Seul Ki Lim

Formaldehyde induces apoptosis through decreased Prx 2 via p38 MAPK in lung epithelial cells.

Formaldehyde (FA) is an important substance that induces sick house syndrome and diseases, such as asthma and allergies. Oxidative stress is involved in the development of respiratory disease, and diverse antioxidants may protect respiratory tract cells from apoptosis. Peroxiredoxin is a pivotal endogenous antioxidant. In the present study, FA induced death in A549 cells, a lung epithelial cell line, in a dose-dependent manner. FA also increased lipid peroxide formation (LPO) in A549 cells, suggesting a role for oxidative stress. Additionally, FA decreased peroxiredoxin 2 (Prx 2) protein levels after a 24 or 48h exposure to FA. We also examined whether the FA-induced decrease in Prx 2 was associated with apoptosis. Prx 2 overexpression protected against FA-induced cell apoptosis but not necrosis. Prx 2 overexpression blocked FA-induced increase in Bax, a pro-apoptotic molecule, and a decrease in Bcl-2, an anti-apoptotic molecule. Prx 2 overexpression also protected against FA-induced activation of some special apoptosis-associated proteins [caspase-3, caspase-9, and polypeptide poly (ADP-ribose) polymerase (PARP)]. Furthermore, we examined the signaling molecules involved in the FA-induced decrease in Prx 2 expression. The FA-induced decrease in Prx 2 and increase in cell apoptosis was restored by treatment with SB203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by SP600125 [a c-jun-N-terminal kinase (JNK) inhibitor]. Also, FA-induced events were blocked by treatment with p38 siRNA, but not by scrambled siRNA. Indeed, FA increased p38 MAPK activation, suggesting a role for p38 MAPK in FA action. In conclusion, FA mediated apoptosis in lung epithelial cells by decreasing Prx 2 via p38 MAPK.

Cannabinoid receptor 1 mediates high glucose-induced apoptosis via endoplasmic reticulum stress in primary cultured rat mesangial cells.

The endocannabinoid system in animals and humans is involved in the onset of diverse diseases, including obesity and diabetic nephropathy, which is a major end-stage renal disease characterized by high glucose (HG)-induced apoptosis of mesangial cells. Endocannabinoids induce physiological and behavioral effects by activating two specific receptors, cannabinoid receptor 1 (CB(1)R) and cannabinoid receptor 2 (CB(2)R). However, the pathophysiology of CB(1)R in diabetic nephropathy has not been elucidated. We investigated the effects of HG on CB(1)R expression and its signaling pathways in primary cultured rat mesangial cells. HG significantly increased CB(1)R mRNA and protein levels in a time-dependent manner and induced CB(1)R internalization. NF-κB and cPLA(2) were involved in the HG-induced increase in CB(1)R levels. Using a CB(1)R antagonist (AM251) and CB(1) siRNA transfection, we showed that HG-induced CB(1)R is linked to apoptosis. Specifically, HG inhibited the expression of GRP78, but induced increases in endoplasmic reticulum (ER) stress proteins, including phosphorylated (p)-protein kinase-like ER-associated kinase, p-eukaryotic initiation factor 2α, p-activating transcription factor-4, and C/EBP homologous protein. In addition, HG increased the Bax/Bcl-2 ratio and increased the amounts of cleaved poly(ADP-ribose) polymerase and caspase-3. These apoptotic effects were prevented by AM251 and by the downregulation of CB(1)R expression by small interfering RNA. We propose a mechanism by which blockade of CB(1)R attenuates HG-induced apoptosis in rat mesangial cells. Our findings suggest that blockade of CB(1)R may be a potential therapy in diabetic nephropathy.

Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retianl pigment epithelial cells

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB1R in the ARPE‐19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE‐19 cells. In contrast, it upregulated the expression of CB1R mRNA and protein. HG‐induced internalization of CB1R in HEK 293 cells and ARPE‐19 cells was blocked by overexpression of FAAH 1 and treatment with the CB1R blocker, AM 251. HG‐induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG‐induced expression of CB1R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG‐induced apoptosis. High glucose increased the Bax/Bcl‐2 ratio and levels of cleaved PARP, cleaved caspase‐9 and caspase‐3, and reduced cell viability. HG‐induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB1R‐specific antagonist AM 251 and CB1R siRNA transfection. In conclusion, HG‐induced apoptosis in ARPE‐19 cells by inducing CB1R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB1R blockade through the recovery of FAAH 1 expression may be a potential anti‐diabetic therapy for the treatment of diabetic retinopathy. J. Cell. Physiol. 227: 569–577, 2012.

