Unlike many central neuronal systems which express predominantly the PAC1
null (neither HIP nor HOP) receptor isoform, nearly all postganglionic sympathetic neurons of the SCG have been shown to express the PAC1(short)
HOP1 receptor variant. As PAC1(short)
HOP1 receptor activation and integration of multiple intracellular signaling cascades have been suggested to be important in transducing the many trophic properties of PACAP, microarray studies of sympathetic PAC1(short)
HOP1 receptor signaling can be especially revealing of the downstream mechanisms and gene targets underlying these events. Both acute and chronic treatment paradigms were performed in these studies to better assess the early and late PACAP-mediated gene responses, respectively. Although semiquantitative PCR analyses of cDNA templates from treated cultures for increased VIP and PACAP mRNA expression [47
] confirmed PACAP-mediated responses prior to sample submission for microarray analyses, the induction of several other transcripts on the genechips further corroborated the specificity of the peptide response. Consistent with previous work, for example, PACAP stimulated transcripts related to sympathetic NPY and catecholamine production [39
]. Chronic 96 h PACAP27 treatments increased neuropeptide Y transcript expression 1.5-fold (p
< 0.0005) which agreed well with previous Northern blot measurement; in parallel, tyrosine hydroxylase mRNA levels were also augmented 1.4-fold (p
< 0.0005), but the changes just narrowly missed criteria for inclusion in the data set. Significantly, mRNA for GTP cyclohydrolase, a rate-limiting enzyme for the synthesis of tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase and catecholamine biosynthesis, was increased 2-fold throughout the acute and chronic PACAP treatment paradigms. These results suggested that PACAP may facilitate or maintain the differentiated sympathetic catecholaminergic/NPY phenotype at multiple regulatory sites. The expression of other transcripts for catecholamine biosynthetic enzymes, including dopa decarboxylase, dopamine
-hydroxylase and phenylethanolamine-N-methyl transferase, was unchanged by PACAP.
Among the over 200 genes under either acute and/or chronic PACAP regulation, only 3 transcripts, VIP, Ecel1/DINE and Areg, demonstrated continued increased expression upon long term PACAP treatment. Interestingly, all 3 transcripts have been associated with injury-induced plasticity and function. Among trophic peptides, VIP has been well shown to promote neuronal survival under a variety of adverse conditions and to be induced in many injury paradigms in the peripheral nervous system [11
]. The membrane-bound zinc metallopeptidase Ecel1/DINE is preferentially expressed in the nervous system and highly induced in several axotomy systems, presumably for neuroprotection [31
]. More recently, Areg has been proposed to behave as an autoregulatory survival factor in sensory neurons [45
]. Hence, the observed increases in sympathetic Ecel1/DINE and Areg expression in these studies appeared consistent with PACAP trophic programs.
While any collection of transcripts within the current data sets can be constructed for functional attributions, only certain transcript groups will be emphasized for conciseness. The PACAP-regulated transcripts encoding peptides, growth factors/cytokines and processing enzymes are prodigious in number and functional diversity. PACAP was self-regulatory, consistent with previous work [27
]. In addition to augmenting VIP transcripts, PACAP induced efficaciously a number of other sympathetic peptides, including galanin, substance P and somatostatin. The regulated and transient developmental expression of somatostatin and enkephalins in sympathetic ganglion has been described [25
]. Although the expression of other PACAP-induced peptides, especially TRH and neuromedin U, has not been well described in sympathetic neurons, these peptide transcripts may have been identified as consequences of temporal postnatal development. Alternatively, as with enkephalin mRNA, the detection of regulated transcripts may be independent of peptide production [25
]. The various sympathetic peptides demonstrated different transcript expression patterns in response to PACAP. As described above, VIP transcript expression was among few that continued to increase upon chronic PACAP treatment. The heightened expression of most peptide mRNAs was sustained with chronic peptide exposure (i.e., galanin and somatostatin); the expression of others, including PACAP and substance P, diminished with treatment time. While the mechanisms underlying the variable peptide expression patterns were unclear, similar temporal dynamics have been observed in axotomy studies [7
]. The increase in peptide production was complemented by increased expression of a number of posttranslational processing enzymes. Whether Ecel1/DINE expression related to peptide processing or degradation was unclear [33
], but the subtilisin/kex-like proprotein convertase PC1, a endoproteolytic enzyme, has been well studied to cleave at dibasic amino acids and release the processed peptide from its precursor molecule [55
