A JNK inhibitor SP600125 induces defective cytokinesis and enlargement in P19 embryonal carcinoma cells

While analyzing the role of c‐Jun NH₂‐terminal kinase (JNK) in neurogenesis in P19 embryonal carcinoma cells, we noticed that treatment with SP600125, a JNK inhibitor, increased the cell size markedly. SP600125‐induced enlargement of P19 cells was time‐ and dose‐dependent. The increased cell size in response to SP600125 was also detected in B6mt‐1 embryonic stem cells. SP600125 treatment inhibited cell growth and increased DNA contents, indicating the inhibition of cell proliferation resulting from endoreduplication. Concurrently, the gene expression of p21, a regulator of G2/M arrest as well as G1 arrest, was increased in cells treated with SP600125. The increased cell size in response to SP600125 was detected even in P19 cells treated with colcemide, an inhibitor of cell cycle progression at the metaphase. The present study suggests that treatment with SP600125 progresses the cell cycle, skipping cytokinesis in P19 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by diallyl disulfide (DADS)

Angiogenesis is a crucial step in the growth and metastasis of cancers. The activation of endothelial cells and their further behaviour are very critical during angiogenesis. We analyzed the effect of diallyl disulfide (DADS) on angiogenesis in in vitro models using human umbilical vein endothelial cells (HUVECs). DADS significantly inhibited endothelial cell migration, invasion and tube formation. (3)H-thymidine proliferation assay clearly showed the inhibitory effect of DADS on the proliferation of HUVECs in vitro. The role of metalloproteinases has been shown to be important in angiogenesis; therefore, zymography was performed to determine whether DADS affected protease activity. Gelatin zymographic analysis showed the inhibitory effect of DADS on the activation of matrix metalloproteinases-MMP-2 and MMP-9. These findings suggest that DADS acts as an angiogenesis inhibitor by inhibiting the activation of matrix metalloproteinases during endothelial morphogenesis.     

Homocysteine modulates the proteolytic potential of human vascular endothelial cells

Pathological levels of homocysteine induce a metalloproteinase-dependent degradation of the elastic structures in arterial wall. This elastolytic process is preferentially localized toward the internal elastic laminae and in the first layers of the media, suggesting endothelium could participate in extracellular matrix degradation induced by homocysteine. Therefore, we studied the effects of homocysteine on proteolytic potential of endothelial cells. Human umbilical vein endothelial cells were cultured with concentrations of homocysteine matching human physiological (10 microM) and pathological (50, 100, and 250 microM) plasma homocysteine levels. Pathological levels of homocysteine increased the secretion of elastolytic metalloproteinase-2 and -9, but not of metalloproteinase-3 and -7. Homocysteine also increased the expression of human tissue kallikrein, a potential activator of matrix metalloproteinase-2 and -9, while the expression of urokinase plasminogen activator was not altered. These results suggest vascular endothelial cells could participate in the subendothelial degradation of the arterial elastic structures occurring in hyperhomocysteinemia.     

The effect of a native collagen gel substratum on the synthesis of collagen by bovine brain capillary endothelial cells

Cultured capillary endothelial cells, derived from bovine brain, and maintained on a plastic substratum synthesized predominantly interstitial collagens of which approximately 75 per cent were secreted into the medium. When grown on a native hydrated collagen type I gel, although no marked alteration in the 'collagen synthetic pattern' was observed, the overall level of collagen synthesis was increased by approximately 100 per cent. More dramatic, however, was the alteration in the distribution of these molecules between medium and cell layer. Interstitial collagens produced by cells grown on collagen gels were almost exclusively associated with the cell layer or collagenous gel. These studies, thus, demonstrate that an extracellular matrix may exert a considerable influence on the cellular synthetic activities and possibly cellular polarity of capillary endothelial cells.     

c-Jun NH2-terminal kinase (JNK)-dependent nuclear translocation of apoptosis-inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI-231 B lymphoma cells

WEHI-231 B lymphoma cells have been employed for analysis of antigen-induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis-inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI-231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan-caspase inhibitor BD-fmk blocked mIg-mediated increase in cells with sub-G1 DNA content, whereas it did not affect mIg-mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant-negative form of c-Jun NH2-terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI-231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl-xL, but not by BD-fmk. Moreover, AIF-deficient clones via small interfering RNA (siRNA)-mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF-deficient clones displayed an enhanced sensitivity to mIg-mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti-apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction.     

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2)

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.     

Ontogeny and regulation of matrix metalloproteinase activity in the zebrafish embryo by in vitro and in vivo zymography

Remodeling of the extracellular matrix (ECM) during development, angiogenesis, wound healing, tumor metastasis, and other morphogenetic processes depends on the exquisitely regulated activities of matrix metalloproteinases (MMPs). Yet very little is known about the activity patterns of these proteases in vivo. We have employed fluorescent MMP-substrates, both in vitro and in vivo, to characterize patterns of MMP activity in the zebrafish embryo. Qualitatively similar patterns of degradation are detected using native Type I or Type IV collagen substrates, suggesting that multiple MMPs are being regulated concomitantly. MMP activity is observed primarily in ECM-rich structures predicted to be undergoing active remodeling, such as the perichordal sheath and somite boundaries. Patterns of Type I and Type IV collagen hydrolysis are similar, but not identical in embryos of any given stage. Conventional gelatin zymography shows MMPs present in embryos as early as 3-somites (11 h) and our in vivo assays detect Type IV collagen degradation at somite boundaries as early as 4-somites (11.5 h). However, we are unable to detect significant in vitro activity using homogenates made from embryos prior to Prim-16 (31 h). Mixed lysate assays demonstrate that this is the result of endogenous inhibitors present in early embryos, suggesting a model of matrix remodeling regulated by spatially heterogeneous MMP inhibition.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

Adenosine is a negative regulator of NF-kappaB and MAPK signaling in human intestinal epithelial cells

Previous studies suggest that adenosine possesses anti-inflammatory properties, however, the mechanisms by which adenosine affects immune function remain unclear, particularly in the intestine. In this study, we hypothesized that adenosine directly affects pro-inflammatory gene expression in intestinal epithelial cells through modulation of NF-kappaB signaling. HT-29 cells were treated with adenosine prior to incubation with various stimuli and pro-inflammatory gene expression and signal transduction analyzed. Adenosine pretreatment resulted in a reduction in IL-8 expression and secretion in response to TNF-alpha, IL-1, LPS, and PMA. This effect was paralleled by inhibition of kappaB-driven luciferase expression and a reduction in recruitment of NF-kappaB to the IL-8 promoter. Pretreatment of HT-29 cells also resulted in reduced ERK, p38, and JNK MAPK phosphorylation, following TNF-alpha treatment. The observed effects in this study occurred independently of known surface adenosine receptors. This study identifies adenosine as a potent negative regulator of pro-inflammatory signaling in intestinal epithelial cells.