Small GTPase Ras and Rho expression in rat osteoblasts during spaceflight.

Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24-h treatment with 1alpha,25 dihydroxyvitamin D(3). The quantitative RT-PCR determined the mRNA levels of signaling molecules upstream and downstream Ras. The small GTPase is activated by guanine nucleotide exchange protein (GEF) and deactivated by GTPase-activating protein (GAP). When external stimuli are transduced into intracellular signals, various pathways are recruited: focal adhesion kinase (FAK) is associated with integrin-beta, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C-gamma (PLC-gamma) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC-gamma1 and -gamma2 in the flight cultures were increased 150% and 250% of the ground controls. The SOS mRNA levels in the flight cultures were increased 520% and 320% of the ground controls. Signals via G protein-coupled receptors are transmitted through PLC-beta and Ras GRF (another Ras GEF). Activated Ras then stimulates Raf, mitogen-activated protein kinase (MAPK) cascades. The mRNA levels of Raf, extracellular signal-regulated protein kinase of MAPK family (ERK-1 and -2), and PLC-beta were increased during spaceflight. Rho GAP expression in the flight cultures was increased twofold of the ground controls. Since Rho GAP deactivates Rho, microgravity may suppress Rho signals, regulating actin filament rearrangement. Microgravity signals may involve two pathways (G protein-coupled receptor-mediated pathway and tyrosine phosphorylation-mediated pathway) that activate Ras, Raf, and MAPK cascades in rat osteoblasts.

Platelet-activating factor receptor signals in rat osteoblasts during spaceflight.

The platelet-activating factor (PAF) is a lipid mediator. The G-protein-coupled receptor of PAF (PAF-R) is activated by inflammatory and stressful conditions in numerous cell types. PAF/PAF-R is involved in apoptotic and antiapoptotic processes. We examined microgravity effects on the expression of PAF-R and second messengers in rat osteoblasts. The PAF-R signals are transmitted via arachidonic acid, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase. Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized on board. PAF-R gene expression in flight cultures increased to 2-6-fold higher than in ground controls. Gene expression of the G-protein alpha subunit Galphaq in flight cultures increased to 3-fold and higher than in ground controls. It is known that Galphaq stimulates the effecter PLCbeta, activating PKC. The mRNA levels of PKCdelta and PKCtheta in flight cultures were increased to 2-5-fold higher than in ground controls. The PKCalpha mRNA level in flight cultures was increased to 3-fold higher than in ground controls on the 4th day. Gene expression of catalytic and regulatory subunits of protein kinase A was suppressed in flight cultures. PKCdelta and PKCtheta are novel PKCs that can be target substrates of caspases. The PAF-R gene may act as a mechano-sensitive gene that is involved in the apoptotic and antiapoptotic processes of osteoblasts under microgravity.

Coinduction of GTP cyclohydrolase I and inducible NO synthase in rat osteoblasts during space flight: apoptotic and self-protective response?

The mechanism underlying space flight-induced osteopenia is unknown. In osteoblasts, the inducible nitric oxide (NO) synthase (iNOS) is involved in the early response to mechanical strain and induction of apoptosis. GTP cyclohydrolase I (GTPCH) is a key enzyme that is essential for iNOS activity. The coordinate expression of GTPCH prevents apoptosis that is induced by iNOS/NO. The purpose of this study was to investigate the effects of space flight on the expression of apoptotic/anti-apoptotic molecules iNOS and GTPCH in rat osteoblasts. Rat osteoblasts were cultured aboard a space shuttle and solubilized on the 4th and 5th days of the mission. The mRNA levels for iNOS and GTPCH in the flight cultures were increased to at least 120-fold and threefold higher than the ground (1 x g) controls, respectively. The amount of cellular DNA per flight culture vessel was 53% and 58% of the ground controls on the 4th and 5th days, respectively. However, the increasing rate of the DNA amount from the 4th to the 5th day was not different between the flight cultures and the ground controls. Morphologically, the cells grew in space as well as on the ground. Co-expression of GTPCH and iNOS may indicate a self-protective mode of action in osteoblasts against the harmful stress under microgravity.

Osteoblast responsiveness to 1alpha,25-dihydroxyvitamin D3 during spaceflight.

Rat osteoblasts were cultured aboard a space shuttle for 4 and 5 days. Cells were treated with 1 nM 1alpha,25-dihydroxyvitamin D(3) (VD) for the last 1 day. The conditioned media were harvested. Cells were solubilized with guanidine solution on board. We examined microgravity effects on the production/expression of osteocalcin, bone sialoprotein (BSP), and VD receptor (VD-R) in osteoblasts. Under VD treatment, the osteocalcin protein level was 243 +/- 117 and 1,718 +/- 534 pg/microg cellular DNA in flight cultures and ground controls, respectively. Without VD treatment, the osteocalcin protein level was not different between flight cultures and ground controls. The osteocalcin mRNA level in the VD-treated flight cultures was as low as 16% of that in ground controls. The VD-R mRNA level in the VD-treated flight cultures was also decreased to 16% of that in ground controls. Microgravity would suppress the VD-inducible production of osteocalcin but not the basal productivity. The BSP mRNA level was increased by microgravity. VD/VD-R binds to the vitamin D-responsive element (VDRE) on the target genes. The rat osteocalcin gene is positively regulated via "enhancer" VDRE, whereas the rat BSP gene is negatively regulated via "repressor " VDRE. Microgravity might modulate osteoblast responsiveness to VD through the suppression of VD-R.

Inhibition of HSP70 and a collagen-specific molecular chaperone (HSP47) expression in rat osteoblasts by microgravity.

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

Does microgravity induce apoptotic signal in rat osteoblasts via cjun-n-terminal kinase?

The mechanism of space flight-induced bone mass loss is unknown. We conducted experiments aboard the Space Shuttle using primary cultured osteoblasts. During flight, quadruplicate cultures were treated with 1 nM of 1alpha,25 dihydroxyvitamin D3 for one day, then fixed with guanidine isothiocyanate solution on board. After return to the Earth, the mRNA levels were analyzed by quantitative RT-PCR. Microgravity increased the mRNA levels of JNK, c-Jun N-terminal kinase or stress-activated protein kinase, in rat osteoblasts, as compared to the 1G ground control. Microgravity decreased the mRNA levels of the principal heat shock protein, Hsp 70. JNK is known to play a key role particularly in the stress-activated cell apoptosis. Data suggested apoptotic and anti-apoptotic effects of microgravity on rat osteoblast.