]. Many expressed peptides, including VIP, PACAP, galanin, neuromedin U, and TRH, are ∀-amidated for full biological activity, and the increase in PAM expression, an essential ∀-amidating enzyme, appeared sustained to maintain bioactive peptide production [50
The number of sympathetic peptide transcripts induced was matched by an equally diverse group of growth factors, cytokines and chemokines. The increased expression of IL-6, follistatin, TGF∀, BDNF, FGF14 and amphiregulin among others, represented at least 4 different receptor/intracellular signaling pathways implicated in neuronal survival, differentiation and plasticity. Of particular note, the transcripts for the neurotrophin BDNF and its cognate TrkB tyrosine kinase receptor were upregulated coordinately 3.0- and 1.7-fold, respectively, following acute PACAP treatment. While PACAP regulation of BDNF expression has been implicated in central neuronal systems [49
], many previous studies have shown that sympathetic neurons do not respond to TrkB signaling [2
]. The current observations appeared novel and suggested that sympathetic BDNF and TrkB expression may be induced coordinately, under specific developmental or physiological states, to promote survival, synaptic plasticity or related neurotrophic functions. Unlike the transient increase in TrkB mRNA which returned to basal levels after chronic peptide treatment, the increase in BDNF transcripts declined 50% to a different expression plateau during the same period. Significantly, other components of Trk signaling were also regulated, but in a reciprocal manner by PACAP. After long term PACAP treatment, expression of the neurotrophin low affinity p75 receptor transcript was diminished 1.5-fold from untreated control levels. In attenuating the proapoptotic or axonal repulsion signals attributed to p75 signaling, the response appeared to reinforce the trophic effects of BDNF and TrkB. Contrary to the TrkB response, PACAP also diminished TrkA transcript expression upon chronic peptide treatment. Although the decrement in TrkA mRNA was small (1.2-fold decrease) and did not meet one of the criteria for inclusion in the data set, the decrease was significant (p
< 0.0167). Other neurotrophin signaling components, including NGF, NT-3 and TrkC, were not regulated by PACAP by microarray analyses.
PACAP also appeared to have complex regulatory effects on other receptor signaling cascades. From acute or chronic peptide treatments, PACAP increased transcript expression of IL-1
, IL-6, IL-1 receptor-like 1 (Il1rl1), and the downstream cytokine signaling effector janus kinase 2 (Jak2). While IL-6 belongs to the ciliary neurotrophic factor (CNTF)/leukemia inhibitor factor (LIF)/cardiotrophin-1 (CT-1) family of neuropoietic cytokines, to implicate roles in neurophenotypic differentiation and plasticity [17
], the sympathetic effects of IL-6, in the absence of a soluble IL-6 receptor, appeared modest in previous studies to challenge current understandings its physiological contributions [38
]. Within the same pathway, PACAP also augmented transcripts for suppressor of cytokine signaling 2 (Socs2); but whether the increase represented transient compensatory mechanisms to dampen cytokine signaling or an independent means to facilitate neuronal fiber outgrowth remains to be further elucidated [62
]. PACAP-mediated increases in TGF∀, follistatin and Smad1 transcripts implicated modulation of bone morphogenetic protein (BMP) signaling elements. Surprisingly, PACAP not only increased the expression of adenylyl cyclase 7 (AC7), but also protein kinase C*, which binds and phosphorylates AC7 for cyclase activation [43
]. While this may present one basis for sustained PAC1
receptor intracellular signaling, PACAP also induced protein kinase inhibitor-
(Pkib) and several cyclic AMP- and MAPK-selective phosphodiesterase/phosphatase transcripts suggesting mechanisms to balance the responses.
The transcriptional factor/immediate early gene transcripts affected by acute PACAP stimulation appeared equally extensive in scope, and two of the gene targets, Plagl1 (LOT1/Zac1) and Ier3 (PRG1), were especially notable from their response magnitude and associations with PACAP/PAC1
receptor function. Other PACAP-regulated transcriptional factors also included CREB/CREM, Fos, Fra-1/2, Cebpb and Cited1/2, which may have been anticipated from cyclic AMP and related downstream signaling events. Together, the plethora of transcriptional factors induced may be consistent with the diversity in altered cellular programs necessary for PACAP-mediated neurotrophic responses. While the expression of most PACAP-induced transcriptional factor/immediate early gene transcripts was transient reflecting the immediacy of the response, PACAP sustained the induction of Atf3 transcripts, a transcriptional factor augmented after axotomy and other neuronal injuries [61
]. Atf3, in conjunction with c-jun expression, has been associated with neuronal survival and regeneration responses, and the ability for PACAP to induce Atf3 may have echoed those neurotrophic functions [48
]. If the analytical criteria were relaxed, the transcriptional factors induced by PACAP would have included multiple Jun members, including c-jun (>1.2-fold induction at p
< 0.0300) to suggest additional mechanisms for PACAP-mediated fiber outgrowth.