PRMT3 Regulates Hepatic Lipogenesis Through Direct Interaction With LXRα

Arginine methylation is responsible for diverse biological functions and is mediated by protein arginine methyltransferases (PRMTs). Nonalcoholic fatty liver disease (NAFLD) is accompanied by excessive hepatic lipogenesis via liver X receptor α (LXRα). Thus we examined the pathophysiological role of PRMTs in NAFLD and their relationship with LXRα. In this study, palmitic acid (PA) treatment increased PRMT3, which is correlated with the elevation of hepatic lipogenic proteins. The expression of lipogenic proteins was increased by PRMT3 overexpression, but decreased by PRMT3 silencing and use of the PRMT3 knockout (KO) mouse embryonic fibroblast cell line. PRMT3 also increased the transcriptional activity of LXRα by directly binding with LXRα in a methylation-independent manner. In addition, PA treatment translocated PRMT3 to the nucleus. In animal models, a high-fat diet increased the LXRα and PRMT3 expressions and binding, which was not observed in LXRα KO mice. Furthermore, increased PRMT3 expression and its binding with LXRα were observed in NAFLD patients. Taken together, LXRα and PRMT3 expression was increased in cellular and mouse models of NAFLD and human patients, and PRMT3 translocated into the nucleus bound with LXRα as a transcriptional cofactor, which induced lipogenesis. In conclusion, PRMT3 translocation by PA is coupled to the binding of LXRα, which is responsible for the onset of fatty liver.

Activation of PRMT1 and PRMT5 mediates hypoxia- and ischemia-induced apoptosis in human lung epithelial cells and the lung of miniature pigs: the role of p38 and JNK mitogen-activated protein kinases.

Severe hypoxic and ischemic injury leads to primary graft dysfunction after lung transplantation. Arginine methylation, which is responsible for the regulation of a variety of biological functions, is mediated by protein arginine methylation transferases (PRMTs). This study examined the role of hypoxia in PRMT activation in A549 human lung epithelial cells, as well as the role of ischemia in PRMT activation in the lung of miniature pigs. In A459 cells, hypoxia increased the expression of PRMT1 and PRMT5, and overexpression of PRMT1 and PRMT5 induced apoptosis. The transfection of PRMT1 and PRMT5 small interfering RNA (siRNA) prevented hypoxia-inducible factor (HIF)-1α expression and apoptosis in A549 cells. Hypoxia-induced expression of PRMT1 and PRMT5 was blocked by p38 and JNK mitogen-activated protein kinase (MAPK) inhibitors, but not by an inhibitor of extracellular signal-regulated kinases (ERK) 1/2. In the lungs of miniature pigs, ischemia stimulated PRMT1 and PRMT5 expression and induced phosphorylation of p38 MAPK (p-p38), phosphorylation of JNK (p-JNK), and apoptotic molecules. These results demonstrate that PRMT1 and PRMT5 are involved in hypoxia and ischemia-induced apoptosis via p-p38 MAPK and p-JNK in in vitro and in vivo models.

The high glucose-induced stimulation of B1R and B2R expression via CB1R activation is involved in rat podocyte apoptosis

AIMS We examined renal kallikrein-kinin system (KKS) apoptosis and its related signaling pathway in rat podocytes. In addition, we studied the relationship of cannabinoid receptor 1 (CB(1)R) with high glucose and BK receptors. MAIN METHODS Cell viability was determined by an MTT assay and apoptosis by DNA fragmentation assay, while gene expression was investigated by RT-PCR. Protein expression was analyzed by Western blot analysis. A chemical inhibitor or siRNA transfection was used to inhibit B1R, B2R, and CB(1)R signaling. KEY FINDINGS High glucose (25 mM) treatment decreased cell viability and increased DNA fragmentation. High glucose-induced DNA fragmentation and PARP and caspase-3 activations were blocked by both [des-Arg(10)]-HOE 140 (a B1R antagonist) and HOE 140 (a B2R antagonist). High glucose also increased Akt phosphorylation, ER stress-related protein expression, and NF-κB/I-κB phosphorylation in podocytes, which was blocked by both [des-Arg(10)]-HOE 140 and HOE 140. In addition, B1R and B2R siRNA transfections prevented high glucose-induced Akt and NF-κB activations in rat podocytes. Moreover, AM251 (a CB(1)R antagonist) treatment and CB(1)R siRNA transfection blocked the high glucose-induced stimulation of BK receptor expression, Akt activation, and NF-κB activation. SIGNIFICANCE Our study suggests that hyperglycemia induces apoptosis via the stimulation of B1R and B2R expression through CB(1)R activation in rat podocytes in vitro, which is associated with the development of diabetic nephropathy.

Effect of protopanaxadiol derivatives in high glucose-induced fibronectin expression in primary cultured rat mesangial cells: Role of mitogen-activated protein kinases and Akt

A lot of anti-diabetic agents using natural plants have been extensively studied. Ginsenosides are known to be used as a remedy for diabetes in Asian countries and American Societies. Diabetic nephropathy is a major complication of diabetes mellitus. Extracellular matrix in mesangial cells is mainly composed of fibronectin and the increase of fibronectin is a hallmark of diabetic nephropathy. Protopenaxadiol (PPD) is a major component of total ginseng. Thus, we examined the regulatory mechanism of PPD derivatives-induced preventive effect of fibronectin expression in mesangial cells cultivated under diabetic condition. In present study, ginsenoside Rb1 prevented the high glucose-induced increase of fibronectin expression in mesangial cells. Ginsenoside Rb2 and Rg3 also mildly inhibited it. However, ginsenoside Rc and Rd did not prevent the high glucose-induced increase of fibronectin expression in mesangial cells. In addition, ginsenoside Rb1 prevented high glucose-induced phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK, JNK/SAPK, and Akt. These results suggest that ginsenoside Rb1 is the most powerful component of PPD derivatives. In conclusion, ginsenoside Rb1 prevented high glucose-induced increase of fibronectin expression via the inhibition of MAPK-Akt signaling cascade.

