The effect of step-wise increased stretching on rat calvarial osteoblast collagen production

Mechanical forces regulate the function of bone cells. In this paper, the effects of cyclic stretching on osteoblasts derived from rat calvaria were studied at a magnitude occurring in physiological loaded bone tissue. A four-point bending apparatus was used to apply cyclic stretching on osteoblasts. Stretching at 500 microepsilon for 2-24 h resulted in an increase in matrix synthesis(P<0.01). In contrast, the cyclic stretching at 1000 and 1500 microepsilon for 2-24 h inhibited osteoblast collagen production (P<0.01). We also described our new loading method to increase strain magnitude step-by-step. The strain magnitude increased by 500 microepsilon increments from 500 to 1500 microepsilon every 2 or 12 h, respectively. Results showed that osteoblasts could absorb large amount of proline for collagen synthesis when stretched at 500 microepsilon. However, not all the absorbed proline was used to synthesize collagen. Some of it was stored in cells. When the suitable signal (500 microepsilon) was changed to an inhibiting signal (1000 microepsilon), cells responded to it accordingly and released proline to medium. These results demonstrate that the response of osteoblasts is dependent on the magnitude of the strain applied and cells can adjust their bio-chemical response to adapt to the changing environmental stimulation.     

Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants

Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase. These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.     

Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles

Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.     

Adenosine 3'',5''-cyclic monophosphate-dependent release of prolactin from GH3 pituitary tumour cells. A quantitative analysis.

The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Analysis of the mechanical behavior of chondrocytes in unconfined compression tests for cyclic loading

Experimental evidence indicates that the biosynthetic activity of chondrocytes is associated with the mechanical environment. For example, excessive, repetitive loading has been found to induce cell death, morphological and cellular damage, as seen in degenerative joint disease, while cyclic, physiological-like loading has been found to trigger a partial recovery of morphological and ultrastructural aspects in osteoarthritic human articular chondrocytes. Mechanical stimuli are believed to influence the biosynthetic activity via the deformation of cells. However, the in situ deformation of chondrocytes for cyclic loading conditions has not been investigated experimentally or theoretically. The purpose of the present study was to simulate the mechanical response of chondrocytes to cyclic loading in unconfined compression tests using a finite element model. The material properties of chondrocytes and extracellular matrix were considered to be biphasic. The time-histories of the shape and volume variations of chondrocytes at three locations (i.e., surface, center, and bottom) within the cartilage were predicted for static and cyclic loading conditions at two frequencies (0.02 and 0.1 Hz) and two amplitudes (0.1 and 0.2 MPa). Our results show that cells at different depths within the cartilage deform differently during cyclic loading, and that the depth dependence of cell deformation is influenced by the amplitude of the cyclic loading. Cell deformations under cyclic loading of 0.02 Hz were found to be similar to those at 0.1 Hz. We conclude from the simulation results that, in homogeneous cartilage layers, cell deformations are location-dependent, and further are affected by load magnitude. In physiological conditions, the mechanical environment of cells are even more complex due to the anisotropy, depth-dependent inhomogeneity, and tension-compression non-linearity of the cartilage matrix. Therefore, it is feasible to speculate that biosynthetic responses of chondrocytes to cyclic loading depend on cell location and load magnitude.     

Aging enhances a mechanically-induced reduction in tendon strength by an active process involving matrix metalloproteinase activity

Age-associated and degenerative loss of functional integrity in soft tissues develops from effects of cumulative and subtle changes in their extracellular matrix (ECM). The highly ordered tendon ECM provides the tissue with its tensile strength during loading. As age and exercise collide in the high incidence of tendinopathies, we hypothesized that aged tendons fail due to cumulative damage resulting from a combination of diminished matrix repair and fragmentation of ECM proteins induced by prolonged cyclical loading, and that this is an active cell-mediated process. We developed an equine tendon explant model to examine the effect of age on the influence of prolonged cyclical loading at physiologically relevant strain rates (5% strain, 1 Hz for 24 h) on tissue mechanical properties, loss of ECM protein and matrix metalloproteinase (MMP) expression. We show significantly diminished mechanical strength of cyclically loaded tissue compared to controls (39.7 +/- 12%, P 相似文献   

Dependence of alignment direction on magnitude of strain in esophageal smooth muscle cells

