The regulation of transcription is a fundamental way that mammalian cells restrict and adjust their gene expression in response to changing cellular conditions such as stress or injury. In the case of primary afferent nociceptors, gene expression of ion channel -receptors such TRPV1 can be increased or decreased depending on changes in the cellular environment. Recruitment of RNA polymerase II for gene-specific transcription of TRPV1 DNA into RNA is envisioned to require a unique set of transcription factors that direct a subset of sensory neurons to express TRPV1. Our findings reported here support the hypothesis that the P2-promoter is a major site for the regulation of TRPV1 RNA transcription. In addition to being adjacent to TRPV1 transcriptional (RNA) start sites confirmed by 5'-RACE and EST database , we have also reported that the P2-promoter directs transcription in a cell-type specific manner. Moreover, P2-promoter activity is increased in DRG cultures enriched in sensory neurons but lacking in cells such as 3T3 fibroblasts  or HEK293 cells (unpublished observations). We now report that factors Sp1/Sp4, acting at a specific GC-box binding site, play a critical role in controlling TRPV1 RNA transcription in sensory neurons.
Transcription factors Sp1 and Sp4 regulate TRPV1 P2-promoter activity
Two functionally distinct GC-box binding sites have been identified within the P2-promoter. We propose that Sp1 and Sp4 function to regulate activation of TRPV1 transcription primarily through binding to GC-box "a" in the P2-promoter. Evidence to support this hypothesis includes: Detection of Sp1 and Sp4 protein bound to native DRG chromatin structure of the TRPV1 P2-promoter region GC-box a + b (Figure 1); Complete loss of promoter activity in DRG neurons and NGF-treated PC12 cells following deletion of GC-box "a" alone or both GC-box "a + b" (Figure 2,3); Increased promoter activity in DRG neurons co-transfected with Sp1 (Figure 2A,4); Blockade of NGF/Sp1- dependent promoter activity in PC12 cells using an inhibitor of Sp1-like binding, mithramycin-a (Figure 5); Decreased promoter activity in DRG neurons/NGF treated PC12 cells with siSp1 and decrease of Sp1-dependent promoter activity with siSp1 and siSp4 in PC12 cells (Figure 6).
Factors Sp1/Sp4 regulate TRPV1 RNA transcription in sensory neurons
Building on our observations of Sp1/Sp4 - dependent changes in promoter activity, we then determined whether manipulation of Sp1/Sp4 mRNA expression would direct concomitant changes in endogenous levels of TRPV1 mRNA in sensory neurons. As shown in Figure 7, we demonstrated that over-expression of Sp1/Sp4 mRNA in cultured DRG neurons was associated with an increase in TRPV1 mRNA. Conversely, we demonstrated a decrease in TRPV1 mRNA in sensory neurons under conditions of siSp4 and siSp1/Sp4 RNA knockdown (Figure 8B). Importantly, in both experimental series, changes in Sp4 were associated with the largest change in TRPV1 mRNA content. This suggests that although both Sp1 and Sp4 are bound to the P2-promoter site and are purported to have similar DNA binding properties in vitro, Sp4 may provide the dominant contribution to activate TRPV1 transcription in sensory neurons.
The structure and function of Sp1-like transcription factors
Sp1, one of the first transcription factors to be isolated , is the founding member of an expanding family of Sp1-like/Kruppel-like transcription factors that share common structural features such as glutamine-rich N-terminal activation domains and C-terminal zinc-finger DNA binding domains [31–33]. Members of this Sp1-like family are distinguished by their ability to bind GC- and / or GT-rich DNA regions within promoter regions and to activate gene transcription. Sp1 is also one of the best-characterized transcription factors [31, 32]. Although Sp1 was initially considered to be 'ubiquitously' expressed, its level of expression actually differs greatly (up to a 100 fold) during development and between tissue/cell types [32, 34]. A multitude of expressed genes common to all cells have been proposed to be regulated by Sp1 by virtue of containing GC/GT box binding sites within or adjacent to their promoter region. However, the actual number of genes critically dependent upon Sp1 are much fewer, suggesting sophisticated roles in its maintenance of differentiated cell types and tissue function. The role of Sp1 in nociception is unstudied as null mice completely lacking Sp1 die early in embryogenesis , whereas, Sp3 -/- null mice are growth-retarded and die at birth due to respiratory failure [18, 36]. Nevertheless, in addition to TRPV1, other Sp1-regulated genes may be involved in nociception such as the NR1 promoter [18, 36], DRG-specific expression of H-Antigen  and expression of 12 (S) lipoxygenase - products that have been shown to mediate bradykinin induced TRPV1 activation [5, 38].
