Yasuhiro Kumei

Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization

It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5- to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3- to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4- to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.

CELL DIFFERENTIATION AND p38MAPK CASCADE ARE INHIBITED IN HUMAN OSTEOBLASTS CULTURED IN A THREE-DIMENSIONAL CLINOSTAT

SummaryA three-dimensional (3D) clinostat is a device for multidirectional G force generation. By controlled rotation of two axes, a 3D clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10−3 G over time. We cultured a human osteoblast cell line in a 3D clinostat and examined the growth properties and differentiation of the cells, including morphology, histological detection of calcification, and mitogenactivated protein kinase (MAPK) cascades. In a normal 1G condition, alkaline phosphatase (AIPase) activity was detected on day 7 of culture, bone nodules were formed on day 12, and calcium deposits were seen on day 20. In the 3D clinostat, the cell looked larger and bulged. AIPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found in the 3D clinostat up to day 21. The expression levels of core-binding factor A1 (a transcription factor for bone formation) and osteocalcin (a bone matrix protein) increased in the control culture but decreased in culture in 3D clinostat. Phosphorylation of p38MARK (p38) was repressed in culture in 3D clinostat, whereas total p38 as well as total and phosphorylated forms of extracellular signal-regulated kinases and stress-activated protein kinase/jun N-terminal kinase were not changed in the 3D clinostat. When a p38 inhibitor, SB 203580, was added to the culture medium in a normal 1 G environment, AIPase activity and formation of bone nodules and calcium deposits were strongly inhibited. On the other hand, they were inhibited only partially by a MARK kinase inhibitor, U-0126. On the basis of these results, it is concluded that (1) osteoblast differentiation is inhibited in culture in a 3D clinostat and (2) this inhibition is mainly due to the suppression of p38 phophorylation.

Antagonism between apoptotic (Bax/Bcl-2) and anti-apoptotic (IAP) signals in human osteoblastic cells under vector-averaged gravity condition.

Abstract: A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl‐2. Released cytochrome c then activates the initiator caspase, caspase‐9, which can be blocked by the anti‐apototic (IAP) family of molecules. The effector caspase, caspase‐3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector‐averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector‐averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose‐gel electrophoresis. The mRNA levels for Bax, Bcl‐2, XIAP, and caspase‐3 genes were analyzed by semi‐quantitative RT‐PCR. Twenty‐four hours after starting clinostat rotation, the ratios of Bax/Bcl‐2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti‐apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector‐averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase‐3 mRNA levels were not affected by vector‐averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.

Microgravity Signal Ensnarls Cell Adhesion, Cytoskeleton, and Matrix Proteins of Rat Osteoblasts

Abstract:  Rat osteoblasts were cultured for 4 or 5 days aboard the Space Shuttle and solubilized during spaceflight. Post‐flight analyses by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) determined the relative mRNA levels of matrix proteins, adhesion molecules, and cytoskeletal proteins including osteopontin (OP), osteonectin (ON), CD44, α‐tubulin, actin, vimentin, fibronectin (FN), and β1‐integrin. The mRNA levels of OP and α‐tubulin in the flight cultures were decreased by 30% and 50% on day 4 and day 5 of flight, as compared to the ground controls. In contrast, the CD44 mRNA levels in the flight cultures increased by 280% and 570% of the ground controls on day 4 and day 5. The mRNA levels of ON and FN in the flight cultures were slightly increased as compared to ground controls. The mRNA levels of actin, vimentin, or β1‐integrin did not change in spaceflight conditions. The matrix proteins, adhesion molecules, and cytoskeletal proteins may form dynamic network complexity with signaling molecules as an adaptive response to perturbation of mechanical stress under microgravity.

