigation was to determine if mechanical stretching of a cell will alter nuclear protein import and the mechanisms that may be responsible. Vascular smooth muscle cells that were mechanically stretched exhibited an increase in proliferating cell nuclear antigen expression, cell number, and cell size within 24-48 h. Cells were microinjected with marker proteins for nuclear import. Nuclear protein import was significantly stimulated in stretched cells when compared with control. This was associated with an increase in the expression of nuclear pore proteins as detected by Western blots. Inhibition of the MAPK pathway blocked the stretch-induced stimulation of both cell proliferation and nuclear protein import. We conclude that nuclear protein import and nuclear pore density can adapt to mechanical stimuli during the process of cell growth through a MAPK-mediated mechanism.     

Effect of hypergravity stimulus on XTH gene expression in Arabidopsis thaliana.

There are several reports indicating that hypergravity and microgravity influence the mechanical properties of cell walls in shoots, resulting in changes in the growth rate. The mechanical properties of cell walls in dicots are mainly determined by the physicochemical properties of xyloglucan, a matrix polysaccharide. An increase in the molecular mass of xyloglucan correlated with a decrease in cell wall extensibility. Hypergravity is known to increase the molecular mass of xyloglucan. The cell wall enzyme, xyloglucan endotransglucosylase/hydrolase (XTH) is involved in xyloglucan metabolism. Using Arabidopsis, it was examined whether or not the expression of XTH genes in the floral stem and rosette leaf is influenced by hypergravity. RT-PCR analysis revealed that the expression of XTH genes changes in response to hypergravity of 300 g.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

A close-up view of wood structure and properties across a growth ring of Norway spruce (Picea abies [L] Karst.)

A growth ring of an adult Norway spruce (Picea abies [L] Karst.) was analyzed to a high resolution at the single cell level with respect to structural and mechanical changes during the growth period. For this purpose structural characterization was performed by means of light microscopy, scanning electron microscopy and wide angle X-ray diffraction for investigating the geometry of cells, their cell wall fractions and cellulose microfibril angles (MFA). The mechanical properties were determined in microtensile tests on individual tracheids which had been taken from sequentially cut tangential slices. The results revealed pronounced differences in tensile stiffness between earlywood and latewood cells but only minor differences in tensile stiffness between the cell walls of both tissue types. These comparatively small changes in cell wall stiffness across the growth ring were caused by slight changes in MFA. The findings suggest that trees mainly vary cell size to optimize water transport and mechanical stability during the growth period and that modification of the cell wall organisation plays a minor role.     

Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties

Cellular morphogenesis involves changes to cellular size and shape which in the case of walled cells implies the mechanical deformation of the extracellular matrix. So far, technical challenges have made quantitative mechanical measurements of this process at subcellular scale impossible. We used micro-indentation to investigate the dynamic changes in the cellular mechanical properties during the onset of spatially confined growth activities in plant cells. Pollen tubes are cellular protuberances that have a strictly unidirectional growth pattern. Micro-indentation of these cells revealed that the initial formation of a cylindrical protuberance is preceded by a local reduction in cellular stiffness. Similar cellular softening was observed before the onset of a rapid growth phase in cells with oscillating growth pattern. These findings provide the first quantitative cytomechanical data that confirm the important role of the mechanical properties of the cell wall for local cellular growth processes. They are consistent with a conceptual model that explains pollen tube oscillatory growth based on the relationship between turgor pressure and tensile resistance in the apical cell wall. To further confirm the significance of cell mechanics, we artificially manipulated the mechanical cell wall properties as well as the turgor pressure. We observed that these changes affected the oscillation profile and were able to induce oscillatory behavior in steadily growing tubes.     

Measuring,reversing, and modeling the mechanical changes due to the absence of Fibulin-4 in mouse arteries

Mice with a smooth muscle cell (SMC)-specific deletion of Fibulin-4 (SMKO) show decreased expression of SMC contractile genes, decreased circumferential compliance, and develop aneurysms in the ascending aorta. Neonatal administration of drugs that inhibit the angiotensin II pathway encourages the expression of contractile genes and prevents aneurysm development, but does not increase compliance in SMKO aorta. We hypothesized that multidimensional mechanical changes in the aorta and/or other elastic arteries may contribute to aneurysm pathophysiology. We found that the SMKO ascending aorta and carotid artery showed mechanical changes in the axial direction. These changes were not reversed by angiotensin II inhibitors, hence reversing the axial changes is not required for aneurysm prevention. Mechanical changes in the circumferential direction were specific to the ascending aorta; therefore, mechanical changes in the carotid do not contribute to aortic aneurysm development. We also hypothesized that a published model of postnatal aortic growth and remodeling could be used to investigate mechanisms behind the changes in SMKO aorta and aneurysm development over time. Dimensions and mechanical behavior of adult SMKO aorta were reproduced by the model after modifying the initial component material constants and the aortic dilation with each postnatal time step. The model links biological observations to specific mechanical responses in aneurysm development and treatment.     

Mechanical strain affects dura mater biological processes: implications for immature calvarial healing

The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.     

