TDAG51 promotes transcription factor FoxO1 activity during LPS-induced inflammatory responses.

Tight regulation of Toll-like receptor (TLR)-mediated inflammatory responses is important for innate immunity. Here, we show that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)-induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow-derived macrophages (BMMs). LPS-induced inflammatory mediator production was significantly decreased in TDAG51-deficient BMMs. In TDAG51-deficient mice, LPS- or pathogenic Escherichia coli infection-induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14-3-3ζ to FoxO1 was competitively inhibited by the TDAG51-FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double-deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51- or FoxO1-deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS- or pathogenic E. coli infection-induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a regulator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS-induced inflammatory response.

FOXO1 represses lymphatic valve formation and maintenance via PRDM1.

Intraluminal lymphatic valves (LVs) contribute to the prevention of lymph backflow and maintain circulatory homeostasis. Several reports have investigated the molecular mechanisms which promote LV formation; however, the way in which they are suppressed is not completely clear. We show that the forkhead transcription factor FOXO1 is a suppressor of LV formation and maintenance in lymphatic endothelial cells. Oscillatory shear stress by bidirectional flow inactivates FOXO1 via Akt phosphorylation, resulting in the upregulation of a subset of LV-specific genes mediated by downregulation of a transcriptional repressor, PRDM1. Mice with an endothelial-specific Foxo1 deletion have an increase in LVs, and overexpression of Foxo1 in mice produces a decrease in LVs. Genetic reduction of PRDM1 rescues the decrease in LV by Foxo1 overexpression. In conclusion, FOXO1 plays a critical role in lymph flow homeostasis by preventing excess LV formation. This gene might be a therapeutic target for lymphatic circulatory abnormalities.

FOXO1 regulates developmental lymphangiogenesis by upregulating CXCR4 in the mouse-tail dermis.

Lymphangiogenesis plays important roles in normal fetal development and postnatal growth. However, its molecular regulation remains unclear. Here, we have examined the function of forkhead box protein O1 (FOXO1) transcription factor, a known angiogenic factor, in developmental dermal lymphangiogenesis using endothelial cell-specific FOXO1-deficient mice. FOXO1-deficient mice showed disconnected and dilated lymphatic vessels accompanied with increased proliferation and decreased apoptosis in the lymphatic capillaries. Comprehensive DNA microarray analysis of the causes of in vivo phenotypes in FOXO1-deficient mice revealed that the gene encoding C-X-C chemokine receptor 4 (CXCR4) was the most drastically downregulated in FOXO1-deficient primary lymphatic endothelial cells (LECs). CXCR4 was expressed in developing dermal lymphatic capillaries in wild-type mice but not in FOXO1-deficient dermal lymphatic capillaries. Furthermore, FOXO1 suppression impaired migration toward the exogenous CXCR4 ligand, C-X-C chemokine ligand 12 (CXCL12), and coordinated proliferation in LECs. These results suggest that FOXO1 serves an essential role in normal developmental lymphangiogenesis by promoting LEC migration toward CXCL12 and by regulating their proliferative activity. This study provides valuable insights into the molecular mechanisms underlying developmental lymphangiogenesis.

Upregulation of IFN-ß induced by Sema4D-dependent partial Erk1/2 inhibition promotes NO production in microglia.

Interactions between Sema4D and its receptors, PlexinB1 and CD72, induce various functions, including axon guidance, angiogenesis, and immune activation. Our previous study revealed that Sema4D is involved in the upregulation of nitric oxide production in microglia after cerebral ischemia. In this study, we investigated the underlying mechanisms of the enhancement of microglial nitric oxide production by Sema4D. Primary microglia expressed PlexinB1 and CD72, and cortical microglia expressed CD72. Sema4D promoted nitric oxide production and slightly inhibited Erk1/2 phosphorylation in microglia. Partial Erk1/2 inhibition enhanced microglial nitric oxide production. Inhibition of Erk1/2 phosphorylation induced the expression of Ifn-ß mRNA, and IFN-ß promoted nitric oxide production in microglia. In the ischemic cortex, the expression of Ifn-ß mRNA was downregulated by Sema4D deficiency. These findings indicated that the enhancement of nitric oxide production by Sema4D is involved in partial Erk1/2 inhibition and upregulation of IFN-ß.

Endothelial specific deletion of FOXO1 alters pericyte coverage in the developing retina.

Pericytes are mural cells that cover small blood vessels. While defects in pericyte coverage are known to be involved in various vessel related pathologies, including diabetic retinopathy, the molecular mechanisms underlying pericyte coverage are not fully understood. In this study, we investigated the contribution of the forkhead transcription factor FOXO1 in endothelial cells to pericyte coverage in the developing retina. We observed retinal pericytes in tamoxifen-inducible endothelium-specific Foxo1 deletion mice. Tamoxifen was injected at postnatal day 1-3 and the retinas were harvested at P21. Our results demonstrated that Foxo1 deletion in the endothelium affected arteriole pericyte morphology without altering pericyte number, proliferation, and apoptosis. We hypothesized that abnormal pericyte morphogenesis in the knockout retina was caused by impaired pericyte differentiation. FOXO1 silencing by siRNA in the primary artery endothelium further revealed that THBS1 (thrombospondin 1), which promotes pericyte differentiation via TGFß activation, was reduced in the FOXO1-deficient endothelium. Immunohistochemistry of FOXO1 knockout mice showed reduced numbers of phospho-Smad3+ arteriole pericytes compared with wild-type mice. In addition, endothelium-pericyte co-culture analysis revealed that pericytes cultured with FOXO1-deficient endothelial cells failed to differentiate sufficiently; this failure was partially rescued by the addition of recombinant THBS1 to the supernatant. The findings suggest that endothelial FOXO1 contributes to pericyte differentiation via regulation of THBS1 expression. This study provides new insights into the molecular mechanism of pericyte coverage in the context of endothelium-derived regulation and highlights a new therapeutic target for pericyte-related pathology.

Changes in L-arginine metabolism by Sema4D deficiency induce promotion of microglial proliferation in ischemic cortex.

Cerebral ischemia induces neuroinflammation and microglial activation, in which activated microglia upregulate their proliferative activity and change their metabolic states. In activated microglia, l-arginine is metabolized competitively by inducible nitric oxide synthase (iNOS) and arginase (Arg), which then synthesize NO or polyamines, respectively. Our previous study demonstrated that Sema4D deficiency inhibits iNOS expression and promotes proliferation of ionized calcium-binding adaptor molecule 1 (Iba1)-positive (Iba1+) microglia in the ischemic cortex, although the underlying mechanisms were unclear. Using middle cerebral artery occlusion, we tested the hypothesis that Sema4D deficiency alters the balance of l-arginine metabolism between iNOS and Arg, leading to an increase in the production of polyamines, which are an essential factor for cell proliferation. In the peri-ischemic cortex, almost all iNOS+ and/or Arg1+ cells were Iba1+ microglia. In the peri-ischemic cortex of Sema4D-deficient (Sema4D-/-) mice, the number of iNOS+ Arg1- Iba1+ microglia was smaller and that of iNOS- Arg1+ Iba1+ microglia was greater than those of wild-type (WT) mice. In addition, urea and polyamine levels in the ischemic cortex of Sema4D-/- mice were higher than those of WT mice; furthermore, the presence of Sema4D inhibited polyamine production in primary microglia obtained from Sema4D-/- mice. Finally, microglia cultured under polyamine putrescine-supplemented conditions demonstrated increased proliferation rates over non-supplemented controls. These findings indicate that Sema4D regulates microglial proliferation at least in part by regulating the competitive balance of l-arginine metabolism.

