MicroRNA-30 inhibits antiapoptotic factor Mcl-1 in mouse and human hematopoietic cells after radiation exposure.

We previously reported that microRNA-30 (miR-30) expression was initiated by radiation-induced proinflammatory factor IL-1ß and NFkB activation in mouse and human hematopoietic cells. However, the downstream effectors of miR-30 and its specific role in radiation-induced cell death are not well understood. In the present study, we evaluated effects of radiation on miR-30 expression and activation of intrinsic apoptotic pathway Bcl-2 family factors in in vivo mouse and in vitro human hematopoietic cells. CD2F1 mice and human CD34+ cells were exposed to different doses of gamma-radiation. In addition to survival studies, mouse blood, bone marrow (BM) and spleen cells and human CD34+ cells were collected at 4 h, and 1, 3 and 4 days after irradiation to determine apoptotic and stress response signals. Our results showed that mouse serum miR-30, DNA damage marker γ-H2AX in BM, and Bim, Bax and Bak expression, cytochrome c release, and caspase-3 and -7 activation in BM and/or spleen cells were upregulated in a radiation dose-dependent manner. Antiapoptotic factor Mcl-1 was significantly downregulated, whereas Bcl-2 was less changed or unaltered in the irradiated mouse cells and human CD34+ cells. Furthermore, a putative miR-30 binding site was found in the 3' UTR of Mcl-1 mRNA. miR-30 directly inhibits the expression of Mcl-1 through binding to its target sequence, which was demonstrated by a luciferase reporter assay, and the finding that Mcl-1 was uninhibited by irradiation in miR-30 knockdown CD34+ cells. Bcl-2 expression was not affected by miR-30. Our data suggest miR-30 plays a key role in radiation-induced apoptosis through directly targeting Mcl-1in hematopoietic cells.

Human pregnancy specific beta-1-glycoprotein 1 (PSG1) has a potential role in placental vascular morphogenesis.

Previous studies suggest that human pregnancy specific beta-1-glycoproteins (PSGs) play immunomodulatory roles during pregnancy; however, other possible functions of PSGs have yet to be explored. We have observed that PSGs induce transforming growth factor beta 1 (TGFB1), which among its other diverse functions inhibits T-cell function and has proangiogenic properties. The present study investigates a potential role for PSG1, the most abundant PSG in maternal serum, as a possible inducer of proangiogenic growth factors known to play an important role in establishment of the vasculature at the maternal-fetal interface. To this end, we measured TGFB1, vascular endothelial growth factors (VEGFs) A and C, and placental growth factor (PGF) protein levels in several cell types after PSG1 treatment. In addition, tube formation and wound healing assays were performed to investigate a possible direct interaction between PSG1 and endothelial cells. PSG1 induced up-regulation of both TGFB1 and VEGFA in human monocytes, macrophages, and two human extravillous trophoblast cell lines. We did not observe induction of VEGFC or PGF by PSG1 in any of the cells tested. PSG1 treatment resulted in endothelial tube formation in the presence and absence of VEGFA. Site-directed mutagenesis was performed to map the essential regions within the N-domain of PSG1 required for functional activity. We found that the aspartic acid at position 95, previously believed to be required for binding of PSGs to cells, is not required for PSG1 activity but that the amino acids implicated in the formation of a salt bridge within the N-domain are essential for PSG1 function.

Binding of pregnancy-specific glycoprotein 17 to CD9 on macrophages induces secretion of IL-10, IL-6, PGE2, and TGF-beta1.