The response of cells in vitro to mechanical forces has been the subject of much research using devices to exert controlled mechanical stimulation on cultured cells or isolated tissue. In this study, esophageal smooth muscle cells were seeded on flexible polyurethane membranes to form a confluent cell layer. The cells were then subjected to uniform cyclic stretch of varying magnitudes at a frequency of approximately five cycles per minute in a custom made mechatronic bioreactor, providing similar strains experienced in the in vivo mechanical environment of the esophagus. The results show that the orientation response is dependent on the magnitude of cyclic stretch applied. Smooth muscle cells showed parallel alignment to the force direction at low cyclic strains (2%) compared to the hill‐valley morphology of static controls. At higher strains (5% and 10% magnitude), the cells exhibited a consistent alignment perpendicular to the strain. To our knowledge, this is the first time that the alignment direction's dependence on strain magnitude has been demonstrated. MTS analysis indicated that cell metabolism was reduced when mechanical strain was applied, and proliferation was inhibited by mechanical strain. Protein expression indicates a decrease in smooth muscle α‐actin, indicative of changes in cell phenotype, an increase in vimentin, which is associated with increased cell motility, and an increase in desmin, indicating differentiation in stimulated cells. Biotechnol. Bioeng. 2009;102: 1703–1711. © 2008 Wiley Periodicals, Inc.     

The functional response of U937 macrophage-like cells is modulated by extracellular matrix proteins and mechanical strain.

Extracellular matrix proteins (ECMs) play a significant role in the transfer of mechanical strain to monocyte-derived macrophages (MDMs) affecting morphological changes in a foreign body reaction. This study investigated how the functional responses of U937 macrophage-like cells differed when subjected to 2 dynamic strain types (nonuniform biaxial or uniform uniaxial strain) while cultured on siloxane membranes coated with either collagen type I or RGD peptide repeats (ProNectin). Biaxial strain caused an increase in intracellular esterase and acid phosphatase (AP) activities, as well as monocyte-specific esterase (MSE) protein levels in cells that were seeded on either uncoated surfaces (shown previously) or collagen, but not ProNectin. Released AP activity, but not released esterase activity, was increased on all surfaces. Biaxial strain increased IL-6, but not IL-8 on all surfaces. When cells were subjected to uniaxial strain, intracellular esterase increased on coated surfaces only, whereas intracellular AP activity was unaffected. Both esterase and AP released activities increased on all surfaces. Uniaxial strain increased the release of IL-6 on all surfaces, but IL-8 on coated surfaces only. This study demonstrated for the first time that ECM proteins could specifically modulate cellular responses to different types of strain. Using this approach with an in vitro cell system may help to unravel the complex function of MDMs in the foreign-body reaction.     

Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation

Accumulating data show that oxygen tension can have an important effect on cell function and fate. We used the human pre-osteoblastic cell line SV-HFO, which forms a mineralizing extracellular matrix, to study the effect of low oxygen tension (2%) on osteoblast differentiation and mineralization. Mineralization was significantly reduced by 60-70% under 2% oxygen, which was paralleled by lower intracellular levels of reactive oxygen species (ROS) and apoptosis. Following this reduction in ROS the cells switched to a lower level of protection by down-regulating their antioxidant enzyme expression. The downside of this is that it left the cells more vulnerable to a subsequent oxidative challenge. Total collagen content was reduced in the 2% oxygen cultures and expression of matrix genes and matrix-metabolizing enzymes was significantly affected. Alkaline phosphatase activity and RNA expression as well as RUNX2 expression were significantly reduced under 2% oxygen. Time phase studies showed that high oxygen in the first phase of osteoblast differentiation and prior to mineralization is crucial for optimal differentiation and mineralization. Switching to 2% or 20% oxygen only during mineralization phase did not change the eventual level of mineralization. In conclusion, this study shows the significance of oxygen tension for proper osteoblast differentiation, extra cellular matrix (ECM) formation, and eventual mineralization. We demonstrated that the major impact of oxygen tension is in the early phase of osteoblast differentiation. Low oxygen in this phase leaves the cells in a premature differentiation state that cannot provide the correct signals for matrix maturation and mineralization.     

Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes

ATP is actively released into the extracellular environment from a variety of cell types in response to mechanical stimuli. This is particularly true in bone where mechanically induced ATP release leads to immediate early gene activation to regulate bone remodelling; however there is no consensus as to which mechanical stimuli stimulate osteoblasts the most. To elucidate which specific type(s) of mechanical stimuli induce ATP release and gene activation in human osteoblasts, we performed an array of experiments using different mechanical stimuli applied to both monolayer and 3D cultures of the same osteoblast cell type, SaOS-2. ATP release from osteoblasts cultured in monolayer significantly increased in response to turbulent fluid flow, laminar fluid flow and substrate strain. No significant change in ATP release could be detected in 3D osteoblast cultures in response to cyclic or static compressive loading of osteoblast-seeded scaffolds, whilst turbulent fluid flow increased ATP release from 3D cultures of osteoblasts to a greater degree than observed in monolayer cultures. Cox-2 expression quantified using real time PCR was significantly lower in cells subjected to turbulent fluid flow whereas c-fos expression was significantly higher in cells subjected to strain. Load-induced signalling via c-fos was further investigated using a SaOS-2 c-fos luciferase reporter cell line and increased in response to substrate strain and turbulent fluid flow in both monolayer and 3D, with no significant change in response to laminar fluid flow or 3D compressive loading. The results of this study demonstrate for the first time strain-induced ATP release from osteoblasts and that turbulent fluid flow in 3D up regulates the signals required for bone remodelling.     

