Ryosuke Fukuda

TLR3 induction by anticancer drugs potentiates poly I:C-induced tumor cell apoptosis

Toll‐like receptor 3 (TLR3) has gained recognition as a novel molecular target for cancer therapy because TLR3 activation by its synthetic ligand poly I:C directly causes tumor cell death. Recently, we reported that tumor suppressor p53 increases the expression of TLR3 in several tumor cell lines. Another study also showed that interferon‐α (IFN‐α) up‐regulates TLR3 expression. We thus hypothesized that various anticancer drugs such as p53‐activating reagents and IFNs may potentiate poly I:C‐induced tumor cell death through the up‐regulation of TLR3 expression. Here, we screened several anticancer drugs that, together with poly I:C, effectively cause tumor cell death in colon carcinoma HCT116 cells. We found that the DNA‐damaging reagent 5‐fluorouracil (5‐FU) increased TLR3 mRNA expression and potentiated poly I:C‐induced apoptosis in HCT116 p53+/+ cells but had only minimal effect in p53−/− cells, indicating a p53‐dependent pathway. On the other hand, IFN‐α increased poly I:C‐induced apoptosis and the TLR3 mRNA level in HCT116 p53+/+ and p53−/− cell lines. Furthermore, the combination of poly I:C, 5‐FU and IFN‐α induced the highest apoptosis in HCT116 p53+/+ and p53−/− cells. Taken together, these data suggest that the anticancer drugs increased TLR3 expression and subsequently potentiated poly I:C‐induced apoptosis likely via p53‐dependent and ‐independent pathways. Considering that the p53 status in malignant cells is heterogeneous, this combination approach may provide a highly effective tumor therapy. (Cancer Sci 2010)

Mild Electrical Stimulation at 0.1-ms Pulse Width Induces p53 Protein Phosphorylation and G2 Arrest in Human Epithelial Cells

Background: A controlled approach as opposed to conventional toxic drugs to activate p53 is applicable for tumors and metabolic and inflammatory diseases. Results: A 0.1-ms pulse width mild electrical stimulation (MES) activated p53 function in epithelial cell lines. Conclusion: MES induced p53 phosphorylation via p38 MAPK signaling and G2 cell cycle arrest without cell death. Significance: MES works as a non-cytotoxic and controllable p53 activator. Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53−/− cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

MEF/ELF4 transactivation by E2F1 is inhibited by p53

Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unex-plored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

Mild Electrical Stimulation and Heat Shock Ameliorates Progressive Proteinuria and Renal Inflammation in Mouse Model of Alport Syndrome

Alport syndrome is a hereditary glomerulopathy with proteinuria and nephritis caused by defects in genes encoding type IV collagen in the glomerular basement membrane. All male and most female patients develop end-stage renal disease. Effective treatment to stop or decelerate the progression of proteinuria and nephritis is still under investigation. Here we showed that combination treatment of mild electrical stress (MES) and heat stress (HS) ameliorated progressive proteinuria and renal injury in mouse model of Alport syndrome. The expressions of kidney injury marker neutrophil gelatinase-associated lipocalin and pro-inflammatory cytokines interleukin-6, tumor necrosis factor-α and interleukin-1β were suppressed by MES+HS treatment. The anti-proteinuric effect of MES+HS treatment is mediated by podocytic activation of phosphatidylinositol 3-OH kinase (PI3K)-Akt and heat shock protein 72 (Hsp72)-dependent pathways in vitro and in vivo. The anti-inflammatory effect of MES+HS was mediated by glomerular activation of c-jun NH2-terminal kinase 1/2 (JNK1/2) and p38-dependent pathways ex vivo. Collectively, our studies show that combination treatment of MES and HS confers anti-proteinuric and anti-inflammatory effects on Alport mice likely through the activation of multiple signaling pathways including PI3K-Akt, Hsp72, JNK1/2, and p38 pathways, providing a novel candidate therapeutic strategy to decelerate the progression of patho-phenotypes in Alport syndrome.

Podocyte p53 Limits the Severity of Experimental Alport Syndrome

Alport syndrome (AS) is one of the most common types of inherited nephritis caused by mutation in one of the glomerular basement membrane components. AS is characterized by proteinuria at early stage of the disease and glomerular hyperplastic phenotype and renal fibrosis at late stage. Here, we show that global deficiency of tumor suppressor p53 significantly accelerated AS progression in X-linked AS mice and decreased the lifespan of these mice. p53 protein expression was detected in 21-week-old wild-type mice but not in age-matched AS mice. Expression of proinflammatory cytokines and profibrotic genes was higher in p53(+/-) AS mice than in p53(+/+) AS mice. In vitro experiments revealed that p53 modulates podocyte migration and positively regulates the expression of podocyte-specific genes. We established podocyte-specific p53 (pod-p53)-deficient AS mice, and determined that pod-p53 deficiency enhanced the AS-induced renal dysfunction, foot process effacement, and alteration of gene-expression pattern in glomeruli. These results reveal a protective role of p53 in the progression of AS and in maintaining glomerular homeostasis by modulating the hyperplastic phenotype of podocytes in AS.

