The Expression of Transcription Factors Mecp2 and CREB Is Modulated in Inflammatory Pelvic Pain

Early activation of transcription factors is one of the epigenetic mechanisms contributing to the induction and maintenance of chronic pain states. Previous studies identified the changes in a number of nociception-related genes, such as calcitonin gene-related peptide (CGRP), substance P (SP), and brain-derived neurotropic factor (BDNF) in the pelvic organs after transient colonic inflammation. The gene and protein expression of these neuropeptides could be modulated by transcription factors Methyl-CpG-binding protein 2 (Mecp2) and cAMP response element-binding protein (CREB). In this study, we aimed to evaluate time-dependent changes in the expression levels of Mecp2 and CREB in the lumbosacral (LS) spinal cord and sensory ganglia after inflammation-induced pelvic pain in rat. Adult Sprague-Dawley rats were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce transient colonic inflammation. LS (L6-S2) spinal cord segments and respective dorsal root ganglias (DRGs) were isolated from control and experimental animals at 1, 2, 6, 24 h and 3 days post-TNBS treatment. Immunohistochemical (IHC) labeling and Western blotting experiments were performed to assess the expression of Mecp2, CREB and their phosphorylated forms. Total Mecp2 expression, but not phosphorylated p-Mecp2 (pS421Mecp2) expression was detected in the cells of the spinal dorsal horn under control conditions. Colonic inflammation triggered a significant decrease in the number of Mecp2-expressing neurons in parallel with elevated numbers of pS421Mecp2-expressing cells at 2 h and 6 h post-TNBS. The majority of Mecp2-positive cells (80 ± 6%) co-expressed CREB. TNBS treatment caused a transient up-regulation of CREB-expressing cells at 1 h post-TNBS only. The number of cells expressing phosphorylated CREB (pS133CREB) did not change at 1 h and 2 h post-TNBS, but was down-regulated by three folds at 6 h post-TNBS. Analysis of DRG sections revealed that the number of Mecp2-positive neurons was up-regulated by TNBS treatment, reaching three-fold increase at 2 h post-TNBS, and eight-fold increase at 6 h post-TNBS (p ≤ 0.05 to control). These data showed early changes in Mecp2 and CREB expression in the dorsal horn of the spinal cord and sensory ganglia after colonic inflammation, suggesting a possible contribution Mecp2 and CREB signaling in the development of visceral hyperalgesia and pelvic pain following peripheral inflammation

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Kidney injury molecule-1 is up-regulated in renal epithelial cells in response to oxalate in vitro and in renal tissues in response to hyperoxaluria in vivo.

Oxalate is a metabolic end product excreted by the kidney. Mild increases in urinary oxalate are most commonly associated with Nephrolithiasis. Chronically high levels of urinary oxalate, as seen in patients with primary hyperoxaluria, are driving factor for recurrent renal stones, and ultimately lead to renal failure, calcification of soft tissue and premature death. In previous studies others and we have demonstrated that high levels of oxalate promote injury of renal epithelial cells. However, methods to monitor oxalate induced renal injury are limited. In the present study we evaluated changes in expression of Kidney Injury Molecule-1 (KIM-1) in response to oxalate in human renal cells (HK2 cells) in culture and in renal tissue and urine samples in hyperoxaluric animals which mimic in vitro and in vivo models of hyper-oxaluria. Results presented, herein demonstrate that oxalate exposure resulted in increased expression of KIM-1 m RNA as well as protein in HK2 cells. These effects were rapid and concentration dependent. Using in vivo models of hyperoxaluria we observed elevated expression of KIM-1 in renal tissues of hyperoxaluric rats as compared to normal controls. The increase in KIM-1 was both at protein and mRNA level, suggesting transcriptional activation of KIM-1 in response to oxalate exposure. Interestingly, in addition to increased KIM-1 expression, we observed increased levels of the ectodomain of KIM-1 in urine collected from hyperoxaluric rats. To the best of our knowledge our studies are the first direct demonstration of regulation of KIM-1 in response to oxalate exposure in renal epithelial cells in vitro and in vivo. Our results suggest that detection of KIM-1 over-expression and measurement of the ectodomain of KIM-1 in urine may hold promise as a marker to monitor oxalate nephrotoxicity in hyperoxaluria

Genome wide analysis of differentially expressed genes in HK-2 cells, a line of human kidney epithelial cells in response to oxalate.

Nephrolithiasis is a multi-factorial disease which, in the majority of cases, involves the renal deposition of calcium oxalate. Oxalate is a metabolic end product excreted primarily by the kidney. Previous studies have shown that elevated levels of oxalate are detrimental to the renal epithelial cells; however, oxalate renal epithelial cell interactions are not completely understood. In this study, we utilized an unbiased approach of gene expression profiling using Affymetrix HG_U133_plus2 gene chips to understand the global gene expression changes in human renal epithelial cells [HK-2] after exposure to oxalate. We analyzed the expression of 47,000 transcripts and variants, including 38,500 well characterized human genes, in the HK2 cells after 4 hours and 24 hours of oxalate exposure. Gene expression was compared among replicates as per the Affymetrix statistical program. Gene expression among various groups was compared using various analytical tools, and differentially expressed genes were classified according to the Gene Ontology Functional Category. The results from this study show that oxalate exposure induces significant expression changes in many genes. We show for the first time that oxalate exposure induces as well as shuts off genes differentially. We found 750 up-regulated and 2276 down-regulated genes which have not been reported before. Our results also show that renal cells exposed to oxalate results in the regulation of genes that are associated with specific molecular function, biological processes, and other cellular components. In addition we have identified a set of 20 genes that is differentially regulated by oxalate irrespective of duration of exposure and may be useful in monitoring oxalate nephrotoxicity. Taken together our studies profile global gene expression changes and provide a unique insight into oxalate renal cell interactions and oxalate nephrotoxicity

Mitogen Activated Protein kinase signal transduction pathways in the prostate

Abstract The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.</p