Reciprocal Regulation of Shh Trafficking and H2O2 Levels via a Noncanonical BOC-Rac1 Pathway.

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

H2O2 and Engrailed 2 paracrine activity synergize to shape the zebrafish optic tectum.

Although a physiological role for redox signaling is now clearly established, the processes sensitive to redox signaling remains to be identified. Ratiometric probes selective for H2O2 have revealed its complex spatiotemporal dynamics during neural development and adult regeneration and perturbations of H2O2 levels disturb cell plasticity and morphogenesis. Here we ask whether endogenous H2O2 could participate in the patterning of the embryo. We find that perturbations of endogenous H2O2 levels impact on the distribution of the Engrailed homeoprotein, a strong determinant of midbrain patterning. Engrailed 2 is secreted from cells with high H2O2 levels and taken up by cells with low H2O2 levels where it leads to increased H2O2 production, steering the directional spread of the Engrailed gradient. These results illustrate the interplay between protein signaling pathways and metabolic processes during morphogenetic events.

Bidirectional transfer of homeoprotein EN2 across the plasma membrane requires PIP2.

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates in the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms, but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer within HeLa cells in culture, we demonstrate a core role of phosphatidylinositol (4,5)-bisphosphate (PIP2) together with cholesterol in the translocation of the homeobox protein engrailed-2 (EN2) across the plasma membrane. By using drug and enzyme treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement for PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased in a paracrine-deficient mutant of EN2. We propose that the bidirectional plasma membrane translocation events that occur during homeoprotein secretion and internalization are parts of a common process.

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment.

Mitotic spindles assemble from two centrosomes, which are major microtubule-organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an "inside-out" mechanism, ending with establishment of the poles. We used HSET (kinesin-14) as a tool to shift meiotic spindle assembly toward a mitotic "outside-in" mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic-like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique "inside-out" mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.

Nerves, H2O2 and Shh: Three players in the game of regeneration.

The tight control of reactive oxygen species (ROS) levels is required during regeneration. H2O2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H2O2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H2O2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H2O2 levels to their original state. A similar regulatory loop between H2O2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity.

A soft cortex is essential for asymmetric spindle positioning in mouse oocytes.

At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during meiosis I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos-MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.

Link between adipose tissue angiogenesis and fat accumulation in severely obese subjects.

BACKGROUND: White adipose tissue (WAT) can rapidly expand or regress under different nutritional conditions. The role of angiogenesis in the expandability of human adipose tissue is established. However, whether sc and omental WAT (scWAT and oWAT) angiogenesis could influence fat distribution and metabolic diseases is not known. AIM: The aim of this study was to analyze whether the capacity of angiogenesis in scWAT and oWAT correlates with fat accumulation and fat loss, fat distribution, adipocyte hypertrophy, and metabolic disorders in obese subjects. METHODS: Samples of scWAT and oWAT were obtained during bariatric surgery in 29 obese nondiabetic subjects. Vascular density and inflammatory infiltrate were analyzed by immunohistochemistry, and expression of angiogenic genes was analyzed by quantitative PCR. These parameters were correlated with anthropometric and metabolic parameters. RESULTS: Vascular density of scWAT correlated positively with body mass index, whereas vascular density of the oWAT correlated with waist circumference. There was no correlation of markers of angiogenesis and metabolic disorders. The number of vessels per adipocyte and the expression level of receptor 2 of vascular endothelial growth factor correlated with adipocyte area in scWAT and oWAT. Finally, weight loss after bariatric surgery correlated negatively with adipocyte hypertrophy and vascular density and positively with inflammation and angiogenesis of WAT. CONCLUSION: Angiogenesis may influence WAT expansion and plasticity but does not appear to be involved in the development of insulin resistance in subjects with severe obesity.

Data supporting a new physiological role for brain apelin in the regulation of hypothalamic oxytocin neurons in lactating rats.