Weissella cibaria WIKIM28 ameliorates atopic dermatitis-like skin lesions by inducing tolerogenic dendritic cells and regulatory T cells in BALB/c mice.

The occurrence of atopic dermatitis (AD), a chronic inflammatory skin disease, has been increasing steadily in children and adults in recent decades. In this study, we evaluated the ability of the lactic acid bacterium Weissella cibaria WIKIM28 isolated from gatkimchi, a Korean fermented vegetable preparation made from mustard leaves, to suppress the development of AD induced by 2,4-dinitrochlorobenzene in a murine model. Oral administration of W. cibaria WIKIM28 reduced AD-like skin lesions, epidermal thickening, and serum immunoglobulin E levels. Furthermore, the production of type 2 helper T (Th2) cytokines such as interleukin (IL)-4, IL-5, and IL-13 decreased in peripheral lymph node cells. Moreover, the intake of W. cibaria WIKIM28 increased the proportion of CD4+CD25+Foxp3+ regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) and IL-10 levels in polyclonally stimulated MLN cells. In conclusion, the oral administration of W. cibaria WIKIM28 isolated from gatkimchi ameliorated AD-like symptoms by suppressing allergic Th2 responses and inducing Treg responses. These results suggest that W. cibaria WIKIM28 may be applicable as a probiotic for the prevention and amelioration of AD.

High glucose stimulates the expression of erythropoietin in rat glomerular epithelial cells.

It has been reported that the levels of erythropoietin are associated with diabetes mellitus. Glomerular epithelial cells, located in the renal cortex, play an important role in the regulation of kidney function and hyperglycemia-induced cell loss of glomerular epithelial cells is implicated in the onset of diabetic nephropathy. This study investigated the effect of high glucose on erythropoietin and erythropoietin receptor expression in rat glomerular epithelial cells. We found that 25 mM D-glucose, but not mannitol or L-glucose, stimulated erythropoietin mRNA and protein expression in a time dependent manner (>4 h) in rat glomerular epithelial cells. In addition, 25 mM glucose, but not mannitol or L-glucose, also increased the phosphorylation of erythropoietin receptor, suggesting a role for erythropoietin receptor phosphorylation in erythropoietin synthesis. We conclude that high glucose stimulates erythropoietin production and erythropoietin receptor phosphorylation in rat glomerular epithelial cells.

Both B1R and B2R act as intermediate signaling molecules in high glucose‐induced stimulation of glutamate uptake in ARPE cells

Bradykinin (BK) is a potent modulator of biological processes in the retina, and retinal pigment epithelial cells (RPE) and the regulation of glutamate are believed to be important in the pathogenesis of diabetic retinopathy. However, the mechanism by which BK regulates glutamate uptake in RPE cells in diabetic retinopathy is unknown. Here, we examined the involvement of BK receptors in high glucose‐induced dysfunction of glutamate uptake in human ARPE cells. High glucose stimulated glutamate uptake and the expression of excitatory amino acid transporter‐4 (EAAT4) mRNA, and these were blocked by treatment with small interfering RNA (siRNA) for BK1 receptor (B1R) and BK2 receptor (B2R), but not scrambled siRNA, supporting an involvement of B1R and B2R in this process. High glucose‐stimulated glutamate uptake was also blocked by the B1R antagonist [des‐Arg10]‐HOE 140 and the B2R antagonist HOE 140. High glucose increased B1R and B2R mRNA and protein expression in a time‐dependent manner, increased B1R and B2R translocation from the cytosol to the nucleus, and stimulated kininogen, kallikrein, and kininase I mRNA expression. We examined whether BK receptors were involved in high glucose‐induced signaling pathways. High glucose stimulated arachidonic acid release, cytosolic phospholipase A2 and cyclooxygenase‐2 proteins, nuclear factor‐κB activation, and inhibitor‐κB activation; these events were blocked by treatment with B1R and B2R siRNAs, but not scrambled siRNA. In addition, high glucose‐induced stimulation of glutamate uptake was blocked by the cyclooxygenase‐2 inhibitors arachidonyl trifluoromethyl ketone, mepacrine, 5‐bromo‐2‐(4‐fluorophenyl)‐3‐[4‐(methyl‐sulfonyl)phenyl]‐thiophene, and N‐[2‐cyclohexyloxy‐4‐nitrophenyl] methane‐sulfonamide, and by the nuclear factor‐κB inhibitors pyrrolidine dithiocarbamate and SN‐50. J. Cell. Physiol. 221: 677–687, 2009.