Intermolecular Decarboxylative Direct C-3 Arylation of Indoles with Benzoic Acids

A palladium catalyzed C−H activation of indoles and a silver catalyzed decarboxylative C−C activation of ortho substituted benzoic acids are synergistically combined to synthesize indoles arylated exclusively in the C-3 position. This novel decarboxylative C−H arylation methodology is compatible with electron-donating and -withdrawing substituents in both coupling partners

Modulation of Fibroblast Growth Factor Signaling Is Essential for Mammary Epithelial Morphogenesis

<div><p>Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that <i>Spry2</i> gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that <i>Spry2</i> is expressed at various stages of the developing mammary gland. Targeted removal of <i>Spry2</i> function from mammary epithelium leads to accelerated epithelial invasion. <i>Spry2</i> is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, <i>Spry2</i> gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of <i>Spry2</i> expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by <i>Spry2</i> is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.</p></div

Gain of Spry2 function in the mammary epithelium reduces FGF signaling activities, epithelial invasion and branching in vitro.

<p>(<b>A</b>) <i>Spry2</i> expression, as measured by qPCR, in control and mutant MECs. MECs were prepared similar to the scheme described in Fig. 3B. Adenovirus-transduced control (<i>Spry2</i>^+/+) and mutant (<i>Spry2-</i>GOF) MECs were GFP⁺ and were purified by FACS. Purified cells were used for RNA harvest and qPCR assays. (<b>B–C</b>) Expression of the FGF signaling target genes <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i> in control and mutant organoids in response to 24-hour treatment of FGF2 (200 ng/ml, <b>B</b>) or FGF10 (200 ng/ml, <b>C</b>). Expression is relative to that of the control samples. Values shown are the mean ± standard deviation (SD) of three independent experiments. Statistically significant differences of p<0.05 (t test) were observed between expression of control and mutant samples for all genes except for <i>Etv5</i>. (<b>D–F</b>) in vitro branching assay in which control (<b>D</b>) and <i>Spry2-</i>GOF mutant organoids (<b>E</b>) were subjected to cultures in basal medium containing FGF2. When stimulated by FGF2 at progressively higher concentrations from 0.05 nM to 0.5 nM, a progressively higher percentage of MECs underwent branching, but a plateau was reached at 1.0 nM. In addition to forming branched structures at a lower than normal percentage, <i>Spry2-</i>GOF mutant structures were overall smaller than those derived from control MECs. Data were from experiments repeated three times or more. At least 100–150 organoids were examined for each treatment conditions. (<b>F</b>) Quantitative comparisons of control and mutant MECs in their ability to undergo epithelial branching in vitro. Values shown are the mean ± SD for each data point: *P<0.0005, unpaired, two-tailed Student’s <i>t</i> tests. Scale bars: 100 μm. (<b>G–R</b>) Mammary epithelial responses to beads pre-soaked in BSA (<b>G–J</b>) or FGF10 (<b>K–R</b>) during a 72-hour time course. Heparin acrylic beads of ∼100 μm in diameter were juxtaposed with mammary organoids at a distance of ∼100–150 μm. Control (con) organoids were on the left and experimental (exp) ones, including <i>Spry2</i>^Δ/Δ (<b>K–N</b>) and <i>Spry2</i>-GOF ones (<b>O–R</b>), were on the right side of heparin beads. Note <i>Spry2</i>^Δ/Δ organoids (n = 4) migrated faster than control ones and reached the bead by 48 hours (<b>K–N</b>) rather than 72 hours that controls took (<b>K–R</b>); by contrast, <i>Spry2</i>-GOF organoids (n = 5) moved much slower than control and had not reached FGF10-beads by 72 hours (<b>O–R</b>). Scale bars: 100μm.</p

Hypoxia induces caspase3 expression without affecting cell viability in microglia.

A. Bar graph shows the viability of BV-2 cells after hypoxia for 12 h compared to control by MTS. Note no significant difference was observed in the two groups. B. PI- Annexin V staining determined by FACS analysis in microglia. C. Western blotting of caspase3 protein expression in BV-2 cells exposed to hypoxia for 2, 4, 6, 8 and 12 h and control (c). The left panel in C shows specific bands of cleaved caspase3 (17 kDa) and β-actin (43 kDa). The right panel in C is a bar graph showing significant changes in the optical density following hypoxic exposure (normalized with β-actin). Note significant increase in cleaved caspase3 expression after hypoxic treatment of varying durations in BV-2 cells, especially at 8 h. D. Confocal images showing cleaved caspase3 expression in control BV-2 cells and those exposed to hypoxia for 8 h. Weak cleaved caspase3 expression is detected in the control BV-2 cells with enhanced immunofluorescence intensity after 8 h of hypoxic exposure. The experiments have been repeated at least in triplicate. Significant differences between control and hypoxic BV-2 cells are calculated using Student’s t-test and expressed as *p .05 and **p .01. The values represent the mean ± SD in triplicate. Scale bar in D = 20 μm.</p

Conditional removal of <i>Spry2</i> function from mammary epithelium causes accelerated epithelial invasion.