An improved quantitative RT-PCR fluorescent method for analysis of gene transcripts in the STS-65 space shuttle experiment

We describe a reverse transcription polymerase chain reaction (RT-PCR) technique using fluorescent dUTP to examine changes in mRNA level in samples. In this procedure, the amplified product is identified by the fluorescent detection system in an automated DNA sequencer, and if the MW of the DNA/RNA or the fluorescent dye is different, several samples can be analyzed in a single lane. The basis for this technique is similar to that of radiolabeled methods, and we applied this technique for the comparison of the expression level of the rat c-myc gene in osteoblasts exposed to microgravity and unit gravity conditions. Using the fluorescent- and radiolabeled methods, the level of rat c-myc mRNA were compared quantitatively and the results demonstrated that the c-myc expression level was not altered by microgravity. Therefore, this fluorescent RT-PCR technique is useful for gene expression analysis particularly when the samples are limited, such as in space flight experiments. The method also allows for rapid assessment of mRNA changes in many samples simultaneously.

Inhibition of HSP70 and a Collagen‐Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

Abstract: Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1α, 25 dihydroxyvitamin D3 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‐β1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 ×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%, ∼ 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‐β1 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‐β1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

Expression of PDGF-β receptor, EGF receptor, and receptor adaptor protein Shc in rat osteoblasts during spaceflight

A number of studies have indicated that microgravity induces osteopenia and modulates functions of mammalian cells. However, the molecular mechanisms underlying these effects of microgravity are still unknown. Rat osteoblasts were cultured for 4 and 5 days during Shuttle-Spacelab flight, and fixed by guanidine isothiocyanate solution on board after treatment with 1α, 25 (OH)2 vitamin D3. The mRNA levels for platelet-derived growth factor (PDGF)-β receptor, epidermal growth factor (EGF) receptor, the growth factor receptor adaptor protein Shc, and c-fos were determined using the method of quantitative reverse transcription-polymerase chain reaction. The mRNA levels for EGF receptor were not altered by microgravity. However, the mRNA levels for PDGF-β receptor, Shc, and c-fos were decreased to 62, 55 and 25% on the 4th day of flight, and 47, 40, and 43% on the 5th day, respectively, as compared to the corresponding ground controls. Expression of the growth factor receptor and the receptor adaptor protein was modulated in rat osteoblasts during spaceflight. Data suggest that signal transduction via growth factor receptors in rat osteoblasts is impaired by microgravity. Dysfunction of osteoblasts might be involved in spaceflight-induced osteopenia.

Coinduction of GTP Cyclohydrolase I and Inducible NO Synthase in Rat Osteoblasts during Space Flight

Abstract: 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 × 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.

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

Abstract:  Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24‐h treatment with 1α,25 dihydroxyvitamin D3. 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‐β, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C‐γ (PLC‐γ) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC‐γ1 and ‐γ2 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‐β 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‐β 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.

Hypergravity Stimulates Osteoblast Phenotype Expression: A Therapeutic Hint for Disuse Bone Atrophy

Abstract: Physiological actions of osteoblasts are disordered by gravity unloading. We investigated the possibility that the appropriate level of hypergravity could improve osteoblast functions that are susceptible to mechanical unloading. We evaluated hypergravity effects on the 1α,25‐dihydroxyvitamin D3 (VD)‐inducible osteocalcin expression of primary rat osteoblasts. Cell culture plates were centrifuged for 24 h at 3, 6, 12, 24, and 48g in a 37°C incubator. The mRNA levels were analyzed by quantitative RT‐PCR. The mRNA levels for osteocalcin and vitamin D receptor (VD‐R) at 12g were enhanced to 187% and 228% of the 1g control, respectively. However, the excess hypergravity conversely decreased osteocalcin expression. Osteocalcin gene expression was enhanced by VD/VD‐R through the vitamin D‐responsive element in the promoter. The increased osteocalcin expression might reflect the augmented VD‐R expression. Alternatively, Runx2, a master gene of osteoblast differentiation, might be responsible for the osteocalcin induction, since the Runx2 mRNA levels were also increased to 247% of control at 12g. Another VD‐inducible osteoblast phenotype, alkaline phosphatase, was also upregulated at 12g and 24g. The appropriate level of hypergravity enhanced the VD‐inducible expression of osteocalcin, a typical phenotype of osteoblast differentiation. These data suggest molecular features to prevent disuse bone atrophy of long‐term bed‐rest patients.