BMP antagonists and FGF signaling contribute to different domains of the neural plate in Xenopus

In ectodermal explants from Xenopus embryos, inhibition of BMP signaling is sufficient for neural induction, leading to the idea that neural fate is the default state in the ectoderm. Many of these experiments assayed the action of BMP antagonists on animal caps, which are relatively naïve explants of prospective ectoderm, and different results have led to debate regarding both the mechanism of neural induction and the appropriateness of animal caps as an assay system. Here we address whether BMP antagonists are only able to induce neural fates in pre-patterned explants, and the extent to which neural induction requires FGF signaling. We suggest that some discrepancies in conclusion depend on the interpretations of sox gene expression, which we show not only marks definitive neural tissue, but also tissue that is not yet committed to neural fates. Part of the early sox2 domain requires FGF signaling, but in the absence of organizer signaling, this domain reverts to epidermal fates. We also reinforce the evidence that ectodermal explants are naïve, and that explants that lack any dorsal prepattern are readily neuralized by BMP antagonists, even when FGF signaling is inhibited.     

2-Keto-4-methylthiobutyrate, an intermediate in the methionine salvage pathway, is a good substrate for CtBP1

In the present work, we have studied the kinetic properties of the catalytic domain of CtBP1, a co-repressor belonging to the d-2-hydroxyacid dehydrogenase family and known to reduce pyruvate in the presence of NADH. CtBP1 acted on a variety of alpha-keto acids, for which it displayed biphasic curves with inhibition at elevated concentrations, as observed with other dehydrogenases of the same family. Based on catalytic efficiencies, the best substrate was 2-keto-4-methylthiobutyrate, an intermediate of the methionine salvage pathway. It was about 20-fold better than 2-ketoisocaproate and glyoxylate, and 80-fold better than pyruvate. From these data we conclude that 2-keto-4-methylthiobutyrate may be an important regulator of CtBP activity, possibly linking gene repression to the activity of the methionine salvage and spermine synthesis pathways.     

cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitor of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.     

BMP4 regulates vascular progenitor development in human embryonic stem cells through a smad‐dependent pathway

The signals that direct pluripotent stem cell differentiation into lineage‐specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum‐free condition, hESCs sequentially differentiated into CD34+CD31?, CD34+CD31+, and then CD34?CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFβ suppressed BMP4‐induced CD34+CD31+ cell development, and promoted CD34+CD31? cells that failed to give rise to either endothelial or smooth muscle cells. The BMP‐Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad‐dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells. J. Cell. Biochem. 109: 363–374, 2010. © 2009 Wiley‐Liss, Inc.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

Time‐dependent contribution of BMP,FGF, IGF,and HH signaling to the proliferation of mesenchymal stroma cells during chondrogenesis

Early loss of up to 50% of cells is common for in vitro chondrogenesis of mesenchymal stromal cells (MSC) in pellet culture, reducing the efficacy and the tissue yield for cartilage engineering. Enhanced proliferation could compensate for this unwanted effect, but relevant signaling pathways remain largely unknown. The aim of this study was to identify the contribution of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin‐like growth factor (IGF), and hedgehog (HH) signaling toward cell proliferation during chondrogenesis and investigate whether a further mitogenic stimulation is possible and promising. Human MSC were subjected to chondrogenesis in the presence or absence of pathway inhibitors or activators up to Day 14 or from Days 14 to 28, before proliferation, DNA and proteoglycan content were quantified. [3H]‐thymidine incorporation revealed arrest of proliferation on Day 3, after which cell division was reinitiated. Although BMP signaling was essential for proliferation throughout chondrogenesis, IGF signaling was relevant only up to Day 14. In contrast, FGF and HH signaling drove proliferation only from Day 14 onward. Early BMP4, IGF‐1, or FGF18 treatment neither prevented early cell loss nor allowed further mitogenic stimulation. However, application of the HH‐agonist purmorphamine from Day 14 increased proliferation 1.44‐fold (p < 0.05) and late BMP4‐application enhanced the DNA and proteoglycan content, with significant effects on tissue yield. Conclusively, a differential and phase‐dependent contribution of the four pathways toward proliferation was uncovered and BMP4 treatment was promising to enhance tissue yield. Culture forms less prone to size limitations by nutrient/oxygen gradients and a focus on early apoptosis prevention may be considered as the next steps to further enhance chondrocyte formation from MSC.     

Assembly of the human origin recognition complex occurs through independent nuclear localization of its components

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.     

PGF2alpha increases FGF-2 and FGFR2 trafficking in Py1a rat osteoblasts via clathrin independent and importin beta dependent pathway

Previous studies showed that prostaglandin F2alpha (PGF2alpha) stimulated fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2alpha increased the formation of clathrin-coated structures in Py1a rat osteoblasts, they were not involved in FGF-2 and FGFR2 trafficking. PGF2alpha increased binding of FGF-2 and FGFR2 and co-localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF-2 and FGFR2 were in close spatial correlation with importin beta, further supporting nuclear import of the FGF-2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin beta protein labeling in response to PGF2alpha. Similar to PGF2alpha, phorbol 12-myristate 13-acetate (PMA) also increased importin beta protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF-2/FGFR2/importin beta nuclear trafficking.     

Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis

Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.