Transient receptor potential vanilloid 1 activation induces autophagy in thymocytes through ROS-regulated AMPK and Atg4C pathways.

Autophagy is a highly conserved process involved in lymphocyte development and differentiation. Herein, we demonstrated for the first time that triggering of TRPV1 by the specific agonist CPS induces autophagy in mouse thymocytes. TRPV1-dependent autophagy required [Ca(2+)](i )and ROS generation, resulting in AMPK activation. CPS specifically increased Atg4C mRNA expression and induced oxidation of Atg4C protein by ROS generation. TRPV1-triggered autophagy was Atg6/Beclin-1-dependent, as demonstrated by the use of Beclin-1(+/-) transgenic mice, and involved ROS- and AMPK-mediated up-regulation of Beclin-1 expression. Autophagy is activated as a prosurvival process, as its inhibition triggered apoptosis of thymocytes: this effect was accompanied by down-regulation of Atg4C, Bcl-X(L), and Irgm1 mRNA expression, decreased Bcl-X(L) and Beclin-1 protein levels, and caspase-3 activation, suggesting the existence of a molecular interplay between autophagic and apoptotic programs. TRPV1 activation by CPS altered the expression of CD4 and CD8α antigens, inducing the development of DP(dull). Interestingly, we found that CPS induces autophagy of DP(dull) cells, and inhibition of CPS-induced autophagy by the 3-MA autophagic inhibitor induces apoptosis of DP(dull) cells, suggesting the presence of an interplay between autophagic survival and apoptotic cell death. Overall, our findings suggest that DP(dull) cells constitute a distinct thymocyte subpopulation involved in the homeostatic control of cellularity and in the responses to chemical stress signals during thymocyte maturation, via regulating autophagy and apoptosis in a TRPV1-dependent manner

OnabotulinumtoxinA intradetrusorial injections modulates bladder expression of NGF, TrkA, p75 and TRPV1 in patients with detrusor overactivity

OnabotulinumtoxinA (BoNT/A) intradetrusorially injected in patients with neurogenic and idiopathic detrusor overactivity (DO) significantly decreased NGF levels and TRPV1 expression in bladder and urine. To date no information exist on gene expression of TrkA and p75 (NGF receptors) in bladder tissues of patients treated with BoNT/A. In this study we investigated BoNT/A-induced changes in gene expression of TRKA and p75, and of NGF and TRPV1 in bladder tissue of patients with neurogenic and idiopathic DO. MATERIAL & METHODS: 18 patients with neurogenic DO and 7 with idiopathic DO underwent clinical, urodynamic and cystoscopic evaluation with bladder biopsy specimen before (T0) and one month (T1) after intradetrusor BoNT/A injection (300 U in neurogenic DO and 100 U in idiopathic DO). Changes in gene expression of NGF, TRPV1, TRKA and p75 were investigated with Real Time PCR (qPCR) at T0 and T1. Changes in NGF-protein content (ng/mg) were measured by ELISA assay at T0 and T1. Data are expressed as post/pre ratio. RESULTS: Clinical and urodynamic significant improvements were observed in all patients at T1. We observed a significant increase in NGF (1.68±0.2 folds), TRPV1 (1.83±0.3 folds), TRKA (1.36±0.2 folds) and p75 (2.02±0.4 folds) expression in all patients and a significant decrease in NGF protein bladder content (689±113 ng/total mg vs. 252±67 ng/total mg). (Fig. 1) We found a linear correlation between changes in NGF and TRKA (p=0.005), NGF and TRPV1 (p=0.02), TRKA and TRPV1 (p=0.0002). Patients injected with 300 BoNT/A U had a higher increase in NGF, TRPV1 and TRKA expression than those who received 100 U. No significant correlation was found between p75 and changes in NGF expression (p=0.17). CONCLUSIONS: We confirm the previously reported activity of BoNT/A in reducing NGF bladder tissue content in patients with neurogenic and idiopathic DO. The novel finding of this study was the significant neurotoxin- induced up-regulation of bladder NGF, TrkA and TRPV1 in the short time. This may represent a compensatory change aimed at re-establishing NGF bladder levels and activity. The linear correlation existing among NGF, TrkA and TRPV1 overexpression after treatment demonstrated a strictly linked activity of these transmitters/receptors. p75 expression was highly variable and reflected the fact it is a "pan-neuroreceptor"

