Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane

Intracellular calcium transient ([Ca²⁺]_i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca²⁺]_i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca²⁺]_i transient in osteoblasts. Fluorescent dyes DiBAC₄(3) and fura-2 AM were respectively used to detect membrane potential and [Ca²⁺]_i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca²⁺]_i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca²⁺]_i transients. Replacing extracellular Na⁺ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca²⁺]_i transients. However, voltage-activated K⁺ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca²⁺ or blocking of L-type voltage-sensitive Ca²⁺ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca²⁺]_i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca²⁺ mobilization wherein FSS activated SACs to promote Na⁺ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca²⁺ influx though L-VSCCs switched on [Ca²⁺]_i response in osteoblasts.     

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.     

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.     

The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation

Ca²⁺ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca²⁺ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca²⁺ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca²⁺ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca²⁺ uptake which were influenced by the amplitude of Ca²⁺ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.     

Formation of focal adhesions on fibronectin promotes fluid shear stress induction of COX-2 and PGE2 release in MC3T3-E1 osteoblasts.

Mechanical loading of bone is important for the structural integrity of the skeleton and the maintenance of bone mass. Mechanically loading bone generates fluid shear stress (FSS) across the surface of bone cells resulting in the induction of cyclooxygenase-2 (COX-2) and release of prostaglandins, both of which are necessary for mechanically induced bone formation. However, the mechanisms by which cells transduce FSS-induced signals across the membrane and into the cell remain poorly understood. Focal adhesions, which are specialized sites of attachment between cells and the extracellular matrix, play a role in signal transduction and have been proposed to function as mechanosensors. To directly test whether focal adhesions mediate mechanotransduction in bone cells, we inhibited the formation of focal adhesions by 1). culturing MC3T3-E1 osteoblasts on bovine serum albumin (BSA), which does not contain integrin binding sites or by 2). treating cells cultured on fibronectin with soluble Arg-Gly-Asp-Ser (RGDS) peptide to specifically block integrin-fibronectin interactions. We then subjected the cells to FSS and measured COX-2 induction and PGE(2) release. Both COX-2 induction and PGE(2) release in response to FSS were significantly decreased when osteoblasts were treated with soluble RGDS peptide compared with controls. However, RGDS peptide treatment did not affect FSS-induced ERK phosphorylation. Interestingly, osteoblasts cultured on BSA to suppress focal adhesion formation secreted fibronectin and increased focal adhesion formation over time, which correlated with the induction of COX-2 in response to FSS. Together, these results suggest that fibronectin-induced formation of focal adhesions promotes FSS-induced PGE(2) release and upregulation of COX-2 protein.     

Fo Shou San,an Ancient Chinese Herbal Decoction,Protects Endothelial Function through Increasing Endothelial Nitric Oxide Synthase Activity

Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca²⁺ levels in HUVECs, and the Ca²⁺ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca²⁺ and a reduction of ROS.     

Deformation of the Bowditch staircase in Ca2+-overloaded mammalian cardiac tissue — a calcium phenomenon?

