Atherosclerosis is readily observed in areas where disturbed flow is formed, while the atheroprotective region is found in areas with steady laminar flow (L-flow). It has been established that L-flow protects endothelial cells against endothelial dysfunction, including apoptosis and inflammation. It has also been reported that extracellular signal-regulated kinase 5 (ERK5) regulated endothelial integrity and protected endothelial cells from vascular dysfunction and disease under L-flow. However, the molecular mechanism by which L-flow-induced ERK5 activation inhibits endothelial apoptosis has not yet been determined. Transcription factor p53 is a major pro-apoptotic factor which contributes to apoptosis in various cell types. In this study, we found that 15-deoxy-Δ(12,14)-prostaglandin J2 induced p53 expression and that endothelial apoptosis was reduced under the L-flow condition. This anti-apoptotic response was reversed by the biochemical inhibition of ERK5 activation. It was also found that activation of ERK5 protected endothelial apoptosis in a C terminus of Hsc70-interacting protein (CHIP) ubiquitin ligase-dependent manner. Moreover, molecular interaction between ERK5-CHIP and p53 ubiquitination were addressed with a CHIP ubiquitin ligase activity assay. Taken together, our data suggest that the ERK5-CHIP signal module elicited by L-flow plays an important role in the anti-apoptotic mechanism in endothelial cells.
Laminar flow; Endothelial apoptosis; ERK5; CHIP; p53
High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload.
Methods and results
Analysis of human biopsy samples by immunohistochemistry showed decreased nuclear HMGB1 expression in failing hearts compared with normal hearts. Nuclear HMGB1 decreased in response to both endothelin-1 (ET-1) and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to the cytoplasm. Overexpression of nuclear HMGB1 attenuated ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild-type (WT) mice. Cardiac hypertrophy after TAC was attenuated in HMGB1-Tg mice and the survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of foetal cardiac genes was decreased in HMGB1-Tg mice compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice and significantly attenuated DNA damage after TAC was attenuated in HMGB1-TG mice.
These results suggest that the maintenance of stable nuclear HMGB1 levels prevents hypertrophy and heart failure by inhibiting DNA damage.
HMGB1; Heart failure; Acetylation; Translocation; Pressure overload
Cardiomyocyte apoptosis is one of the key events in the development and progression of heart failure, and a crucial role for ICER (inducible cAMP early repressor) in this process has been previously reported. ERK5 is known to inhibit cardiac apoptosis after myocardial infarction (MI), especially in hyperglycemic states, via association with CHIP ubiquitin (Ub) ligase and subsequent up-regulation of CHIP ligase activity, which induces ICER ubiquitination and subsequent protein degradation. The regulatory mechanism governing ERK5/CHIP interaction is unknown.
We previously demonstrated increased p90RSK activation in the diabetic heart. As a logical extension of this work, we now investigate whether p90RSK activation inhibits ERK5-mediated CHIP activation, and subsequently increases ICER levels and apoptosis.
Methods and Results
p90RSK activation inhibits ERK5/CHIP association and CHIP Ub ligase activity. p90RSK and CHIP share a common binding site in the ERK5 C-terminal domain (aa571-807). Overexpression of either p90RSK or an ERK5 fragment (aa571-807) inhibits ERK5/CHIP association, suggesting that p90RSK and CHIP competes for ERK5 binding and that p90RSK activation is critical for inhibiting ERK5/CHIP interaction. We also identified ERK5-S496 as being directly phosphorylated by p90RSK, and demonstrated that an ERK5-S496A mutant significantly impairs Angiotensin II-mediated inhibition of CHIP activity and subsequent increase in ICER levels. In vivo, either cardiac specific depletion of ERK5 or overexpression of p90RSK inhibits CHIP activity and accelerates cardiac apoptosis after MI--a phenomenon fully reversible by activating ERK5.
These data suggest a role for p90RSK in inhibiting CHIP activity and promoting cardiac apoptosis through binding to and phosphorylation of ERK5-S496.
MAP kinase pathway; ubiquitin; diabetes mellitus (DM); myocardial infarction (MI); apoptosis
Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense. However, overactive response is detrimental to the host. Thus inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in the normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. Here, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae (NTHi)- and TNF-α-induced NF-κB-dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA binding activity. Moreover, expression of EVI1 itself is induced by NTHi and TNF-α in a NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus our study may not only provide important insights into the novel role of EVI1 in negatively regulating NF-κB-dependent inflammation, but may also shed light on the future development of novel anti-inflammatory strategies.
