The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE₂ release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE₂ release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE₂ release. In fact, PGE₂ release was increased threefold in the polymerization inhibited cells and this PGE₂ release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE₂ release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE₂ release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton. mechanotransduction; cell mechanics     

Mechanical loading by fluid shear stress enhances IGF-1 receptor signaling in osteoblasts in a PKCzeta-dependent manner

Maintenance of optimal bone physiology requires the coordinated activity of osteoclasts that resorb old bone and osteoblasts that deposit new bone. Mechanical loading of bone and the resulting movement of interstitial fluid within the spaces surrounding bone cells is thought to play a key role is maintaining optimal bone mass. One way in which fluid movement may promote bone formation is by enhancing osteoblast survival. We have shown previously that application of fluid flow to osteoblasts in vitro confers a protective effect by inhibiting osteoblast apoptosis (Pavalko et al., 2003, J. Cell Physiol., 194: 194-205). To investigate the cellular mechanisms that regulate the response of osteoblasts to fluid shear stress, we have examined the possible interaction between fluid flow and growth factors in MC3T3-E1 osteoblast-like cells. We found that insulin-like growth factor-I (IGF-I) was significantly more effective at preventing TNF-alpha-induced apoptosis when cells were first subjected to mechanical loading by exposure to either unidirectional or oscillatory fluid flow compared to cells that were maintained in static culture. Additionally, downstream signaling in response to treatment with IGF-I, including ERK and Akt activation, was enhanced in cells that were subjected to fluid flow, compared to cells maintained in static culture. Furthermore, we found that PKC activity is essential for fluid shear stress sensitization of IGF-IR, since a specific inhibitor of PCKzeta function blocked the flow-enhanced IGF-I-activated Akt and ERK phosphorylation. Together, our results suggest that fluid shear stress may regulate IGF-I signaling in osteoblasts in a PKC-zeta-dependent manner.     

Response of osteoblasts to low fluid shear stress is time dependent

The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation.     

Computational simulations predict a key role for oscillatory fluid shear stress in de novo valvular tissue formation

Previous efforts in heart valve tissue engineering demonstrated that the combined effect of cyclic flexure and steady flow on bone marrow derived stem cell-seeded scaffolds resulted in significant increases in engineered collagen formation [Engelmayr et al. Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues. Biomaterials 2006; 27(36): 6083–95]. Here, we provide a new interpretation for the underlying reason for this observed effect. In addition, another related investigation demonstrated the impact of fluid flow on DNA content and quantified the fluid-induced shear stresses on the engineered heart valve tissue specimens [Engelmayr et al. A Novel Flex-Stretch-Flow Bioreactor for the Study of Engineered Heart Valve Tissue Mechanobiology]. Annals of Biomedical Engineering 2008, 36, 1–13]. In this study, we performed more advanced CFD analysis with an emphasis on oscillatory wall shear stresses imparted on specimens when mechanically conditioned by a combination of cyclic flexure and steady flow. Specifically, we hypothesized that the dominant stimulatory regulator of the bone marrow stem cells is fluid-induced and depends on both the magnitude and temporal directionality of surface stresses, i.e., oscillatory shear stresses (OSS) acting on the developing tissues. Therefore, we computationally quantified the (i) magnitude of fluid-induced shear stresses as well as (ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter. Noting that sample cyclic flexure induces a high degree of OSS, we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: (1) No Flow, No Flexure (control group), (2) Steady Flow-alone, (3) Cyclic Flexure-alone and (4) Combined Steady flow and Cyclic Flexure environments. Indeed we found that the coexistence of both OSS and appreciable shear stress magnitudes explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress.     

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.     

