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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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29 records · Page 2
All sourcesNASA NTRSDOE OSTI
NASA NTRS2000

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

Non-NASA Center↗
NASA NTRS1997

Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain

Mechanical force applied to bone produces two localized mechanical signals on the cell: deformation of the extracellular matrix (substrate strain) and extracellular fluid flow. To study the effects of these stimuli on osteoblasts, MC3T3-E1 cells were grown on type I collagen-coated plastic plates and subjected to four-point bending. This technique produces uniform levels of physiological strain and fluid forces on the cells. Each of these parameters can be varied independently. Osteopontin (OPN) mRNA expression was used to assess the anabolic response of MC3T3-E1 cells. When fluid forces were low, neither strain magnitude nor strain rate was correlated with OPN expression. However, higher-magnitude fluid forces significantly increased OPN message levels independently of the strain magnitude or rate. These data indicate that fluid forces, and not mechanical stretch, influence OPN expression in osteoblasts and suggest that fluid forces induced by extracellular fluid flow within the bone matrix may play an important role in bone formation in response to mechanical loading.

Non-NASA Center↗
NASA NTRS1997

1,25 (OH)2D3 enhances PTH-induced Ca2+ transients in preosteoblasts by activating L-type Ca2+ channels

We previously demonstrated electrophysiologically that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] shifts the activation threshold of L-type Ca2+ channels in osteoblasts toward the resting potential and prolongs mean open time. Presently, we used single-cell Ca2+ imaging to study the combined effects of 1,25(OH)2D3 and parathyroid hormone (PTH) during generation of Ca2+ transients in fura 2-loaded MC3T3-E1 cells. Pretreatment with 1,25(OH)2D3 concentrations, which alone did not produce Ca2+ transients, consistently enhanced Ca2+ responses to PTH. Enhancement was dose dependent over the range of 1 to 10 nM and was blocked by pretreatment with 5 microM nitrendipine during pretreatment. A 1,25(OH)2D3 analog that activates L-type channels and shifts their activation threshold also enhanced PTH responses. In contrast, an analog devoid of membrane Ca2+ effects did not enhance PTH-induced Ca2+ transients. The PTH-induced Ca2+ transient involved activation of a dihydropyridine-insensitive cation channel that was inhibited by Gd3+. Together, these data suggest that 1,25(OH)2D3 increases osteoblast responsiveness to PTH through rapid modification of L-type Ca2+ channel gating properties, whose activation enhances Ca2+ entry through other channels such as the PTH-responsive, Gd(3+)-sensitive cation channel.

Non-NASA Center↗
NASA NTRS2001

Parathyroid hormone enhances fluid shear-induced [Ca2+]i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca2+ channels

Osteoblasts respond to both fluid shear and parathyroid hormone (PTH) with a rapid increase in intracellular calcium concentration ([Ca2+]i). Because both stimuli modulate the kinetics of the mechanosensitive cation channel (MSCC), we postulated PTH would enhance the [Ca2+]i response to fluid shear by increasing the sensitivity of MSCCs. After a 3-minute preflow at 1 dyne/cm2, MC3T3-E1 cells were subjected to various levels of shear and changes in [Ca2+]i were assessed using Fura-2. Pretreatment with 50 nM bovine PTH(1-34) [bPTH(1-34)] significantly enhanced the shear magnitude-dependent increase in [Ca2+]i. Gadolinium (Gd3+), an MSCC blocker, significantly inhibited the mean peak [Ca2+]i response to shear and shear + bPTH(1-34). Nifedipine (Nif), an L-type voltage-sensitive Ca2+ channel (VSCC) blocker, also significantly reduced the [Ca2+]i response to shear + bPTH(1-34), but not to shear alone, suggesting VSCC activation plays an interactive role in the action of these stimuli together. Activation of either the protein kinase C (PKC) or protein kinase A (PKA) pathways with specific agonists indicated that PKC activation did not alter the Ca2+ response to shear, whereas PKA activation significantly increased the [Ca2+]i response to lower magnitudes of shear. bPTH(1-34), which activates both pathways, induced the greatest [Ca2+]i response at each level of shear, suggesting an interaction of these pathways in this response. These data indicate that PTH significantly enhances the [Ca2+]i response to shear primarily via PKA modulation of the MSCC and VSCC.

Non-NASA Center↗
NASA NTRS2002

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

NASA Discipline Neuroscience↗
NASA NTRS1994

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

NASA Discipline Cell Biology↗
NASA NTRS2000

Cell movement is guided by the rigidity of the substrate

Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.

NASA Discipline Cell Biology↗
NASA NTRS1994

Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition.

Non-NASA Center↗
NASA NTRS1993

The identification of a naturally occurring cell surface growth inhibitor related to a previously described bovine sialoglycopeptide

A 66-kDa sialoglycoprotein has been identified as the parental membrane molecule of an earlier described sialoglycopeptide (SGP), an 18-kDa molecule released by protease treatment of intact bovine cerebral cortex cells that was shown to be a potent inhibitor of cellular proliferation. The 66-kDa parental sialoglycoprotein (p-SGP) was purified approximately 2,400-fold, to apparent homogeneity, from bovine cerebral cortex cell membranes by its release during incubation with 3 M NaCl, preparative isoelectric focusing and lectin affinity chromatography. Although a membrane-associated molecule, the p-SGP appeared to be tightly bound to the cell membrane, since it was not released during incubations in the absence of 3 M NaCl. Incubation of the membrane preparations with 3 M urea proved to be too harsh, and the antigenicity required to follow the purification of the p-SGP was abolished. Analyses by SDS-PAGE, under reducing and nonreducing conditions, suggested that the p-SGP membrane component was a single polypeptide without subunit structure. The p-SGP was shown to be structurally related to the SGP fragment by immunoblots with IgG raised to the SGP inhibitor, and functionally related to the SGP by its ability to inhibit Swiss 3T3 proliferation at concentrations strikingly similar to that previous measured with the SGP fragment.

Non-NASA Center↗
NASA NTRS2003

Effects of broad frequency vibration on cultured osteoblasts

Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0-3000 microstrain peak-to-peak followed by 0.33 s resting time, (2) "broad frequency vibration" of low amplitude strain (standard deviation of 300 microstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S + V). The cells were harvested on day 4 or 8. We found that the S + V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S + V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts. Copyright 2002 Elsevier Science Ltd.

Non-NASA Center↗
NASA NTRS1993

Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells

We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

NASA Program Space Biology↗