Uniaxial Cyclic Stretch Research Articles

Changes in the distribution of tyrosine phosphorylated focal proteins caused by uniaxial cyclic stretch in endothelial cells

Changes in the distribution of tyrosine phosphorylated focal proteins caused by uniaxial cyclic stretch in endothelial cells

N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3–75 h) after seeding and continued for 24 h. Stretch stimulation in 3 h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24–75 h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell–cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell–cell adhesion, cardiomyocytes cultured for 72 h were replated by trypsin treatment and exposed to stretch 3 h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.

Intracellular signaling specificity in response to uniaxial vs. multiaxial stretch: implications for mechanotransduction.

Several lines of evidence suggest that muscle cells can distinguish between specific mechanical stimuli. To test this concept, we subjected C(2)C(12) myotubes to cyclic uniaxial or multiaxial stretch. Both types of stretch induced an increase in extracellular signal-regulated kinase (ERK) and protein kinase B (PKB/Akt) phosphorylation, but only multiaxial stretch induced ribosomal S6 kinase (p70(S6k)) phosphorylation. Further results demonstrated that the signaling events specific to multiaxial stretch (p70(S6k) phosphorylation) were elicited by forces delivered through the elastic culture membrane and were not due to greater surface area deformations or localized regions of large tensile strain. Experiments performed using medium that was conditioned by multiaxial stretched myotubes indicated that a release of paracrine factors was not sufficient for the induction of signaling to p70(S6k). Furthermore, incubation with gadolinium(III) chloride (500 microM), genistein (250 microM), PD-98059 (250 microM), bisindolylmaleimide I (20 microM), or LY-294002 (100 microM ) did not block the multiaxial stretch-induced signaling to p70(S6k). However, disrupting the actin cytoskeleton with cytochalasin D did block the multiaxial signaling to p70(S6k), with no effect on signaling to PKB/Akt. These results demonstrate that specific types of mechanical stretch activate distinct signaling pathways, and we propose that this occurs through direct mechanosensory-mechanotransduction mechanisms and not through previously defined growth factor/receptor binding pathways.

Uni-axial cyclic stretch induces Cbfa1 expression in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin beta1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin beta1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.

Uniaxial cyclic stretch induces osteogenic differentiation and synthesis of bone morphogenetic proteins of spinal ligament cells derived from patients with ossification of the posterior longitudinal ligaments

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To elucidate this mechanism, we investigated the effects of in vitro sinusoidal cyclic stretch (120% peak to peak, at 1 Hz) on cultured spinal ligament cells derived from OPLL and non-OPLL patients. The mRNA expressions of alkaline phosphatase (ALP), osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, and BMP receptors as well as ALP activity in cell layers and production of BMPs into the conditioned medium were significantly increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. A stretch-activated Ca2+ channel blocker, Gd3+, the voltage-dependent L-type Ca2+ channel blockers diltiazem and nifedipine, and Ca2+-free medium suppressed stretch-induced ALP activity, which suggests a role of Ca2+ influx in the signal transduction of mechanical stress to the osteogenic response of OPLL cells. Our study provides first evidences that mechanical stress plays a key role in the progression of OPLL through the induction of osteogenic differentiation in spinal ligament cells and the promotion of the autocrine/paracrine mechanism of BMPs in this lesion.

Role of prostaglandin I2 in the gene expression induced by mechanical stress in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments, and mechanical stress has been suggested to play an important role in the progression of OPLL. To identify the genes that participate in OPLL, the differential display reverse transcription-polymerase chain reaction (RT-PCR) method was used. A 283-base pair cDNA fragment corresponding to prostaglandin I2 (PGI2) synthase was highly expressed in OPLL cells compared with non-OPLL cells. To examine the effect of mechanical stress on the expression of PGI2 synthase, cells were subjected to uniaxial cyclic stretch (0.5 Hz, 20% stretch), and PGI2 synthase mRNA expression was assessed by quantitative RT-PCR. Cyclic stretch induced an increase in PGI2 synthase in OPLL cells in a time-dependent manner, whereas no change was observed in non-OPLL cells. Cyclic stretch for 9 h also induced a 2.86x increase in PGI2 production. Beraprost (a stable PGI2 analog) and dibutyryl cAMP (a membrane-permeable cAMP analog) increased the mRNA expression of alkaline phosphatase (ALP) as a marker for osteogenic differentiation up to 240 and 200%, respectively, in OPLL cells, whereas no change was observed in non-OPLL cells. The increases in ALP mRNA induced by beraprost and cyclic stretch were both inhibited by SQ22536, a potent adenylate cyclase inhibitor. These data suggest that the increase in PGI2 synthase induced by mechanical stress plays a key role in the progression of OPLL, at least in part through the induction of osteogenic differentiation in spinal ligament cells via the PGI2/cAMP system.