Apelin is a bioactive peptide identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ in 1998. The present data show that apelin modulates the activity of magnocellular and parvocellular oxytocin (OXY) neurons in the lactating rat. A combination of in situ hybridization and immunohistochemistry demonstrated the presence of apelin receptor mRNA in hypothalamic OXY neurons. Double immunofluorescence labeling then revealed the colocalization of apelin with OXY in about 20% of the hypothalamic OXY-positive neurons. Intracerebroventricular apelin administration inhibited the activity of magnocellular and parvocellular OXY neurons, as shown by measuring the c-fos expression in OXY neurons or by direct electrophysiological measurements of the electrical activity of these neurons. This effect was correlated with a decrease in the amount of milk ejected. Thus, apelin inhibits the activity of OXY neurons through a direct action on apelin receptors expressed by these neurons in an autocrine and paracrine manner. In conclusion, these findings highlight the inhibitory role of apelin as an autocrine/paracrine peptide acting on OXY neurons during breastfeeding.

Pathologic calcification of adult vascular smooth muscle cells differs on their crest or mesodermal embryonic origin.

Vascular calcifications can occur in the elderly and in patients suffering from various diseases. Interestingly, depending on the pathology, different regions of the arterial system can be affected. Embryonic observations have clearly indicated that vascular smooth muscle cell (VSMC) origin is notably heterogeneous. For instance, in the aorta, VSMCs colonizing the aortic arch region derive from cardiac neural crest cells, whereas those populating the descending aorta derive from the mesoderm. We examined here whether the embryonic origin of aortic VSMCs would correlate with their ability to mineralize. Under hyperphosphatemic conditions that induce vascular calcifications, we performed ex vivo aortic explant cultures as well as in vitro VSMC cultures from wild-type mice. Our data showed that VSMC embryonic origin affects their ability to mineralize. Indeed, the aortic arch media made up of VSMCs of neural crest origin calcifies significantly earlier than the descending aorta composed of VSMCs, which are mesoderm-derived. Similar results were obtained with cultured VSMCs harvested from both aortic regions. We also demonstrated that in a mouse model deficient in matrix Gla protein, a potent calcification inhibitor, developing extensive and spontaneous medial calcifications of the aorta, lesions initiate in the aortic arch. Subsequently, calcifications progress outside the aortic arch region and ultimately spread all over the entire arterial tree, including the descending aorta. Altogether, our results support an unsuspected correlation between VSMCs of embryonic origin and the timing of appearance of calcifications.

Traditional anthropometric parameters still predict metabolic disorders in women with severe obesity.

It is well established that fat distribution rather than the total quantity of fat is the major determinant of cardiovascular risk in overweight subjects. However, it is not known whether the concept of fat distribution still makes sense in severely obese subjects. Particularly, the role of visceral fat accumulation and/or of adipocyte hypertrophy in insulin resistance (IR) has not been studied in this population. Therefore, the aim of this study was to clarify the determinants of metabolic disorders in severely obese women. We performed a cross-sectional study in 237 severely obese women (BMI >35 kg/m(2)). We assessed total body fat mass and fat distribution by anthropometric measurements (BMI and waist-to-hip ratio (WHR)) and by dual-energy X-ray absorptiometry (DXA). In 22 women, we measured subcutaneous and visceral adipocyte size on surgical biopsies. Mean BMI was 44 +/- 7 kg/m(2) (range 35-77), mean age 37 +/- 11 years (range 18-61). Lipid parameters (triglycerides, high-density lipoprotein cholesterol) and IR markers (fasting insulin and homeostasis model assessment (HOMA) index) correlated with fat distribution, whereas inflammatory parameters (C-reactive protein, fibrinogen) correlated only with total fat mass. An association was observed between android fat distribution and adipocyte hypertrophy. Visceral adipocyte hypertrophy was associated with both IR and hypertension, whereas subcutaneous fat-cell size was linked only to hypertension. Our results obtained in a large cohort of women showed that fat distribution still predicts metabolic abnormalities in severe obesity. Furthermore, we found a cluster of associations among fat distribution, metabolic syndrome (MS), and adipocyte hypertrophy.

Angiogenesis associated with visceral and subcutaneous adipose tissue in severe human obesity.

