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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
NASA NTRS2004

Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

Meyers, Valerie E.↗
DOE OSTI2021

The PI3K/mTOR inhibitor Gedatolisib eliminates dormant breast cancer cells in organotypic culture, but fails to prevent metastasis in preclinical settings

Dormant, disseminated tumor cells (DTCs) are thought to be the source of breast cancer metastases several years or even decades after initial treatment. To date, a selective therapy that leads to their elimination has not been discovered. While dormant DTCs resist chemotherapy, evidence suggests that this resistance is driven not by their lack of proliferation, but by their engagement of the surrounding microenvironment, via integrin-β1-mediated interactions. Because integrin-β1-targeted agents have not been translated readily to the clinic, signaling nodes downstream of integrin-β1 could serve as attractive therapeutic targets in order to sensitize dormant DTCs to therapy. By probing a number of kinases downstream of integrin-β1, we determined that PI3K inhibition with either a tool compounds or a compound (PF-05212384; aka Gedatolisib) in clinical trials robustly sensitizes quiescent breast tumor cells seeded in organotypic bone marrow cultures to chemotherapy. These results motivated the preclinical study of whether Gedatolisib—with or without genotoxic therapy—would reduce DTC burden and prevent metastases. Despite promising results in organotypic culture, Gedatolisib failed to reduce DTC burden or delay, reduce or prevent metastasis in murine models of either triple-negative or estrogen receptor-positive breast cancer dissemination and metastasis. This result held true whether analyzing Gedatolisib on its own (vs. vehicle-treated animals) or in combination with dose-dense doxorubicin and cyclophosphamide (vs. animals treated only with dose-dense chemotherapies). These data suggest that PI3K is not the node downstream of integrin-β1 that confers chemotherapeutic resistance to DTCs. More broadly, they cast doubt on the strategy to target PI3K in order to eliminate DTCs and prevent breast cancer metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Vinculin is required for neuronal mechanosensing but not for axon outgrowth

Integrin receptors are transmembrane proteins that bind to the extracellular matrix (ECM). In most animal cell types integrins cluster together with adaptor proteins at focal adhesions that sense and respond to external mechanical signals. In the central nervous system (CNS), ECM proteins are sparsely distributed, the tissue is comparatively soft and neurons do not form focal adhesions. Thus, how neurons sense tissue stiffness is currently poorly understood. Here, we found that integrins and the integrin-associated proteins talin and focal adhesion kinase (FAK) are required for the outgrowth of neuronal processes. Vinculin, however, whilst not required for neurite outgrowth was a key regulator of integrin-mediated mechanosensing of neurons. During growth, growth cones of axons of CNS derived cells exerted dynamic stresses of around 10–12 Pa on their environment, and axons grew significantly longer on soft (0.4 kPa) compared to stiff (8 kPa) substrates. Depletion of vinculin blocked this ability of growth cones to distinguish between soft and stiff substrates. These data suggest that vinculin in neurons acts as a key mechanosensor, involved in the regulation of growth cone motility.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Kindlin-3 mutation in mesenchymal stem cells results in enhanced chondrogenesis

Identifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation, and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation, similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers, including SOX9, under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated β{sub 3} integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2001

Modulation of lens cell adhesion molecules by particle beams

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

NASA Discipline Radiation Health↗
NASA NTRS1996

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

NASA Discipline Number 40-20↗
DOE OSTI2021

Tunicamycin-induced endoplasmic reticulum stress inhibits chemoresistance of FaDu hypopharyngeal carcinoma cells in 3D collagen I cultures and in vivo

Highlights: • 3D collagen I culture induces chemoresistance of FaDu hypopharyngeal carcinoma cells. • Tunicamycin-induced ER stress inhibits chemoresistance in collagen I cultures. • Tunicamycin-induced ER stress potentiates anticancer efficacy in vivo. • Tunicamycin-induced ER stress inhibits TGF-β1 and modifies integrin β1 glycosylation. The prognosis in patients with advanced head and neck squamous cell carcinoma (HNSCC) is widely affected by the resistance to chemotherapy. As a culture scaffold, collagen I was showed to promote CSC (cancer stem cell) properties of cancer cells which could be used as in vitro models to study the chemoresistance in HNSCC. Endoplasmic reticulum (ER) stress is a cellular stress condition which could affect tumor progression and promote the anti-tumor effects of certain drugs. However, the impact of ER stress on collagen I induced CSC properties and chemoresistance of HNSCC cells has not been addressed. In this study we investigated the effects of tunicamycin (TM) induced ER stress on the stemness and sensitivity to chemotherapeutic drugs of FaDu hypopharyngeal carcinoma cells in 3D (three-dimensional) collagen I cultures and mouse xenograft models. Our study revealed that Collagen I scaffold promoted CSC properties and increased G1 population of FaDu cells in 3D cultures, accompanied by maturation of integrin β1 and enhanced activated TGF-β1 concentration. Compared to 2D (two-dimensional) cultured cells, cells in 3D Collagen I scaffold exhibited significantly increased resistance to chemotherapeutic drugs of cisplatin and paclitaxel. Further analysis revealed that TM induced ER stress preferentially attenuated chemoresistance of FaDu cells in 3D collagen I, downregulated their CSC properties and TGF-β1 concentration and resulted in deglycosylation of integrin β1. TM was further evaluated in the mouse xenograft models and showed significant tumor growth inhibition in combination with paclitaxel than either TM or paclitaxel alone. Taken together, Our findings suggest that TM-induced ER stress potentiates anticancer efficacy of FaDu cells in 3D cultures and in vivo, and highlight implications for targeting chemotherapy-resistant cancer stem cells under ER stress conditions.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1994

Dermal Aged and Fetal Fibroblasts Realign in Response to Mechanical Strain

Integrins specifically recognize and bind extracellular matrix components, providing physical anchor points and functional setpoints. Focal adhesion complexes, containing integrin and cytoskeletal proteins, are potential mechanoreceptors, poised to distribute applied forces through the cytoskeleton. Pursuing the hypothesis that cells both perceive and respond to external force, we applied a stretch/relaxation regimen to normal human fetal and aged dermal fibroblast monolayers cultured on flexible membranes. The frequency and magnitude of the applied force is precisely controlled by the Flexercell Unit(Trademark). A protocol of stretch (20% elongation of the monolayer) at a frequency of 6 cycles/min caused a progressive change from a randomly distributed pattern of cells to a symmetric, radial distribution with cells aligned parallel to the applied force. We have coined the term 'orienteering' as the process of active alignment of cells in response to applied force. Cytochalasin D was added in graded doses to investigate the role of the actin cytoskeleton in force perception and transmission. A clear dose response was found; at high concentrations orienteering was abolished; and the drug's impact was reversible. The two cell strains used were similar in their alignment behavior and in their responses to cytochalasin D. Orienteering was influenced by cell density, and the cell strains studied differed in this respect. Fetal cells, unlike their aged counterparts, failed to orient at high cell density. In both cell strains, mid-density cultures aligned rapidly and sparse cultures lagged. These results indicate that both cell-cell adhesion and cytoskeleton integrity are critical in mediating the orienteering response. Differences between these two cell strains may relate to their expression of extracellular matrix molecules (fibronectin, collagen type 1) integrins and their relative binding affinities.

Sawyer, Christine↗
NASA NTRS2004

Cellular Mechanisms Underlying Bone-Forming Cell Proliferative Response to Hypergravity

Life on Earth has evolved under the continuous influence of gravity (1-g). As humans explore and develop space, however, we must learn to adapt to an environment with little or no gravity. Studies indicate that lack of weightbearing for vertebrates occurring with immobilization, paralysis, or in a microgravity environment may cause muscle and bone atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) (consisting of molecules such as collagen, fibronectin, and laminin) in mechanosensitive tissues. We test for the presence of gravity-sensitive pathways in bone-forming cells (osteoblasts) using hypergravity applied by a cell culture centrifuge. Stimulation of 50 times gravity (50-g) increased proliferation in primary rat osteoblasts for cells grown on collagen Type I and fibronectin, but not on laminin or uncoated surfaces. Survival was also enhanced during hypergravity stimulation by the presence of ECM. Bromodeoxyuridine incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. Reverse transcription-polymerase chain reaction was used to test for all possible integrins. Our combined results indicate that beta1 and/or beta3 integrin subunits may be involved. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signalling pathways which are sensitive to g-level. Further research to define the mechanisms involved will provide direction so that we may better adapt and counteract bone atrophy caused by the lack of weightbearing.