EVI1; NF-κB; NTHi; p65 acetylation; inflammation
Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense. However, overactive response is detrimental to the host. Thus inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in the normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. Here, we show that EVI1 negatively regulatesnontypeable Haemophilus influenzae(NTHi)- and TNF-α-induced NF-κB-dependent inflammationin vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310,thereby inhibiting its DNA binding activity. Moreover, expression of EVI1 itself is induced by NTHi and TNF-αin a NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus our study may not only provide important insights into the novel role of EVI1 in negatively regulating NF-κB-dependent inflammation, but may also shed light onthe future development of novel anti-inflammatory strategies.
EVI1; NF-κB; NTHi; p65 acetylation; inflammation
Respiratory systems are constantly being challenged by pathogens. Lung epithelial cells serve as a first line of defense against microbial pathogens by detecting pathogen-associated molecular patterns (PAMPs) and activating downstream signaling pathways, leading to a plethora of biological responses required for shaping both the innate and adaptive arms of the immune response. Acute-phase proteins (APPs), such as type 1 plasminogen activator inhibitor (PAI-1), play important roles in immune/inflammatory responses. PAI-1, a key regulator for fibrinolysis and coagulation, acts as an APP during acute phase response (APR) such as acute tissue injury (ALI), inflammation, and sepsis. However, the role of PAI-1 in the pathogenesis of these diseases still remains unclear, especially in bacterial pneumonia. In this study, we showed that PAI-1 expression is upregulated following nontypeable Haemophilus influenzae (NTHi) infection. PAI-1 knockout (KO) mice failed to generate early immune responses against NTHi. Failure of generating early immune responses in PAI-1 KO mice resulted in reduced bacterial clearance and prolonged disease process, which in turn led to enhanced inflammation at late stage of infection. Moreover, we also found that NTHi induces PAI-1 via activation of TLR2-MyD88-MKK3-p38 MAPK signaling pathway. These data suggest that PAI-1 plays critical role in earl host defense response against NTHi infection. Our study thus reveals a novel role of PAI-1 in infection caused by NTHi, one of the most common gram-negative bacterial pathogens in respiratory systems.
Nontypeable Haemophilus influenzae; type 1 plasminogen activator inhibitor; inflammation; pneumonia; innate immune response
Disturbed flow-mediated PKCζ–PIASy association is critical for p53 SUMOylation and induces p53 nuclear export and endothelial cell apoptosis.
Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow–mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow–mediated peroxynitrite (ONOO−) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53–Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO− increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301–410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ–PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53−/− mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ–PIASy interaction during d-flow–mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.
Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production.These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production.
Cilostazol; Proliferation; ROS; AMPK; HO-1
Sumoylation is a covalent modification, which is mediated by small ubiquitin-like modifier (SUMO) polypeptides. A growing body of evidence has shown that sumoylation affects the functional properties of many substrates in the regulation of cellular processes. Recent reports indicate the critical role of sumoylation in human diseases including familial dilated cardiomyopathy, suggesting that targeting of sumoylation would be of considerable interest for novel therapies. Even though hundreds of SUMO substrates have been identified, their pathophysiological roles remain to be determined. Among them, ERK5-sumoylation has recently been linked to diabetes and implicated in endothelial dysfunction and cardiomyocyte apoptosis in vivo. These findings support the idea that ERK5-sumoylation is a novel therapeutic target for the treatment of diabetes-related cardiovascular diseases.
SUMO; sumoylation; ERK5; diabetes; cardiovascular disease
NADPH oxidase; T cells; Rac; p47phox phosphorylation; neointimal formation
Diabetes (DM) contributes to the exacerbation of left ventricle (LV) dysfunction after myocardial infarction (MI). Activation of ERK5, an atypical mitogen activated protein kinase with transcriptional activity, inhibits apoptosis and LV dysfunction after doxorubicin treatment. SUMOylation has been proposed as a negative regulator of various transcription factors. In the current study, we investigated the role of ERK5-SUMOylation in ERK5 transcriptional activity as well as on DM-mediated exacerbation of LV dysfunction and apoptosis after MI. ERK5 wild type transcriptional activity was inhibited by Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), but not in the ERK5-SUMOylation-site defective mutant (K6R/K22R). H2O2 and high glucose, two well-known mediators of diabetes, induced ERK5-SUMOylation, and the K6R/K22R mutant, dominant negative form of Ubc9, and siRNA-PIAS1 reversed H2O2-mediated reduction of ERK5 transcriptional activity in cardiomyocytes, indicating the presence of SUMOylation-dependent ERK5 transcriptional repression. Constitutively active form of MEK5α (CA-MEK5α) inhibited ERK5-SUMOylation independent of kinase activity, but dependent on MEK5-ERK5 association. To investigate the pathological role of ERK5-SUMOylation in DM mice after MI, we utilized cardiac specific CA-MEK5α transgenic mice (CA-MEK5α-Tg). MI was induced in streptozotocin (STZ)-injected (DM + MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery. The ERK5-SUMOylation was increased in the DM + MI, but not in the MI group. ERK5-SUMOylation, the exacerbation of LV dysfunction, and the number of TUNEL positive cells in DM + MI was significantly inhibited in CA-MEK5α-Tg mice. Of note, we could not detect any difference of cardiac function after MI in non-diabetic CA-MEK5α-Tg and non-transgenic littermate control mice. These results demonstrated that ERK5 transcriptional activity is subject to down regulation by diabetes-dependent SUMOylation, which resulted in a pro-apoptotic condition contributing to poor post-MI LV function.