The responses of osteoblasts to fluid shear stress depend on substrate chemistries

Natural bone tissue receives chemical and mechanical stimuli in physiological environment. The effects of material chemistry alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the synergistic influences of material chemistry and flow shear stress (FSS) on biological functions of osteoblasts. Self-assembled monolayers (SAMs) on glass slides with functional groups of OH, CH₃, and NH₂ were employed to provide various material chemistries, while FSS (12 dynes/cm²) was produced by a parallel-plate fluid flow system. Material chemistry alone had no obvious effects on the expressions of ATP, nitric oxide (NO), and prostaglandin E₂ (PGE₂), whereas FSS stimuli alone increased the production of those items. When both material chemistry and FSS were loaded, cell proliferation and the expressions of ATP, NO and PGE₂ were highly dependent on the material chemistry. Examination of the focal adhesion (FA) formation and F-actin organization of osteoblasts before FSS exposure indicates that the FA formation and F-actin organization followed similar chemistry-dependence. The inhibition of FAs and/or disruption of F-actins eliminated the material dependence of FSS-induced ATP, PGE₂ and NO release. A possible mechanism is proposed: material chemistry controls the F-actin organization and FA formation of osteoblasts, which further modulates FSS-induced cellular responses.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

A novel in vitro loading system to produce supraphysiologic oscillatory fluid shear stress

A multi-well fluid loading (MFL) system was developed to deliver oscillatory subphysiologic to supraphysiologic fluid shear stresses to cell monolayers in vitro using standard multi-well culture plates. Computational fluid dynamics modeling with fluid-structure interactions was used to quantify the squeeze film fluid flow between an axially displaced piston and the well plate surface. Adjusting the cone angle of the piston base modulated the fluid pressure, velocity, and shear stress magnitudes. Modeling results showed that there was near uniform fluid shear stress across the well with a linear drop in pressure across the radius of the well. Using the MFL system, RAW 264.7 osteoclastic cells were exposed to oscillatory fluid shear stresses of 0, 0.5, 1.5, 4, 6, and 17 Pa. Cells were loaded 1 h per day at 1 Hz for two days. Compared to sub-physiologic and physiologic levels, supraphysiologic oscillatory fluid shear induced upregulation of osteoclastic activity as measured by tartrate-resistant acid phosphatase activity and formation of mineral resorption pits. Cell number remained constant across all treatment groups.     

TNFR1 and TNFR2 signaling interplay in cardiac myocytes

Tumor necrosis factor alpha (TNFalpha) plays a major role in chronic heart failure, signaling through two different receptor subtypes, TNFR1 and TNFR2. Our aim was to further delineate the functional role and signaling pathways related to TNFR1 and TNFR2 in cardiac myocytes. In cardiac myocytes isolated from control rats, TNFalpha induced ROS production, exerted a dual positive and negative action on [Ca(2+)] transient and cell fractional shortening, and altered cell survival. Neutralizing anti-TNFR2 antibodies exacerbated TNFalpha responses on ROS production and cell death, arguing for a major protective role of the TNFR2 pathway. Treatment with either neutralizing anti-TNFR1 antibodies or the glutathione precursor, N-acetylcysteine (NAC), favored the emergence of TNFR2 signaling that mediated a positive effect of TNFalpha on [Ca(2+)] transient and cell fractional shortening. The positive effect of TNFalpha relied on TNFR2-dependent activation of the cPLA(2) activity, independently of serine 505 phosphorylation of the enzyme. Together with cPLA(2) redistribution and AA release, TNFalpha induced a time-dependent phosphorylation of ERK, MSK1, PKCzeta, CaMKII, and phospholamban on the threonine 17 residue. Taken together, our results characterized a TNFR2-dependent signaling and illustrated the close interplay between TNFR1 and TNFR2 pathways in cardiac myocytes. Although apparently predominant, TNFR1-dependent responses were under the yoke of TNFR2, acting as a critical limiting factor. In vivo NAC treatment proved to be a unique tool to selectively neutralize TNFR1-mediated effects of TNFalpha while releasing TNFR2 pathways.     

