Transcription factors Sp 1 and Sp 4 regulate TRPV 1 gene expression in rat sensory neurons

Background: The capsaicin receptor, transient receptor potential vanilloid type -1 (TRPV1) directs complex roles in signal transduction including the detection of noxious stimuli arising from cellular injury and inflammation. Under pathophysiologic conditions, TRPV1 mRNA and receptor protein expression are elevated in dorsal root ganglion (DRG) neurons for weeks to months and is associated with hyperalgesia. Building on our previous isolation of a promoter system for the rat TRPV1 gene, we investigated the proximal TRPV1 P2-promoter by first identifying candidate Sp1-like transcription factors bound in vivo to the P2-promoter using chromatin immunoprecipitation (ChIP) assay. We then performed deletion analysis of GC-box binding sites, and quantified promoter activity under conditions of Sp1 / Sp4 over-expression versus inhibition/knockdown. mRNA encoding Sp1, Sp4 and TRPV1 were quantified by qRT-PCR under conditions of Sp1/Sp4 over-expression or siRNA mediated knockdown in cultured DRG neurons. Results: Using ChIP analysis of DRG tissue, we demonstrated that Sp1 and Sp4 are bound to the candidate GC-box site region within the endogenous TRPV1 P2-promoter. Deletion of GC-box “a” or “a + b” within the P2promoter resulted in a complete loss of transcriptional activity indicating that GC-box “a” was the critical site for promoter activation. Co-transfection of Sp1 increased P2-promoter activity in cultured DRG neurons whereas mithramycin-a, an inhibitor of Sp1-like function, dose dependently blocked NGF and Sp1-dependent promoter activity in PC12 cells. Co-transfection of siRNA directed against Sp1 or Sp4 decreased promoter activity in DRG neurons and NGF treated PC12 cells. Finally, electroporation of Sp1 or Sp4 cDNA into cultures of DRG neurons directed an increase in Sp1/Sp4 mRNA and importantly an increase in TRPV1 mRNA. Conversely, combined si-RNA directed knockdown of Sp1/Sp4 resulted in a decrease in TRPV1 mRNA. Conclusion: Based on these studies, we now propose a model of TRPV1 expression that is dependent on Sp1-like transcription factors with Sp4 playing a predominant role in activating TRPV1 RNA transcription in DRG neurons. Given that increases of TRPV1 expression have been implicated in a wide range of pathophysiologic states including persistent painful conditions, blockade of Sp1-like transcription factors represents a novel direction in therapeutic strategies. Background Identification of receptors/ion channels that respond to noxious stimuli has been at the forefront of a new understanding of peripheral pain transduction. A seminal finding was the isolation of TRPV1 (capsaicin receptor, VR1) [1] which functions as an integrator of multiple noxious stimuli [2-5] and is essential for the detection of inflammatory pain/hyperalgesia [6,7]. TRPV1 is not only selectively expressed in a subset of primary afferent nociceptors, but its expression is also dynamically regulated. Nociceptor expression of TRPV1 mRNA and receptor protein is lost over a period of days when target-tissue derived trophic factors such as Nerve Growth Factor (NGF) are reduced [8,9]. In contrast, conditions that increase trophic factors as a result of inflammation or tissue-nerve injury result in an increase in TRPV1 mRNA and/or receptor protein expression [10-13]. In part, these reports suggest that a transcription-dependent mechanism drives persistent TRPV1 mediated pain and hyperalgesia. To advance our understanding of how TRPV1 transcription is enhanced under pathophysiologic conditions, * Correspondence: schumacm@anesthesia.ucsf. edu † Contributed equally University of California, San Francisco Department of Anesthesia and Perioperative Care 513 Parnassus Ave. Rm. S436 (Box0427), University of California, San Francisco 94143-0427 USA Chu et al. Molecular Pain 2011, 7:44 http://www.molecularpain. com/content/7/1/44 MOLECULAR PAIN © 2011 Chu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. we initially isolated and characterized a dual promoter system for the rat TRPV1 gene [14]. These studies revealed that the proximal P2-promoter directed celltype specific activity that was positively regulated by the trophic factor NGF [14]. Building on these observations, we now investigate the role of regulatory sites within the P2-promoter and attempt to identify candidate transcription factors that control the activity of the P2promoter and apparently regulate the transcription of TRPV1 RNA in sensory neurons. Based on transcription factor binding studies in dorsal root ganglion (DRG), luciferase-based transcriptional assays in cultured sensory neurons and NGF treated PC12 cells and quantitative measurements of mRNA encoding Sp1-like factors and TRPV1, we propose a model of TRPV1 gene expression that is dependent on the action of at least two members of the Sp1-like transcription factor family, Sp1 and Sp4, acting at a specific GC-box binding site.