Other transcripts in the microarray data sets can be organized within distinct pathways for specific functions, and one group of note is composed of transcripts for arginase 1 (Arg1), ornithine decarboxylase (ODC) and antizyme inhibitor 1 (Azin1) which participate in polyamine biosynthesis. Arginase 1 catalyzes the conversion of arginine to ornithine, and the rate limiting ODC enzyme facilitates ornithine catalysis to putrescine as substrate for downstream synthesis of the polyamines spermidine and spermine. Azin1 is highly similar to ODC and in binding to the antizyme, facilitates ODC activity and prevents ODC degradation. The application of exogenous polyamines to injured SCG or facial motoneurons in vivo
facilitated neurite outgrowth and regeneration which could be recapitulated upon upregulation of Arg1 expression in neurons cultured on nonpermissive substrates [7
]. In conjunction with their fiber outgrowth and regeneration properties, activation of the polyamine pathway has been suggested to be antiapoptotic and promote central neuron survival after insults [18
]. Hence, the ability for PACAP/PAC1
receptor signaling to upregulate polyamine biosynthesis can be significant within the totality of the neurotrophic program.
As adrenal medullary chromaffin cells are also derived from the neural crest, there were expectations that the PACAP-responsive genes from sympathetic and pheochromocytoma cells would be similar. Indeed, upon inspection of results from previous studies [26
], some targets such as Erg1, Fos, Klf4, Ier3 and Odc1 appear common between PC12 and sympathetic neurons, and may have unique functional dimensions in many neuronal systems requiring more detailed studies. Yet, the similarities were limited suggesting that PACAP-regulated genes may be cell type-specific. This was especially evident in our more recent PACAP array studies with retinal neuroblasts which had little resemblance to our sympathetic data sets (data not shown). Alternatively, the culture conditions between PC12 and primary neurons may be sufficiently different to impact target responses. Surprisingly however, the PACAP-regulated transcripts in the currents studies, bore marked similarities to the changes in gene expression following axotomy [7
]. Many of the peptide, growth factor/cytokine, transcriptional factor, and cell survival genes between the two studies were identical. Allowing some relaxation in the fold-change criterion, more than 40% of the altered transcripts identified following SCG axotomy were also regulated in the same direction in the PACAP-treated sympathetic cultures. Although not as abundant, many of the PACAP-regulated transcripts in sympathetic neurons were also common to genes regulated in dorsal root ganglia after sciatic nerve transection [14
]. Given that the in vivo
axotomy microarray studies almost certainly reflected altered gene expression not only in neurons but also in nonneuronal cells, the similarities in gene data sets between injured neurons and PACAP-treated sympathetic neuronal cultures appeared even more striking.
There may be several underlying reasons for the parallels. For one, PACAP/PAC1
receptor signaling pathways may intersect with those following neuronal injury. As PAC1
receptor activation can stimulate elements related to cytokine signaling, for example, a significant subset of the injury-induced transcripts would be shared by PACAP signaling. However, another possibility is that some alterations in neuronal gene expression after injury reflect secondary responses to PACAP function. PACAP expression in sympathetic neurons can be tonically inhibited by target-derived factors [47
], and the release of that inhibition after axotomy may stimulate PACAP production and signaling as an integral component to initiate a comprehensive injury response. Given the exceptional abilities for PACAP to drive diverse transcript expression with high efficacy, the mechanism has important attractions. Nevertheless, the compendia of sympathetic transcripts regulated by PACAP recapitulate those for neuronal differentiation, repair and regeneration. The PAC1(short)
HOP1 receptor is able to engage multiple intracellular signaling cascades and the current data set suggest that the number of downstream effectors may be broader than previously suspected. Some important consequences of that signaling may be reflected in the number and diversity of targeted genes which position PACAP uniquely in a nexus of cellular strategies to promote neuronal survival and regeneration. In understanding and harnessing PACAP/PAC1
receptor function, there may be important therapeutic avenues to stimulate endogenous cellular repair.