Calcium regulates the PI3K-Akt pathway in stretched osteoblasts

Mechanical loading plays a vital role in maintaining bone architecture. The process by which osteoblasts convert mechanical signals into biochemical responses leading to bone remodeling is not fully understood. The earliest cellular response detected in mechanically stimulated osteoblasts is an increase in intracellular calcium concentration ([Ca 2+] i). In this study, we used the clonal mouse osteoblast cell line MC3T3-E1 to show that uniaxial cyclic stretch induces: (1) an immediate increase in [Ca 2+] i, and (2) the phosphorylation of critical osteoblast proteins that are implicated in cell proliferation, gene regulation, and cell survival. Our data suggest that cyclic stretch activates the phosphoinositide 3-kinase (PI3K) pathway including: PI3K, Akt, FKHR, and AFX. Moreover, cyclic stretch also causes the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase. Attenuation in the level of phosphorylation of these proteins was observed by stretching cells in Ca 2+-free medium, using intra- (BAPTA-AM) and extracellular (BAPTA) calcium chelators, or gadolinium, suggesting that influx of extracellular calcium plays a significant role in the early response of osteoblasts to mechanical stimuli.

Molecular events caused by cyclic stretch in spinal ligament cells from patients with ossification of the posterior longitudinal ligament

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Although mechanical stress is thought to be an important factor in the progression of OPLL, the mechanism by which mechanical stress promotes the ossification of ligaments in OPLL is still unknown. Therefore, we investigated osteogenic responses induced by mechanical stress in OPLL cells and pathways required for transmitting mechanical signals in these processes. Cells obtained from spinal ligament tissues of OPLL and non-OPLL patients (NOP) were subjected to uni-axial cyclic stretch. Messenger RNA expressions of alkaline phosphatase (ALP) and osteopontin, bone morphogenetic protein-2 (BMP-2), and ALP activity in OPLL cells were significantly increased by cyclic stretch, whereas no change was observed in non-OPLL cells. Ca 2+ channel blockers suppressed stretch-induced ALP activity to resting state level. Furthermore, conditioned medium of OPLL cell cultures subjected to cyclic stretch increased the ALP activity of other OPLL cells. These results suggested that mechanical stress plays a key role in the progression of OPLL through the induction of osteogenic differentiation in ligament cells, Ca 2+ mobilization and promotion of the autocrine/paracrine mechanism of BMP-2 in this lesion.

Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor kappaB in human fibroblast cells.

The effect of uni-axial cyclic mechanical stretch on the activation of the transcription factor nuclear factor kappaB (NF-kappaB) was investigated in a human fibroblast cell line (TIG-1). In response to uni-axial cyclic stretch, NF-kappaB was found to be translocated into the nucleus. The NF-kappaB was first detectable 2 min after the onset of stretch and then peaked at 4 min and returned to the basal level within 10 min. To investigate whether NF-kappaB is activated following the translocation into the nucleus, we measured the luciferase activity in the cells transfected with pNF-kappaB-luciferase. The activity of luciferase increased 4 min after the initiation of cyclic stretch, peaked at 15 min (6.4-fold increase), and decreased gradually. We examined the involvement of the stretch-activated (SA) channel in the stretch-induced NF-kappaB activation. The application of Gd3+, a blocker of the SA channel, or the removal of extracellular Ca2+ inhibited both the translocation into the nucleus and the activation of NF-kB, which suggests that NF-kappaB is activated by uni-axial cyclic stretch via SA channel activation in human lung fibroblasts.

Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells.

Mechanical stretch affects the healing and remodeling process of the anterior cruciate ligament (ACL) after surgery in important ways. In this study, the effects of mechanical stress on gene expression of type I and III collagen by cultured human ACL cells and roles of transforming growth factor (TGF)-beta1 in the regulation of mechanical strain-induced gene expression were investigated. Uniaxial cyclic stretch was applied on ACL cells at 10 cycles/min with 10% length stretch for 24 h. mRNA expression of the type I and type III collagen was increased by the cyclic stretch. TGF-beta1 protein in the cell culture supernatant was also increased by the stretch. In the presence of anti-TGF-beta1 antibody, stretch-induced increase in type I and type III mRNA expression was markedly ablated. The results suggest that the stretch-induced mRNA expression of the type I and type III collagen is mediated via an autocrine mechanism of TGF-beta1 released from ligament cells.

Uniaxial Cyclic Stretch Induces Focal Adhesion Kinase (FAK) Tyrosine Phosphorylation Followed by Mitogen-Activated Protein Kinase (MAPK) Activation

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 ± 0.51, n = 3), and 20 min (2.98 ± 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd3+ or extracellular Ca2+ removal which may inhibit intracellular Ca2+ increase caused by the activation of cation selective (Ca2+-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation.