<p>(<b>A–H</b>) The mammary branching tree from the #4 glands at the postnatal stages indicated, as revealed by Carmine Red staining of glands in wholemount. (<b>A–C</b>) glands from control (M-Cre;<i>Spry2</i>^fl/+) mice; (<b>D–F</b>) glands from mutant (M-Cre;<i>Spry2</i>^fl/Δ) mice. (A–F) Arrows indicate the extent of ductal penetration in the fat pad. Dotted white line illustrates the epithelial invasion front. (<b>G, H</b>) Quantitative comparisons of ductal penetration and branch point formation between control and mutant glands. At 6 weeks, ductal penetration measurements were 3.8±1.3 (control, n = 10) and 6.5±0.6 (mutant, n = 6); at 8 weeks, the measurements were 8.5±1.5 (control, n = 4) and 10.3±0.8 (mutant, n = 6); at 13 weeks, they were 17.4±1.5 (control, n = 8) and 17.8±1.1 (mutant, n = 14). Measurements of branching points were 2.1±0.7 (control) and 1.7±0.6 (mutant) at 6 weeks, 2.2±0.1 (control) and 2.2±0.2 (mutant) at 8 weeks, and 1.4±0.1 (control) and 1.7±0.2 (mutant) at 13 weeks. Values shown are the mean ± SD for each data point: *, P<0.05, unpaired, two-tailed Student’s <i>t</i> test. N is the number of mammary glands examined. (<b>I</b>, <b>J</b>) Assays for β-GAL activity in wholemount of control (<b>I</b>, M-Cre;<i>Spry2</i>^fl/+;<i>R</i>^fl/+) and mutant (<b>J</b>, M-Cre;<i>Spry2</i>^fl/Δ;<i>R</i>^fl/+) glands at 6-weeks of age. The dashed boxes demarcate the portions of branching trees that are shown at higher magnification in insets. β-GAL expression marks cells derived from those in which MMTV-Cre-mediated recombination occurred. Note that β-GAL-positive <i>Spry2</i> null cells were well represented in the distal branching network, including TEBs of mutant glands (<b>J</b>, n = 18). Scale bars: 2.5 mm.</p

Spry2 expression is reduced in the MMTV-PyMT mouse model.

<p>(<b>A–B</b>) <i>Spry2</i> expression at different stages of cancer progression in the MMTV-PyMT mouse model. (<b>A</b>) <i>Spry2</i> expression in normal ductal epithelium (nor) and PyMT epithelium during the hyperplastic adenoma (hyp) and advanced carcinoma (car) stages relative to that in the distal un-invaded stroma of virgin female mice at 5-weeks of age. Analysis was based on data published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092735#pone.0092735-KourosMehr1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092735#pone.0092735-KourosMehr2" target="_blank">[40]</a>. Epithelium/distal stroma (Cy5/Cy3) expression ratios are shown for three independent experiments (lanes 1–3) and their respective means (M) using the color scale shown, with black indicating no difference in expression, red indicating relative enrichment in normal or cancer epithelium and green representing higher expression in the distal stroma. (<b>B</b>) <i>Spry2</i> mRNA expression was measured by qPCR using RNA harvested from mammary glands at the above stages. Values were normalized against actin expression and <i>Spry2</i> expression in normal glands was set as base value against which other stages were compared. (<b>C</b>) Model diagram depicting <i>Spry2</i> function in regulation of FGF signaling during mammary gland epithelial branching. The terminal end buds (TEBs) develop at the onset of puberty (three weeks after birth) at the distal tip of each primary duct. In the following six weeks the TEBs invade the stroma in a proximal (Pr)-to-distal (Di) direction until the whole fat-pad is occupied by around nine weeks of age. The epithelial network is further elaborated by lateral branches on the side of primary ducts. Regulation of FGF signaling levels is essential for normal epithelial branching morphogenesis in the mammary gland. Stromal FGF10 and FGF2 activate FGFR2 and FGFR1 in the epithelium and stimulate SPRY2 expression. SPRY2 in turn fine-tunes FGF signaling level (yellow bell-shape) and regulates epithelial invasion and branching into the stroma. In the absence of SPRY2 function in the mutant mammary glands, FGF signaling level (green bell-shape) is higher than normal due to the loss of a negative regulator. As a consequence, epithelial invasion into the fat-pad is accelerated (green arrow) during branching morphogenesis. Conversely, SPRY2 expression is augmented in <i>Spry2-</i>GOF mammary glands, due to intrinsic transcriptional regulation and expression from the transgene (curved red arrow) and FGF signaling level (red bell-shape) is reduced as a result. As a consequence, epithelial invasion is stunted (red arrow) during mammary branching at late puberty.</p

Pups generated from self-crosses of <i>Spry2</i> heterozygous mice.

