Kun Gao

5′-AMP-Activated Protein Kinase Attenuates Adriamycin-Induced Oxidative Podocyte Injury through Thioredoxin-Mediated Suppression of the Apoptosis Signal-Regulating Kinase 1–P38 Signaling Pathway

Oxidative stress–induced podocyte injury is one of the major mechanisms underlying the initiation and progression of glomerulosclerosis. 5′-AMP-activated protein kinase (AMPK), a serine/threonine kinase that senses intracellular energy status and maintains energy homeostasis, is reported to have antioxidative effects. However, little is known about its application and mechanism. In this study, we investigated whether and how AMPK affected oxidative podocyte injury induced by Adriamycin (ADR; Wako Pure Chemical, Osaka, Japan). Exposure of podocytes to ADR resulted in cell injury, which was preceded by increased reactive oxygen species (ROS) generation and P38 activation. Prevention of oxidative stress with the antioxidant N-acetyl-cysteine and glutathione or inhibition of P38 with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] attenuated cell injury. Activation of AMPK with three structurally different AMPK activators also protected podocytes from ADR-elicited cell injury. This effect was associated with strong suppression of oxidative stress-sensitive kinase apoptosis signal-regulating kinase 1 (ASK1) and P38 without obvious influence on ROS level. Further analyses revealed that AMPK promoted thioredoxin (Trx) binding to ASK1. Consistently, AMPK potently suppressed the expression of thioredoxin-interacting protein (TXNIP), a negative regulator of Trx, whereas it significantly enhanced the activity of Trx reductases that convert oxidized Trx to reduced form. In further support of a key role of Trx, downregulation or inhibition of Trx exaggerated but downregulation of TXNIP attenuated the cell injury. These results indicate that AMPK prevents oxidative cell injury through Trx-mediated suppression of ASK1-P38 signaling pathway. Our findings thus provide novel mechanistic insights into the antioxidative actions of AMPK. AMPK could be developed as a novel therapeutic target for treatment of oxidative cell injury.

Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels - pivotal roles in hemichannel-mediated cell injury.

ABSTRACT Connexin hemichannels regulate many cell functions. However, the molecular mechanisms involved remain elusive. Hemichannel opening causes loss of ATP, we therefore speculated a potential role for AMPK in the biological actions of hemichannels. Activation of hemichannels by removal of extracellular Ca2+ led to an efflux of ATP and a weak activation of AMPK. Unexpectedly, dysfunction of hemichannels markedly potentiated AMPK activation, which was reproduced by promotion of extracellular ATP degradation or inhibition of P2 purinoceptors but counteracted by exogenous ATP. Further analysis revealed that ATP induced a purinoceptor-dependent activation of Akt and mTOR. Suppression of Akt or mTOR augmented AMPK activation, whereas activation of Akt by transfection of cells with myristoylated Akt, a constitutively active form of Akt, abolished AMPK activation. In a pathological model of hemichannel opening triggered by Cd2+, disclosure of hemichannels similarly enhanced AMPK activity, which protected cells from Cd2+-induced cell injury through suppression of mTOR. In summary, our data point to a channel-mediated mechanism for the regulation of AMPK through a purinergic signaling pathway. Furthermore, we define AMPK as a pivotal molecule that underlies the regulatory effects of hemichannels on cell survival.

Connexin43 Hemichannel-Mediated Regulation of Connexin43

Background Many signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd2+), we tested this hypothesis. Principal Findings Incubation of Cx43-transfected LLC-PK1 cells with Cd2+ led to an increased expression of Cx43. This effect of Cd2+ was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd2+ on JNK activation and Cx43 expression. Indeed, Cd2+ induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd2+. Conclusions Collectively, our results thus indicate that Cd2+-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.

p53 Protein-mediated Up-regulation of MAP Kinase Phosphatase 3 (MKP-3) Contributes to the Establishment of the Cellular Senescent Phenotype through Dephosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2)

Background: Growth arrest is a hallmark of cellular senescence. Results: Loss of cell proliferation in senescent cells was associated with impaired ERK1/2 activation, which was caused by p53-mediated elevation of MKP-3. Conclusion: The p53/MKP-3/ERK1/2 cascade contributed to the establishment of the senescent phenotype. Significance: Our study provides novel insights into the actions and mechanisms of p53 on cellular senescence. Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced β-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogen-activated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-α. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence.

Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels.

Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well‐known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non‐junctional connexin43 (Cx43) hemichannels, pore‐forming α‐haemolysin and channels involved in ATP release under hypotonic conditions.

A novel TXNIP-based mechanism for Cx43-mediated regulation of oxidative drug injury.

Gap junctions (GJs) play an important role in the regulation of cell response to many drugs. However, little is known about their mechanisms. Using an in vitro model of cytotoxicity induced by geneticin (G418), we explored the potential signalling mechanisms involved. Incubation of cells with G418 resulted in cell death, as indicated by the change in cell morphology, loss of cell viability and activation of caspase‐3. Before the onset of cell injury, G418 induced reactive oxygen species (ROS) generation, activated oxidative sensitive kinase P38 and caused a shift of connexin 43 (Cx43) from non‐phosphorylated form to hyperphosphorylated form. These changes were largely prevented by antioxidants, suggesting an implication of oxidative stress. Downregulation of Cx43 with inhibitors or siRNA suppressed the expression of thioredoxin‐interacting protein (TXNIP), activated Akt and protected cells against the toxicity of G418. Further analysis revealed that inhibition of TXNIP with siRNA activated Akt and reproduced the protective effect of Cx43‐inhibiting agents, whereas suppression of Akt sensitized cells to the toxicity of G418. Furthermore, interference of TXNIP/Akt also affected puromycin‐ and adriamycin‐induced cell injury. Our study thus characterized TXNIP as a presently unrecognized molecule implicated in the regulatory actions of Cx43 on oxidative drug injury. Targeting Cx43/TXNIP/Akt signalling cascade might be a promising approach to modulate cell response to drugs.