Vercoutere, W.↗
NASA NTRS2002

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

NASA Discipline Number 40-20↗
DOE OSTI2021

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin β3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Scavenger receptor MARCO contributes to macrophage phagocytosis and clearance of tumor cells

Highlights: • The MARCO receptor on macrophages was positively correlated with the ability to phagocyte tumor cells. • Macrophage MARCO affects cytoskeleton rearrangement and pseudopodia formation through the SYK-PI3K-Rac1 pathway. • Integrin is the ligand of MARCO on the tumor cells. Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the β5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structure-guided design of pure orthosteric inhibitors of αIIbβ3 that prevent thrombosis but preserve hemostasis

A prevailing dogma is that inhibition of vascular thrombosis by antagonizing platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis, thus causing serious bleeding and increased morbidity and mortality. It is speculated that these adverse outcomes result from drug-induced activating conformational changes in αIIbβ3 but direct proof is lacking. Here, we report the structure-guided design of peptide Hr10 and a modified form of the partial agonist drug tirofiban that act as “pure” antagonists of αIIbβ3, i.e., they no longer induce the conformational changes in αIIbβ3. Both agents inhibit human platelet aggregation but preserve clot retraction. Hr10 and modified tirofiban are as effective as partial agonist drugs in inhibiting vascular thrombosis in humanized mice, but neither causes serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbβ3 may thus provide safer alternatives for human therapy, and valuable tools to probe structure–activity relationships in integrins.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1996

The Cytoskeleton: Mechanical, Physical, and Biological Interactions

This workshop, entitled "The Cytoskeleton: Mechanical, Physical, and Biological Interactions," was sponsored by the Center for Advanced Studies in the Space Life Sciences at the Marine Biological Laboratory. This Center was established through a cooperative agreement between the MBL and the Life Sciences Division of the National Aeronautics and Space Administration. To achieve these goals, the Center sponsors a series of workshops on various topics in the life sciences. Elements of the cytoskeleton have been implicated in the effects of gravity on the growth of plants fungi. An intriguing finding in this regard is the report indicating that an integrin-like protein may be the gravireceptor in the internodal cells of Chara. Involvement of the cytoskeleton in cellular graviperception of the basidiomycete Flammulina velutipes has also been reported. Although the responses of mammalian cells to gravity are not well documented, it has been proposed that integrins can act as mechanochemical transducers in mammalian cells. Little is known about the integrated mechanical and physical properties of cytoplasm, this workshop would be the best place to begin developing interdisciplinary approaches to the effects of mechanical stresses on cells and their most likely responsive cytoplasmic elements- the fibrous proteins comprising the cytoskeleton.

Source record↗
NASA NTRS2000

Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

Globus. Ruth K.↗
NASA NTRS2003

Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense hypergravity and activate distinct matrix-dependent FAK signaling pathways that can enhance proliferation. Our results also imply that brief exposures to hypergravity accelerate cell adhesion and spreading processes via the focal adhesion-signaling axis. These results support the role of the ECM/integrin-signaling axis in osteoblast response to hypergravity loading.

Parra, M.↗
NASA NTRS2003

The impact of skeletal unloading on bone formation

Skeletal unloading leads to decreased bone formation and decreased bone mass. Bone resorption is uncoupled from bone formation, contributing to the bone loss. During space flight bone is lost principally from the bones most loaded in the 1 g environment. Determining the mechanism(s) by which loading of bone is sensed and translated into a signal(s) controlling bone formation remains the holy grail in this field. It seems likely that matrix/cell interactions will underlie much of the mechanocoupling. Integrins are a prime mediator of such interactions. The role for systemic hormones such as PTH, GH and 1,25(OH)2D compared to locally produced factors such as IGF-I, PTHrP, BMPs and TGF beta in modulating the cellular response to load remains unclear. Our studies demonstrate that skeletal unloading leads to resistance to the anabolic actions of IGF-I on bone as a result of failure of IGF-I to activate its own signaling pathways. This is associated with a reduction in integrin expression, suggesting crosstalk between these two pathways. As the mechanism(s) by which bone responds to changes in mechanical load with changes in bone formation is further elucidated, applications of this knowledge to other etiologies of osteoporosis are likely to develop. Skeletal unloading provides a perturbation in bone mineral homeostasis that can be used to understand the mechanisms by which bone mineral homeostasis is maintained, and that such understanding will lead to effective treatment for disuse osteoporosis in addition to preventive measures for the bone loss that accompanies space travel.

NASA Discipline Musculoskeletal↗