Heonyong Park

Apoptosis signal-regulating kinase 1 controls the proapoptotic function of death-associated protein (Daxx) in the cytoplasm.

Although Daxx (death-associated protein) was first reported to mediate the apoptotic signal from Fas to JNK in the cytoplasm, other data suggested that Daxx is mainly located in the nucleus as a transcriptional regulator. Here, we demonstrated that cellular localization of Daxx could be determined by the relative concentration of a proapoptotic kinase, apoptosis signal-regulating kinase 1 (ASK1) by using immunofluorescence and transcriptional reporter assay. ASK1 sequestered Daxx in the cytoplasm and inhibited the repressive activity of Daxx in transcription. In addition, Daxx was bound to the activated Fas only in the presence of ASK1, accelerating the Fas-mediated apoptosis. These results suggest that Daxx requires ASK1 for its cytoplasmic localization and Fas-mediated signaling. Taken together, we could conclude that ASK1 controls the dual function of Daxx as a transcriptional repressor in the nucleus and as a proapoptotic signal mediator in the cytoplasm.

Novel regulatory interactions and activities of mammalian tRNA synthetases

Aminoacyl‐tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs, thereby ensuring the faithful translation of genetic code. In addition to their enzymatic function, these enzymes have been discovered to regulate various cellular functions such as tRNA export, ribosomal RNA synthesis, apoptosis, inflammation and angiogenesis in mammalian. The insights into the noncanonical activities of these enzymes have been obtained from their unique cellular localization, interacting partners, isoform generation and expression control. Mammalian ARSs also form a macromolecular protein complex with a few auxiliary factors. Although the physiological significance of this complex is poorly understood, it also supports the potential of mammalian ARSs as sophisticated multifunctional proteins for regulating various cellular procedures. In this review, the novel regulatory activities of mammalian ARSs will be discussed in different biological processes.

X-linked inhibitor of apoptosis protein controls α5-integrin-mediated cell adhesion and migration

The association of integrins with caveolin-1 regulates cell adhesion. However, the vascular ramifications of this association remain to be clearly determined. We recently reported that the X chromosome-linked inhibitor of apoptosis protein (XIAP)-caveolin-1 interaction is critical to endothelial cell survival. Thus, we hypothesized that XIAP performs a crucial function in integrin/caveolin-1-mediated endothelial cell survival. In this study, we demonstrated that XIAP is recruited into the alpha(5)-integrin complex via caveolin-1 binding and mediates cell adhesion. We also determined that XIAP is critical to shear stress-stimulated ERK activation in an alpha(5)-integrin-dependent manner but is not important to VEGF-induced ERK activation. This differential activation of ERK is partly attributable to unique localizations of the receptors. Furthermore, we confirmed that XIAP is an essential molecule in the efficient recruitment of focal adhesion kinase (FAK) into the alpha(5)-integrin-associated complex. This alpha(5)-integrin-caveolin-1-XIAP-FAK multicomplex regulates endothelial cell migration via a mechanism that involves shear-dependent ERK activation. Together, our results indicate that XIAP stabilizes the alpha(5)-integrin-associated focal adhesion complex, thereby further regulating endothelial cell adhesion and migration. The findings of this study provide us with greater insight into the molecular mechanisms underlying the control of vascular function by integrins.

XIAP is essential for shear stress-enhanced Tyr-576 phosphorylation of FAK.

In endothelial cells, X-chromosome linked inhibitor of apoptosis protein (XIAP) regulates cell survival, migration and adhesion. We have recently found that XIAP recruits focal adhesion kinase (FAK) into integrin-associated focal adhesions, controlling cell migration. However, little is understood about the molecular mechanisms by which FAK modulation is controlled by XIAP. In this study, we show that XIAP modulates FAK activity through the control of FAK phosphorylation. In bovine aortic endothelial cells (BAEC), phosphorylation of Tyr-576 in FAK is elevated by laminar shear stress. This elevated phosphorylation appears to be responsible for shear stress-stimulated ERK activation. We found that XIAP knockdown reduces shear stress-enhanced phosphorylation of Tyr-576 and induces shear stress-triggered translocation of FAK into nucleus. Nuclear translocation of FAK reduces contact between FAK and Src, a kinase which phosphorylates Tyr-576. This spatial segregation of FAK from Src decreases Tyr-576 phosphorylation and thus shear-stimulated ERK activation. Taken together, our results demonstrate that XIAP plays a key role in shear stress-stimulated ERK activation by maintaining the Src-accessible location of FAK.

Pinosylvin at a high concentration induces AMPK-mediated autophagy for preventing necrosis in bovine aortic endothelial cells

Pinosylvin is a known functional compound of the Pinus species. Pinosylvin at low concentrations (∼ pmol/L) was reported to promote cell proliferation in endothelial cells. However, this study found that pinosylvin at a high concentration (100 μmol/L) induces cell death in bovine aortic endothelial cells. Therefore, we examined how pinosylvin was associated with apoptosis, autophagy, and necrosis. Pinosylvin at a high concentration appeared to promote caspase-3 activation, nuclear condensation, and the flip-flop of phosphatidylserine, indicating that pinosylvin induces apoptosis. However, based on flow cytometry data obtained from double-staining with annexin V and propidium iodide, pinosylvin was shown to inhibit necrosis, a postapoptotic process. Pinosylvin induced LC3 conversion from LC3-I to LC3-II and p62 degradation, which are important indicators of autophagy. In addition, AMP-activated protein kinase (AMPK) appeared to be activated by pinosylvin, and an AMPK inhibitor was markedly shown to reduce the LC3 conversion. The inhibitory effect of an AMPK inhibitor was reversed by pinosylvin. These results suggest that pinosylvin induces autophagy via AMPK activation. Further, necrosis was found to be promoted by an autophagy inhibitor and then restored by pinosylvin, while the caspase-3 inhibitor had no effect on necrosis. These findings indicate that pinosylvin-induced autophagy blocks necrotic progress in endothelial cells.

