Masanori Kitamura

Suppression of Apoptosis by All-trans-Retinoic Acid DUAL INTERVENTION IN THE c-JUN N-TERMINAL KINASE-AP-1 PATHWAY

Retinoic acid induces apoptosis of various cells, whereas little is known about its anti-apoptotic potential. In this report, we describe an anti-apoptotic property of all-trans-retinoic acid (t-RA) in mammalian cells. Mesangial cells exposed to hydrogen peroxide (H2O2) exhibited shrinkage of the cytoplasm, membrane blebbing, condensation of nuclei, and DNA fragmentation. Pretreatment with t-RA attenuated the morphologic and biochemical hallmarks of apoptosis. t-RA also inhibited apoptosis of mesangial cells triggered by pyrrolidine dithiocarbamate, whereas it did not prevent tumor necrosis factor-α-induced apoptosis. The anti-apoptotic effect against H2O2 was similarly observed in NRK49F fibroblasts, but not in Madin-Darby canine kidney epithelial cells and ECV304 endothelial cells. Mesangial cells exposed to H2O2 undergo apoptosis via the activator protein 1 (AP-1)-dependent pathway. We found that t-RA abrogated the H2O2-induced expression of c-fos/c-jun and activation of AP-1. Furthermore, t-RA inhibited H2O2-triggered activation of c-Jun N-terminal kinase (JNK), and dominant-negative inhibition of JNK attenuated the H2O2-induced apoptosis. These data disclosed the novel potential of retinoic acid as an inhibitor of apoptosis. The anti-apoptotic action of t-RA was ascribed, at least in part, to dual suppression of the cell death pathway mediated by JNK and AP-1.

Unexpected transcriptional induction of monocyte chemoattractant protein 1 by proteasome inhibition: involvement of the c-Jun N-terminal kinase-activator protein 1 pathway.

Proteasome inhibitors, the well-known inhibitors of NF-κB, are recently considered therapeutic agents for inflammation. However, the anti-inflammatory properties of these agents have not been fully evaluated. In this report we describe a novel effect of proteasome inhibitors on the expression of monocyte chemoattractant protein 1 (MCP-1) in mesangial cells. We found that proteasome inhibitor MG132 dose-dependently induced expression of MCP-1 at the transcriptional level. The stimulatory effect was similarly observed with other proteasome inhibitors (proteasome inhibitor 1 and lactacystin) and in other cell types (NRK fibroblasts). The 5′-flanking region of the MCP-1 gene contains multiple AP-1 sites. To explore the mechanisms involved, we examined the effects of proteasome inhibition on the AP-1 pathway. Northern blot analysis showed that MG132 rapidly induced the expression of c-jun, but not c-fos. Immunoblot analysis showed that MG132 prevented degradation of c-Jun protein. Kinase assay revealed that c-Jun N-terminal kinase (JNK) was rapidly activated by MG132. Consistent with these results, a reporter assay showed that AP-1 activity was up-regulated after treatment with MG132. Curcumin, a pharmacological inhibitor of the JNK-AP-1 pathway, abrogated the induction of MCP-1 by MG132. Similarly, stable transfection with a dominant-negative mutant of c-Jun attenuated both MG132-induced activation of AP-1 and expression of MCP-1. The transcriptional activation by proteasome inhibitors was observed not only in MCP-1, but also in other AP-1-dependent genes, including stromelysin and mitogen-activated protein kinase phosphatase 1. These data revealed that proteasome inhibition triggered the expression of MCP-1 and other genes via the multistep induction of the JNK-c-Jun/AP-1 pathway.

Transcriptional Induction of Mitogen-activated Protein Kinase Phosphatase 1 by Retinoids SELECTIVE ROLES OF NUCLEAR RECEPTORS AND CONTRIBUTION TO THE ANTIAPOPTOTIC EFFECT

All-trans-retinoic acid (t-RA) inhibits hydrogen peroxide (H2O2)-induced apoptosis by inhibiting the c-Jun N-terminal kinase (JNK)-activator protein 1 (AP-1) pathway. In this report, we examined the involvement of mitogen-activated protein kinase phosphatase 1 (MKP-1) in suppression of JNK and the antiapoptotic effect of t-RA and the roles of nuclear receptors in the regulation of MKP-1 by t-RA. We found that not only t-RA, but also a selective agonist of retinoic acid receptor (RAR), a selective agonist of retinoid X receptor (RXR), and a pan-agonist of RAR and RXR all induced MKP-1 at the transcriptional level. Activation of RAR was required for all of these triggering effects, but activation of RXR was required only for the RXR agonist-induced MKP-1 expression. Among the three RAR subtypes, RARα and RARγ, but not RARβ, mediated the t-RA-induced MKP-1 expression. The antiapoptotic effect of t-RA on H2O2-induced apoptosis in several cell types was correlated with the inducibility of MKP-1 by t-RA. Inhibition of MKP-1 by vanadate enhanced JNK phosphorylation and attenuated the antiapoptotic effect of t-RA. Furthermore, overexpression of MKP-1 inhibited H2O2-induced JNK phosphorylation and apoptosis. To our knowledge, this is the first to demonstrate that 1) MKP-1 is inducible by retinoids at the transcriptional level, 2) RXR and individual RAR subtypes have different roles in this process, and 3) the induced MKP-1 plays a significant role in mediating both JNK inhibition and the antiapoptotic effect of t-RA in oxidative stress.