Regulation of ovarian steroidogenesis. The disparity between 125I-labelled choriogonadotropin binding cyclic adenosine 3',5'-monophosphate formation and progesterone synthesis in the rat ovary.

The binding of 125I-labelled human choriogonadotropin, formation of cyclic adenosine 3',5'-monophosphate (cyclic AMP), and synthesis of progesterone were examined in ovarian cells from immature rats. Collagenase dispersed ovarian cells were found to respond specifically to lutropin-like activity. The equilibrium dissociation constant (Kd) for the binding of 125I-for the binding of 125I-labelled choriogonadotropin was 1.7-10(-10) M. Progesterone synthesis was increased at least 40-fold and cyclic AMP formation 10-fold in response to maximum hormonal stimulation. The concentration of choriogonadotropin which stimulated progesterone synthesis maximally in Eagle's minimum essential medium--0.1% gelatin (2 ng/ml), resulted in minimal (less than 30% of maximum) increases in cyclic AMP accumulation and hormone binding. Similarly, binding of choriogonadotropin was not saturated at a hormone concentration (50 ng/ml) that stimulated maximal cyclic AMP formation. These results are consistent with the existence of receptor reserve in the ovarian cell. A marked shift in the dose vs. response relationship for progesterone synthesis occurred when fetal calf serum was used to supplement Eagle's minimum essential medium, however. Under these experimental conditions, progesterone synthesis reached a maximum at a hormone concentration of the same order of magnitude as did cyclic AMP formation. It is concluded that the degree of spare receptor effect observed may depend not only on an absolute amount of excess receptor, but also on the readiness of the system to respond in a given fashion.     

Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I,osteopontin, and osteocalcin gene expression in chicken osteoblasts

Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)₂ D₃ showed a 2–10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for α₁(I) and α₂(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)₂ D₃ at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)₂ D₃ treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)₂ D₃ in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.     

Effect of thyroliberin on the concentration of adenosine 3'':5''-phosphate and on the activity of adenosine 3'':5''-phosphate-dependent protein kinase in prolactin-producing cells in culture.

1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.     

Synergistic action of static stretching and BMP-2 stimulation in the osteoblast differentiation of C2C12 myoblasts

Static stretching is a major type of mechanical stimuli utilized during distraction osteogenesis (DO), a general surgical method for the lengthening of bone. The molecular signals that drive the regenerative process in DO include a variety of cytokines. Among these, bone morphogenic protein (BMP, -2 and -4) has been reported to exhibit strongly enhanced expression following the application of mechanical strain during the distraction phase. We hypothesize that mechanical stretching enhances osteoblast differentiation in DO by means of interaction with BMP-2 induced cytokine stimulation. C2C12 pluripotential myoblasts were exposed to stretching load and the resulting cell proliferation and osteoblast differentiation were then examined. The application of static stretching force resulted in significant cell proliferation at day 3, although with variable intensity according to the magnitude of stretching. A combined treatment of stretching load with BMP-2 stimulation significantly increased alkaline phosphatase (ALP) activity and up-regulated the gene expression of osteogenic markers (ALP, type I collagen, osteopontin, osteocalcin, cbfa1, osterix and dlx5). Results obtained with the combined treatment yielded more activity than just the BMP-2 treatment or stretching alone. These results reveal that specific levels of static stretching force increase cell proliferation and effectively stimulate the osteoblast differentiation of C2C12 cells in conjunction with BMP-2 stimulation, thus indicating a synergistic interaction between mechanical strain and cytokine signaling.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Strain and strain rate activation of G proteins in human endothelial cells

The endothelium is known to sense and respond to its physical environment, but the underlying mechanisms and early events of endothelial cell mechanotransduction are not well understood. The present study measured G protein activation by mechanical strain in human umbilical vein endothelial cells (HUVEC) directly by photoincorporation of a hydrolysis resistant, radiolabeled GTP analog. Ten percent uniaxial strain at a strain rate of 20% s(-1) over 1min activated a 38kDa Galpha subunit 167+/-17% relative to controls, while 2% cyclic strain failed to significantly activate the protein (117+/-19%). A single cycle of 10% strain at 20% s(-1) strain rate activated the Galpha subunit 152+/-25%, while activation at the same strain but lower strain rate (0.3% s(-1)) was not significantly different from controls (116+/-12%). Western blot analysis identified the 38kDa protein as Galpha(q/11). These results demonstrate the rapid activation of G proteins in HUVEC by cyclic uniaxial strain in a strain- and strain rate-dependent manner.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.     

NO inhibits stretch-induced MAPK activity by cytoskeletal disruption

Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at -27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization.