Modeled Microgravity Research Articles

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 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal-related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

Hypoxia suppresses runx2 independent of modeled microgravity.

Bone loss is a consequence of skeletal unloading as seen in bed rest and space flight. Unloading decreases oxygenation and osteoblast differentiation/function in bone. Previously we demonstrated that simulation of unloading in vitro, by culturing differentiating mouse osteoblasts in a horizontal rotating wall vessel (RWV), results in suppressed expression of runx2, a master transcriptional regulator of osteoblast differentiation. However, the RWV is able to reproduce in a controlled fashion at least two aspects of disuse that are directly linked, model microgravity and hypoxia. Hypoxia in the RWV is indicated by reduced medium oxygen tension and increased expression of GAPDH and VEGF. To uncouple the role of model microgravity from hypoxia in suppressed runx2 expression, we cultured osteoblasts under modeled microgravity (oxygenated, horizontal RWV rotation), hypoxia (vertical RWV rotation), or both conditions (horizontal RWV rotation). The expression, DNA binding activity and promoter activity of runx2, was suppressed under hypoxic but not normoxic modeled microgravity RWV conditions. Consistent with a role for hypoxia in suppression of runx2, direct exposure to hypoxia alone is sufficient to suppress runx2 expression in osteoblasts grown in standard tissue culture plates. Taken together, our findings indicate that hypoxia associated with skeletal unloading could be major suppressor of runx2 expression leading to suppressed osteoblast differentiation and bone formation.

Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes.

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis.

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.

Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity.

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.

Modeled microgravity inhibits apoptosis in peripheral blood lymphocytes.

Microgravity interferes with numerous lymphocyte functions (expression of cell surface molecules, locomotion, polyclonal and antigen-specific activation, and the protein kinase C activity in signal transduction). The latter suggests that gravity may also affect programmed cell death (PCD) in lymphocyte populations. To test this hypothesis, we investigated spontaneous, activation- and radiation-induced PCD in peripheral blood mononuclear cells exposed to modeled microgravity (MMG) using a rotating cell culture system. The results showed significant inhibition of radiation- and activation-induced apoptosis in MMG and provide insights into the potential mechanisms of this phenomenon.

Microgravity as a novel environmental signal affecting Salmonella enterica serovar Typhimurium virulence.

The effects of spaceflight on the infectious disease process have only been studied at the level of the host immune response and indicate a blunting of the immune mechanism in humans and animals. Accordingly, it is necessary to assess potential changes in microbial virulence associated with spaceflight which may impact the probability of in-flight infectious disease. In this study, we investigated the effect of altered gravitational vectors on Salmonella virulence in mice. Salmonella enterica serovar Typhimurium grown under modeled microgravity (MMG) were more virulent and were recovered in higher numbers from the murine spleen and liver following oral infection compared to organisms grown under normal gravity. Furthermore, MMG-grown salmonellae were more resistant to acid stress and macrophage killing and exhibited significant differences in protein synthesis than did normal-gravity-grown cells. Our results indicate that the environment created by simulated microgravity represents a novel environmental regulatory factor of Salmonella virulence.