Chai-Fei Li

Key gravity-sensitive signaling pathways drive T cell activation

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity‐dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real‐time RT‐PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF‐κB, CREB, ELK, AP‐1, and STAT after crosslinking the T‐cell receptor contributes to T‐cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T‐cell activation. Since the majority of the genes were regulated by NF‐κB, CREB, and AP‐1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down‐regulated in vg. In contrast, PI3‐K, PKC, and its upstream regulator pLAT were not significantly down‐regulated by vectorless gravity. Since NF‐κB, AP‐1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T‐cell activation in altered gravity.

A Short Pulse of Mechanical Force Induces Gene Expression and Growth in MC3T3-E1 Osteoblasts via an ERK 1/2 Pathway†

Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4–30g corresponds to physiological levels of 40–300 μstrain. Short‐term gravity loading (15 minutes) induced a 15‐fold increase in expression of growth‐related immediate early gene c‐fos, a 5‐fold increase in egr‐1, and a 3‐fold increase in autocrine bFGF. The non‐growth‐related genes EP‐1, TGF‐β, and 18s were unaffected by gravity loading. Short‐term physiological loading induced extracellular signal‐regulated kinase (ERK 1/2) phosphorylation in a dose‐dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g‐Loading did not activate P38 MAPK or c‐jun N‐terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c‐fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK‐specific inhibitors. The long‐term consequence of a single 15‐minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity‐induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3‐E1 osteoblasts primarily through an ERK 1/2‐mediated pathway.

Arachidonic Acid Activates Phosphatidylinositol 3-Kinase Signaling and Induces Gene Expression in Prostate Cancer

Essential fatty acids are not only energy-rich molecules; they are also an important component of the membrane bilayer and recently have been implicated in induction of fatty acid synthase and other genes. Using gene chip analysis, we have found that arachidonic acid, an omega-6 fatty acid, induced 11 genes that are regulated by nuclear factor-kappaB (NF-kappaB). We verified gene induction by omega-6 fatty acid, including COX-2, IkappaBalpha, NF-kappaB, GM-CSF, IL-1beta, CXCL-1, TNF-alpha, IL-6, LTA, IL-8, PPARgamma, and ICAM-1, using quantitative reverse transcription-PCR. Prostaglandin E(2) (PGE(2)) synthesis was increased within 5 minutes of addition of arachidonic acid. Analysis of upstream signal transduction showed that within 5 minutes of fatty acid addition, phosphatidylinositol 3-kinase (PI3K) was significantly activated followed by activation of Akt at 30 minutes. Extracellular signal-regulated kinase 1 and 2, p38 and stress-activated protein kinase/c-Jun-NH(2)-kinase were not phosphorylated after omega-6 fatty acid addition. Thirty minutes after fatty acid addition, we found a significant 3-fold increase in translocation of NF-kappaB transcription factor to the nucleus. Addition of a nonsteroidal anti-inflammatory drug (NSAID) caused a decrease in COX-2 protein synthesis, PGE(2) synthesis, as well as inhibition of PI3K activation. We have previously shown that NSAIDs cause an inhibition of arachidonic acid-induced proliferation; here, we have shown that arachidonic acid-induced proliferation is also blocked (P < 0.001) by PI3K inhibitor LY294002. LY294002 also significantly inhibited the arachidonic acid-induced gene expression of COX-2, IL-1beta, GM-CSF, and ICAM1. Taken together, the data suggest that arachidonic acid via conversion to PGE(2) plays an important role in stimulation of growth-related genes and proliferation via PI3K signaling and NF-kappaB translocation to the nucleus.

The role of FGF-2 and BMP-2 in regulation of gene induction, cell proliferation and mineralization

IntroductionThe difficulty in re-growing and mineralizing new bone after severe fracture can result in loss of ambulation or limb. Here we describe the sequential roles of FGF-2 in inducing gene expression, cell growth and BMP-2 in gene expression and mineralization of bone.Materials and methodsThe regulation of gene expression was determined using real-time RTPCR (qRTPCR) and cell proliferation was measured by thymidine incorporation or fluorescent analysis of DNA content in MC3T3E1 osteoblast-like cells. Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization.ResultsFibroblast growth factor-2 (FGF-2) had the greatest ability to induce proliferation after 24 hours of treatment when compared to transforming growth factor beta (TGFβ, insulin-like growth factor-1 (IGF-1), bone morphogenic protein (BMP-2), platelet derived growth factor (PDGF) or prostaglandin E2 (PGE2). We found that FGF-2 caused the most significant induction of expression of early growth response-1 (egr-1), fgf-2, cyclo-oxygenase-2 (cox-2), tgfβ and matrix metalloproteinase-3 (mmp-3) associated with proliferation and expression of angiogenic genes like vascular endothelial growth factor A (vegfA) and its receptor vegfr1. We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp). In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth.ConclusionsThe ability of FGF-2 to re-program a mineralizing gene expression profile to one of proliferation suggests that FGF-2 plays a critical role of osteoblast growth in early fracture repair while BMP-2 is instrumental in stimulating mineralization.

The Rel/NF‐κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long‐term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti‐CD28 on board the ISS. An on‐board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg‐ and 1g‐activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down‐regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF‐κB, CREB, and SRF gene targets were down‐regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti‐CD3/anti‐CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.

Fibroblast growth factor-2 is an immediate-early gene induced by mechanical stress in osteogenic cells.

Fifteen minutes of physiological MS induces FGF‐2 in osteogenic cells. Here, we show that MS induced proliferation in both MC3T3‐E1 and BMOp cells isolated from Fgf2+/+ mice; Fgf2−/− BMOp cells required exogenous FGF‐2 for a normal proliferation response. The induction of fgf‐2 is mediated by PKA and ERK pathways.

Glycosylation regulates turnover of cyclooxygenase-2

Cyclooxygenase‐2 (COX‐2) catalyzes the rate‐limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H2. COX‐2 exists as 72 and 74 kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn580. In this study, Asn580 was mutated to determine the biological significance of this variable glycosylation. COS‐1 cells transfected with the mutant gene were unable to express the 74 kDa glycoform and were found to accumulate more COX‐2 protein and have five times greater COX‐2 activity than cells expressing both glycoforms. Thus, COX‐2 turnover appears to depend upon glycosylation of the 72 kDa glycoform.

Spaceflight alters expression of microRNA during T-cell activation

Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long‐term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR‐21 was significantly up‐regulated 2‐fold during early T‐cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative realtime PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co‐up‐regulated with miR‐21. These genes share common promoter regions with pre‐mir‐21; as the miR‐21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T‐cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T‐cell activation itself may induce a sequence of gene expressions that is self‐limited by miR‐21.—Hughes‐Fulford, M., Chang, T. T., Martinez, E. M., Li, C.‐F. Spaceflight alters expression of microRNA during T‐cell activation. FASEB J. 29, 4893–4900 (2015). www.fasebj.org