Sustentacular cells provide both structural and metabolic support for the olfactory sensory neurons of the olfactory epithelium. These glial-like supporting cells have a multitude of functions including the physical and chemical insulation of OSNs , active phagocytosis of dead cells , and the regulation of the extracellular ionic environment [42, 44, 45]. Here we report the induction of phosphorylation of the transcription factor CREB in sustentacular cell nuclei in response to odorant-mediated signal transduction in OSNs. Interestingly, we found CREB activation in sustentacular cells to be inhibited by pre-incubation with purinergic receptor antagonists, suggesting the involvement of extracellular nucleotides in mediating intercellular communication between OSNs and their neighbouring counterparts, the sustentacular cells.
The concept that stimulus detection from extracellular signals, such as hormones, growth factors, and neuronal activity, modulates transcriptional events to produce long-term changes is well established . In many cases changes in transcription occur through the reversible phosphorylation of transcription factors [47–49]. The transcription factor CREB can be activated by many different pathways, such as the neurotransmitter-mediated PKA pathway, calcium-calmodulin-dependent kinases, or growth factor-sensitive kinase pathways [50–52]. CREB is known to be activated by phosphorylation at Ser-133 and in this study we used an antibody specific to CREB phosphorylated at Ser-133 to investigate the activation of this transcription factor.
To date, CREB activation in OSNs via odorant stimulation has already been examined to a certain extent. For example, it has been shown that ERK phosphorylation and CRE-mediated transcription occurred in OSNs in response to short-term treatment with the odorant citralva . Moreover, odorant exposure stimulated the survival of OSNs in an in vivo model of apoptosis through the activation of MAPK and CREB . In the present study, we show that exposure of mice to a mixture of 100 different odorants leads to an increase in the levels of p42/44 MAPK phosphorylation in OSNs in vivo. Interestingly, CREB phosphorylation occurred not only in OSNs, as was observed for MAPK activation, but also in the sustentacular cells of the olfactory epithelium. The induction of CREB phosphorylation in sustentacular cells was significantly reduced on inhibition of the canonical olfactory signaling cascade, using an inhibitor of ACIII, an enzyme central to odorant-mediated signal transduction which occurs only in OSNs. Odorant-dependent CREB phosphorylation in sustentacular cells therefore seems to be controlled by an intact signaling cascade in olfactory sensory neurons.
Interestingly, on inhibition of purinergic receptor signaling using general purinergic receptor antagonists, CREB phosphorylation remained evident in the nuclei of OSNs and immature OSNs not expressing OMP, but was reduced in the nuclei of sustentacular cells.
Sustentacular cells express P2Y purinergic receptors, in particular subtype P2Y2 [25, 29] and they respond to P2R agonists ATP and UTP with increases in intracellular calcium. P2Y receptors belong to a G-protein coupled receptor family, and stimulation of these receptors by ATP activates diverse intracellular signaling pathways, including the activation of PLC, PKC, and CaM kinase . In sustentacular cells, purinergic signaling induces PLC activation resulting in the formation of IP3, leading to the release of Ca2+ from IP3-sensitive Ca2+ stores , which could possibly lead to Ca2+-mediated CREB activation. Alternatively, the activation of PKC leads to the induction of ERK/MAP kinase signaling pathways and CREB phosphorylation at Ser-133 has been shown to be induced by ERK1/2 . Further work will be undertaken in order to dissect the particular mechanism of CREB activation via purinergic receptors in this system.
P2Y receptors can be activated by ATP, which plays an important role in intercellular communication. In the CNS, ATP has been shown to be activity-dependently released from neurons and is also released from astrocytes. Extracellular ATP can spread to neighboring astrocytes and activate membrane receptors, generating an increase in intracellular Ca2+ concentration. Thus, in the CNS, ATP plays an important role in the communication between neurons and astrocytes . For example, Takasaki et. al. found that ATP, through the activation of P2Y receptors, the elevation of intracellular Ca2+ concentration, the activation of CaM kinase and the phosphorylation of CREB, induces the upregulation of BDNF exon IV mRNA synthesis in astrocytes . Moreover, in the olfactory system, ATP has been shown to be released by OSNs and their axons [27, 28], and from sympathetic and from trigeminal nerve fibres [56–58] and ATP was shown to modulate the odorant sensitivity of OSNs .
We found that induction of CREB phosphorylation in the nuclei of the sustentacular cells was mimicked by exogenous ATP application. Pre-incubation with an inhibitor of ACIII reduced the levels of CREB phosphorylation in the sustentacular cell nuclei, however, this inhibition did not completely abolish the phosphorylation of CREB, indicating that the ATP-induced phosphorylation of CREB in sustentacular cells is partially dependent on ACIII signaling. Most of the 8 functional P2Y receptors identified to date act via G-protein coupling to activate phospholipase C, leading to the production of IP3 and mobilization of Ca2+ from intracellular stores ; however, some P2Y receptors couple to adenylate cyclase and increase the production of cAMP . Interestingly, phosphorylated CREB in both mature and immature OSNs induced following ATP treatment was completely blocked by pre-incubation with the inhibitor of ACIII, pointing to a different mechanism of ATP-mediated CREB activation in these cells as compared to sustentacular cells. ATP induced CREB phosphorylation in the nuclei of the immature OSNs was reduced upon inhibition of adenylate cyclase signaling, but the odorant induced CREB phosphorylation was adenylate cyclase independent, indicating that different mechanisms are involved. One of these mechanisms is likely to be the activity-dependent ATP signaling from OSNs, the other mechanism and the signal or transmitter involved is unknown at present.
It has previously been reported that odorant treatment may have a damaging effect, causing cell death and leading to the release of ATP in the OE. However, it has also been shown that odorants can have a protective effect on OSNs, enhancing neuronal survival via MAPK and CREB activation . In this study we used concentrations of odorant previously shown to initiate physiological responses in OSNs . Furthermore, the effects we observe in this study are blocked by the specific inhibitor of adenylate cyclase (SQ22536) and therefore are unlikely to be due to unspecific cell death-induced ATP release and more likely to be due to specific olfactory signaling. Therefore, odorant treatment induces the activation of OSNs via a specific olfactory signal transduction cascade involving ACIII, and this signaling is essential for the purinergic receptor-mediated activation of CREB in sustentacular cells.