Tip-cell behavior is regulated by transcription factor FoxO1 under hypoxic conditions in developing mouse retinas.

Forkhead box protein O1 (FoxO1) is a transcription factor and a critical regulator of angiogenesis. Various environmental stimuli, including growth factors, nutrients, shear stress, oxidative stress and hypoxia, affect FoxO1 subcellular localization and strongly influence its transcriptional activity; however, FoxO1-localization patterns in endothelial cells (ECs) during development have not been clarified in vivo. Here, we reported that FoxO1 expression was observed in three layers of angiogenic vessels in developing mouse retinas and that among these layers, the front layer showed high levels of FoxO1 expression in the nuclei of most tip ECs. Because tip ECs migrate toward the avascular hypoxic area, we focused on hypoxia as a major stimulus regulating FoxO1 subcellular localization in tip cells. In cultured ECs, FoxO1 accumulated into the nucleus under hypoxic conditions, with hypoxia also inducing expression of tip-cell-specific genes, including endothelial-specific molecule 1 (ESM1), which was suppressed by FoxO1 knockdown. Additionally, in murine models, EC-specific FoxO1 deletion resulted in reduced ESM1 expression and suppressed tip-cell migration during angiogenesis. These findings indicated roles for FoxO1 in tip-cell migration and that its transcriptional activity is regulated by hypoxia.

Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor.

Absence of Sema4D improves oligodendrocyte recovery after cerebral ischemia/reperfusion injury in mice.

Sema4D, originally identified as a negative regulator of axon guidance during development, is involved in various physiological and pathological responses. In this study, we evaluated the effect of Sema4D-deficiency on oligodendrocyte restoration after the cerebral ischemia/reperfusion using direct ligation of the middle cerebral artery followed by reperfusion. In both Sema4D(+/+) wild-type and Sema4D(-/-) null mutant mice, the peri-infarct area showed a decrease in the number of oligodendrocytes at 3 days post-reperfusion. Subsequently, the number of oligodendrocytes was observed to gradually recover in both groups. Sema4D-deficient mice, however, showed an enhanced recovery of oligodendrocytes and an upregulation of oligodendrocyte progenitor cells at days 14 and 28 of reperfusion. Cell proliferation identified by incorporation of bromodeoxyuridine was enhanced in Sema4D(-/-) mice from days 3 to 14 post-reperfusion compared to the Sema4D(+/+) mice. Furthermore, apoptotic cell death of oligodendrocytes was reduced at days 7 post-reperfusion in Sema4D(-/-) mice compared to Sema4D(+/+) mice. These findings indicate that enhanced proliferation of progenitor cells and survival of oligodendrocytes resulted in improved oligodendrocyte recovery in Sema4D(-/-) mice. This may provide a new approach for neurorestorative treatment in patients with stroke, which aims to manipulate endogenous oligodendrogenesis and thereby to promote brain repair after stroke.

Effect of Sema4D on microglial function in middle cerebral artery occlusion mice.

Cerebral ischemia evokes neuroinflammatory response. Inflammatory stimulation induces microglial activation, such as changes of their morphology from ramified to ameboid, expression of iNOS and cytokines, and the elevation of proliferative activity. Activated microglia play important roles in pathogenesis of cerebral ischemia. A previous study indicated that Sema4D promoted iNOS expression in cultured microglia; however, roles of Sema4D on microglial activation in ischemic injury remains unclear. We investigated the effect of Sema4D-deficiency on microglial activation by using permanent middle cerebral artery occlusion (MCAO) in mice. In this study, ischemia-induced activated microglia were classified into activated-ramified microglia and ameboid microglia based on their morphology. We demonstrated that the rate of iNOS expression in activated-ramified microglia was lower than that in ameboid microglia, while the most proliferating microglia were activated-ramified microglia but not ameboid microglia after cerebral ischemia. Sema4D-deficiency decreased the number of ameboid microglia and iNOS-expressing activated-ramified microglia in the peri-ischemic cortex. These changes by Sema4D-deficiency contributed to the reduction of NO production that was estimated by nitrite concentration in ischemic cortex. On the other hand, Sema4D-deficiency promoted proliferation of microglia in the peri-ischemic cortex. Importantly, ischemia-induced apoptosis and postischemic behavioral abnormality were moderated in Sema4D(-/-) mice. These findings suggest that Sema4D promotes cytotoxic activation of microglia and inhibits functional recovery after cerebral ischemia.