流体剪切力对成骨细胞LEF-1蛋白表达的影响

目的：探讨MC3T3-E1细胞在流体剪切力作用下LEF-1的表达。方法：通过流体剪切加载系统对MC3T3-E1爬片细胞施加12dyn/cm的流体剪切力,分别作用0h,2h,4h,8h,12h,用RT-PCR方法检测细胞受力前后LEF-1 mRNA表达的变化;应用免疫荧光双标记法检测不同时间点流体剪切力作用下MC3T3-E1细胞中的LEF-1 mRNA表达改变。结果：RT-PCR和免疫荧光双标记法的结果表明12dyn/cm 8h流体剪切力作用下的MC3T3-E1细胞LEF-1 mRNA的表达较其它各组明显增强。结论：通过流体剪切力力学刺激,激活了成骨细胞LEF-1/TCF1转录活动,LEF-1 mRNA的表达增强可能是成骨细胞经典Wnt信号通路对剪切应力的应答反应。     

The role of endothelial glycocalyx components in mechanotransduction of fluid shear stress

The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). In this study, enzymes were used to selectively degrade glycocalyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI(2)) production were determined. Depletion of HS, HA, and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI(2) production. The results may be interpreted in terms of a glypican-caveolae-eNOS mechanism for shear-induced NO transduction, with PGI(2) being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not.     

MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.     

TGF-beta1 calcium signaling in osteoblasts

Transforming growth factor-beta1 (TGF-beta1) action is known to be initiated by its binding to multiple cell surface receptors containing serine/threonine kinase domains that act to stimulate a cascade of signaling events in a variety of cell types. We have previously shown that TGF-beta1 and BMP-2 treatment of primary human osteoblasts (HOBs) enhances cell-substrate adhesion. In this report, we demonstrate that TGF-beta1 elicits a rapid, transient, and oscillatory rise in the intracellular Ca(2+) concentration, [Ca(2+)](i), that is necessary for enhancement of cell adhesion in HOBs but does not alter the phosphorylation state of Smad proteins. This rise in [Ca(2+)](i) in HOB is not observed in the absence of extracellular calcium or when the cells are treated with the L-type Ca(2+) channel blocker, nifedipine, but is stimulated upon treatment with the L-type Ca(2+) channel agonist, Bay K 8644, or under high K(+) conditions. The rise in [Ca(2+)](i) is severely attenuated after treatment of the cells with thapsigargin, a selective endoplasmic reticulum Ca(2+) pump inhibitor. TGF-beta1 enhancement of HOB adhesion to tissue culture polystyrene is also inhibited in cells treated with nifedipine. These data suggest that intracellular Ca(2+) signaling is an important second messenger of the TGF-beta1 signal transduction pathway in osteoblast function.     

Effect of LIMK2 RNAi on reorganization of the actin cytoskeleton in osteoblasts induced by fluid shear stress

The biomechanical characteristics of bone tissue and its cells under mechanical stress are significant for bone biomechanics research, but the mechanism of mechanotransduction is still unknown. It has been established that the actin cytoskeleton of osteoblasts plays an important role in this process. However, the structure of the actin cytoskeleton is reorganized when loaded with mechanical stress, which results in changes in cell stiffness. These phenomena suggest that an actin-cytoskeleton-induced feedback regulation mechanism may be involved in the mechanotransduction of osteoblasts, but this has not yet been proven. The aim of this study was to explore the role of LIMK2 in the reorganization of the actin cytoskeleton induced by fluid shear stress in osteoblasts by using RNA interference. Balb/c mouse primary osteoblasts were divided into four groups. Cells in Groups 1 and 3 were transfected with negative control RNA, while cells in Groups 2 and 4 were transfected with a specific siRNA designed to silence the LIMK2 gene. Twenty-four hours after transfection, cells in Groups 1 and 2 were loaded with fluid shear stress at 12 dyne/cm2 while cells in Groups 3 and 4 were not. Compared with Group 1, the mean fluorescence density of the actin cytoskeleton in the other three groups was 28.9%, 45.7%, and 33.0%, respectively. These results indicate that LIMK2 plays an important role in the reorganization of the actin cytoskeleton induced by fluid shear stress.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

The role of shear stress in atherosclerosis

Atherosclerotic lesions preferentially localize near side branches or curved vessels. During the last few decades, research has been shown that low or low and oscillating shear stress is associated with plaque location. Despite ample evidence, the precise mechanism is unknown. This is mainly because of a lack of appropriate animal models. We describe two novel methods to study the hypothesis that shear stress acts through endothelial gene expression or shear stress acts through localizing of inflammation. Both literature evidence and own findings support a role for both mechanisms in atherosclerosis.