Pregnancy-specific glycoproteins (PSGs) are a family of secreted proteins produced by the placenta, which are believed to have a critical role in pregnancy success. Treatment of monocytes with three members of the human PSGs induces interleukin (IL)-10, IL-6, and transforming growth factor-beta(1) (TGF-beta(1)) secretion. To determine whether human and murine PSGs have similar functions and use the same receptor, we treated wild-type and CD9-deficient macrophages with murine PSG17N and human PSG1 and -11. Our data show that murine PSG17N induced secretion of IL-10, IL-6, prostaglandin E(2), and TGF-beta(1) and that CD9 expression is required for the observed induction of cytokines. Therefore, the ability of PSG17 to induce anti-inflammatory cytokines parallels that of members of the human PSG family, albeit human and murine PSGs use different receptors, as CD9-deficient and wild-type macrophages responded equally to human PSGs. We then proceeded to examine the signaling mechanisms responsible for the CD9-mediated response to PSG17. Inhibition of cyclooxygenase 2 significantly reduced the PSG17N-mediated increase in IL-10 and IL-6. Further characterization of the response to PSG17 indicated that cyclic adenosine monophosphate-dependent protein kinase A (PKA) is involved in the up-regulation of IL-10 and IL-6, and it is not required for the induction of TGF-beta(1). Conversely, treatment of macrophages with a PKC inhibitor reduced the PSG17-mediated induction of TGF-beta(1), IL-6, and IL-10 significantly. The induction of anti-inflammatory cytokines by various PSGs supports the hypothesis that these glycoproteins have an essential role in the regulation of the maternal immune response in species with hemochorial placentation.

Circulating IL-18 Binding Protein (IL-18BP) and IL-18 as Dual Biomarkers of Total-Body Irradiation in Mice.

We have previously reported that circulating interleukin-18 (IL-18) can be used as a radiation biomarker in mice, minipigs and nonhuman primates. In this study, we further determined the serum levels of IL-18 binding protein (IL-18BP), a natural endogenous antagonist of IL-18, in CD2F1 mice 1-13 days after total-body gamma irradiation (TBI) with different doses (5-10 Gy). We compared the changes in blood lymphocyte, neutrophil and platelet counts as well as the activation of the proapoptotic executioner caspase-3 and caspase-7, and the expression of the inflammatory factor cyclooxygenase 2 (COX-2) in spleen cells, with the changes of IL-18BP and IL-18 in mouse serum. We also evaluated the significance, sensitivity and specificity of alterations in radiation-induced IL-18BP. IL-18 increased from day 1-13 after TBI in a dose-dependent manner that was paralleled with an increase in IL-18 receptor alpha (IL-18Rα) in irradiated mouse spleen cells. IL-18BP rapidly increased (25-63 fold) in mouse serum on day 1 after different doses of TBI. However, it returned to baseline within 3 days after 5-7 Gy doses and within 7 days after 8 Gy dose, and was unaltered thereafter. In contrast, high doses of radiation (9 and 10 Gy) significantly sustained a higher level of IL-18BP in mouse serum and later induced a second phase of increase in IL-18BP on day 9-13 after irradiation, which coincided with the onset of animal mortality. Consistent with this observation, highly activated caspase-3 and -7 in 8-10 Gy irradiated mouse spleen cells exhibited reduced or no activity 24 h after 5 Gy, although radiation induced an inflammatory response, as shown by COX-2 expression in all irradiated cells. Our data suggest that the radiation-induced differential elevation of IL-18 and IL-18BP in animal serum is a dynamic and discriminative indicator of the severity of injury after exposure to ionizing radiation. These findings support the inclusion of the dual biomarkers IL-18BP and IL-18 in the development of a multifactorial strategy for radiation dose and injury assessment.

Delta-tocotrienol suppresses radiation-induced microRNA-30 and protects mice and human CD34+ cells from radiation injury.

We reported that microRNA-30c (miR-30c) plays a key role in radiation-induced human cell damage through an apoptotic pathway. Herein we further evaluated radiation-induced miR-30 expression and mechanisms of delta-tocotrienol (DT3), a radiation countermeasure candidate, for regulating miR-30 in a mouse model and human hematopoietic CD34+ cells. CD2F1 mice were exposed to 0 (control) or 7-12.5 Gy total-body gamma-radiation, and CD34+ cells were irradiated with 0, 2 or 4 Gy of radiation. Single doses of DT3 (75 mg/kg, subcutaneous injection for mice or 2 µM for CD34+ cell culture) were administrated 24 h before irradiation and animal survival was monitored for 30 days. Mouse bone marrow (BM), jejunum, kidney, liver and serum as well as CD34+ cells were collected at 1, 4, 8, 24, 48 or 72 h after irradiation to determine apoptotic markers, pro-inflammatory cytokines interleukin (IL)-1ß and IL-6, miR-30, and stress response protein expression. Our results showed that radiation-induced IL-1ß release and cell damage are pathological states that lead to an early expression and secretion of miR-30b and miR-30c in mouse tissues and serum and in human CD34+ cells. DT3 suppressed IL-1ß and miR-30 expression, protected against radiation-induced apoptosis in mouse and human cells, and increased survival of irradiated mice. Furthermore, an anti-IL-1ß antibody downregulated radiation-induced NFκBp65 phosphorylation, inhibited miR-30 expression and protected CD34+ cells from radiation exposure. Knockdown of NFκBp65 by small interfering RNA (siRNA) significantly suppressed radiation-induced miR-30 expression in CD34+ cells. Our data suggest that DT3 protects human and mouse cells from radiation damage may through suppression of IL-1ß-induced NFκB/miR-30 signaling.

