The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

BACKGROUND: By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. METHODS: Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. RESULTS: Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. CONCLUSION: We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132

Urokinase Plasminogen Activator Induces Pro-Fibrotic/M2 Phenotype in Murine Cardiac Macrophages

<div><p>Objective</p><p>Inflammation and fibrosis are intertwined in multiple disease processes. We have previously found that over-expression of urokinase plasminogen activator in macrophages induces spontaneous macrophage accumulation and fibrosis specific to the heart in mice. Understanding the relationship between inflammation and fibrosis in the heart is critical to developing therapies for diverse myocardial diseases. Therefore, we sought to determine if uPA induces changes in macrophage function that promote cardiac collagen accumulation.</p> <p>Methods and Results</p><p>We analyzed the effect of the uPA transgene on expression of pro-inflammatory (M1) and pro-fibrotic (M2) genes and proteins in hearts and isolated macrophages of uPA overexpressing mice. We found that although there was elevation of the pro-inflammatory cytokine IL-6 in hearts of transgenic mice, IL-6 is not a major effector of uPA induced cardiac fibrosis. However, uPA expressing bone marrow-derived macrophages are polarized to express M2 genes in response to IL-4 stimulation, and these M2 genes are upregulated in uPA expressing macrophages following migration to the heart. In addition, while uPA expressing macrophages express a transcriptional profile that is seen in tumor–associated macrophages, these macrophages promote collagen expression in cardiac but not embryonic fibroblasts.</p> <p>Conclusions</p><p>Urokinase plasminogen activator induces an M2/profibrotic phenotype in macrophages that is fully expressed after migration of macrophages into the heart. Understanding the mechanisms by which uPA modulates macrophage function may reveal insights into diverse pathologic processes.</p> </div

SR-uPA^+/0 macrophages are polarized to adopt M2 phenotype.

<p><b>A.</b> Expression of M2 genes measured by <i>qrtPCR</i> in SR-uPA^+/0 and NTG macrophages. Columns represent the mean of arbitrary <i>C</i> units normalized to <i>Gapdh</i>, error bars are S.D., n = 5 mice per genotype. <b>B.</b> Increase in expression of markers of M2 activation in response to IL-4 (10 ng/ml) in SR-uPA^+/0 and NTG bone marrow derived macrophages. Columns represent means and error bars represent S.E.M. <i>P</i> values are calculated by Mann-Whitney as data were non-parametric, n = 5–6 mice per genotype. <b>C.</b> Production of TNF-alpha protein after serial treatment with IL-4 followed by LPS in SR-uPA^+/0 and NTG macrophages. Circles represent the mean of duplicate samples from individual mice, n  = 3 mice per genotype. <b>D.</b> Fold increase in expression of TNF-alpha mRNA after treatment with LPS or LPS+IL-4 in SR-uPA^+/0 and NTG macrophages, n  = 4 mice per genotype. <b>E.</b> Arginase activity in conditioned media from SR-uPA^+/0 and NTG macrophages. N = 4 mice per genotype <b>F.</b> Immunoblot for Arg1 in protein extract from SR-uPA^+/0 and NTG macrophages.</p

Cardiac IL-6 is elevated but does not mediate uPA-induced cardiac fibrosis.

<p><b>A.</b> IL-6 concentrations in explant culture media from hearts of SR-uPA^+/0 and NTG mice. N  = 5 mice per genotype. <b>B.</b> Collagen content of hearts from SR-uPA^+/0 and NTG littermates at 15 weeks of age. Solid circles represent mice heterozygous for IL-6; open circles are mice homozygous (+/+ or −/−) for IL-6. Bars are medians. N  = 3–5 mice per heterozygous or homozygous genotype. <b>C.</b> Macrophage accumulation in hearts of SR-uPA^+/0 and NTG littermates at 7 weeks of age. Solid circles represent mice heterozygous for IL-6; open circles are mice homozygous (+/+ or −/−) for IL-6. Bars are medians. N  = 3–5 mice per heterozygous or homozygous genotype. Representative heart sections stained with picrosirius red of <b>D.</b> NTG mice, <b>E.</b> SR-uPA^+/0<i>il6</i>^+/+ and <b>F.</b> SR-uPA^+/0<i>il6</i>^−/− mice. Bars represent 100 µm.</p

Transcriptome analysis of SR-uPA^+/0 versus NTG macrophages.