Analysis of Foxo1-regulated genes using Foxo1-deficient pancreatic ß cells.

Several reports have suggested that Foxo1, a key regulator in differentiation, growth and metabolism, is involved in pancreatic ß-cell function. However, detailed analyses have been hampered by a lack of Foxo1-deficient ß cells. To elucidate Foxo1's function in ß cells, we produced a ß-cell line with inducible Foxo1 deletion. We generated a conditional knockout mouse line, in which Cre recombinase deletes the Foxo1 gene. We then established a ß-cell line from an insulinoma induced in this knockout mouse by the ß-cell-specific expression of simian virus 40 T antigen. In this cell line, designated MIN6-Foxo1flox/flox, adenovirus-mediated Cre expression ablates the Foxo1 gene, generating MIN6-Foxo1-KO cells. Using these knockout and floxed cell lines, we found that Foxo1 ablation enhanced the glucose-stimulated insulin secretion (GSIS) at high glucose concentrations and enhanced ß-cell proliferation. We also conducted DNA microarray analyses of MIN6-Foxo1-KO cells infected with either an adenovirus vector expressing a constitutively active FOXO1 or a control vector and identified several Foxo1-regulated genes, including some known to be related to ß-cell function. These cells should be useful for further studies on Foxo1's roles in ß-cells and may lead to novel strategies for treating the impaired insulin secretion in type 2 diabetes mellitus.

Sema4D as an inhibitory regulator in oligodendrocyte development.

The specific functions of intrinsic regulators of OL differentiation are poorly understood. Sema4D, originally found as a negative regulator of axon guidance, is mainly expressed by oligodendrocytes in the postnatal brain, and our previous study revealed that the lack of Sema4D induced an increase in the number of oligodendrocytes in the cerebral cortex, suggesting that Sema4D may function as an intrinsic regulator of oligodendrocyte development. In this study, we assessed the effects of Sema4D deficiency and of the exogenous addition of Sema4D on oligodendrocyte differentiation. Sema4D deficiency induced an increase in the number of oligodendrocytes in the cerebral cortex at postnatal day 14 and later, without increase in the number of oligodendrocyte progenitor cells. This increase was also observed in cultured oligodendrocytes obtained from Sema4D-deficient mice. Then we investigated whether Sema4D deficiency can increase the proliferation of the progenitor cells or influence the apoptosis. Apoptotic oligodendrocytes were markedly reduced in number in the developing cerebral cortex and in cultured oligodendrocytes obtained from Sema4D-deficient mice, although no significant change was found in proliferation of oligodendrocyte progenitor cells. Exogenous addition of Sema4D prevented the oligodendrocytes from this reduction of apoptosis, and further enhanced apoptosis in oligodendrocytes. Thus, Sema4D may act as an intrinsic inhibitory regulator of oligodendrocyte differentiation by promoting apoptosis.

FoxO1 is involved in the antineoplastic effect of calorie restriction.