Sp1-like factors share a high degree of homology, but they do not appear to have merely redundant functional attributes. Despite the ability of Sp1, 3 and 4 to bind identical 'GC-box' consensus targets in vitro, a more selective pattern of Sp1-like binding and transcriptional activation is actually observed when studied in the context of a complex genomic sequence, complex chromatin structure or diverse cellular environments. This is consistent with our observations that neither Sp3 nor Sp4 appear to exhibit the same profile of expression/activation as found for Sp1. Under control conditions, we observed only trace amounts of endogenous Sp3 protein bound to DRG TRPV1 P2-promoter. However, over-expression of Sp3 resulted in an increase in TRPV1 promoter activity (Figure 4). In contrast, co-expression of Sp3 with Sp1 resulted in a reduction of Sp1-mediated TRPV1 promoter activation. Therefore, as opposed to a synergistic effect reported between the tandem binding of Sp1, increased levels of Sp3 may serve a negative regulatory role in TRPV1 transcription. Such a role is consistent with a previous report showing that Sp3 inhibits the transcriptional activation of Sp1 .
Does transcription factor Sp4 play a unique role in regulating nociceptor phenotype?
Sp4 (HF-1b) is distinguished from Sp1 and Sp3 by a restrictive pattern of expression in neuronal cell types [39, 40]. Depending on the cellular context, there is also evidence that Sp4 can function as a transcriptional activator but without the capacity to act in a synergistic manner as exhibited by Sp1. Sp4 has also been reported to be expressed in other sensory-neuronal systems such as the cGMP - phosphodiesterase beta subunit that is exclusively expressed in rod photoreceptors . There are no published reports focused on the consequence of reduced Sp4 expression on behaviors related to peripheral nociception. Although Sp4 is highly expressed in several subregions of the brain, Sp4 -/- null mice showed only selective structural defects in the hippocampus . Behavioral testing of mice with reduced levels of Sp4 (2-5% of residual activity) showed structural defects in the hippocampus as well as contextual memory deficits and sensori - motor gating abnormalities . Mice modified with a deletion of the Sp4 N-terminal activation domain appear normal at birth but the majority later died by 1 month of age [44, 45]. Importantly, Sp4 -/- null mice appear to be predisposed to cardiac arrhythmias leading to sudden death . When Sp4 was reduced to 60% of expressed levels in neural crest derived cells (primary sensory, sympathetic and parasympathetic neurons), physiologic defects in atrial and atrial-ventricular conduction were found, despite a lack of morphological changes in cardiac tissues . This may reflect the loss of specific ion channels and/or other elements that are essential to signal transduction including synaptic structure and dendritic remodeling . Although it remains to be studied what affect the Sp4 null phenotype will have on the regulation of peripheral pain transduction, our findings here suggest Sp4 playing a critical role in the gene expression of TRPV1 mRNA in DRG neurons.
Recruitment of Sp1-like transcription factors is dependent on post-translational modification
In addition to a diverse pattern of tissue expression, Sp1 and Sp4 are subjected to a complex array of post-translational modifications that regulate target site (DNA) binding and/or recruitment of other transcription factors at gene promoter sites. These modifications include glycosylation, phosphorylation, acetylation and sumolyation [16, 48–50]. Post-translational modification of transcription factors is a primary way by which extracellular signaling events have long-term consequences on target gene expression. For example, Sp1 was found to be a critical downstream regulator of NR1 promoter activity in response to NGF activation of the extracellular signal regulated kinase -1 (ERK-1) cascade and the action of phosphitidylinositol 3-kinase (PI3 K) . It is interesting to consider that NGF-induced activation of PI3 K has been shown to not only translocate PI3 K to the nucleus for transcriptional activation, but also to direct NGF-induced sensitization of TRPV1 thermal hyperalgesia through receptor modification and increased trafficking of TRPV1 to the plasma membrane [51–53]. It is plausible that such NGF dependent pathways could drive both acute (receptor sensitization) and chronic (increased transcription/translation) changes in nociceptor phenotype.
NGF has emerged as a key inflammatory mediator directing both acute and persistent pain and hyperalgesia [12, 13]. As previously published by us and others, elevated concentrations of NGF drive an increase in TRPV1 mRNA expression in cultured DRG neurons and an increased number of TRPV1 mRNA positive DRG neurons in vivo
[54–57]. This is consistent with our earlier findings that NGF stimulates P2-promoter activity . In this present study, NGF-dependent P2-promoter activity requiring the presence of an intact GC-box "a" (Figure 2D) is blocked by a known inhibitor of Sp1 function (Figure 4) and can be decreased by Sp1-siRNA (Figure 5B). Taken together, these findings support the hypothesis that Sp1, in part, mediates NGF-dependent TRPV1 transcription. In the central nervous system, Sp1-like transcription factors direct protective regulatory responses under cellular stress and injury . Sp1 has also been shown to direct the expression of NGF dependent cell survival genes . In fact, Sp1 and Sp3 are oxidative stress-induced transcription factors in cortical neurons that function to reduce apoptosis and positively regulate neuronal survival . However, it remains to be studied what role Sp1 and its related family of transcription factors play under conditions of peripheral inflammation and nerve injury.