eIF2α-Independent Inhibition of TNF-α-Triggered NF-κB Activation by Salubrinal

Salubrinal is a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2α (eIF2α). In previous reports, salubrinal was shown to have the potential to inhibit the activation of nuclear factor-κB (NF-κB) by several stimuli. However, the effects of salubrinal on NF-κB signaling are largely unknown. In this study, we investigated whether and how salubrinal affects NF-κB activation induced by tumor necrosis factor (TNF)-α and interleukin (IL)-1β. We found that salubrinal selectively blocked TNF-α- but not IL-1β-induced activation of NF-κB. This inhibitory effect occurred upstream of transforming growth factor (TGF)-β-activated kinase 1 (TAK1). Further experiments revealed that salubrinal blocked TNF-α-triggered NF-κB activation independent of its action on eIF2α because knockdown of eIF2α by small interfering RNA (siRNA) did not reverse the inhibitory effect of salubrinal on NF-κB. Moreover, guanabenz, a selective inhibitor of the regulatory subunit of protein phosphatase (PP) 1, also preferentially inhibited TNF-α-triggered activation of NF-κB. These findings raise the possibility that salubrinal may selectively block TNF-α-triggered activation of the NF-κB pathway through inhibition of the PP1 complex.

AMPK Suppresses Connexin43 Expression in the Bladder and Ameliorates Voiding Dysfunction in Cyclophosphamide-induced Mouse Cystitis.

Bladder voiding dysfunction is closely related to local oxidation, inflammation, and enhanced channel activities. Given that the AMP-activated protein kinase (AMPK) has anti-oxidative, anti-inflammatory and channel-inhibiting properties, we examined whether and how AMPK affected bladder activity. AMPK activation in rat bladder smooth muscle cells (BSMCs) using three different AMPK agonists resulted in a decrease in connexin43 (Cx43) expression and function, which was associated with reduced CREB phosphorylation, Cx43 promoter activity and mRNA expression, but not Cx43 degradation. Downregulation of CREB with siRNA increased Cx43 expression. A functional analysis revealed that AMPK weakened BSMC contraction and bladder capacity. AMPK also counteracted the IL-1β- and TNFα-induced increase in Cx43 in BSMCs. In vivo administration of the AMPK agonist AICAR attenuated cyclophosphamide-initiated bladder oxidation, inflammation, Cx43 expression and voiding dysfunction. Further analysis comparing the responses of the wild-type (Cx43+/+) and heterozygous (Cx43+/−) Cx43 mice to cyclophosphamide revealed that the Cx43+/− mice retained a relatively normal micturition pattern compared to the Cx43+/+ mice. Taken together, our results indicate that AMPK inhibits Cx43 in BSMCs and improves bladder activity under pathological conditions. We propose that strategies that target AMPK can be developed as novel therapeutic approaches for treating bladder dysfunction.

Suramin inhibits antibody binding to cell surface antigens and disrupts complement-mediated mesangial cell lysis

Suramin inhibits immune responses and protects cells against inflammatory cell injury. However, little is known about its mechanisms. Using an inxa0vitro model of glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we investigated the potential protective effects and mechanisms of suramin on immunologic cell injury. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused complement-dependent cell lysis, which was blocked by suramin and its structural analogue NF023 and NF049, but not by PPADS, an antagonist of purinergic receptors. Addition of exogenous ATP also failed to affect MC lysis. Further analysis revealed that suramin interfered with antibody binding to cell membrane antigens and suppressed antibody-induced phosphorylation of several proteins, including p38. Inhibition of p38 with chemical inhibitor significantly attenuated cell injury. Collectively, our results indicate that suramin protects cells against antibody-initiated and complement-dependent cell injury through inhibition of antibody binding to cell surface antigens and suppression of p38 activation. Our study thus provides novel mechanistic insights into the actions of suramin and suggests that suramin might be used to treat certain immune diseases.

Qiguiyishen decoction reduced the accumulation of extracellular matrix in the kidneys of rats with adriamycin-induced nephropathy

OBJECTIVEnTo investigate the effect of Qiguiyishen decoction (QGYS) on the severity of nephropathy.nnnMETHODSnTwenty-four rats were randomly divided into four groups (I, II, III, IV) according to the random number table. Group I as control group did not establish nephropathy model. Groups II, III, and IV were intravenously administered Adriamycin (7.5 mg/kg) through the tail vein to establish nephropathy model. QGYS was prepared with the extracts of Huangqi (Radix Astragali Mongolici), Danggui (Radix Angelicae Sinensis), Niuxi (Radix Achyranthis Bidentatae), and Chuanxiong (Rhizoma Chuanxiong). Group IV was administered QGYS (2 mL x kg(-1) x d(-1)), group III was administered benazepril (10 mL x kg(-1) x d(-1)), and group I, II was administered water (2 mL x kg(-1) x d(-1)) once daily for eight weeks.nnnRESULTSnQGYS reduced the excretion of urinary protein and N-acetyl-beta-D-glucosaminidase and alleviated the accumulation of extracellular matrix (ECM) in renal tissue. Additionally, QGYS effectively regulated the levels of transforming growth factor, tissue inhibitor of matrix metalloproteinase, and matrix metalloproteinases in the kidney of the rats.nnnCONCLUSIONnQGYS may reduce the accumulation of ECM in the kidneys of rats with Adriamycin-induced nephropathy.