The FoxO transcription factors may be involved in the antiaging effect of calorie restriction (CR) in mammals. To test the hypothesis, we used FoxO1 knockout heterozygotic (HT) mice, in which the FoxO1 mRNA level was reduced by 50%, or less, of that in wild-type (WT) mouse tissues. The WT and HT mice were fed ad libitum (AL) or 30% CR diets from 12 weeks of age. Aging- and CR-related changes in body weight, food intake, blood glucose, and insulin concentrations were similar between the WT and HT mice in the lifespan study. The response to oxidative stress, induced by intraperitoneal injection of 3-nitropropionic acid (3-NPA), was evaluated in the liver and hippocampus at 6 months of age. Several of the selected FoxO1-target genes for cell cycle arrest, DNA repair, apoptosis, and stress resistance were up-regulated in the WT-CR tissues after 3-NPA injection, while the effect was mostly diminished in the HT-CR tissues. Of these gene products, we focused on the nuclear p21 protein level in the liver and confirmed its up-regulation only in the WT-CR mice in response to oxidative stress. The lifespan did not differ significantly between the WT and HT mice in AL or CR conditions. However, the antineoplastic effect of CR, as indicated by reduced incidence of tumors at death in the WT-CR mice, was mostly abrogated in the HT-CR mice. The present results suggest a role for FoxO1 in the antineoplastic effect of CR through the induction of genes responsible for protection against oxidative and genotoxic stress.

Foxo1 is essential for in vitro vascular formation from embryonic stem cells.

The forkhead transcription factors regulate the correct organization of vascular system. One of them, Foxo1 is an important physiological regulator of endothelial cell morphology in response to VEGF, while underlying mechanisms are largely unknown. In order to elucidate the cellular function of Foxo1, we used a three-dimensional culture system for the differentiation of Flk1-expressing mesodermal precursor cells derived from ES cells to cord forming endothelial cells and associating vascular smooth muscle cells. While Foxo1(+/+) endothelial cells organized into long vessel-like structures associated with smooth muscle cells, Foxo1(-/-) endothelial cells could form only short sprouts. Foxo1(-/-) endothelial cells have punctate accumulation of filamentous actin, thick circumferential bundles of microtubules with small spikes at the tip of cells, and no interaction with smooth muscle cells. Our results suggest the involvement of Foxo1 in cytoskeletal remodeling of endothelial cells and recruitment of smooth muscle cells during vascular development.

Different roles of Foxo1 and Foxo3 in the control of endothelial cell morphology.

Foxo1, a member of the Foxo subfamily of forkhead box transcription factors, is known to be essential for progression of normal vascular development in the mouse embryos. In the cultures of endothelial cells derived from embryonic stem cells, Foxo1-deficient endothelial cells exhibit an abnormal morphological response to vascular endothelial growth factor-A (VEGF-A), which is characterized by a lack of cell elongation, yet the molecular mechanisms governing endothelial cell morphology under angiogenic stimulation remain unknown. Here, we report that transforming growth actor-beta also induces endothelial cell elongation in collaboration with Foxo1 and VEGF-A. Furthermore, tetracycline-regulated induction of Foxo3, another member of the Foxo subfamily, into Foxo1-null endothelial cells failed to restore abnormal morphological response to VEGF-A at an early differentiation stage. In contrast, Foxo1 and Foxo3 exerted the same function at a late differentiation stage, i.e. enhancement of VEGF responsiveness and promotion of cell elongation. Our results provide evidence that endothelial cell morphology is regulated by several mechanisms in which Foxo1 and Foxo3 express distinct functional properties depending on differentiation stages.

Sema4D deficiency results in an increase in the number of oligodendrocytes in healthy and injured mouse brains.

Semaphorins, a family of secreted and membrane-bound proteins, are known to function as repulsive axon guidance molecules. Sema4D, a class 4 transmembrane-type semaphorin, is expressed by oligodendrocytes in the central nervous system, but its role is unknown. In this study, the effects of Sema4D deficiency on oligodendrocytes were studied in intact and ischemic brains of adult mice. As observed in previous studies, Sema4D marked by beta-galactosidase in Sema4D mutant mice was localized exclusively on myelin-associated glycoprotein (MAG)-positive oligodendrocytes but not on NG2-positive oligodendrocyte progenitor cells (OPCs). Although there was no difference in the number of the latter cells between Sema4D-deficient and wild-type mice, the number of MAG-positive cells was significantly increased in the cerebral cortex of both nonischemic and postischemic brains of Sema4D-deficient mice. Cell proliferation, observed by using bromodeoxyuridine incorporation, was evident in the MAG-positive cells that developed after cerebral ischemia. These data indicate that Sema4D is involved in oligodendrogenesis during development and during recovery from ischemic injury.