ERK5; SUMOylation; diabetes; myocardial infarction; apoptosis
Toll-like receptor 2 (TLR2) plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. Streptococcus pneumoniae (S. pneumoniae) and nontypeable Haemophilus influenzae (NTHi) were previously reported to synergize with each other to induce inflammatory responses. Despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface TLR2.
Here we provide direct evidence that S. pneumoniae synergizes with NTHi to upregulate TLR2 expression in lung and middle ear of the mice. Pneumolysin (PLY) appears to be the major virulence factor involved in this synergism. Moreover, S. pneumoniae PLY induces TLR2 expression via a TLR4-MyD88-NF-κB-dependent signaling pathway. Interestingly, tumor suppressor CYLD acts as a negative regulator of S. pneumoniae-induced TLR2 up-regulation via negative-crosstalk with NF-κB signaling.
Our study thus provides novel insights into the regulation of TLR2 expression in mixed bacterial infections.
Up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the lung airway epithelium is associated with the epithelium-leukocyte interaction, critical for the pathogenesis of various lung airway inflammatory diseases such as asthma. However, little is known about how ICAM-1 is up-regulated in human airway epithelial cells. In this study, we show that tumor TNF-α induces monocyte adhesion to A549 human lung airway epithelium and also up-regulation of ICAM-1 expression. These effects were significantly diminished by pre-treatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase-like flavoenzyme. In addition, the level of reactive oxygen species (ROS) was increased in response to TNF-α in A549 cells, suggesting a potential role of ROS in the TNF-α-induced signaling to ICAM-1 expression and monocyte adhesion to airway epithelium. Further, we found out that expression of RacN17, a dominant negative mutant of Rac1, suppressed TNF-α-induced ROS generation, ICAM-1 expression, and monocyte adhesion to airway epithelium. These findings suggest that Rac1 lies upstream of ROS generation in the TNF-α-induced signaling to ICAM-1 expression in airway epithelium. Finally, pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, reduced TNF-α-induced ICAM-1 expression and both DPI and RacN17 significantly diminished NF-κB activation in response to TNF-α. Together, we propose that Rac1-ROS-linked cascade mediate TNF-α-induced ICAM-1 up-regulation in the airway epithelium via NF-κB-dependent manner.
intercellular adhesion molecule-1; NF-κB; rac GTP-binding proteins; reactive oxygen species; respiratory mucosa; tumor necrosis factor-α
Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis.
Lung injury initiates a series of wound-healing responses, which if unregulated, can lead to fibrosis. Li et al. show that the deubquitinase CYLD has a key role in the prevention of fibrosis by inhibiting transforming growth factor β-signalling through the direct deubiquitination of the protein kinase Akt.
Non-typeable Haemophilus influenza (NTHi) is an important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are characterized by inflammation, which is mainly mediated by nuclear transcription factor kappaB (NF-κB)-dependent production of inflammatory mediators. The deubiquitinating enzyme cylindromatosis (CYLD), loss of which was originally reported to cause a benign human syndrome called cylindromatosis, has been identified as a key negative regulator for NF-κB in vitro. However, little is known about the role of CYLD in bacteria-induced inflammation in vivo. Here, we provided direct evidence for the negative role of CYLD in NTHi-induced inflammation of the mice in vivo. Our data demonstrated that CYLD is induced by NTHi in the middle ear and lung of mice. NTHi-induced CYLD, in turn, negatively regulates NTHi-induced NF-κB activation through deubiquitinating TRAF6 and 7 and down-regulates inflammation. Our data thus indicate that CYLD acts as a negative regulator for NF-κB-dependent inflammation in vivo, hence protecting the host against detrimental inflammatory response to NTHi infection.