Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.     

Dll4-selective Notch signaling induces ephrinB2 gene expression in endothelial cells

The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation. Here, we show that Notch stimulation instructively induces arterial characteristics in endothelial cells (EC). Forced expression of Notch intracellular domain (NICD, activated form of Notch) induced mRNA expression for a subset of arterial-specific markers such as ephrinB2, connexin40, and HERP1 only in EC but not other cell lines. In co-culture experiments using EC and either Dll4- or Jagged1-expressing cells, we found that Dll4 stimulation but not Jagged1 markedly induced ephrinB2 expression. An inducible expression of HERP1 and HERP2 by NICD has no measurable effects on expression of ephrinB2 and venous marker EphB4 although either HERP1 or HERP2 overexpression exerts potent inhibitory effects on EphB4 expression without ephrinB2 induction. We also found no functional interaction between Notch and TGF-beta-ALK1 signalings in an induction of ephrinB2 expression. These results suggest that Dll4-stimulated Notch signaling induces a part of arterial characteristics only in EC via HERP-independent mechanism. Our data provide new insight into the molecular mechanism of ligand-selective Notch activation during differentiation of arterial EC.     

SirT1 inhibition reduces IGF-I/IRS-2/Ras/ERK1/2 signaling and protects neurons

Sirtuins are known to protect cells and extend life span, but our previous studies indicated that S. cerevisiae Sir2 can also increase stress sensitivity and limit life-span extension. Here we provide evidence for a role of the mammalian Sir2 ortholog SirT1 in the sensitization of neurons to oxidative damage. SirT1 inhibition increased acetylation and decreased phosphorylation of IRS-2; it also reduced activation of the Ras/ERK1/2 pathway, suggesting that SirT1 may enhance IGF-I signaling in part by deacetylating IRS-2. Either the inhibition of SirT1 or of Ras/ERK1/2 was associated with resistance to oxidative damage. Markers of oxidized proteins and lipids were reduced in the brain of old SirT1-deficient mice, but the life span of the homozygote knockout mice was reduced under both normal and calorie-restricted conditions. These results are consistent with findings in S. cerevisiae and other model systems, suggesting that mammalian sirtuins can play both protective and proaging roles.     

EphB4 signaling is capable of mediating ephrinB2-induced inhibition of cell migration

Signaling between the ligand ephrinB2 and the respective receptors of the EphB class is known to play a vital role during vascular morphogenesis and angiogenesis. The relative contribution of each EphB receptor type present on endothelial cells to these processes remains to be determined. It has been shown that ephrinB2-EphB receptor signal transduction leads to a repulsive migratory behavior of endothelial cells. It remained unclear whether this anti-migratory effect can be mediated by EphB4 signaling alone or whether other EphB receptors are necessary. It also remained unclear whether the kinase activity of EphB4 is pivotal to its action. To answer these questions, we developed a cellular migration system solely dependent on ephrinB2-EphB4 signaling. Using this system, we could show that EphB4 activation leads to the inhibition of cell migration. Furthermore we identified PP2, a known inhibitor of kinases of the Src family, and PD 153035, a known inhibitor of EGF receptor kinase, as inhibitors of EphB4 kinase activity. Using PP2, the inhibition of cell migration by ephrinB2 could be relieved, demonstrating that the kinase function of EphB4 is of prominent importance in this process. These results show that EphB4 activation is not only accompanying ephrinB2 induced repulsive behavior of cells, but is capable of directly mediating this effect.     

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.     

Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

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Dimorphic effects of Notch signaling in bone homeostasis

Notch signaling is a key mechanism in the control of embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and, specifically, in bone homeostasis, remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis owing to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by upregulating the genes encoding cyclin D, cyclin E and Sp7 (osterix). The intracellular domain of Notch1 also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all Notch signaling in osteoblasts, generated by deletion of the genes encoding presenilin-1 and presenilin-2 in bone, is associated with late-onset, age-related osteoporosis, which in turn results from increased osteoblast-dependent osteoclastic activity due to decreased osteoprotegerin mRNA expression in these cells. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.     

Sphingosine-1-phosphate signaling controlling osteoclasts and bone homeostasis

Bone is a dynamic organ that is continuously turned over during growth, even in adults. During bone remodeling, homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. However, in pathological conditions such as osteoporosis, osteopetrosis, arthritic joint destruction, and bone metastasis, this equilibrium is disrupted. Since osteoclasts are excessively activated in osteolytic diseases, the inhibition of osteoclast function has been a major therapeutic strategy. It has recently been demonstrated that sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. By means of intravital multiphoton imaging of bone tissues, we have recently revealed that the reciprocal action of S1P controls the migration of osteoclast precursors between bone tissues and blood stream. Imaging technologies have enabled us to visualize the in situ behaviors of different cell types in intact tissues. In this review we also discuss future perspectives on this new method in the field of bone biology and medical sciences in general. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation

Bone is constantly resorbed and formed throughout life by coordinated actions of osteoclasts and osteoblasts. Here we show that Smurf1, a HECT domain ubiquitin ligase, has a specific physiological role in suppressing the osteogenic activity of osteoblasts. Smurf1-deficient mice are born normal but exhibit an age-dependent increase of bone mass. The cause of this increase can be traced to enhanced activities of osteoblasts, which become sensitized to bone morphogenesis protein (BMP) in the absence of Smurf1. However, loss of Smurf1 does not affect the canonical Smad-mediated intracellular TGFbeta or BMP signaling; instead, it leads to accumulation of phosphorylated MEKK2 and activation of the downstream JNK signaling cascade. We demonstrate that Smurf1 physically interacts with MEKK2 and promotes the ubiquitination and turnover of MEKK2. These results indicate that Smurf1 negatively regulates osteoblast activity and response to BMP through controlling MEKK2 degradation.     

Phosphate starvation signaling: a threesome controls systemic P(i) homeostasis

Systemic signaling between roots and shoots is required to maintain mineral nutrient homeostasis for optimal metabolism under varying environmental conditions. Recent work has revealed molecular components of a signaling module that controls systemic phosphate homeostasis, modulates uptake and transport in Arabidopsis. This module comprises PHO2, a protein that controls protein stability, the phloem-mobile microRNA-399 and a ribo-regulator that squelches the activity of miR399 towards PHO2 by a novel mechanism. This advance is a significant step for the design of future rational approaches to improve crop phosphate use efficiency.     

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.Eph/ephrin work in the laboratory of the authors is supported by a grant from the Deutsche Forschungsgemeinschaft (Au83/3–2 within the SPP1069 "Angiogenesis")     

Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes

The serine/threonine kinase Akt2 has been implicated in insulin-regulated glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. However, it remains unclear whether activation of Akt2 is sufficient since a role for alternate signaling pathways has been proposed. Here we have engineered 3T3-L1 adipocytes to express a rapidly inducible Akt2 system based on drug-inducible heterodimerization. Addition of the dimerizer rapalog resulted in activation of Akt2 within 5 min, concomitant with phosphorylation of the Akt substrates AS160 and GSK3. Comparison with insulin stimulation revealed that the level of Akt2 activity observed with rapalog was within the physiological range, reducing the likelihood of off-target effects. Transient activation of Akt2 also increased glucose transport and GLUT4 translocation to the plasma membrane. These results show that activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes to an extent similar to insulin.     

KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis

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CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca²⁺-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca²⁺ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca²⁺ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca²⁺ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS–YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca²⁺ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

One-sentence summary: The immunity-related Ca²⁺ channel CYCLIC NUCLEOTIDE-GATED CHANNEL 2 modulates auxin homeostasis and balances cellular auxin perception by influencing auxin biosynthesis.