Impaired basal thermal homeostasis in rats lacking capsaicin-sensitive peripheral small sensory neurons.

We studied the effects of selective loss of capsaicin-sensitive primary sensory neurons on thermosensation and thermoregulation in rats. Neonatal capsaicin treatment in rats caused a remarkable decrease in the number of small-diameter neurons in the dorsal root ganglion (DRG) compared with their number in the control rats. Gene expression analysis for various thermo-sensitive transient receptor potential (TRP) channels indicated marked reductions in the mRNA levels of TRPV1 (70%), TRPM8 (46%) and TRPA1 (64%), but not of TRPV2, in the DRG of capsaicin-treated rats compared with those in the control rats. In addition to the heat and cold insensitivity, capsaicin-treated rats showed lower rectal core temperature, higher skin temperature and decreased sensitivity to ambient temperature alteration under normal housing at room temperature, suggesting impaired thermosensation and change in thermoregulation in the rats. Uncoupling protein 1 (UCP1) expression and the thermogenic ability in brown adipose tissues were attenuated in the capsaicin-treated rats. These results indicate a critical role of capsaicin-sensitive sensory neurons in both heat and cool sensation and hence in basal thermal homeostasis, which is balanced by heat release and production including UCP1 thermogenesis, following sensation of the ambient temperature.

Semaphorin 4D inhibits collagen synthesis of rat pulp-derived cells.

OBJECTIVE: Semaphorins are a group of secreted and membrane-associated molecules that play important roles in axon navigation. Several semaphorin family molecules are expressed in the pharyngeal arches and tooth germs. We analysed the expression of membrane-associated Semaphorin 4D (Sema4D) in tooth germs, and examined its potential role in regulating the differentiation of rat incisor pulp-derived cells in vitro. DESIGN: mRNA expression was examined by in situ hybridisation. The effects of Sema4D on rat pulp-derived cells were examined by adenovirus-mediated overexpression in vitro. RESULTS: Both epithelial and mesenchymal cells of tooth germs expressed Sema4D at the early bell stage. Later, the odontoblasts predominantly expressed Sema4D, while the epithelial expression greatly decreased. The overexpression of Sema4D in rat incisor pulp-derived cells strongly inhibited mineralisation. This inhibition was preceded by a reduction of collagen fibre production at the level of mRNA synthesis. CONCLUSIONS: These results indicate that Sema4D is expressed in both epithelial and mesenchymal cells of the tooth germs. Sema4D represses collagen synthesis of pulp-derived cells, indicating it might negatively regulate odontoblast differentiation.

Sema4D-plexin-B1 implicated in regulation of dendritic spine density through RhoA/ROCK pathway.

Plexin-B1, Sema4D receptor, mediates retraction and extension signals in axon guidance by associating with PDZ-containing Rho guanine nucleotide exchange factors (PDZ-RhoGEFs) which can activate a small Rho GTPase RhoA. RhoA is implicated in spine formation by rearranging actin cytoskeleton. Exogenous application of Sema4D to cultured neurons caused activation of RhoA, increase of spine density and changes in spine shape. Sema4D-induced changes in spine density were blocked by either Rho-kinase (a downstream of RhoA, ROCK) inhibitor Y-27632 or by overexpression of plexin-B1 mutant lacking the C-terminus which no longer associates with PDZ-RhoGEFs. This study suggests that Sema4D-plexin-B1 play a crucial role in spine formation by regulating RhoA/ROCK pathway.