FERM domain promotes resveratrol-induced apoptosis in endothelial cells via inhibition of NO production

Focal adhesion kinase (FAK) consists of an N-terminal band 4.1; ezrin, radixin, moesin (FERM) domain; tyrosine kinase domain; and C-terminal FA targeting domain. Here we show that ectopically expressed FERM is largely located in the cytosolic fraction under quiescent conditions. We further found that this ectopically expressed FERM domain aggravates endothelial cell apoptosis triggered by 100 μM resveratrol, whereas FERM had no effect on apoptosis induced by TNF-α. We determined that resveratrol at low doses (<20 μM) promotes phosphorylation (S1177) of eNOS via an AMPK-dependent pathway. The presence of the FERM domain blocked this resveratrol-stimulated eNOS phosphorylation and NO production. Thus, the pro-apoptotic activity of cytosolic FERM domain is at least partially mediated by down-regulation of NO, a critical cell survival factor. Consistently, we found that the apoptosis induced by cytosolic FERM in the presence of resveratrol was reversed by an NO donor, SNAP. In conclusion, FERM located in the cytosolic fraction plays a pivotal role in aggravating cell apoptosis through diminishing NO production.

XIAP reverses various functional activities of FRNK in endothelial cells

In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

Extract from Acanthopanax senticosus prevents LPS-induced monocytic cell adhesion via suppression of LFA-1 and Mac-1

A crude extract from Acanthopanax senticosus (AS) has drawn increased attention because of its potentially beneficial activities, including anti-fatigue, anti-stress, anti-gastric-ulcer, and immunoenhancing effects. We previously reported that AS crude extract exerts anti-inflammatory activity through blockade of monocytic adhesion to endothelial cells. However, the underlying mechanisms remained unknown, and so this study was designed to investigate the pathways involved. It was confirmed that AS extract inhibited lipopolysaccharide (LPS)-induced adhesion of monocytes to endothelial cells, and we found that whole extract was superior to eleutheroside E, a principal functional component of AS. A series of PCR experiments revealed that AS extract inhibited LPS-induced expression of genes encoding lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) in THP-1 cells. Consistently, protein levels and cell surface expression of LFA-1 and Mac-1 were noticeably reduced upon treatment with AS extract. This inhibitory effect was mediated by the suppression of LPS-induced degradation of IκB-α, a known inhibitor of nuclear factor-κB (NF-κB). In conclusion, AS extract exerts anti-inflammatory activity via the suppression of LFA-1 and Mac-1, lending itself as a potential therapeutic galenical for the prevention and treatment of various inflammatory diseases.

Apoptotic Effect of Pinosylvin at a High Concentration Regulated by c-Jun N-Terminal Kinase in Bovine Aortic Endothelial Cells

Pinosylvin is a stilbenoid found in the Pinus species. Pinosylvin at ~pM to ~nM concentrations induces cell proliferation, cell migration and anti-inflammatory activity in endothelial cells. However, it was recently reported that pinosylvin at high concentrations (50 to 100 μM) induces cell death in bovine aortic endothelial cells. In this study, we conducted a series of experiments to discover how pinosylvin at a high concentration (50 μM) induces endothelial cell death. Pinosylvin at the high concentration was shown to induce endothelial cell apoptosis through enhancing caspase-3 activity, flip-flop of phosphatidyl serine, and nuclear fragmentation. We found that pinosylvin at the high concentration additively increased caspase-3 activity enhanced by serum-starvation or treatment with 100 μM etoposide. We also determined that pinosylvin at the high concentration promoted activations of c-Jun N-terminal kinase (JNK) and endothelial nitric oxide synthetase (eNOS). We further ran a series of experiments to find out which signaling molecule plays a critical role in the pinosylvin-induced apoptosis. We finally found that SP-600125, a JNK inhibitor, had an inhibitory effect on the pinosylvin-induced endothelial cell death, but L-NAME, an eNOS inhibitor, had no effect. These data indicate that JNK is involved in the pinosylvin-induced apoptosis. Collectively, pinosylvin at high doses induces cell apoptosis via JNK activation.

Effect of Alliin on Vascular Functions

Little is known about the cardiovascular roles of alliin, a functional component in garlic that has been used as food material. Thus, we examined a broad range of cardiovascular activities of alliin in this study. From our in vitro experiments, alliin was determined to act as a stimulant to induce endothelial cell proliferation and endothelial cell migration. Since endothelial cell proliferation and migration are highly associated with angiogenesis and wound healing, alliin is suggested as a regulator to control angiogenesis and wound healing. In addition, alliin was elucidated to prevent lipopolysaccharide (LPS)-induced adhesion of THP-1 leukocytes to endothelial cells and LPS-induced homotypic THP-1 cell aggregation. These inhibitory effects indicate that alliin is likely to act as an anti-atherosclerotic and anti-thrombotic factor, because leukocytic adhesion to endothelial cells and homotypic leukocyte aggregation are highly associated with atherosclerosis and thrombosis, respectively. Our additional findings show that alliin has no effect on the production of nitric oxide (NO), an important vasoregulator. In conclusion, alliin is suggested as a regulator for controlling various cardiovascular functions.