Stretch-induced morphological changes of human endothelial cells depend on the intracellular level of Ca 2+ rather than of cAMP

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca 2+] i. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs. Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca 2+] i resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca 2+] i and resulted in cell shape changes. On the other hand, 2′5′-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca 2+] i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca 2+] i rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca 2+ rather than cAMP.

1415 単軸繰返し負荷下での培養血管内皮細胞ストレスファイバーの配向に関する理論的予測

1415 単軸繰返し負荷下での培養血管内皮細胞ストレスファイバーの配向に関する理論的予測

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts.

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

Uni-axial cyclic stretch induces c-src activation and translocation in human endothelial cells via SA channel activation

The kinase activity of c-src increased and peaked at 15 min after an application of uni-axial cyclic stretch in HUVECs followed by a translocation of c-src to Triton-insoluble fraction. Suppression of c-src by an antisense S-oligodeoxynucleotide inhibited the stretch-induced tyrosine phosphorylation and morphological changes. The stretch-induced increase in c-src activity was inhibited by FK506, a specific inhibitor for calcineurin, by Gd 3+, a blocker for stretch activated channels, and by the extracellular Ca 2+ depletion suggesting the involvement of SA channels. These results strongly suggest c-src plays an important role in the downstream of SA channel activation followed by the morphological changes.

Pp125FAK is required for stretch dependent morphological response of endothelial cells.

In this study, critical signaling pathway required for the stretch induced morphological changes of human umbilical endothelial cells (HUVECs) was investigated. Uniaxial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual morphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the stretch axis. We found that protein tyrosine phosphorylation of cellular proteins increased and peaked at 20 min in response to cyclic stretch. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ blocked the tyrosine phosphorylation of the proteins, suggesting that stretch-activated (SA) ion channels regulated stretch specific tyrosine phosphorylation. The major phosphorylated proteins had molecular masses of approximately 120-135 kDa, and 70 kDa. Immunoprecipitation experiments revealed that paxillin, focal adhesion kinase (pp125FAK) and pp130CAS were included in the 70 kDa and 120-135 kDa bands, respectively. The morphological change was inhibited by herbimycin A and genistein, inhibitors of tyrosine kinases, suggesting that tyrosine phosphorylation was required for the morphological change. In addition, the kinase activation of pp125FAK was observed in response to cyclic stretch. Moreover, suppression of pp125FAK expression by the antisense phosphorothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibition of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of tyrosine kinase(s) by an increase in intracellular Ca2+ and pp125FAK play a critical role in the unique morphological change specifically observed in endothelial cells subjected to uni-axial cyclic stretch.

Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch.

The present work was designed to elucidate the involvement of Ca(2+)-permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicone membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and > 90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+]i) transiently increased in response to uniaxial stretch, and the basal [Ca2+]i gradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.

Up-regulation of COX2 expression by uni-axial cyclic stretch in human lung fibroblast cells.

The effect of uni-axial cyclic mechanical stretch on the expression of cyclooxygenases (COX) was investigated in a human lung fibroblast cell line (TIG-1). In response to uni-axial cyclic stretch, the level of COX2 mRNA significantly increased and peaked at 3 h (9.09 +/- 3.82-fold, mean +/- standard error, n = 6, compared with that at 1 h). The level of the expression of COX2 protein peaked at 6 h, whereas the level of COX1 protein was not significantly changed. The involvement of stretch-activated (SA) channel was investigated in the stretch-induced COX2 production. The application of Gd3-, a blocker for SA channel, or the removal of extracellular Ca2+ inhibited the production of COX2 mRNA without any effect on the production of COX1 or GAPDH mRNA. These data strongly suggest that COX2 expression is up-regulated by uni-axial cyclic stretch via the activation of SA channel in human lung fibroblasts.

Up-regulation of Integrin β3Expression by Cyclic Stretch in Human Umbilical Endothelial Cells

The effect of uni-axial cyclic mechanical stretch on the expression of the adhesion protein integrin was investigated. Human umbilical endothelial cells (HUVECs) cultured on fibronectin coated silicon membranes were subjected to uni-axial cyclic stretch. The level of expression of integrin β3mRNA was found to be increased and peaked at 4 hours in response to cyclic stretch using a semiquantitative RT-PCR method. The increased level of the integrin mRNA from stretched HUVECs remained higher than that from non-stretched controls. The amount of integrin β3also increased and peaked at 12 hr. Immuno-fluorescent microscopy revealed that the amount of integrin β3adhesions increased in stretched HUVECs compared with that in non-stretched HUVECs. These results suggest that uni-axial cyclic stretch up-regulates the expression of integrin β3. This increase in integrin β3may enhance the adhesiveness to the substratum and contribute to the protection of HUVECs against being peeled off from the vessel wall.