Restriction of intracellular Salmonella replication by restoring TFEB-mediated xenophagy.

Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular Salmonella typhimurium infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden. ABBREVIATIONS: 3-MA: 3-methyladenine; BafA1: bafilomycin A1; CFU: colony-forming units; DQ-BSA: dye quenched-bovine serum albumin; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; FM 4-64: pyridinium,4-(6-[4-{diethylamino}phenyl]-1,3,5-hexatrienyl)-1-(3[triethylammonio] propyl)-dibromide; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; MAPILC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; RFP: red fluorescent protein; SCVs: Salmonella-containing vacuoles; SD: standard deviation; SDS: sodium dodecyl sulfate; SEM: standard mean error; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TFEB: transcription factor EB.

Modulation of Autophagy by a Small Molecule Inverse Agonist of ERRα Is Neuroprotective.

Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson's disease (PD), XCT 790 exerted neuroprotective effects in the dopaminergic neurons of nigra by inducing autophagy to clear toxic protein aggregates and, in addition, ameliorated motor co-ordination deficits. Using a chemical biology approach, we unrevealed the role of ERRα in regulating autophagy and can be therapeutic target for neurodegeneration.

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity.

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.

Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes.

INTRODUCTION: The farnesoid X receptor (FXR) is critically involved in the regulation of the hepato-biliary system. Recent data suggest a role for FXR in modulating other metabolic pathways and vascular function. AIM: To investigate whether long-term administration of the selective FXR agonist INT-747 ameliorates erectile function, we tested it in two animal models of metabolic derangements: a rabbit model of high-fat diet (HFD)-induced metabolic syndrome (MetS) and a rat model of streptozotocin (STZ)-induced type 1 diabetes. METHODS: HFD rabbit or STZ rats with or without chronic INT-747 dosing (10 mg/kg/day for 12 weeks). INT-747 addition to rabbit penile smooth muscle cells (rpSMCs). MAIN OUTCOME MEASURE: Effects of INT-747 on metabolic features and erectile function in animal models and clarification of mechanism of action in isolated cells. RESULTS: INT-747 dosing normalized visceral adiposity and glucose intolerance in HFD rabbits. INT-747 increased penile FXR expression and partially restored endothelial nitric oxide synthase and dimethylarginine dimethylaminohydrolase 1 expression as well as impaired nitric oxide (NO)-dependent relaxation (improved responsiveness to acetylcholine and electrical field stimulation). INT-747 was also effective in regulating NO downstream events, as shown by increased sodium nitroprusside-induced relaxation. Because phosphodiesterase type 5 and protein kinase G (PKG) were unaltered by INT-747, we analyzed the calcium-sensitizing RhoA/ROCK pathway. HFD increased, and INT-747 normalized, RhoA membrane translocation/activation. RhoA/ROCK signaling inhibition by INT-747 was confirmed in rpSMCs by confocal microscopy, MYPT1-phosphorylation, cytoskeleton remodeling, cell migration, and smooth muscle-related genes expression. In STZ rats, FXR penile expression was not altered but was significantly upregulated by INT-747 dosing. In this model, INT-747 improved penile erection induced by electrical stimulation of cavernous nerve and hypersensitivity to intracavernous injection of a ROCK-inhibitor, Y-27632, without improving hyperglycemia. CONCLUSION: In HFD rabbits, INT-747 dosing improved glucose sensitivity and MetS-associated erectile dysfunction, via upregulation of NO transmission and inhibition of RhoA/ROCK pathway. In STZ rats, INT-747 restored in vivo penile erection and sensitivity to ROCK inhibition, independently of effects on glycemia.

Sex steroid receptors in male human bladder: expression and biological function.

INTRODUCTION: In male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity. AIM: To investigate the effects of changing sex steroids on bladder smooth muscle. METHODS: ER α, ER ß, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca(2+) ](i) was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed. MAIN OUTCOME MEASURES: The effects of classical (ER α, ER ß) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17 ß-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol. RESULTS: Aromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17 ß-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole. CONCLUSION: Our data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway.

