Epigallocatechin-3-gallate alleviates bladder overactivity in a rat model with metabolic syndrome and ovarian hormone deficiency through mitochondria apoptosis pathways.

Metabolic syndrome (MetS) and ovarian hormone deficiency could affect bladder storage dysfunction. Epigallocatechin-3-gallate (EGCG), a polyphenolic compound in green tea, has been shown to protect against ovarian hormone deficiency induced overactive bladder (OAB). The present study investigated oxidative stress induced by MetS and bilateral ovariectomy (OVX), and elucidated the mechanism underlying the protective effect of EGCG (10 umol/kg/day) on bladder overactivity. Rats were fed with high fat high sugar (HFHS) diet to induce MetS and received ovariectomy surgery to deprive ovarian hormone. By dieting with HFHS for 6 months, rats developed MetS and OAB. MetS + OVX deteriorated bladder storage dysfunction more profound than MetS alone. MetS and MetS + OVX rats showed over-expression of inflammatory and fibrosis markers (1.7~3.8-fold of control). EGCG pretreatment alleviated storage dysfunction, and protected the bladders from MetS and OVX - induced interstitial fibrosis changes. Moreover, OVX exacerbated MetS related bladder apoptosis (2.3~4.5-fold of control; 1.8~2.6-fold of Mets group), enhances oxidative stress markers (3.6~4.3-fold of control; 1.8~2.2-fold of Mets group) and mitochondrial enzyme complexes subunits (1.8~3.7-fold of control; 1.5~3.4-fold of Mets group). EGCG pretreatment alleviated bladder apoptosis, attenuated oxidative stress, and reduced the mitochondrial and endoplasmic reticulum apoptotic signals. In conclusions, HFHS feeding and ovarian hormone deficiency enhances the generation of oxidative stress mediated through mitochondrial pathway. EGCG reduced the generation of oxidative stress and lessened bladder overactivity.

Elucidating Mechanisms of Bladder Repair after Hyaluronan Instillation in Ketamine-Induced Ulcerative Cystitis in Animal Model.

Ketamine-induced ulcerative cystitis (KIC) initially damaged the bladder mucosa and induced contracted bladder thereafter. Hyaluronan (hyaluronic acid; HA) instillation to the bladder has been used to treat KIC. The present study investigated bladder injury by urothelial defect and HA degeneration and bladder repair by urothelium proliferation and differentiation. This work was based on the hypothesis that HA treatment altered the bladder urothelial layer and the expression of hyaluronan-metabolizing enzymes and/or HA receptors in KIC. Cystometrogram study and tracing analysis of voiding behavior revealed that the ketamine-treated rats exhibited significant bladder hyperactivity with an increase in micturition frequency and a decrease in bladder capacity. The expression of inflammatory and fibrosis markers was also increased in the ketamine-treated group. Moreover, ketamine administration decreased the expression of urothelial barrier-associated protein, altered HA production, and induced abnormal urothelial differentiation, which might attribute to urothelial lining defects. However, HA instillation ameliorated bladder hyperactivity, lessened bladder mucosa damage, and decreased interstitial fibrosis. HA instillation also improved the level of HA receptors (CD44, Toll-like receptor-4, and receptor for HA-mediated motility) and HA synthases 1 to 3 and decreased the expression of hyaluronidases in the urothelial layer of bladder, resulting in enhanced mucosal regeneration. These findings suggested that HA could modulate inflammatory responses, enhance mucosal regeneration, and improve urothelial lining defects in KIC.

KMUP-1 attenuates high glucose and transforming growth factor-ß1-induced pro-fibrotic proteins in mesangial cells.

We have previously demonstrated that KMUP-1, a xanthine-based nitric oxide enhancer, attenuates diabetic glomerulosclerosis, while increasing renal endothelial nitric oxide synthase expression in rats. However, the anti­fibrotic mechanisms of KMUP­1 treatment in diabetic nephropathy in terms of cell biology and transforming growth factor-ß1 (TGF­ß1) remain unclear. Therefore, the present study involved investigating the effects of KMUP­1 on high glucose (HG) or TGF­ß1­induced pro­fibrotic proteins in mouse mesangial (MES13) cells, and the effects of KMUP­1 on streptozotocin (STZ)­induced diabetic rats. It was identified that KMUP­1 (10 µM) attenuated HG (30 mM)­induced cell hypertrophy while attenuating TGF­ß1 gene transcription and bioactivity in MES13 cells. In addition, KMUP­1 attenuated TGF­ß1 (5 ng/ml)­induced Smad2/3 phosphorylation while attenuating HG or TGF­ß1­induced collagen IV and fibronectin protein expression. Furthermore, KMUP­1 attenuated HG­decreased Suv39h1 and H3K9me3 levels. Finally, KMUP­1 attenuated diabetes-induced collagen IV and fibronectin protein expression in STZ­diabetic rats at 8 weeks. In conclusion, KMUP­1 attenuates HG and TGF­ß1­induced pro­fibrotic proteins in mesangial cells and attenuation of TGF­ß1­induced signaling and attenuation of HG­decreased Suv39h1 expression may be two of the anti-fibrotic mechanisms of KMUP­1.