Circulating interleukin-18 as a biomarker of total-body radiation exposure in mice, minipigs, and nonhuman primates (NHP).

We aim to develop a rapid, easy-to-use, inexpensive and accurate radiation dose-assessment assay that tests easily obtained samples (e.g., blood) to triage and track radiological casualties, and to evaluate the radioprotective and therapeutic effects of radiation countermeasures. In the present study, we evaluated the interleukin (IL)-1 family of cytokines, IL-1ß, IL-18 and IL-33, as well as their secondary cytokines' expression and secretion in CD2F1 mouse bone marrow (BM), spleen, thymus and serum in response to γ-radiation from sublethal to lethal doses (5, 7, 8, 9, 10, or 12 Gy) at different time points using the enzyme-linked immune sorbent assay (ELISA), immunoblotting, and cytokine antibody array. Our data identified increases of IL-1ß, IL-18, and/or IL-33 in mouse thymus, spleen and BM cells after total-body irradiation (TBI). However, levels of these cytokines varied in different tissues. Interestingly, IL-18 but not IL-1ß or IL-33 increased significantly (2.5-24 fold) and stably in mouse serum from day 1 after TBI up to 13 days in a radiation dose-dependent manner. We further confirmed our finding in total-body γ-irradiated nonhuman primates (NHPs) and minipigs, and demonstrated that radiation significantly enhanced IL-18 in serum from NHPs 2-4 days post-irradiation and in minipig plasma 1-3 days post-irradiation. Finally, we compared circulating IL-18 with the well known hematological radiation biomarkers lymphocyte and neutrophil counts in blood of mouse, minipigs and NHPs and demonstrated close correlations between these biomarkers in response to radiation. Our results suggest that the elevated levels of circulating IL-18 after radiation proportionally reflect radiation dose and severity of radiation injury and may be used both as a potential biomarker for triage and also to track casualties after radiological accidents as well as for therapeutic radiation exposure.

Genistein nanoparticles protect mouse hematopoietic system and prevent proinflammatory factors after gamma irradiation.

Previous studies demonstrated that genistein protects mice from radiation-induced bone marrow failure. To overcome genistein's extremely low water solubility, a nanoparticle suspension of genistein has been formulated for more rapid dissolution. In the current study, we evaluated the radioprotective effects of a nanoparticle formulation of genistein on survival and hematopoietic recovery in mice exposed to total-body gamma irradiation. A single intramuscular injection of a saline-based genistein nanosuspension (150 mg/kg) administered to CD2F1 mice 24 h before 9.25 Gy (60)Co radiation exposure resulted in a 30-day survival rate of 95% compared to 25% in vehicle-treated animals. In mice irradiated at 7 Gy, the genistein nanosuspension increased mouse bone marrow cellularity from approximately 2.9% (vehicle treated) to 28.3% on day 7 postirradiation. Flow cytometry analysis demonstrated decreased radiation-induced hematopoietic stem and progenitor cell (HSPC, Lineage(-)/cKit(+)) death from 77.0% (vehicle) to 43.9% (genistein nanosuspension) with a significant recovery of clonogenicity 7 days after irradiation. The genistein nanosuspension also attenuated the radiation-induced elevation of proinflammatory factors interleukin 1 beta (IL-1ß), IL-6 and cyclooxygenase-2 (COX-2) in mouse bone marrow and spleen, which may contribute to protecting HSPCs.

