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.

Figure 1

Induction of TAC1 by capsaicin does not occur in all TRPV1 expressing cells. fluorescent immunohistochemical analysis of 10 μm sections from mouse neonate DRG explants with (iii and iv) anti-TRPV1 and (v and vi) anti-SP antibody (i, iii, v) before and (ii, iv, vi) after 24 hours incubation with 10 μM capsaicin. Ai and ii represent merged images showing cellular co-localisation of TRPV1 and SP (yellow). White arrow heads highlight cells that express SP but not TRPV1 after capsaicin treatment. B, and C, graphical analysis of the size distribution (in microns) of neurones within DRG explants expressing (B) SP and (C) TRPV1 before (White bar) and after (black bar) capsaicin treatment (n = 3, total number SP/TRPV1 immunostaining cells counted and measured = 284, *; p < 0.05).

Figure 2

The ECR2-TAC1prom transgene can be activated by cell depolarisation. A, Diagramatic representation (not to scale) demonstrating the linear relationships of the components of each of the different constructs used in the current study. Construction of these reporter vectors has been previously described[16]. pA; SV40 polyadenylation sequence, lacZ; gene encoding β galactosidase marker protein, hβgprom; human beta globin promoter, TAC1prom; TAC1 promoter, ECR2; evolutionary conserved region 2, bent black arrow; indicates the transcriptional start site of the LacZ marker gene. B; bar graph demonstrating the proportion of primary DRG neurones that express βgal (as assayed using X-gal) following their transfection with pECR2-TAC1prom-LacZ and cultured in the presence of forskolin or KCl (n > 3, *;p < 0.05, n.s., not significant). Proportions are adjusted relative to a control plasmid containing the CMV promoter that was transfected at the same time to normalise transfection efficiencies. C, graphical analysis of the size distribution (diameter in microns) of neurones within ECR2-TAC1prom-LacZ transgenic DRG explants showing the proportion of cells expressing the β-gal before and after treatment with 30 mM KCl demonstrating a shift in the proportion of larger diameter cells expressing SP and the receptor (n = 3, No cells counted/measured = 201, *; p < 0.05).

Figure 3

Capsaicin can induce the expression of the NK1 receptor in DRG neurones that also express SP. A. fluorescent immunohistochemical analysis of 10 μm sections from mouse neonate DRG explants with (iii and iv) anti-NK1 and (v and vi) anti-SP antibody (i, iii, v) before and (ii, iv, vi) after 24 hours incubation with 10 μM capsaicin. Ai and ii represent merged images showing cellular co-localisation of NK1 and SP (yellow). B, graphical analysis of the size distribution (in microns) of neurones within mouse neonate DRG explants showing the proportion of cells expressing both SP and NK1 expression before and after capsaicin treatment demonstrating a shift in the proportion of larger diameter cells expressing SP and the receptor (n = 3, No cells counted/measured = 242, *; p < 0.05).

Figure 4

Activation of the NK1 receptor induces expression of the ECR2-Tac1prom-LacZ transgene. A. Fluorescent immunohistochemical analysis of 10 μm sections from ECR2-TAC1prom-LacZ transgenic DRG explants with (iii and iv) anti-SP and (v and vi) anti-βgal antibody (i, iii, v) before and (ii, iv, vi) after 24 hours incubation with 100 nM NK1 agonist [Sar9, Met(02)11]-SP. Ai and ii represent merged images showing cellular co-localisation of NK1 and SP (yellow). B graphical analysis of the size distribution (in microns) of neurones within ECR2-TAC1prom-LacZ transgenic DRG explants showing the proportion of cells expressing both SP and βgal expression before and after [Sar9, Met(01)11]-SP treatment demonstrating a shift in the proportion of larger diameter cells expressing SP and the receptor (n = 3, No cells counted/measured = 407, *; p < 0.05).

Figure 5

Induction of TAC1prom by LPS is independent of ECR2. A; immunohistochemical analysis of the expression of (i) TLR4 and (ii) SP and in mouse neonate DRG. iv; merged images and cells co-expressing SP and TRL4 are highlighted in yellow (white arrows). Scale bar = 23 microns. B; bar graph demonstrating the proportion of primary DRG neurones that express βgal (as assayed using X-gal) following their transfection with each of the constructs shown in figure 2A and cultured in the absence or presence of LPS (n > 3). Proportions are adjusted relative to a control plasmid containing the CMV promoter that was transfected at the same time to normalise transfection efficiencies. C; statistical analysis of data from the previous graph demonstrating that average induction rates for TAC1prom-LacZ by LPS is not significantly affected by the presence of ECR2. D; graphical analysis of the size distribution (in microns) of neurones within ECR2-TAC1prom-LacZ transgenic DRG explants analysing transgene expression following culture in the absence or presence of LPS (n = 3) demonstrating a lack of a shift in the proportion of larger diameter cells expressing βgal (n = 3, no. βgal cells counted and measured = 170).

Figure 6

Diagrammatical representation of the hypothetical genomic and cellular events that affect the activation of TAC1prom in sensory neurones treated with KCl, capsaicin or LPS. This model is based on the conclusions of the current study and the literature. See the discussion section in the main text for a detailed description.