Histone methyltransferase Suv39h1 attenuates high glucose-induced fibronectin and p21(WAF1) in mesangial cells.

Suppressor of variegation 3-9 homolog 1 (Suv39h1) is a histone methyltransferase that trimethylates lysine 9 of histone H3 (H3K9me3), which results in gene silencing. A previous study found that H3K9me3 and Suv39h1 were decreased in diabetic mouse vascular smooth muscle cells whereas Suv39h1 overexpression attenuated ischemic myocardial injury. Moreover, high glucose (HG) decreased H3K9me3 and Suv39h1 levels in some cells. Thus, we studied the roles of Suv39h1 in HG-induced effects in MES13 (mouse mesangial) cells. We found that HG, chaetocin (a Suv39h1 inhibitor) and Suv39h1 siRNA decreased Suv39h1 while increasing fibronectin and p21(WAF1) protein levels. HG increased mRNA while chaetocin increased transcription of fibronectin and p21(WAF1)genes. Both HG and chaetocin decreased histone H3K9me3 levels at the promoters of fibronectin and p21(WAF1) genes. Additionally, Suv39h1 overexpression attenuated HG-induced fibronectin and p21(WAF1) mRNA and protein expressions while attenuating HG-induced cell hypertrophy. Suv39h1 overexpression also attenuated HG-suppressed histone H3K9me3 levels at the promoters of fibronectin and p21(WAF1) genes. Moreover, LY294002 or the dominant-negative phosphoinositide 3-kinase (PI3K) mutant (Δp85) attenuated HG-decreased Suv39h1 and HG-induced fibronectin and p21(WAF1) protein expressions. We concluded that HG decreased Suv39h1 via the PI3K pathway in mesangial cells. Inhibition of Suv39h1 increased fibronectin and p21(WAF1) expressions. Moreover, Suv39h1 overexpression attenuated HG-induced fibronectin and p21(WAF1) expressions and cell hypertrophy while attenuating HG-suppressed histone H3K9me3 levels at the promoters of fibronectin and p21(WAF1) genes.

Arecoline-induced pro-fibrotic proteins in LLC-PK1 cells are dependent on c-Jun N-terminal kinase.

Areca nut (AN) chewing is associated with chronic kidney disease (CKD). However, the molecular mechanisms of AN-induced CKD are not known. Thus, we studied the effects of arecoline, a major alkaloid of AN, on proximal tubule (LLC-PK1) cells in terms of cytotoxicity, fibrosis, transforming growth factor-ß (TGF-ß) and c-Jun N-terminal kinase (JNK). We found that arecoline dose (0.1-0.5mM) and time (24-72h)-dependently induced cytotoxicity without causing cell death. Arecoline (0.25 mM) also time-dependently (24-72h) increased fibronectin and plasminogen activator inhibitor-1 (PAI1) protein expressions. Arecoline (0.25 mM) time-dependently (24-72h) increased TGF-ß gene transcriptional activity and supernatant levels of active TGF-ß1. Moreover, arecoline (0.25 mM) activated JNK while SP600125 (a JNK inhibitor) attenuated arecoline-induced TGF-ß gene transcriptional activity. SP600125, but not SB431542 (a TGF-ß receptor type I kinase inhibitor), attenuated arecoline-induced fibronectin and PAI1 protein expressions. Finally, tubulointerstitial fibrosis occurred and renal cortical expressions of fibronectin and PAI1 proteins increased in arecoline-fed mice at 24 weeks. We concluded that arecoline induced tubulointerstitial fibrosis in mice while arecoline-induced TGF-ß and pro-fibrotic proteins (fibronectin, PAI1) are dependent on JNK in LLC-PK1 cells.

Ketamine-induced ulcerative cystitis and bladder apoptosis involve oxidative stress mediated by mitochondria and the endoplasmic reticulum.

Ketamine abusers develop severe lower urinary tract symptoms. The major aims of the present study were to elucidate ketamine-induced ulcerative cystitis and bladder apoptosis in association with oxidative stress mediated by mitochondria and the endoplasmic reticulum (ER). Sprague-Dawley rats were distributed into three different groups, which received normal saline or ketamine for a period of 14 or 28 days, respectively. Double-labeled immunofluorescence experiments were performed to investigate tight junction proteins for urothelial barrier functions. A TUNEL assay was performed to evaluate the distribution of apoptotic cells. Western blot analysis was carried out to examine the expressions of urothelial tight junction proteins, ER stress markers, and apoptosis-associated proteins. Antioxidant enzymes, including SOD and catalase, were investigated by real-time PCR and immunofluorescence experiments. Ketamine-treated rats were found to display bladder hyperactivity. This bladder dysfunction was accompanied by disruptions of epithelial cadherin- and tight junction-associated proteins as well as increases in the expressions of apoptosis-associated proteins, which displayed features of mitochondria-dependent apoptotic signals and ER stress markers. Meanwhile, expressions of mitochondria respiratory subunit enzymes were significantly increased in ketamine-treated bladders. Conversely, mRNA expressions of the antioxidant enzymes Mn-SOD (SOD2), Cu/Zn-SOD (SOD1), and catalase were decreased after 28 days of ketamine treatment. These results demonstrate that ketamine enhanced the generation of oxidative stress mediated by mitochondria- and ER-dependent pathways and consequently contributed to bladder apoptosis and urothelial lining defects. Such oxidative stress-enhanced bladder cell apoptosis and urothelial barrier defects are potential factors that may play a crucial role in bladder overactivity and ulceration.