Proteomic analysis of early‐response to mechanical stress in neonatal rat mandibular condylar chondrocytes

The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four‐point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 µstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2‐DE and MALDI‐TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling. J. Cell. Physiol. 223:610–622, 2010. © 2010 Wiley‐Liss, Inc.     

Determination of mechanical stress distribution in Drosophila wing discs using photoelasticity

Morphogenesis, the process by which all complex biological structures are formed, is driven by an intricate interplay between genes, growth, as well as intra- and intercellular forces. While the expression of different genes changes the mechanical properties and shapes of cells, growth exerts forces in response to which tissues, organs and more complex structures are shaped. This is exemplified by a number of recent findings for instance in meristem formation in Arabidopsis and tracheal tube formation in Drosophila. However, growth not only generates forces, mechanical forces can also have an effect on growth rates, as is seen in mammalian tissues or bone growth. In fact, mechanical forces can influence the expression levels of patterning genes, allowing control of morphogenesis via mechanical feedback. In order to study the connections between mechanical stress, growth control and morphogenesis, information about the distribution of stress in a tissue is invaluable. Here, we applied stress-birefringence to the wing imaginal disc of Drosophila melanogaster, a commonly used model system for organ growth and patterning, in order to assess the stress distribution present in this tissue. For this purpose, stress-related differences in retardance are measured using a custom-built optical set-up. Applying this method, we found that the stresses are inhomogeneously distributed in the wing disc, with maximum compression in the centre of the wing pouch. This compression increases with wing disc size, showing that mechanical forces vary with the age of the tissue. These results are discussed in light of recent models proposing mechanical regulation of wing disc growth.     

Heparin-dependent regulation of fibronectin matrix conformation

Extracellular matrix (ECM) conformation is regulated by a variety of stimuli in vivo, including mechanical forces and allosteric binding partners, and these conformational changes contribute to the regulation of cell behavior. Heparin and heparan sulfate, for example, have been shown to regulate the sequestration and presentation of numerous growth factors, including vascular endothelial growth factor, on the heparin 2 binding domain in fibronectin (Fn). However, mechanical force also alters Fn conformation, indicating that the growth factor binding region may be co-regulated by both heparin and mechanical force. Herein, we describe a simple antibody-based method for evaluating the conformation of the heparin 2 binding domain in Fn, and use it to determine the relative contributions of heparin and mechanical strain to the regulation of Fn conformation. We achieved specificity in quantifying conformational changes in this region of Fn by measuring the ratio of two fluorescent monoclonal antibodies, one that is insensitive to Fn conformational changes and a second whose binding is reduced or enhanced by non-equilibrium conformational changes. Importantly, this technique is shown to work on Fn adsorbed on surfaces, single Fn fibers, and Fn matrix fibers in cell culture. Using our dual antibody approach, we show that heparin and mechanical strain co-regulate Fn conformation in matrix fibrils, which is the first demonstration of heparin-dependent regulation of Fn in its physiologically-relevant fibrillar state. Furthermore, the dual antibody approach utilizes commercially available antibodies and simple immunohistochemistry, thus making it accessible to a wide range of scientists interested in Fn mechanobiology.     

Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.     

Mechanical Induction of β1-Integrin-Mediated Calcium Signaling in a Hepatocyte Cell Line

Mechanical stress influences growth, differentiation, and gene expression in a variety of cell types. It is believed that via extracellular matrix the mechanical stimulus is transmitted to integrin receptors which thus play a key role in transducing signals into the cell interior. Here we demonstrate that incubation of suspended hepatocytes with specific antibodies to β1-integrin subunits followed by a short-term mechanical stimulation is sufficient to induce a rise in intracellular Ca²⁺. The results indicate that mechanical loading of individual integrin subunits activates Ca²⁺-specific signal pathways.     

Biomechanics of plant growth

Growth of turgid cells, defined as an irreversible increase in cell volume and surface area, can be regarded as a physical process governed by the mechanical properties of the cell wall and the osmotic properties of the protoplast. Irreversible cell expansion is produced by creating a driving force for water uptake by decreasing the turgor through stress relaxation in the cell wall. This mechano-hydraulic process thus depends on and can be controlled by the mechanical properties of the wall, which in turn are subject to modification by wall loosening and wall stiffening reactions. The biochemical mechanisms of these changes in mechanical wall properties and their regulation by internal signals (e.g., hormones) or external signals (e.g., light, drought stress) are at present incompletely understood and subject to intensive research. These signals act on walls that have the properties of composite materials in which the molecular structure and spatial organization of polymers rather than the distribution of mechanical stresses dictate the allometry of cell and organ growth and thus cell and organ shape. The significance of cell wall architecture for allometric growth can be demonstrated by disturbing the oriented deposition of wall polymers with microtubule-interfering drugs such as colchicine. Elongating organs (e.g., cylindrical stems or coleoptiles) composed of different tissues with different mechanical properties exhibit longitudinal tissue tensions resulting in the transfer of wall stress from inner to peripheral cell layers that adopt control over organ growth. For physically analyzing the growth process leading to seed germination, the same mechanical and hydraulic parameters as in normal growth are principally appropriate. However, for covering the influences of the tissues that restrain embryo expansion (seed coat, endosperm), an additional force and a water permeability term must be considered.