Acute vardenafil administration improves bladder oxygenation in spontaneously hypertensive rats.

INTRODUCTION: In human bladder, phosphodiesterase type 5 (PDE5) is present not only in the muscular wall but also in the vascular beds, suggesting a role for PDE5 inhibitors in favoring bladder blood flow and tissue oxygenation. AIM: To investigate whether acute administration of vardenafil could affect bladder oxygenation in spontaneously hypertensive rats (SHR), an animal model of naturally occurring overactive bladder. MAIN OUTCOME MEASURES: The effect of vardenafil on hypoxia-induced alterations was studied in vivo in SHR by acute dosing (10 mg/kg, 90 minutes before sacrifice) and in vitro in human bladder smooth muscle cells (hBCs). METHODS: Bladder oxygenation was detected using the hypoxyprobe immunostaining. The expression of some hypoxia markers (vascular endothelial growth factor [VEGF] and endothelin-1 type B [ETB] receptor) was also evaluated by immunohistochemistry and Western blot. Gene expression in hBC was quantified by real-time reverse transcription-polymerase chain reaction. RESULTS: Rat bladder PDE5 immunopositivity was detected in the muscular wall and in the endothelial and smooth muscle cells of blood vessels. In SHR bladder, a significant increase of hypoxic cells, VEGF, and ETB expression was observed when compared with their normotensive counterpart Wistar Kyoto rats (WKY). Vardenafil treatment dramatically decreased hypoxyprobe staining, as well as VEGF and ETB expression in SHR bladder up to WKY level. Accordingly, in SHR bladder, vardenafil administration significantly blunted relaxation induced by the selective ETB agonist IRL-1620. In hBCs, experimental hypoxia significantly induced gene expression of hypoxia markers (carbonic anhydrase IX and VEGF), which was not changed by simultaneous treatment with vardenafil. Conversely, the hypoxia-related induction of smooth muscle-specific genes (alphaSMA, SM22alpha, and desmin) was significantly reduced by vardenafil. CONCLUSIONS: SHR showed bladder hypoxia which was significantly reduced by acute vardenafil treatment. Thus, besides relaxing muscular wall, PDE5 inhibition may positively affect urinary vesicle blood perfusion.

Testosterone partially ameliorates metabolic profile and erectile responsiveness to PDE5 inhibitors in an animal model of male metabolic syndrome.

INTRODUCTION: Metabolic syndrome (MetS) is a clustering of cardio-metabolic risk factors (hyperglycemia, hypertension, dyslipidemia, visceral fat accumulation) that is also associated with hypogonadism and erectile dysfunction (ED). AIM: To clarify the relationships among MetS, hypogonadism, and ED, we developed an animal model of MetS. METHODS: Male rabbits fed a high-fat diet (HFD), with or without testosterone (T) supplementation, were compared with control rabbits (fed a standard chow) and with rabbits made hypogonadal by a single injection of a long-acting GnRH-analog, triptorelin. MAIN OUTCOME MEASURES: Evaluation of metabolic disturbances (plasma glucose, cholesterol, triglycerides, testosterone, LH, FSH level, glucose tolerance, mean arterial pressure, visceral fat accumulation), and corpora cavernosa (CC) relaxant capacity (in vitro contractility study) in HFD animals as compared with control, GnRH analog-treated rabbits, and T-supplemented HFD rabbits. RESULTS: HFD rabbits showed all the features of MetS. HFD induced hypogonadotropic hypogonadism is characterized by a reduction of plasma T, FSH, LH levels, testis and seminal vesicles weight, and testicular steroidogenic enzymes. Such a phenotype is similar to that induced by triptorelin administration. A reduced GnRH immunopositivity in hypothalamus suggests a central origin of HFD-related hypogonadism. HFD also induced penile alterations, as demonstrated by a reduction of acetylcholine-and electrical field stimulation-induced CC relaxation, hyper-responsiveness to the NO donor, SNP, and unresponsiveness to PDE5 inhibitors. Similar penile alterations were observed in triptorelin treated rabbit. In HFD, as well as in triptorelin treated rabbits, PDE5 and eNOS mRNA expression quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) were significantly decreased. T administration prevented almost all penile alterations observed in HFD rabbits. T treatment dramatically reduced HFD-induced visceral obesity, partially ameliorating also the metabolic profile. CONCLUSION: We have developed an animal model of MetS associated with hypogonadotropic hypogonadism and penile alterations including unresponsiveness to PDE5 inhibitors. T supplementation was able to partially revert HFD-induced phenotype.