Concentrations of 1–4 mol l^–1 isoproterenol cause both in right ventricular papillary muscles and in enzymatically isolated myocytes of the guinea-pig a Ca^2– overload-induced state which is functionally characterized by biphasic (multiphasic) twitches and biphasic (multiphasic) intracellular calcium transients, respectively, during excitation-contraction coupling. This state was stabilized in the in vitro experiments for some hours by a co-ordination of the interstimulus interval, the temperature of the superfasion fluid and the addition of calcium agonists. The functional stability is the precondition for the reproducibility of the experimental results particularly after the application of long-lasting stimulation programmes. Changes in the shape of biphasic contractions were compared with changes in the time course of biphasic intracellular calcium transients using three manipulations of a different kind: (1) the interruption of the steady pacing rhythm, (2) the variation of the interstimulus interval, (3) the addition of ryanodine. It was shown that: (1) The BOWDITCH staircase in calcium overloaded multicellular preparations is changed in that each individual component of the twitch passes through its own staircase. A homologous behaviour can be observed in the configuration of the phasic and tonic component of biphasic intracellular calcium transients. (2) At different driving frequencies the relative proportion of the two components of a biphasic twitch corresponds to the time integrals of the two components of biphasic intracellular calcium transients. (3) Ryanodine suppresses both the first component of the biphasic twitch and the phasic component of the biphasic intracellular calcium transient.The SR Ca²⁺-ATPase inhibitor thapsigargin increases the second component of the biphasic calcium transient. This supports the hypothesis that the size of the tonic component is in part determined by intracellular calcium reuptake. The results of both kinds of experiments would be compatible with the assumption that in calcium overloaded mammalian cardiac cells calcium reaches the contractile system directly as well as via two intracellular stores (extended two-Ca-store concept).     

Ab initio molecular dynamics of the reactivity of vitamin C toward hydroxyl and $$ {HO}_2/{O}_2^{-} $$ radicals

Vitamin C is one of the most abundant exogenous antioxidants in the cell, and it is of the utmost importance to elucidate its mechanism of action against radicals. In this study, the reactivity of vitamin C toward OH and \( {HO}_2/{O}_2^{-} \) radicals in aqueous medium was analyzed by ab initio molecular dynamics using CPMD code. The simulations led to results similar to those of static studies or experiments for the pair of \( {HO}_2/{O}_2^{-} \) radicals but bring new insights for the reactivity with hydroxyl radical: the reaction takes place before the formation of an adduct and consists of two steps: first an electron is transferred to hydroxyl radical and then the ascorbyl radical loses a proton.

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Graphical Abstract Reactivity of vitamin C toward hydroxyl and \( {HO}_2/{O}_2^{-} \) radicals

     

Protein kinase G and focal adhesion kinase converge on Src/Akt/β-catenin signaling module in osteoblast mechanotransduction

Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca(2+)]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of β-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.     

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.     

Marker-aided selection and validation of various $${ }$$ gene combinations for rice blast resistance in elite rice variety ADT 43

Rice blast caused by fungal pathogen Pyricularia oryzae has a major impact on reducing yield potential of rice. In this study, homozygous plants were selected using microsatellite markers from the \(\hbox {BC}_{3}\hbox {F}_{2}\) population pyramided with four major genes in elite rice variety ADT 43. Background and selected lines with various blast resistance gene combinations were screened under natural conditions to study the effects of various gene combinations. Upon inspection of lines with different gene combinations, the three-gene pyramided line Pi54+Pi33+Pi1 was found to be highly resistant with the score of 3.3 followed by other three-gene pyramided lines Pi54+Pi2+Pi1 and Pi33+Pi2+Pi1, with the scores of 3.9 and 3.8, respectively. Two-gene pyramided lines Pi54+Pi1, Pi33+Pi1 and Pi2+Pi1 were found to be moderately resistant with a mean score of 4.0 each. In the case of monogenic lines, positive plants for Pi54 performed almost equal to three-gene pyramided lines with a mean score of 3.6. Lines with Pi2 and Pi1 were found to be moderately resistant and moderately susceptible with the mean scores of 4.1 and 4.5, respectively.     

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress

Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.     