Quantitative analyses of myelofibrosis by determining hydroxyproline.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are blood malignancies manifested in increased production of red blood cells, white blood cells, and/or platelets. Myelofibrosis is a subtype of MPNs characterized by the formation of scar-like tissues in the bone marrow due to abnormal hematopoiesis. It is considered a disease of both hematopoietic stem cells and stem cell niches. Patients with myelofibrosis have very poor prognosis, and there is no effective treatment so far. Myelofibrosis has routinely been detected by using histochemical staining methods which produce qualitative rather than quantitative results. In this study, we developed a quantitative assay of bone marrow myelofibrosis in JAK2V617F transgenic mice by determining hydroxyproline. METHODS: The JAK2V617F transgenic mice's tissue was collected to detect the bone marrow myelofibrosis. Statistical analyses were performed using the GraphPad Prism program. Differences of samples between two groups were accessed using t tests. P values less than 0.05 (2-tailed) were considered significantly different. RESULTS: We developed a quantitative method for detecting myelofibrosis by analyzing the content of hydroxyproline, a modified amino acid largely restricted to collagen which forms the fibrotic structure in bone marrow tissues. Our study also demonstrated age-dependent development of bone marrow myelofibrosis in JAK2V617F transgenic mice. CONCLUSIONS: In the present study, we have developed a new method for detecting bone marrow myelofibrosis by analyzing hydroxyproline contents. The method is highly sensitive and accurate. It provides more accurate, representative, and quantitative information than histochemical analyses. We believe that this method should find wide applications for analyzing the progression of myelofibrosis and efficacy of drug treatment.

Tea contains potent inhibitors of tyrosine phosphatase PTP1B.

Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 value of 0.4-4 g dry tea leaves per liter of water. Black tea shows the strongest inhibition activities, followed by oolong and then by green tea. Biochemical fractionations demonstrated that the major effective components in tea corresponded to oxidized polyphenolic compounds. This was further verified by the fact that tea catechins became potent inhibitors of PTP1B upon oxidation catalyzed by tyrosinases. When applied to cultured cells, tea extracts induced tyrosine phosphorylation of cellular proteins. Our study suggests that some beneficial effects of tea may be attributed to the inhibition of PTP1B.

Tyrosine phosphorylation of phospholipase D1 by v-Src does not per se result in activation.

The relationship between tyrosine phosphorylation and activation of phospholipase D1 (PLD1) by v-Src was examined. Co-expression of v-Src and PLD1 in COS-7 cells resulted in increased activity and marked tyrosine phosphorylation of PLD1. PLD activity was increased in membranes or immunoprecipitates prepared from these cells. Dephosphorylation of the immunoprecipitated enzyme by tyrosine phosphatase or phosphorylation by c-Src produced no changes in its activity. Tyrosine phosphorylation induced by v-Src caused a shift of the enzyme from the Triton-soluble to the Triton-insoluble fraction. v-Src and PLD1 could be co-immunoprecipitated from cells co-expressing these and were co-localized in the perinuclear region as assessed by immunofluorescence. Mutation of the palmitoylation sites of PLD1 significantly reduced tyrosine phosphorylation by v-Src. It is concluded that tyrosine phosphorylation of PLD1 by v-Src does not per se alter its activity. It is proposed that activation of PLD1 by v-Src in vivo may involve association/colocalization of the two proteins.

Arfaptin 1 inhibits ADP-ribosylation factor-dependent matrix metalloproteinase-9 secretion induced by phorbol ester in HT 1080 fibrosarcoma cells.

Matrix metalloproteinase-9 (MMP-9) is a collagenolytic enzyme secreted by cancer cells and involved in invasiveness and metastasis. Its secretion from human fibrosarcoma HT 1080 cells is markedly enhanced by phorbol 12-myristate 13-acetate (PMA) and abolished by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation. These results support a role for ARF in PMA-stimulated MMP-9 secretion. Overexpression of arfaptin 1, a 39 kDa ARF-binding protein that inhibits in vitro activation of cholera toxin ADP-ribosyltransferase and phospholipase D (PLD) by ARF, inhibited PMA-stimulated MMP-9 and PLD activation. These data are in agreement with previous results demonstrating a significant role for PLD in regulating MMP-9 secretion.

Functional implications of post-translational modifications of phospholipases D1 and D2.

Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(DeltaN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(DeltaN168) when coexpressed in COS 7 cells and that palmitoylation of Cys(240) and Cys(241) was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys(223) and Cys(224) in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(DeltaN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.

Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq.

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha subunits of the heterotrimeric G proteins G(13) and G(q). Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(13)Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.