Vardenafil modulates bladder contractility through cGMP-mediated inhibition of RhoA/Rho kinase signaling pathway in spontaneously hypertensive rats.

INTRODUCTION: Phosphodiesterase type 5 inhibitors (PDE5i), the most widely used drugs for erectile dysfunction, could also improve lower urinary tract symptoms, essentially due to overactive bladder (OAB), a condition hypothesized to be a result of an increased RhoA/Rho-kinase (ROCK) signaling. Phosphorylation/inactivation of RhoA by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) activity has been described in vascular smooth muscle. AIM: The aim of this paper was to investigate whether vardenafil-induced cGMP accumulation reduces RhoA/ROCK signaling in bladder. METHODS: Spontaneously hypertensive rats (SHRs), a strain genetically prone to develop OAB, were treated with vardenafil (10 mg/kg/day) for 2 weeks. Wistar-Kyoto rats (WKY) were used as control. In vitro experiments were performed in human bladder smooth muscle cells (hBCs). MAIN OUTCOME MEASURES: Urodynamic parameters were registered in vivo in anesthetized WKY and SHRs. RhoA/ROCK activity in bladder was evaluated by molecular and functional studies in tissues and cells. RESULTS: The intercontraction interval and bladder capacity, and were decreased in SHRs and restored by vardenafil. The in vitro relaxant effect of the ROCK inhibitor Y-27632 was higher in bladder strips from SHR than from WKY and reduced by vardenafil. Nomega-nitro-L-arginine-methyl-ester (a NO-synthase inhibitor, 40 mg/kg/day during the last week of the 2-week treatment with vardenafil) partially antagonized vardenafil effect on Y-27632 responsiveness. Vardenafil prevented RhoA membrane translocation/activation, decreased ROCK activity, and increased cGMP levels in vivo (rat) and in vitro (hBCs). Exposing hBCs to vardenafil increased Ser(188) RhoA phosphorylation, to the same extent as the PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. Moreover, vardenafil inhibited several RhoA-dependent functions in hBCs, including smooth muscle gene transcription and endothelin-1-induced migration. These effects were reverted by the PKG inhibitor KT 5823, further suggesting a cGMP/PKG-dependency. In hBCs, vardenafil was active in the low nanomolar range. CONCLUSIONS: This is the first study demonstrating that the effect of vardenafil on OAB could be partially determined by a cGMP-dependent RhoA/ROCK signaling inhibition.

Dihydrotestosterone and leptin regulate gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts.