Delta-tocotrienol protects mice from radiation-induced gastrointestinal injury.

We recently demonstrated that natural delta-tocotrienol (DT3) significantly enhanced survival in total-body irradiated (TBI) mice, and protected mouse bone marrow cells from radiation-induced damage through Erk activation-associated mTOR survival pathways. Here, we further evaluated the effects and mechanisms of DT3 on survival of radiation-induced mouse acute gastrointestinal syndrome. DT3 (75-100 mg/kg) or vehicle was administered as a single subcutaneous injection to CD2F1 mice 24 h before 10-12 Gy (60)Co total-body irradiation at a dose rate of 0.6 Gy/min and survival was monitored. In a separate group of mice, jejunum sections were stained with hematoxylin and eosin and the surviving crypts in irradiated mice were counted. Apoptosis in intestinal epithelial cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and bacterial translocation from gut to heart, spleen and liver in irradiated mice were evaluated. DT3 (75 mg/kg) significantly enhanced survival in mice that received 10, 10.5, 11 or 12 Gy TBI. Administration of DT3 protected intestinal tissue, decreased apoptotic cells in jejunum and inhibited gut bacterial translocation in irradiated mice. Furthermore, DT3 significantly inhibited radiation-induced production of pro-inflammatory factors interleukin-1ß and -6 and suppressed expression of protein tyrosine kinase 6 (PTK6), a stress-induced kinase that promotes apoptosis in mouse intestinal cells. Our data demonstrate that administration of DT3 protected mice from radiation-induced gastrointestinal system damage.

Micro-RNA30c negatively regulates REDD1 expression in human hematopoietic and osteoblast cells after gamma-irradiation.

We recently demonstrated that a novel cell stress response gene REDD1 protects human fetal osteoblast cell line (hFOB) cells from γ-radiation-induced premature senescence. Here we show that levels of endogenous REDD1 are very low in human hematopoietic progenitor CD34+ cells regardless of radiation, but highly expressed in differentiated hematopoietic cells (14 day cultured CD34+ cells) in response to radiation, which might be associated with radiation tolerance of the latter cells. To further understand the mechanisms of radiation-induced damage in different cells, microRNA (miRNA)-arrays were performed using purified miRNAs from CD34+ and hFOB cells before and post-irradiation and real-time reverse transcription (RT)-PCR was used to validate the expression profiles of miRNAs in the radiation-damaged cells. The results indicate that γ-radiation downregulated 16 miRNAs in CD34+ cells and 14 in hFOB cells. Radiation-induced upregulation was observed for 15 miRNAs in CD34+ cells and 18 miRNAs in hFOB cells. The profiles of radiation-induced miRNA expression were completely different in CD34+ vs. hFOB cells. Radiation up-regulated miRNA (miR)-30b, miR-30c and miR-30d in CD34+ cells, whereas it inhibited miR-30c expression in hFOB cells. Since miR-30 has potential target sites located in the 3'untranslated region (UTR) of the REDD1 gene and radiation regulated miR-30c expression in both CD34+ and hFOB cells, we further explored the effects of miR-30c on REDD1 expression using miR-30c inhibitor and precursor (pre-miR-30c). The results show that pre-miR-30c transfection suppressed REDD1 expression in 14 day cultured CD34+ cells and hFOB cells and resulted in hFOB cell death. In contrast, inhibition of miR-30c expression significantly enhanced clonogenicity in CD34+ cells. Our data suggest that CD34+ and hFOB cells have different miRNA expression patterns after irradiation and miR-30c plays a key role in radiation-induced cell damage which might be through regulation of REDD1 expression.

REDD1 protects osteoblast cells from gamma radiation-induced premature senescence.

Radiotherapy is commonly used for cancer treatment. However, it often results in side effects due to radiation damage in normal tissue, such as bone marrow (BM) failure. Adult hematopoietic stem and progenitor cells (HSPC) reside in BM next to the endosteal bone surface, which is lined primarily by hematopoietic niche osteoblastic cells. Osteoblasts are relatively more radiation-resistant than HSPCs, but the mechanisms are not well understood. In the present study, we demonstrated that the stress response gene REDD1 (regulated in development and DNA damage responses 1) was highly expressed in human osteoblast cell line (hFOB) cells after γ irradiation. Knockdown of REDD1 with siRNA resulted in a decrease in hFOB cell numbers, whereas transfection of PCMV6-AC-GFP-REDD1 plasmid DNA into hFOB cells inhibited mammalian target of rapamycin (mTOR) and p21 expression and protected these cells from radiation-induced premature senescence (PS). The PS in irradiated hFOB cells were characterized by significant inhibition of clonogenicity, activation of senescence biomarker SA-ß-gal, and the senescence-associated cytokine secretory phenotype (SASP) after 4 or 8 Gy irradiation. Immunoprecipitation assays demonstrated that the stress response proteins p53 and nuclear factor κ B (NFkB) interacted with REDD1 in hFOB cells. Knockdown of NFkB or p53 gene dramatically suppressed REDD1 protein expression in these cells, indicating that REDD1 was regulated by both factors. Our data demonstrated that REDD1 is a protective factor in radiation-induced osteoblast cell premature senescence.

N-glycosylation is required for binding of murine pregnancy-specific glycoproteins 17 and 19 to the receptor CD9.

PROBLEM: Murine pregnancy-specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post-translational modifications for the activity of these highly glycosylated proteins remains undetermined. METHOD OF STUDY: Recombinant PSG17 and PSG19 were generated and purified by affinity chromatography. An expression library was screened to identify the receptor for mouse PSG19. Binding to the receptor by proteins generated in different expression systems and mapping of the binding domain were analyzed by pull-down assays. Analysis of the carbohydrate composition of the receptor-binding domain was performed with the DIG glycan differentiation kit. RESULTS: PSG19 binds to the tetraspanin CD9, specifically to extra cellular loop 2 and can induce secretion of TGFbeta1 by a macrophage cell line. The receptor-binding domain of PSG17 and PSG19 is post-translationally modified by the addition of N-linked carbohydrates and, when expressed in CHO cells, terminal sialic acids are detected. PSGs produced in bacteria do not bind CD9. CONCLUSION: PSG19, as previously determined for PSG17, binds to the second extracellular loop 2 of the tetraspanin CD9. The first immunoglobulin variable-like domain of PSG19 is sufficient for receptor binding and function. Analysis of receptor usage by the remaining 15 murine PSGs will most likely require that the proteins be generated in eukaryotic expression systems, as we have demonstrated that the addition of carbohydrates is essential for PSG-receptor interaction.

Murine pregnancy-specific glycoprotein 23 induces the proangiogenic factors transforming-growth factor beta 1 and vascular endothelial growth factor a in cell types involved in vascular remodeling in pregnancy.

Haemochorial placentation is a unique physiological process in which the fetal trophoblast cells remodel the maternal decidual spiral arteries to establish the fetoplacental blood supply. Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen family. PSGs are produced by the placenta of rodents and primates and are secreted into the bloodstream. PSG23 is one of 17 members of the murine PSG family (designated PSG16 to PSG32). Previous studies determined that PSGs have immunoregulatory functions due to their ability to modulate macrophage cytokine secretion. Here we show that recombinant PSG23 induces transforming growth factor (TGF) beta1, TGFB1, and vascular endothelial growth factor A (VEGFA) in primary murine macrophages and the macrophage cell line RAW 264.7 cells. In addition, we identified new cell types that responded to PSG23 treatment. Dendritic cells, endothelial cells, and trophoblasts, which are involved in maternal vasculature remodeling during pregnancy, secreted TGFB1 and VEGFA in response to PSG23. PSG23 showed cross-reactivity with human cells, including human monocytes and the trophoblast cell line, HTR-8/SVneo cells. We analyzed the binding of PSG23 to the tetraspanin CD9, the receptor for PSG17, and found that CD9 is not essential for PSG23 binding and activity in macrophages. Overall these studies show that PSGs can modulate the secretion of important proangiogenic factors, TGFB1 and VEGFA, by different cell types involved in the development of the placenta.