Evidence for regulatory diversity and auto-regulation at the TAC1 locus in sensory neurones

Abstract

The neuropeptide substance-P (SP) is expressed from the TAC1 gene in sensory neurones where it acts as a key modulator of neurogenic inflammation. The promoter of TAC1 (TAC1prom) plays a central role in the regulation of the TAC1 gene but requires the presence of a second regulatory element; ECR2, to support TAC1 expression in sensory neurones and to respond appropriately to signalling pathways such as MAPkinases and noxious induction by capsaicin. We examined whether the effect of capsaicin on ECR2-TAC1prom activity in larger diameter neurones was cell autonomous or non- cell autonomous. We demonstrate that TRPV1 is not expressed in all the same cells as SP following capsaicin induction suggesting the presence of a non-cell autonomous mechanism for TAC1 up-regulation following capsaicin induction. In addition, we demonstrate that induction of SP and ECR1-TAC1prom activity in these larger diameter neurones can be induced by potassium depolarisation suggesting that, in addition to capsaicin induction, transgene activity may be modulated by voltage gated calcium channels. Furthermore, we show that NK1 is expressed in all SP- expressing cells after capsaicin induction and that an agonist of NK1 can activate both SP and the transgene in larger diameter neurones. These observations suggest the presence of an autocrine loop that controls the expression of the TAC1 promoter in sensory neurones. In contrast, induction of the TAC1 promoter by LPS was not dependent on ECR2 and did not occur in large diameter neurones. These studies demonstrate the diversity of mechanisms modulating the activity of the TAC1 promoter and provide novel directions for the development of new anti-inflammatory therapies.

Differential Activity by Polymorphic Variants of a Remote Enhancer that Supports Galanin Expression in the Hypothalamus and Amygdala: Implications for Obesity, Depression and Alcoholism

“…Using these cells, we were unable to show that GAL5.1 could act as an enhancer of promoter activity. However, we have previously shown that many regulatory sequences are only able to act as enhancers in response to activation of an appropriate signal transduction cascade (Shanley et al, 2010(Shanley et al, , 2011. Thus, we were able to show that GAL5.1 acted as an enhancer of promoter activity following activation of the PKC pathway, which is known to modulate gene expression in the hypothalamus and amygdala, but not PKA or MAPkinase pathways.…”

“…ECR2 could only drive tissue-specific expression in the presence of the endogenous TAC1 promoter whereas GAL5.1 can support expression in the presence of an exogenous weak promoter such as the human b-globin promoter or HSV-TK promoter. These observations suggest that different enhancers possess varying levels of autonomy or interdependence and that the GAL5.1 is functionally self-contained relative to some other previously characterized enhancers (Shanley et al, 2010(Shanley et al, , 2011.…”

“…Thus, it would be interesting to see how GAL5.1 interacted with the GAL promoter regions previously identified (Bacon et al, 2007). Moreover, an interesting contrast can be drawn between GAL5.1 and the ECR2 enhancer shown to control the expression of the TAC1 gene in sensory neurones (Shanley et al, 2010(Shanley et al, , 2011. ECR2 could only drive tissue-specific expression in the presence of the endogenous TAC1 promoter whereas GAL5.1 can support expression in the presence of an exogenous weak promoter such as the human b-globin promoter or HSV-TK promoter.…”

Lipopolysaccharide-induced Pulpitis Up-regulates TRPV1 in Trigeminal Ganglia

“…Alternatively, LPS can directly modulate pulpal afferents. TLR4 is expressed in a subpopulation of dorsal root ganglia neurons that contain substance P (Shanley et al, 2011). Moreover, CD14 and TLR4 are expressed in pulpal afferent terminals and trigeminal nociceptors, mainly in a TRPV1-positive subpopulation (Wadachi and Hargreaves, 2006;Diogenes et al, 2011;Ferraz et al, 2011).…”

CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake

“…Total RNA was extracted using the isolate II RNA minikit (Bioline). Quantitative reverse transcriptase-PCR (QrtPCR) to determine mRNA expression levels of genes flanking mGAL5.1 ( Lrp5, Ppp6r3, Gal, Mlt5 and Cpt1a ) was undertaken using gene specific primers (See table 1 in supplementary data) on derived cDNA using mouse Qrt-PCR primers as previously described 19, 20 using a Roche Light Cycler 480 with Roche SYBR green. All QrtPCR analyses were normalised using mouse primers specific to the Nono housekeeping gene that gave the most stable expression in all neuronal tissues analysed compared to βactin, HPRT or GAPDH genes.…”