INTRODUCTION: The reversal of hypogonadotropic hypogonadism (HH), occurring after discontinuation of testosterone therapy in adolescents with delayed puberty and in a small percentage of adults with congenital HH, suggests a role for androgens in favoring a spontaneous recovery of reproductive function. AIM: We investigated the effect of androgens and leptin on gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts (FNC-B4). METHODS: Quantitative real-time polymerase chain reaction RT-PCR for mRNA expression and radioimmunoassay for GnRH secretion were used. Immunohistochemical studies assessed GnRH protein expression. FNC-B4 migration was analyzed with multiwell Boyden chamber technique. MAIN OUTCOME MEASURES: Effects of the non-aromatizable androgen dihydrotestosterone (DHT) and leptin in FNC-B4 were tested after 24 and 48 hours. RESULTS: Exposure to increasing concentrations of DHT after 24 hours significantly stimulated GnRH mRNA in FNC-B4. This effect was still present after prolonged exposure (48 hours). Similarly, treatment with leptin significantly induced GnRH mRNA after 24 hours, but not at 48 hours. Interestingly, mRNA for leptin receptors (LEPR) was significantly reduced after 48 hours of leptin, while, at this time point, it was stimulated by DHT. Coincubation for 48 hours with leptin and DHT maintained the stimulatory effect on both GnRH and LEPR mRNA, suggesting that DHT could stabilize the leptin effect by preventing downregulation of LEPR. Similar results were obtained for GnRH protein expression analysis. Moreover, both DHT and leptin increased GnRH release into the culture medium. We also found that DHT or leptin treatment significantly increased FNC-B4 basal migration. As we previously found that GnRH stimulates FNC-B4 migration, we hypothesized that this effect could be mediated by DHT- and leptin-induced GnRH release. Accordingly, the GnRH antagonist cetrorelix inhibited DHT- and leptin-induced migration. CONCLUSION: Our results suggest that androgens (adequate hormonal status) could have a positive effect on GnRH neuronal activity by synergizing with leptin (adequate energy status) in the regulatory mechanisms required for reproductive and sexual fitness.

Regulation of mRNAs encoding MMP-9 and MMP-2, and their inhibitors TIMP-1 and TIMP-2 by androgens in the rat ventral prostate.

The relative activities of matrix metalloproteinases (MMPs) and their natural inhibitors (tissue inhibitors of matrix metalloproteinases, TIMPs) determine the extent of matrix degradation in any tissue. Their identification and characterization is key towards understanding remodeling of the prostate in the context of both castration induced atrophy and tumor invasion and metastasis. Although the expression of MMPs and TIMPs in prostate tumors has been reported, their regulation by androgens has not been studied. Here, we show that androgen ablation by castration increases the steady state mRNA levels of MMP-9, MMP-2, TIMP-1 and TIMP-2. Blockade of the androgen receptor using flutamide, however, has differential effects on the steady state mRNA expressions of these genes. We also show that both castration and flutamide treatment cause enhanced expression of a high molecular weight gelatinolytic activity in the rat ventral prostate (RVP). Actinomycin D does not affect the increase in steady state mRNA levels of MMP-9 and TIMP-1. Furthermore we show that actinomycin D alone enhances the steady state mRNA and protein levels of these genes. Using RNA gel shift assay with 3'-UTR of TIMP-1, we show that an RNA binding protein is induced following castration. Taken together our data suggest that the induction of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNAs post-castration could be at least in part due to post-transcriptional stabilization.

The vitamin D receptor agonist elocalcitol upregulates L-type calcium channel activity in human and rat bladder.

Human bladder contraction mainly depends on Ca2+ influx via L-type voltage-gated Ca2+ channels and on RhoA/Rho kinase contractile signaling, which is upregulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho kinase signaling in rat and human bladder. Since in the normal bladder from Sprague-Dawley rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness and increased sensitivity to the L-type Ca2+ channel antagonist isradipine, we investigated whether elocalcitol upregulated L-type Ca2+ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca2+], which was abrogated by the L-type Ca2+ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca2+ currents (I Ca), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist BAY K 8644. Addition of elocalcitol (10(-7) M) increased L-type I Ca size and specific conductance by inducing faster activation and inactivation kinetics than control and BAY K 8644, while determining a significant negative shift of the activation and inactivation curves, comparable to BAY K 8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10(-8) M, 48 h), which also showed increased mRNA and protein expression of pore-forming L-type alpha(1C)-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30 microg.kg(-1).day(-1), 2 wk). In conclusion, elocalcitol upregulated Ca2+ entry through L-type Ca2+ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.

Expression profiling of genes regulated by TGF-beta: differential regulation in normal and tumour cells.

BACKGROUND: TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. RESULTS: Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. CONCLUSION: These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.