Recombinant R-spondin2 and Wnt3a Up- and Down-Regulate Novel Target Genes in C57MG Mouse Mammary Epithelial Cells

R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/β-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. We observed the up- and down-regulation of several previously unidentified target genes, including ones that encode proteins involved in immune responses, effectors of other growth factor signaling pathways and transcription factors. Dozens of these changes were validated by quantitative real time RT-PCR. Time course experiments showed that Rspo2 typically had little or no effect on Wnt-dependent gene expression at 3 or 6 h, but enhanced expression at 24 h, consistent with biochemical data indicating that Rspo2 acts primarily to sustain rather than acutely increase Wnt pathway activation. Up-regulation of gene expression was inhibited by pre-treatment with Dickkopf1, a Wnt/β-catenin pathway antagonist, and by siRNA knockdown of β-catenin expression. While Dickkopf1 blocked Rspo2/Wnt3a-dependent down-regulation, a number of down-regulated genes were not affected by β-catenin knockdown, suggesting that in these instances down-regulation was mediated by a β-catenin-independent mechanism

Comparative Study of Hepatocyte Growth Factor/Scatter Factor and Keratinocyte Growth Factor Effects on Human Keratinocytes

Hepatocyte growth factor/scatter factor (HGF/SF) and keratinocyte growth factor (KGF, also designated FGF-7) are paracrine growth factors secreted by mesenchymal cells and active on a variety of epithelial cell types. In this study, the biologic response of keratinocytes to these paracrine growth factors were compared. Stimulation of mitogenesis, migration, plasminogen activator (PA) activity, and fibronectin production were examined using human foreskin keratinocytes cultured in serum-free MCDB 153 medium. Although the two factors stimulated a similar level of proliferation when cells were maintained for 5 d in 1.8 mM Ca++, the peak effect of KGF, observed at 10 ng/ml, was approximately threefold higher than that of HGF/SF when cells were in medium containing 0.15 mM Ca++. Both agents promoted the migration of cells in low-calcium medium (0.08 mM Ca++). However, the magnitude of the response was approximately twofold greater for HGF/SF at 10 ng/ml than KGF at the same concentration. None of the matrix proteins such as type I collagen, type IV collagen, laminin, or fibronectin either stimulated or supressed HGF/SF-or KGF-stimulated kerationocyte migration. Both factors stimulated PA activity was maximal with the addition of 10 ng/ml of either factor. Neither factor increased the production of fibronectin under conditions in which transforming growth factor-β1 was active. These results indicate that HGF/SF and KGF, both recognized as paracrine growth factors, elicit distinctive patterns of response by keratinocytes, implying that they have different roles in epidermal physiology

Using a Mathematical Model to Analyze the Role of Probiotics and Inflammation in Necrotizing Enterocolitis

Background: Necrotizing enterocolitis (NEC) is a severe disease of the gastrointestinal tract of pre-term babies and is thought to be related to the physiological immaturity of the intestine and altered levels of normal flora in the gut. Understanding the factors that contribute to the pathology of NEC may lead to the development of treatment strategies aimed at re-establishing the integrity of the epithelial wall and preventing the propagation of inflammation in NEC. Several studies have shown a reduced incidence and severity of NEC in neonates treated with probiotics (beneficial bacteria species). Methodology/Principal Findings: The objective of this study is to use a mathematical model to predict the conditions under which probiotics may be successful in promoting the health of infants suffering from NEC. An ordinary differential equation model is developed that tracks the populations of pathogenic and probiotic bacteria in the intestinal lumen and in the blood/tissue region. The permeability of the intestinal epithelial layer is treated as a variable, and the role of the inflammatory response is included. The model predicts that in the presence of probiotics health is restored in many cases that would have been otherwise pathogenic. The timing of probiotic administration is also shown to determine whether or not health is restored. Finally, the model predicts that probiotics may be harmful to the NEC patient under very specific conditions, perhaps explaining the detrimental effects of probiotics observed in some clinical studies. Conclusions/Significance: The reduced, experimentally motivated mathematical model that we have developed suggests how a certain general set of characteristics of probiotics can lead to beneficial or detrimental outcomes for infants suffering from NEC, depending on the influences of probiotics on defined features of the inflammatory response. © 2010 Arciero et al

Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and non-canonical Wnt signaling

This research was originally published in Journal of Biological Chemitry. González-Sancho. Functional Consequences of Wnt-Induced Dishevelled2 Phosphorylation in Canonical and Non-Canonical Signaling. Journal of Biological Chemistry . 2013. 288 9428-9437 © the American Society for Biochemistry and Molecular BiologyEl título del postprint: Functional Consequences of Wnt-Induced Dishevelled2 Phosphorylation in Canonical and Non-Canonical SignalingDishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594–609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ϵ as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser594, Thr595, and Ser597 of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA123238 (to A. M. C. B.) and Postdoctoral Fellowship F32 CA117662 (to C. L. A.). This work was also supported by a fellowship from the Ministerio de Educación, Cultura, y Deportes, of Spain (to J. M. G.-S.), by New York State Department of Health postdoctoral Fellowship NYS C021339 (to Y. T.), and by charitable donations to Strang Cancer Prevention Center. This research also was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institut

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site

AbstractBackground: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal.Results: We report here a high-resolution solution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site.Conclusions: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding

Does the load-sharing classification predict ligamentous injury, neurological injury, and the need for surgery in patients with thoracolumbar burst fractures?: Clinical article.

OBJECT: The load-sharing score (LSS) of vertebral body comminution is predictive of results after short-segment posterior instrumentation of thoracolumbar burst fractures. Some authors have posited that an LSS \u3e 6 is predictive of neurological injury, ligamentous injury, and the need for surgical intervention. However, the authors of the present study hypothesized that the LSS does not predict ligamentous or neurological injury. METHODS: The prospectively collected spinal cord injury database from a single institution was queried for thoracolumbar burst fractures. Study inclusion criteria were acute (\u3c 24 hours) burst fractures between T-10 and L-2 with preoperative CT and MRI. Flexion-distraction injuries and pathological fractures were excluded. Four experienced spine surgeons determined the LSS and posterior ligamentous complex (PLC) integrity. Neurological status was assessed from a review of the medical records. RESULTS: Forty-four patients were included in the study. There were 4 patients for whom all observers assigned an LSS \u3e 6, recommending operative treatment. Eleven patients had LSSs ≤ 6 across all observers, suggesting that nonoperative treatment would be appropriate. There was moderate interobserver agreement (0.43) for the overall LSS and fair agreement (0.24) for an LSS \u3e 6. Correlations between the LSS and the PLC score averaged 0.18 across all observers (range -0.02 to 0.34, p value range 0.02-0.89). Correlations between the LSS and the American Spinal Injury Association motor score averaged -0.12 across all observers (range -0.25 to -0.03, p value range 0.1-0.87). Correlations describing the relationship between an LSS \u3e 6 and the treating physician\u27s decision to operate averaged 0.17 across all observers (range 0.11-0.24, p value range 0.12-0.47). CONCLUSIONS: The LSS does not uniformly correlate with the PLC injury, neurological status, or empirical clinical decision making. The LSSs of only one observer correlated significantly with PLC injury. There were no significant correlations between the LSS as determined by any observer and neurological status or clinical decision making

Correction: Rapid chemokinetic movement and the invasive potential of lung cancer cells; a functional molecular study

This is a correction of an earlier published article

A high-flux source of polarization-entangled photons from a periodically-poled KTP parametric downconverter

We have demonstrated a high-flux source of polarization-entangled photons using a type-II phase-matched periodically-poled KTP parametric downconverter in a collinearly propagating configuration. We have observed quantum interference between the single-beam downconverted photons with a visibility of 99% and a measured coincidence flux of 300/s/mW of pump. The Clauser-Horne-Shimony-Holt version of Bell's inequality was violated with a value of 2.711 +/- 0.017.Comment: 7 pages submitted to Physical Review