Calcium response in single osteocytes to locally applied mechanical stimulus: Differences in cell process and cell body

It is proposed that osteocytes embedded in the bone matrix have the ability to sense deformation and/or damage to the matrix and to feed these mechanical signals back to the adaptive bone remodeling process. When osteoblasts differentiate into osteocytes during the bone formation process, they change their morphology to a stellate form with many slender processes. This characteristic cell shape may underlie the differences in mechanosensitivity between the cell processes and cell body. To elucidate the mechanism of cellular response to mechanical stimulus in osteocytes, we investigated the site-dependent response to quantitatively controlled local mechanical stimulus in single osteocytes isolated from chick embryos, using the technique of calcium imaging. A mechanical stimulus was applied to a single osteocyte using a glass microneedle targeting a microparticle adhered to the cell membrane by modification with a monoclonal antibody OB7.3. Application of the local deformation induced calcium transients in the vicinity of the stimulated point and caused diffusive wave propagation of the calcium transient to the entire intracellular region. The rate of cell response to the stimulus was higher when applied to the cell processes than when applied to the cell body. In addition, a large deformation was necessary at the cell body to induce calcium transients, whereas a relatively small deformation was sufficient at the cell processes, suggesting that the mechanosensitivity of the cell processes was higher than that of the cell body. These results suggest that the cell shape with slender processes contributes to the site-dependent mechanosensitivity in osteocytes.     

Calcium dynamics during fertilization in C. elegans

Background

Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.

Results

Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed.

Conclusion

Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.     

Delay decomposition approach to $$\mathcal {H}_{\infty }$$ filtering analysis of genetic oscillator networks with time-varying delays

In this paper, the \(\mathcal {H}_{\infty }\) filtering problem is treated for N coupled genetic oscillator networks with time-varying delays and extrinsic molecular noises. Each individual genetic oscillator is a complex dynamical network that represents the genetic oscillations in terms of complicated biological functions with inner or outer couplings denote the biochemical interactions of mRNAs, proteins and other small molecules. Throughout the paper, first, by constructing appropriate delay decomposition dependent Lyapunov–Krasovskii functional combined with reciprocal convex approach, improved delay-dependent sufficient conditions are obtained to ensure the asymptotic stability of the filtering error system with a prescribed \(\mathcal {H}_{\infty }\) performance. Second, based on the above analysis, the existence of the designed \(\mathcal {H}_{\infty }\) filters are established in terms of linear matrix inequalities with Kronecker product. Finally, numerical examples including a coupled Goodwin oscillator model are inferred to illustrate the effectiveness and less conservatism of the proposed techniques.     

Calcium signaling as a novel method to optimize the biosynthetic response of chondrocytes to dynamic mechanical loading

Chondrocyte sensitization and desensitization to mechanical stimuli are complex phenomena that have not been fully described. In this study, we investigated the temporal response of chondrocytes to dynamic mechanical loading and whether changes in calcium signaling could be used a predictor of the biosynthetic response. Cell-seeded agarose gels pre-incubated with an intracellular \(\hbox {Ca}^{2+}\) dye (Fluo-4) were subjected to dynamic compressive loading under varying conditions (amplitude and duration). Induced changes in \(\hbox {Ca}^{2+}\) signaling were determined by confocal imaging and matrix biosynthesis by radioisotope incorporation. It was observed that chondrocytes required a minimum amount of stimulation in order to elicit an anabolic response and they quickly became insensitive to the imposed stimulus. The response appeared to be amplitude dependent and could be predicted by measuring resultant changes in \(\hbox {Ca}^{2+}\) signaling. A positive correlation between \(\hbox {Ca}^{2+}\) signaling and matrix synthesis was achieved when changes in \(\hbox {Ca}^{2+}\) signaling was expressed as a relative number of cells experiencing multiple transients. In addition, these changes in \(\hbox {Ca}^{2+}\) signaling were effective at determining optimal recovery period between successive applications of intermittent mechanical loading, in which full mechanosensitivity was achieved when \(\hbox {Ca}^{2+}\) signaling was allowed to return to baseline (control) levels. The use of \(\hbox {Ca}^{2+}\) signaling to predict the effectiveness of a particular mechanical stimulus as well as to determine optimal refractory periods appears to be advantageous over empirical-based approaches. Future work will investigate the process of \(\hbox {Ca}^{2+}\) ion sequestration into intracellular stores to elucidate potential desensitization mechanisms to dynamic mechanical loading.