<p>Interaction networks in which key molecules are transcriptionally differentially regulated in SR-uPA^+/0 vs. NTG macrophages. Ingenuity Pathways Analysis (IPA) was used to make the network diagrams. In each network diagram, nodes represent specific molecules and transcriptional changes in SR-uPA^+/0 vs. nontransgenic macrophages are indicated with blue or red circles (red = upregulated in SR-uPA^+/0 vs. nontransgenic macrophages, green = downregulated).</p

Pro-fibrotic effect of SR-uPA^+/0 macrophages is limited to cardiac fibroblasts.

<p>Fold increase in expression in <i>Col1a1</i> mRNA in response to conditioned media from SR-uPA^+/0 in comparison to NTG macrophages in <b>A.</b> isolated cardiac fibroblasts <b>B.</b> NIH-3T3 cells. Error bars represent S.D. N  = 4–6 mice per genotype. <b>C. </b><i>Col1a1</i> expression (arbitrary <i>C</i> units normalized to <i>Gapdh</i>) in cardiac fibroblasts treated with conditioned media from SR-uPA^+/0 macrophages treated with BEC or vehicle, N  = 4 mice per treatment, error bars represent S.D.</p

SR-uPA^+/0 macrophages adopt full M2 phenotype in the heart.

<p>Picrosirius red stain for collagen in hearts from <b>A.</b> 5 weeks old, <b>B.</b> 7 weeks old, and <b>C.</b> 11 weeks old SR-uPA^+/0 mice. Bars represent 100 µm. <b>D.</b> Fold increase in expression of <i>Arg1</i>, <i>Ym1</i> and <i>Fizz1</i> mRNA in CD-45+ cell fractions from hearts of SR-uPA^+/0 mice at time-points of increasing fibrosis. Star (*) indicates significant <i>P</i> values versus 4–5 week time-point. N  = 4–7 mice per time-point. <b>E.</b> Comparison of <i>Arg1</i> expression from bone marrow macrophages, cardiac macrophages (CD45+) and cardiac CD45 negative fraction (CD45–). Pound (#) indicates significant <i>P</i> values versus other cell types, n  = 6–7 mice per condition. <b>F.</b> Arginase activity in cardiac explant conditioned media from SR-uPA^+/0 and NTG hearts, n  = 7–8 mice per genotype.</p

TGF- 1 Limits Plaque Growth, Stabilizes Plaque Structure, and Prevents Aortic Dilation in Apolipoprotein E-Null Mice

OBJECTIVE: Impairment of transforming growth factor (TGF)-β1 signaling accelerates atherosclerosis in experimental mice. However it is uncertain whether increased TGF-β1 expression would retard atherosclerosis. The role of TGF-β1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-β1 in hyperlipidemic mice affects atherogenesis and aortic dilation. METHODS AND RESULTS: We generated apolipoprotein E-null mice with transgenes that allow regulated overexpression of active TGF-β1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-β1, less aortic root atherosclerosis (P ≤ 0.002), fewer lesions in the thoracic and abdominal aortae (P ≤ 0.01), less aortic root dilation (P < 0.001), and fewer pseudoaneurysms (P = 0.02). Mechanistic studies revealed no effect of TGF-β1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-β1-overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. CONCLUSIONS: When overexpressed in the heart and plasma, TGF-β1 is an anti-atherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall

Plasminogen mediates the atherogenic effects of macrophage-expressed urokinase and accelerates atherosclerosis in apoE-knockout mice

Urokinase-type plasminogen activator (uPA) is expressed at elevated levels in atherosclerotic human arteries, primarily in macrophages. Plasminogen (Plg), the primary physiologic substrate of uPA, is present at significant levels in blood and interstitial fluid. Both uPA and Plg have activities that could affect atherosclerosis progression. Moreover, correlations between increased Plg activation and accelerated atherosclerosis are reported in several human studies. However, a coherent picture of the role of the uPA/Plg system in atherogenesis has not yet emerged, with at least one animal study suggesting that Plg is atheroprotective. We used a transgenic mouse model of macrophage-targeted uPA overexpression in apolipoprotein E-deficient mice to investigate the roles of uPA and Plg in atherosclerosis. We found that macrophage-expressed uPA accelerated atherosclerotic plaque growth and promoted aortic root dilation through Plg-dependent pathways. These pathways appeared to affect lesion progression rather than initiation and to include actions that disproportionately increase lipid accumulation in the artery wall. In addition, loss of Plg was protective against atherosclerosis both in the presence and absence of uPA overexpression. Transgenic mice with macrophage-targeted uPA overexpression reveal atherogenic roles for both uPA and Plg and are a useful experimental setting for investigating the molecular mechanisms that underlie clinically established relationships between uPA expression, Plg activation, and atherosclerosis progression