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.

Retinoids as a potential treatment for experimental puromycin‐induced nephrosis

Puromycin aminonucleoside (PAN)‐induced nephrosis is a model of human minimal change disease. In rats, PAN induces nephrotic‐range proteinuria, renal epithelial cell (podocyte) damage, infiltration of mononuclear leukocytes, and apoptosis of several renal cell types. Retinoic acid (RA) modulates a wide range of biological processes, such as inflammation and apoptosis. Since renal damage by PAN is characterized by inflammatory infiltration and epithelial cell death, the effect of treatment with all‐trans RA (tRA) was examined in the PAN nephrosis model and in the cultured differentiated podocyte. Treatment with tRA 4 days after PAN injection did not inhibit the proteinuria peak but reversed it significantly. However, treatment with tRA both before and 2 days after the injection of PAN protected the glomerular epithelial cells, diminishing the cellular edema and diffuseness of the foot process effacement. Preservation of the podocyte architecture correlated with the inhibition of proteinuria. The anti‐inflammatory effect of tRA was evidenced by the inhibition of PAN‐induced interstitial mononuclear cell infiltration and the decreased renal expression of two molecules involved in monocyte infiltration: fibronectin and monocyte chemoattractant protein‐1. TUNEL assays showed that tRA inhibited the PAN‐induced apoptosis of cultured differentiated mouse podocytes. We conclude that tRA treatment may prevent proteinuria by protecting the podocytes from injury and diminishing the interstitial mononuclear infiltrate in the model of PAN nephrosis. Retinoids are a potential new treatment for kidney diseases characterized by proteinuria and mononuclear cell infiltration.

NF-κB-mediated self defense of macrophages faced with bacteria

NF‐κB is a ubiquitous transcription factor that is extensively exploited by immune cells involved in host defense mechanisms. Macrophages participate in the first line of defense against microorganisms, but little is known about whether and how NF‐κB is involved in the handling of microbes by macrophages. To explore this issue, NF‐κB‐inactive macrophages, NIKMACNR, were created by overexpression of a super‐repressor mutant of IκBα. When co‐cultured with Escherichia coli, the NIKMACNR macrophages exhibited impairment of bactercidal activity. Microscopic analysis revealed that NIKMACNR cells faced with bacteria underwent rapid and fulminant apoptosis. Similary, NIKMACNR macrophages cultured in the presence of a bacterial component, lipopolysaccharide, showed dramatic apoptosis. Inhibition of RNA synthesis or protein synthesis failed to block the apoptosis of NIKMACNR cells, indicating that macrophages possess a pre‐existing, apoptotic pathway that can be triggered by bacteria. Apoptosis was not observed in NIKMACNR macrophages exposed to non‐microbial stimuli including phorbol ester and opsonized zymosan. However, NIKMACNR cells were more susceptible to apoptosis triggered by TNF‐α and reactive oxygen intermediates, both of which are produced abundantly by macrophages when faced with bacteria. These data suggest a critical role for NF‐κB in the survival of macrophages at the site of bacterial infection.

Human renal mesangial cells are a target for the anti-inflammatory action of 9-cis retinoic acid

Mesangial cells play an active role in the inflammatory response to glomerular injury. We have studied in cultured human mesangial cells (CHMC) several effects of 9‐cis retinoic acid (9‐cRA), an activator of both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9‐cRA inhibited foetal calf serum‐induced CHMC proliferation. It also prevented CHMC death induced by the inflammatory mediator H2O2. This preventive effect was not due to any increase in H2O2 catabolism and it persisted even when both catalase and glutathione synthesis were inhibited. Finally, 9‐cRA diminished monocyte adhesion to FCS‐stimulated CHMC. Interestingly, the retinoid also inhibited in FCS‐stimulated cells the protein expression of two mesangial adhesion molecules, fibronectin and osteopontin, but it did not modify the protein expression of intercellular adhesion molecule‐1 and vascular adhesion molecule‐1. All major RARs and RXRs isotypes were expressed in CHMC regardless of the presence or absence of 9‐cRA. Transcripts to RAR‐α, RAR‐β and RXR‐α increased after incubation with 9‐cRA whereas RXR‐γ was inhibited, suggesting a major role for RARs and RXRs in 9‐cRA‐anti‐inflammatory effects. 9‐cRA was toxic only at 50u2003μM (a concentration 50–5000 times higher than required for the effects above). Cell death occurred by apoptosis, whose onset was associated with a pronounced increase in catalase activity and reduced glutathione content, being more effectively induced by all‐trans retinoic acid. Modulation of the oxidant/antioxidant balance failed to inhibit apoptosis. We conclude that mesangial cells might be a target for the treatment of inflammatory glomerulopathies with 9‐cRA.

The Effect of Selective Growth Hormone Defect in the Progression of Glomerulosclerosis

The role of endogenous growth hormone (GH) in the progression of glomerulosclerosis was examined in a new mutant strain of Sprague-Dawley (SD) rats with a selective GH gene defect. Fifteen spontaneous dwarf [GH(-)] rats and 10 SD rats underwent subtotal nephrectomy (Nx) or sham operation. Twelve weeks after Nx, the mean arterial pressure, glomerular filtration rate, urinary protein excretion rate, glomerular size, and frequency of glomerulosclerosis were examined. Marked elevation in mean arterial pressure was seen in both SD/Nx and GH(-)/Nx rats. In both strains, the glomerular filtration rate at 12 weeks after Nx was approximately 50% to 60% of that seen in the respective Sham-operated rat groups. The urinary protein excretion rate increased significantly only in the SD/Nx rats. The glomerular size, expressed as the ratio of glomerular volume to body weight, increased by 200% in the SD/Nx rats compared with the SD/Sham rats, in marked contrast to the 70% increase in the GH(-)/Nx rats compared with the GH(-) rats. The frequency of glomerulosclerosis in the GH(-)/Nx rats (1.0 +/- 0.5%) was significantly lower than that in the SD/Nx rats (16.7 +/- 2.8%). The frequency of glomerulosclerosis correlated with glomerular hypertrophy in both the SD and GH(-) rats (r = 0.88 and 0.67, respectively). These results show that the progression of glomerular sclerosis was markedly attenuated in the GH-defective rats. This attenuation of structural injury was correlated with a marked decrease in glomerular hypertrophy. These studies indicate that this specific growth regulatory peptide of endogenous origin plays an important determining role in the progression of glomerulosclerosis.

PDGF‐BB enhances α1β1 integrin‐mediated activation of the ERK/AP‐1 pathway involved in collagen matrix remodeling by rat mesangial cells

Platelet‐derived growth factor‐BB (PDGF‐BB) has been implicated in the pathogenesis of progressive glomerulonephritis (GN). Previous studies have reported that PDGF‐BB stimulates mesangial cells (MCs)‐induced collagen matrix remodeling through enhancement of α1β1 integrin‐dependent migratory activity. To determine the cell signaling pathway responsible for abnormal MC‐related mesangial matrix remodeling in progressive GN, we studied the involvement of the extracellular signal‐regulated kinase (ERK)/activator protein‐1 (AP‐1) pathway in PDGF‐BB‐enhanced collagen gel contraction. Western blotting and gel shift assay revealed that MC‐induced gel contraction resulted in ERK activation in parallel with that of AP‐1 binding, peaking at 4 h and lasting at least for 24 h. Application of the MEK inhibitor, U0126, and the c‐jun/AP‐1 inhibitor, curcumin, inhibited gel contraction and AP‐1 activity, respectively, dose dependently. PDGF‐BB enhanced not only gel contraction but ERK phosphorylation and AP‐1 activity by MCs. Marked inhibitory effects on PDGF‐BB‐induced gel contraction and ERK/AP‐1 activity were observed in the presence of either function blocking anti‐α1‐ or anti‐β1‐integrin antibody or U0126. Consistently, AP‐1‐inactive MCs expressing a dominant‐negative mutant of c‐jun showed a significant decrease of PDGF‐BB‐induced gel contraction as compared with mock‐transfected MCs. Finally, migration assay showed that ERK/AP‐1 activity is required for PDGF‐BB‐stimulated α1β1 integrin‐dependent MC migration to collagen I. These results indicated that PDGF‐BB enhances α1β1 integrin‐mediated collagen matrix reorganization through the activation of the ERK/AP‐1 pathway that is crucial for MC migration. We conclude that the ERK/AP‐1 pathway plays an important role in PDGF‐BB‐induced α1β1 integrin‐dependent collagen matrix remodeling; therefore, the inhibition of its pathway may provide a novel approach to regulate abnormal collagen matrix remodeling in progressive GN. J. Cell. Physiol. 198: 470–478, 2004© 2003 Wiley‐Liss, Inc.

Retinoic Acid Regulation of Mesangial Cell Apoptosis

Retinoic acid (RA) is recently used for the treatment of experimental glomerular diseases. However, mechanisms underlying its therapeutic effects are largely unknown. We recently reported that RA has the potential for protecting certain cells from particular injury. A typical example is its effect on oxidant-induced apoptosis of mesangial cells. Mesangial cells exposed to hydrogen peroxide undergo apoptosis through activation of the c-Jun N-terminal kinase activator protein 1 pathway. RA dramatically inhibits this process via suppression of c-fos/c-jun expression and inhibition of the c-Jun N-terminal kinase activation. The anti-apoptotic effect of RA is mediated by both nuclear receptor dependent and nuclear receptor independent mechanisms and is, at least in part, mediated by induction of mitogen-activated protein kinase phosphatase 1. In this review, we briefly summarize the current knowledge on molecular mechanisms involved in the anti-apoptotic effects of RA.