Normal human urothelial cell lines express onabotulinumtoxinA SV2 high affinity receptors

INTRODUCTION & OBJECTIVES: Botulinum A toxin (BoNT/A) internalization within the target cells requires the binding with high affinity receptors (Synaptic Vesicle Proteins type 2 -SV2). To date, 3 different isoforms of SV2 have been identified in synaptic and endocrine tissues. Bladder SV2 immunoreactivity has been previously identified in parasympathetic, sympathetic and sensory fibers, but not into bladder urothelial cells. The aim of this study was to evaluate the expression of SV2 in human non neoplastic urothelial cells at genic and protein levels. MATERIAL & METHODS: The study was performed in normal human urothelial cell lines (NHUCs). Expression of the 3 SV2 isoforms (SV2A, -B –C) was investigated by quantitative Real Time PCR (qPCR) and Western blot analysis. In order to identify SV2 subcellular localization, NHUCs were fractionated and proteins from different cellular compartments including membrane, cytoplasm, soluble nuclear and cytoskeleton were separated by SDS-PAGE and probed with anti SV2-A, -B and -C Abs. Mouse brain tissue was used as positive control and ß actin was used as loading control. RESULTS: qPCR demonstrated the presence of SV2-A, -B and -C mRNAs in NHUCs. Higher levels of SV2C (about 1.8 folds) were expressed as compared to SV2A isoform mRNA levels. In addition, SV2B exhibited a 20- and 30-fold reduction in the amount of mRNA levels in comparison with SV2A and SV2C, respectively (Fig. 1). Western blot analysis showed bands with proper molecular weights, likely corresponding to SV2-A, -B and –C, in lysates from NHUCs and mouse brain tissue. All 3 isoforms were found to be accumulated primarily in the cytoplasmic extract, weakly expressed in the membrane fraction and were not detected in soluble nuclear and cytoskeletal compartments. CONCLUSIONS: We demonstrated for the first time the presence of SV2 BoNT/A high affinity receptors on the plasmatic membranes of NHUCs. Recent observations suggested a crucial role for the urothelium in modulating underlying sensory fibers. This may be due not only to the urothelial release of Ach, but also of other neurotransmitters, as ATP, CGRP and Substance P. BoNT/A could act through SV2-mediated internalization on human urothelial cells thus blocking neurotransmitters' exocytosis and controlling afferent transmission and pain. The higher level of SV2C, as compared to the other isoforms, is consistent with the previously described most robust BoNT/A binding activity with SV2C

Present and future of tyrosine kinase inhibitors in renal cell carcinoma: analysis of hematologic toxicity.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of renal cell carcinoma (RCC) patients. The use of these agents requires early and appropriate management of side effects such as hematologic adverse events (HAE), in order to avoid unnecessary dose reductions and transitory or definitive treatment discontinuations. Beyond the increased infective risk, myelosuppression contributes to TKI-related fatigue, thus reducing both patients' quality of life and overall survival (OS). However, the frequency and severity of myelosuppression vary among sunitinib, sorafenib, pazopanib and axitinib, based on their different kinase selectivity. Their activity against fms-related tyrosine kinase 3 (FLT3 or CD135) and c-kit, which are essential for survival and differentiation of hemopoietic progenitor cells, is critical to determine the hematologic toxicity profiles. This review describes the molecular mechanisms underlying the TKI effects exerted on hematopoiesis and immune response and related recent patents, of drugs already approved or still under evaluation in RCC, highlighting the potential impact of these effects on tumor response to treatment