<p>Pups were genotyped on postnatal day 1 (P1; n = 42) and upon weaning on postnatal day 21 (P21; n = 359). Note the actual frequencies (Act.) of both <i>Spry2</i>^Δ/+ and <i>Spry2</i>^+/+ were more than the expected frequencies (Exp.) because a portion of the <i>Spry2</i>^Δ/Δ pups died prior to weaning.</p

Melatonin inhibits TLR4 mRNA and protein expression and caspase-3 activity in hypoxic BV-2 cells.

<p>A. Hypoxia induces TLR4 mRNA expression in hypoxic microglia that is suppressed by melatonin. B. BV-2 cells subjected to hypoxia, followed by western blot analysis of TLR4 and cleaved caspase-3 with or without melatonin treatment. The panel shows specific bands of TLR4 (95 kDa), cleaved caspase-3 (17 kDa) and β-actin (43 kDa). Note TLR4 and cleaved caspase-3 expression in melatonin-treated group is significantly decreased in comparison with the untreated group (normalized with β-actin). The experiments have been repeated at least in triplicate. The statistical significance of differences between different groups was calculated using ANOVA. Significant difference between control vs hypoxia groups is shown as *<i>p</i><0.05 and **<i>p</i><0.01; significant difference between hypoxia vs hypoxia +Melatonin groups is shown as #<i>p</i><0.05 and ##<i>p</i><0.01. The values represent the mean ± SD in triplicate.</p

<i>Spry2</i> null epithelium shows enhanced FGF signaling activities and increased epithelial branching activities.

<p>(<b>A</b>) Expression, as measured by qPCR, of <i>Spry2</i> and target genes of FGF signaling, including <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i>, in response to a 24-hour treatment of FGF2 (10 nM) or FGF10 (10 nM). Expression is relative to that of the untreated samples. Values shown are the mean ± standard deviation (SD) of three independent experiments. Statistically significant differences of p<0.05 (t test) were observed between expression of untreated and treated samples for all genes except for <i>Etv5</i> in response to FGF2 and FGF10 treatment. (<b>B</b>) Schematic diagram depicting the experimental procedure in sample preparation, treatment, and analysis. Mammary organoids were prepared from <i>Spry2</i>^+/+ and <i>Spry2</i>^fl/fl mice and were infected with adenovirus-Cre-GFP, which generated control (<i>Spry2</i>^+/+) and mutant (<i>Spry2</i>^Δ/Δ) organoids, respectively. Transduced cells were then purified by FACS based on their expression of GFP before they were subjected to analyses on gene expression and epithelial morphogenesis in the presence or absence of FGF2 or FGF10. (<b>C–D</b>) Expression, as measured by qPCR, of <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i> in control and mutant MECs in response to 24-hour treatment of FGF2 (200 ng/ml, <b>C</b>) or FGF10 (200 ng/ml, <b>D</b>). Expression is relative to that of the control samples. Statistically significant differences of p<0.05 (t test) were observed between expression of control and mutant samples for all genes except for <i>Etv5</i> in response to FGF2 treatment and <i>Etv4</i> in response to FGF10 treatment. (<b>E–I</b>) in vitro branching assay in which control (<b>E</b>, <b>F</b>) and mutant organoids (<b>G</b>, <b>H</b>) were subjected to cultures in basal medium with (<b>F</b>, <b>H</b>) or without FGF2 (<b>E</b>, <b>G</b>). When stimulated by FGF2 at progressively higher concentrations from 0.025 nM to 0.5 nM, a progressively higher percentage of organoids underwent branching. At 1.0 nM and 2.5 nM, FGF2 did not stimulate a higher percentage of branched organoids to form. In addition to their differences in branching kinetics, <i>Spry2</i>^Δ/Δ organoids overall formed larger branched structures than control organoids. Scale bars: 100 μm. (<b>I</b>) Quantitative comparisons of control and mutant MECs in their ability to undergo epithelial branching in vitro. Data were from experiments repeated three times or more. At least 100–150 organoids were examined for each treatment conditions. Values shown are the mean ± SD for each data point: *P<0.0005, unpaired, two-tailed Student’s <i>t</i> tests.</p

Caspase3 inhibitor reduces release of proinflammatory mediators and NF-κB activation.

Western blotting of TNF-α, IL-1β, iNOS and NF-κB protein expression in BV-2 cells following hypoxic exposure and Z-DEVD-FMK pretreatment. The upper panel shows specific bands of TNF-α (25.6 k Da), IL-1β (17k Da), iNOS (130 kDa), NF-κB /P65 (65k Da) and β-actin (43 kDa. The lower panels are bar graphs showing significant changes in the optical density in protein expression of different groups (normalized with β-actin). Note the decrease in TNF-α, IL-1β, iNOS and NF-κB expression in hypoxia+ Z-DEVD-FMK group compared with hypoxic BV-2 cells. The experiments have been repeated at least in triplicate. The statistical significance of differences between different groups was calculated using ANOVA. Significant difference between control vs hypoxia groups is shown as *pppp<0.01. The values represent the mean ± SD in triplicate.</p