OBJECTIVE: The expansion of adipose tissue is linked to the development of its vasculature. However, the regulation of adipose tissue angiogenesis in humans has not been extensively studied. Our aim was to compare the angiogenesis associated with subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from the same obese patients in an in vivo model. RESEARCH DESIGN AND METHODS: Adipose tissue samples from visceral (VAT) and subcutaneous (SAT) sites, obtained from 36 obese patients (mean BMI 46.5 kg/m(2)) during bariatric surgery, were layered on chick chorioallantoïc membrane (CAM). RESULTS: Both SAT and VAT expressed angiogenic factors without significant difference for vascular endothelial growth factor (VEGF) expression. Adipose tissue layered on CAM stimulated angiogenesis. Angiogenic stimulation was macroscopically detectable, with engulfment of the samples, in 39% and was evidenced by angiography in 59% of the samples. A connection between CAM and adipose tissue vessels was evidenced by immunohistochemistry, with recruitment of both avian and human endothelial cells. The angiogenic potency of adipose tissue was not related to its localization (with an angiogenic stimulation in 60% of SAT samples and 61% of VAT samples) or to adipocyte size or inflammatory infiltrate assessed in adipose samples before the graft on CAM. Stimulation of angiogenesis by adipose tissue was nearly abolished by bevacizumab, which specifically targets human VEGF. CONCLUSIONS: We have established a model to study the regulation of angiogenesis by human adipose tissue. This model highlighted the role of VEGF in angiogenesis in both SAT and VAT.

Anti-angiogenic properties of myo-inositol trispyrophosphate in ovo and growth reduction of implanted glioma.

We investigate here the anti-angiogenic properties of the synthetic compound myo-inositol trispyrophosphate (ITPP). By increasing oxy-haemoglobin dissociation, ITPP has the potential to counteract the effects of hypoxia, a critical regulator of angiogenesis and cancer progression. ITPP inhibited angiogenesis of the chorioallantoic membrane (CAM), as analyzed with an original program dedicated to automated quantification of angiogenesis in this model. ITPP also markedly reduced tumor progression and angiogenesis in an experimental model of U87 glioma cell nodules grafted onto the CAM. These results point out the potential of ITPP for the development of a new class of anti-angiogenic and anti-cancer compounds.

Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2).

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose-response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.

Effects of juvenile hormone on 20-hydroxyecdysone-inducible EcR, HR3, E75 gene expression in imaginal wing cells of Plodia interpunctella lepidoptera.

The IAL-PID2 cells derived from imaginal wing discs of the last larval instar of Plodia interpunctella were responsive to 20-hydroxyecdysone (20E). These imaginal cells respond to 20E by proliferative arrest followed by a morphological differentiation. These 20E-induced late responses were inhibited in presence of juvenile hormone (JH II). From these imaginal wing cells, we have cloned a cDNA sequence encoding a P. interpunctella ecdysone receptor-B1 isoform (PIEcR-B1). The amino acid sequence of PIEcR-B1 showed a high degree of identity with EcR-B1 isoforms of Bombyx mori, Manduca sexta and Choristoneura fumiferana. The pattern of PIEcR-B1mRNA induction by 20E was characterized by a biphasic response with peaks at 2 h and 18 h. The presence of the protein synthesis inhibitor anisomycin induced a slight reduction in level of PIEcR-B1 mRNA and prevented the subsequent declines observed in 20E-treated cells. Therefore, PIEcR-B1 mRNA was directly induced by 20E and its downregulation depended on protein synthesis. An exposure of imaginal wing cells to 20E in the presence of JH II caused an increased expression of Plodia E75-B and HR3 transcription factors but inhibited the second increase of PIEcR-B1 mRNA. These findings showed that in vitro JH II was able to prevent the 20E-induced differentiation of imaginal wing cells. This effect could result from a JH II action on the 20E-induced genetic cascade through a modulation of EcR-B1, E75-B and HR3 expression.

Putative odorant-degrading esterase cDNA from the moth Mamestra brassicae: cloning and expression patterns in male and female antennae.

An esterase cDNA was isolated from the cabbage armyworm Mamestra brassicae antennae by PCR strategy. The full-length cDNA, designated as Mbra-EST, contains a 1638 bp open reading frame encoding a predicted protein of 546 amino acids. This predicted protein presents the structural characteristics of known insect carboxyl-esterases, in particular the Ser-His-Glu catalytic triad. The expression pattern of the gene was studied by RT-PCR, Northern-blot and in situ hybridization. The ribosomal protein rpL8 gene from M. brassicae was also cloned to obtain a normalized tool for the comparative gene expression studies. Mbra-EST transcripts are specifically expressed in the antennae of males and females and in the proboscis of males. In antennae of both sexes, expression is restricted to the olfactory sensilla trichodea, suggesting a role in degradation of odorant acetate compounds, such as pheromones as well as plant volatile acetate components.