The Transcription Factor MEF/Elf4 Is Dually Modulated by p53-MDM2 Axis and MEF-MDM2 Autoregulatory Mechanism

Background: The ETS transcription factor myeloid elf-1-like factor (MEF) activates some genes including lysozyme, interleukin-8, and MDM2, and also influences the cell cycle. Results: MEF protein expression and stability is suppressed by MDM2 in p53-dependent and -independent manner. Conclusion: MEF is targeted by MDM2 for degradation. Significance: The previously unrecognized MEF-MDM2-p53 axis highlights a regulatory balance of these transcription factors. Myeloid Elf-1-like factor (MEF) or Elf4 is an ETS transcription factor that activates innate immunity-associated genes such as lysozyme (LYZ), human β-defensin 2 (HβD2), and interleukin-8 (IL-8) in epithelial cells and is also known to influence cell cycle progression. MEF is transcriptionally activated by E2F1, but the E2F1-mediated transcriptional activation is inhibited by p53 through E2F1-p53 protein interaction. Although the transcriptional activation of MEF has been investigated in depth, its post-translational regulation is not well explored. By overexpressing MEF cDNA in human cell lines, here we show that MEF protein expression is suppressed by p53. By screening a number of E3 ligases regulated by p53, we found that MDM2 is involved in the effect of p53 on MEF. MDM2 is transcriptionally activated by p53 and interacts with MEF protein to enhance MEF degradation. MDM2 reduces MEF protein expression, as well as stability and function of MEF as transcriptional activator. Furthermore, MDM2 was able to down-regulate MEF in the absence of p53, indicating a p53-independent effect on MEF. Notably, MEF transcriptionally activates MDM2, which was previously demonstrated to be the mechanism by which MEF suppresses the p53 protein. These results reveal that in addition to the potential of MEF to down-regulate p53 by transcriptionally activating E3 ligase MDM2, MEF participates with MDM2 in a novel autoregulatory feedback loop to regulate itself. Taken together with the findings on the effect of p53 on MEF, these data provide evidence that the p53-MDM2-MEF axis is a feedback mechanism that exquisitely controls the balance of these transcriptional regulators.

The effect of mild electrical stimulation with heat shock on diabetic KKAy mice

Diabetes mellitus (DM) is a growing health burden worldwide, and the number of patients with metabolic syndrome is increasing as well. The therapeutic strategies for DM and metabolic syndrome, and prevention of disease progression are therefore urgently needed. Insulin resistance is the main pathology in DM and metabolic syndrome. We previously showed that co-treatment of mild electrical stimulation (MES) with heat shock (HS) enhances insulin sensitivity in vitro and in diabetic mouse models. Moreover, MES+HS ameliorated the pathophysiology in human subjects with DM or metabolic syndrome in clinical trials. Despite the growing evidence that MES+HS could be a novel therapeutic approach, it is unclear whether long-term MES+HS treatment from the early stage of disease has preventive effect for DM. Thus, we assessed the effect of MES+HS on the pathophysiology of maturity-onset obesity using diabetic KKAy mice. Here, we showed that long-term treatment of MES+HS alleviated diabetic condition of KKAy mice. Hyperglycemia and insulin resistance were improved by MES+HS treatment. MES+HS also tended to suppress renal hypertrophy and increased the expression of podocyte-specific genes in the kidney. Collectively, our study suggests that long-term treatment of MES+HS is an effective and well-tolerated therapeutic strategy to decelerate the progression of DM and metabolic syndrome at least in part due to enhanced insulin sensitivity and podocyte function. Abbreviations: DM: diabetes mellitus, MES: mild electrical stimulation, HS: heat shock Introduction Type 2 diabetes mellitus (T2DM) is a serious health problem worldwide, and the number of patients with T2DM is rapidly increasing [1]. About 80-90% of T2DM patients are diagnosed as obese. Obesity, caused by the accumulation of visceral fat, is the fundamental cause of the development of metabolic syndrome [2]. Chronic metabolic syndrome exhibits high-blood pressure, hyperglycemia and insulin resistance, leading to T2DM, arteriosclerosis and cardiovascular defects [3]. Especially, progression of T2DM leads to intercurrent diabetic retinopathy and diabetic nephropathy [4,5]. Therefore, treatment that can control the progression of metabolic syndrome and T2DM at the primary stage of these diseases is needed. Previously, we optimized the conditions of mild electrical stimulation (MES) that induce several physiological response signals and molecules such as the insulin receptor (IR)-Akt pathway, MKK3b/6b-p38MAPK, JNK, p53, AMPK and HSP72 [6-9]. Co-treatment with MES and 42°C of heat shock (HS) (MES+HS) ameliorated visceral obesity and diabetic pathophysiology in high-fat diet-induced and leptin receptor mutant (db/db) mice [6]. Moreover, MES+HS treatment reduced the metabolic abnormalities and inflammation in subjects with metabolic syndrome or T2DM in clinical trials [10,11]. The accumulation of IR in the lipid rafts within cell plasma membrane, followed by enhanced sensitivity to insulin, by MES+HS was suggested to contribute to the improvement of insulin resistance in T2DM [12]. Hence, MES+HS can be a useful treatment for metabolic syndrome and T2DM. However, it is unclear whether long-term treatment with MES+HS from the early stage of disease is tolerable and effective in preventing the development of maturityonset obesity and T2DM. Here, we performed long-term treatment (28 weeks) with MES+HS on diabetic KKAy mice. KKAy is a well-established mouse model of metabolic syndrome with maturity-onset obesity, and spontaneously develops hyperglycemia, high blood pressure, and insulin resistance, which are quite similar to those observed in human T2DM [13,14]. The present study showed that long-term treatment with MES+HS decreased the blood glucose levels at the late stage of disease and also improved insulin resistance compared to sham-treated group. Additionally, long-term MES+HS treatment improved renal hypertrophy that is the hallmark of primary stage diabetic nephropathy and increased the expression of glomerular visceral epithelial cells and podocyte marker genes expression. This study revealed the beneficial Correspondence to: Hirofumi Kai, Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto City 862-0973, Japan. Tel: +81-96-371-4405; Fax: +81-96371-4405; E-mail: hirokai@gpo.kumamoto-u.ac.jp

STAT3 inhibition attenuates the progressive phenotypes of Alport syndrome mouse model

Background Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS. Method Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules. Results Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-β) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-β signaling. Conclusion STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS.