Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic–pituitary–adrenal axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN) in response to stress, is secreted into the pituitary portal circulation, resulting in the release of adrenocorticotropic hormone from the anterior pituitary. AVP is synthesized in the PVN and supraoptic nucleus by various stressors. Hypothalamic 4B cells coexpress CRF and AVP. In 4B cells transfected with either a CRF or an AVP promoter-luciferase construct, forskolin increased the transcriptional activity of CRF or AVP. In the present study, we tried to determine whether pituitary adenylate cyclase-activating polypeptide (PACAP) regulates both CRF and AVP genes in the hypothalamic cells, because receptors for PACAP were expressed in the hypothalamic cells. PACAP stimulated activity of both CRF and AVP promoter via protein kinase A pathway. PACAP stimulated interleukin (IL)-6 promoter activity and the levels of IL-6 mRNA and protein. IL-6 stimulated activity of both CRF and AVP promoter in a dose-dependent manner. Finally, we found that the stimulatory effects of PACAP on both activities were significantly inhibited by treatment with anti-IL-6 monoclonal antibody. These data suggest that PACAP is involved in regulating the synthesis of IL-6 mRNA and IL-6 protein, and that the increase in endogenous IL-6 also contributes to stimulate the expression of both CRF and AVP genes. Taken together, these findings indicate that PACAP stimulates the transcription of CRF, AVP, and IL-6 genes in hypothalamic 4B cells.
Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic–pituitary–adrenal (HPA) axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN) in response to stress (Whitnall 1993), is secreted into the pituitary portal circulation, resulting in the release of adrenocorticotropic hormone (ACTH) via the CRF receptor type 1 (CRF1 receptor) from the anterior pituitary (AP). ACTH then stimulates glucocorticoid release from the adrenal glands (Whitnall 1993). Glucocorticoid in turn inhibits hypothalamic PVN production of CRF and pituitary production of ACTH. We previously demonstrated that the protein kinase A (PKA) pathway plays a major role in releasing CRF in hypothalamic tissues (Suda et al. 1985). Other studies also support the hypothesis that stimulation of the PKA pathway in CRF neurons increases CRF expression, and indicate that cyclic AMP (cAMP)-dependent signaling activates CRF gene expression (Seasholtz et al. 1988, Spengler et al. 1992). A functional cAMP-response element (CRE) in the 5′-promoter region of CRF gene takes a part in regulating its gene expression (Seasholtz et al. 1988, Spengler et al. 1992).
AVP is synthesized in both magnocellular and parvocellular regions of the PVN, and in the supraoptic nucleus (SON). Expression of the AVP gene in the SON and PVN is regulated by inflammation, cytokines, and an acute stress (Burbach et al. 2001). Magnocellular AVP neurons in the PVN and SON project to the neural lobe of the pituitary, whereas AVP neurons in the parvocellular region of the PVN project to the external zone of the median eminence (ME) to stimulate ACTH production in the corticotrophs (Vandesande et al. 1977).
Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family, is found in two forms in the mammalian central nervous system (CN): a 38 amino acid peptide (PACAP38) and an N-terminally truncated 27 amino acid peptide (PACAP27; Miyata et al. 1989, 1990). PACAP acts by binding to PACAP receptor 1 (PAC1 receptor) and two VIP-preferring receptors (VPAC1 and VPAC2 receptors). These receptors have different affinities for PACAP and VIP (Harmar et al. 1998). PACAP and the PACAP-selective PAC1 receptors are known to be highly expressed in the hypothalamus, including the parvocellular and magnocellular subdivisions of the PVN, and the SON (Nomura et al. 1996, Shioda et al. 1997a).
PACAP has shown to stimulate cAMP production in the AP (Miyata et al. 1989). PACAP also increases CRF mRNA levels in the parvocellular region of the PVN, suggesting that PACAP is involved in the positive regulation of CRF gene expression (Grinevich et al. 1997). Therefore, it is suggested that PACAP might regulate the HPA axis by stimulation of CRF gene expression in the hypothalamus and through direct effects on pituitary corticotrophs (Boutillier et al. 1994, Aoki et al. 1997, Agarwal et al. 2005). PACAP also activates AVP neurons of the PVN or SON via PAC1 receptor (Shioda et al. 1997b, Nomura et al. 1999).
Interleukin (IL)-6 also shows a variety of biological activities. Plasma IL-6 levels rise in response to both immune activation and non-immune stress (Zhou et al. 1993, Takaki et al. 1994). IL-6 prompts lymphocytic proliferation and differentiation (Schimpl & Wecker. 1972) and induces production of acute-phase proteins in the liver (Castell et al. 1989). In addition, IL-6 stimulates the HPA axis (Venihaki et al. 2001), increasing glucocorticoid levels, which in turn suppress IL-6 production (Karalis et al. 1997, Turnbull & Rivier 1999). Therefore, IL-6 is an important mediator of the interaction between the neuroendocrine and immune systems. IL-6 also plays a role in the regulation of CRF and AVP. For example, IL-6 increases CRF gene expression and secretion in the hypothalamus (Navarra et al. 1991, Vallieres & Rivest 1999). IL-6 is coexpressed with AVP in the SON and PVN neurons (Ghorbel et al. 2003) and also induces AVP secretion in the hypothalamus (Mastorakos et al. 1994).
Little is known about the signaling pathways involved in the control of CRF and AVP transcription in parvocellular PVN neurons due to the lack of availability of homologous hypothalamic cells (Kasckow et al. 2003a). A fetal rat hypothalamic 4B cell line shows characteristics of the parvocellular neurons of the PVN (Kasckow et al. 2003b) and expresses CRF, AVP, and CRF1 and glucocorticoid receptors. In the cells transfected with either a CRF or AVP promoter-luciferase construct, forskolin increased CRF or AVP transcriptional activity in parallel with increases in intracellular cAMP (Nikodemova et al. 2003). In the present study, we asked whether PACAP is involved in the responses of CRF and AVP promoter activities in the hypothalamic 4B cells. We further explored the involvement of PACAP in the regulation of IL-6 gene expression in the hypothalamic cells.
Materials and Methods
Forskolin was purchased from Calbiochem (San Diego, CA, USA). PACAP38 was purchased from the Peptide Institute (Osaka, Japan). H89 was purchased from Seikagaku Corporation (Tokyo, Japan). PD98059 and SQ22536 were purchased from Cabiochem. Rat IL-6 was purchased from PeproTech (London, UK). Anti-rat IL-6 MAB was purchased from Biosource (Camarillo, CA, USA).
Adult male Wistar rats (body weight, BW 280–320 g) were housed in a temperature-controlled room with controlled lighting (light 0600–1800 h) and were given free access to laboratory chow and tap water. The rats were decapitated for tissue collection (hypothalamic tissue and anterior pituitary). This study was carried out in accordance with the Guidelines for Animal Experimentation, Hirosaki University.
Rat hypothalamic 4B cells were incubated in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 U/ml penicillin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were plated at 104 cells/cm2 4 days before each experiment, with the medium being changedforevery 48 h. On day 3, cells were washed and starved overnight with DMEM supplemented with 0.2% BSA. On day 4, cells were incubated in medium with added vehicle, forskolin, with or without medium containing one of various inhibitors or an antagonist.
Constructs and transfection
A 1077 bp restriction fragment containing the CRF promoter (−907 to +170 relative to the proximal transcription start point) and an 829 bp restriction fragment containing the AVP promoter (−803 to +26) were obtained by PCR. The PCR products were then confirmed by sequences. The DNA fragments were used to produce the CRF and AVP promoter-driven luciferase reporter constructs, CRF-907luc and AVP-803luc respectively by a two-step cloning method. First, the DNA fragment was cloned into pGEM-T Easy vector (Promega Corp.), then digested with Kpn I and Hind III, and subcloned into Kpn I and Hind III cloning sites of the pA3-Luc plasmid. A 543 bp restriction fragment containing the IL-6 promoter (−522 to +21) was obtained from rat genomic DNA. This DNA fragment was used to produce the IL-6 promoter-driven luciferase reporter construct, IL-6luc, by the two-step cloning method.
For the luciferase activity assay, cells were placed in 12-well (22 mm diameter) culture trays at 60% confluency. The next day, cells were transfected following the manufacturer’s instructions using the FuGENE 6 Transfection Reagent Kit (Roche Diagnostics). FuGENE 3 μl/1 μg DNA was used. For each well, the total amount of DNA was 0.5 μg. The culture medium was then replaced with DMEM supplemented with 10% FBS. One day before each experiment, cells were washed and starved overnight in DMEM supplemented with 0.2% BSA.
The luciferase assay was performed according to the manufacturer’s protocol. At the end of each experiment, cells were washed twice with PBS without Ca2+ and Mg2+, harvested with PicaGene lysis buffer (Toyo Inki, Tokyo, Japan), and centrifuged at 12 000 r.p.m. for 2 min. For the luciferase assay, 20 μl of each supernatant was used. The reactions were started by the injection of 100 μl luciferin solution, PicaGene buffer. Light output was measured for 20 s at room temperature using a luminometer (Berthold Lumat LB9501, Postfach, Germany). Activity of β-galactosidase was used as an internal control. Cells were treated in triplicate in each experiment, with the average of three independent experiments shown in figures.
Serum-starved 4B cells were pre-incubated for 20 min with 0.1 mM of 3-isobutyl-1-methylxanthine in assay medium and then treated at 37 °C for 20 min with the indicated concentrations of each peptide. The medium was aspirated and cells extracted with 1 ml of 95% ethanol containing 0.1 M HCl. cAMP content was measured in the supernatants using commercial cAMP EIA kits (Amersham Pharmacia Biotech). Cells were treated in triplicate in each experiment, with the average of three independent experiments shown in figures.
Cellular total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Then, cDNAs were synthesized from total RNA (0.5 μg) using random hexamers as primers with the SuperScript First-Strand Synthesis System for reverse transcriptase-PCR (RT-PCR) Kit (Invitrogen Corp.) according to the manufacturer’s instructions.
Reverse transcriptase-PCR (RT-PCR)
PCR was carried out in a programmable thermal controller (Bio-Rad) with the following oligonucleotide primers: CRF-F (5′-TGGATCTCACCTTCCACCTTCTG-3′), CRF-R (5′-CCGATAATCTCCATCAGTTTCCTG-3′); AVP-F (5′-ACATCCGACATGGAGCTG-3′), AVP-R (5′-GCGG-AAAAAACCCTCTCG-3′) (Nagano et al. 1997); β2-Micro-globulin (B2MG)-F (5′-CGGTGACCG- TGATCTTT- CT GGT-3′), B2MG-R (5′-GGTGACGGTTTTGGGCTC-CTT-3′) (Neumann et al. 1997); PAC1 receptor-F (5′-TTT-CATCGGCATCATCATCATCCTT-3′), PAC1 receptor-R (5′-CCTTCCAGCTCCTCCATTTCCTCTT-3′) (Ajpru et al. 2002); VPAC1 receptor-F (5′-TGATTGAGATA-CAGCGTCAG-3′), VPAC1 receptor-R (5′-CCT-CAGAGCAGTGGTCTATC-3′) (Chaudhary & Baumann 2002); VPAC2 receptor-F (5′-TTGGGCTACAGTG-TTTCTCT-3′), VPAC2 receptor-R (5′-ATTAGCAG-CAGTGTGGACTT-3′). Conditions for the CRF were 1× (95 °C, 3 min), 40×(95 °C, 20 s; 62 °C, 30 s; 72 °C, 30 s) and 1×(95 °C, 1 min; 55 °C, 1 min). Conditions for the AVP were 45× (94 °C, 1 min; 54 °C, 1 min; 72 °C, 2 min). Conditions for the B2MG were 1× (95 °C, 3 min), 48× (93 °C, 1 min; 65 °C, 1 min; 72 °C, 1 min) and 1× (72 °C, 5 min). Conditions for the PAC1 receptor were 1× (94 °C, 2 min), 40× (94 °C, 1 min; 58 °C, 1 min; 72 °C, 2 min) and 1× (75 °C, 5 min). Conditions for the VPAC1 and VPAC2 receptors were 1× (94 °C, 5 min), 35× (94 °C, 30 s; 55 °C, 40 s; 72 °C, 70 s), and 1× (72 °C, 7 min). Products were separated by electrophoresis on a 1.2 or 4% (for CRF) agarose gel containing ethidium bromide, followed by quantitative analysis using the NIH image software 1.61 (Bethesda, MD, USA. The results are expressed as corrected arbitrary units. The expected sizes of PCR products for CRF and AVP were 101 and 270 bp respectively. The expected size of PCR products for B2MG was 332 bp. The expected sizes of PCR products for PAC1 receptor were 374 bp (a variant with a single 84 bp insert) and 290 bp (short isoform). The expected sizes of PCR products for VPAC1 and VPAC2 receptors were 509 and 607 bp respectively.
Resulting cDNAs were subjected to real-time PCR as follows. The expression level of rat IL-6 mRNA was evaluated using quantitative real-time PCR based on specific sets of primers and probes (Assays-on-Demand Gene Expression Products, Applied Biosystems, Foster City, CA, USA). B2M was used as a housekeeping gene to normalize values. Each reaction consisted of 1×TaqMan Universal PCR Master Mix (Applied Biosystems), 1×Assays-on-Demand Gene Expression Products (Rn00561420 m1 for rat IL-6; Rn00560865 m1 for rat B2MG), and 2 μl cDNA in a total volume of 50 μl using the following parameters with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems): 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The above assays involved specific sets of primers and TaqMan probe spanning exon/exon junctions and, therefore, should not have been influenced by DNA contamination. Data were collected and recorded by ABI PRISM 7000 SDS Software (Applied Biosystems) and expressed as a function of threshold cycle (CT). Using diluted samples, the amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical.
Relative quantitative gene expression
Relative quantitative gene expression was calculated with the 2−ΔΔCT method (Livak & Schmittgen 2001). In brief, for each sample assayed, the CT for reactions amplifying a gene of interest (rat IL-6) and a housekeeping gene (rat B2MG) was determined. The gene of interest CT for each sample was corrected by subtracting the CT for the housekeeping gene (Δ CT). Untreated controls were chosen as reference samples, and the Δ CT for all experimental samples was reduced by the average Δ CT for the control samples (ΔΔ CT). Finally, experimental mRNA abundance relative to control mRNA abundance was calculated by the formula 2−ΔΔCT.
Western blot analysis
After treatment with PACAP, cells were washed twice with PBS and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and the supernatant was recovered. Samples were boiled and subjected to electrophoresis on a gradient (4–20%) polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane (Daiichi Kagaku, Tokyo, Japan). After blocking by Detector Block blocking buffer (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), the membrane was incubated for 1 h with a rabbit anti-CRE-binding protein (CREB) and anti-phosphorylated (p)-CREB antibodies (Cell Signaling Technology, Beverly, MA, USA), or anti-monoclonal IL-6 (BioSource International Inc.), washed with PBS containing 0.05% Tween 20, and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Daiichi Kagaku). Detection was performed using a chemiluminescent substrate Super-signal WestPico (Pierce Chemical Co., Rock-ford, IL, USA), and the membrane was exposed to BioMax film (Eastman Kodak Co.) followed by quantitative analysis using the NIH image software 1.61. The results are expressed as corrected arbitrary units.
Serum-starved 4B cells were incubated at 37 °C for 48 h with the indicated concentrations of each peptide. The medium was then aspirated and IL-6 levels in the supernatants were measured using commercial IL-6 ELISA kits (Biosource International). All samples from each experiment were determined in the same assay.
All values are expressed as the mean±s.e.m. Statistical analyses of data were performed using one-way ANOVA, followed by Bonferroni/Dunn post hoc test. The level of statistical significance was set at P<0.05.
Expression of CRF, AVP, and receptors for PACAP mRNA in 4B cells
PAC1 receptor gene expression was detected in 4B cells, hypothalamus, and anterior pituitary tissues (Fig. 1A). PAC1 receptor has a short isoform (290 bp) and a variant with a single 84 bp insert (374 bp). Expression levels of VPAC1 receptor were very low in 4B cells, while VPAC2 was detected in 4B cells and rat hypothalamus.
Expression of both CRF and AVP mRNA was examined by RT-PCR. Basal levels of both mRNA were very low or undetectable, while PACAP significantly increased both CRF and AVP mRNA levels from 2 to 24 h after experiment in 4B cells (Fig. 1B). The maximal effect of PACAP was observed at 2 h after experiment.
To determine whether PACAP affected CREB phosphorylation, 4B cells were incubated with PACAP. As shown in Fig. 1C, incubation with 100 nM PACAP significantly increased CREB phosphorylation from 10 to 30 min after the addition.
Effects of PACAP on CRF or AVP 5′-promoter activity in 4B cells
We examined the impact of PACAP on the time- and dose-dependent changes in CRF 5′-promoter activity using 4B cells transfected with CRF-907luc. The time course study showed that PACAP significantly increased CRF 5′-promoter activity (ANOVA; P<0.0001; Fig. 2A(i)). The maximal effect of PACAP occurred at 2 h after experiment, with an approximate fivefold increase in CRF 5′-promoter activity when compared with the basal level in CRF-907luc (P<0.005). PACAP stimulated CRF 5′-promoter activity in a dose-dependent manner with significant enhancing effects of PACAPobserved at 1 nM to 1 μM (ANOVA; P<0.0001) (Fig. 2A(ii)).
Next, we examined the impact of PACAP on the time- and dose-dependent changes in AVP luciferase activity using 4B cells transfected with AVP-803luc (Fig. 2B). The time course study showed that PACAP significantly increased AVP 5′-promoter activity (ANOVA; P<0.0001) (Fig. 2B(i)). The maximal effect of PACAP was observed 2 h after the experiment, with an approximate fourfold increase when compared with the basal level in AVP-803luc (P<0.005). PACAP stimulated AVP luciferase activity in a dose-dependent manner, with significant stimulatory effects at 1 nM to 1 μM (ANOVA; P<0.0001; Fig. 2B(ii)).
Effects of PACAP on cAMP accumulation in 4B cells
Figure 3 shows the levels of cAMP production following PACAP stimulation of 4B cells. Both forskolin and PACAP induced intracellular cAMP production in a dose-dependent manner. Significant stimulatory effects of PACAP were observed at 10 nM to 1 μM.
Effects of a PKA or extracellular signal-related kinases inhibitor on PACAP-induced CRF or AVP 5′-promoter activity in 4B cells
To determine whether PKA pathway is involved in the PACAP-induced CRF or AVP 5′-promoter activity, 4B cells were incubated with H89, a potent PKA inhibitor. As shown in Fig. 4, the stimulatory effects of 100 nM PACAP on both activities were partially inhibited by treatment with 1 μM H89, while these of a smaller dose, 10 nM PACAP were completely done. The effects of 10 nM PACAP on both CRF and AVP promoter activities were not inhibited by 10 μM PD98059, a selective mitogen-activated protein (MAP) kinase kinase extracellular signal-related kinases inhibitor.
Effects of forskolin or PACAP on IL-6 5′-promoter activity in 4B cells
Forskolin or PACAP may stimulate IL-6 gene expression in 4B cells. Therefore, we examined the time- and dose-dependency of forskolin and PACAP on IL-6 5′-promoter activity (Fig. 5A). The time course study revealed gradual increases in IL-6 5′-promoter activity in the presence of 10 μM forskolin. Significant stimulatory effects of forskolin were observed at 0.1–100 μM.
We next examined the effect of PACAP on the time-and dose-dependent changes in IL-6 5′-promoter activity, using 4B cells transfected with IL-6luc (Fig. 5B). The maximal effects of PACAP were observed at 2 and 6 h after experiment, with an approximate threefold increase when compared with the basal level. PACAP stimulated IL-6 5′-promoter activity in a dose-dependent manner with significant stimulatory effects observed at 10 nM to 1 μM.
To determine whether an adenylate cyclase pathway was involved in PACAP-induced IL-6 5′-promoter activity, 4B cells were pre-incubated for 30 min with 100 μM SQ22536, an adenylate cyclase inhibitor. As shown in Fig. 5D, treatment with SQ22536 significantly inhibited IL-6 5′-promoter activity.
Effects of forskolin or PACAP on IL-6 mRNA levels in 4B cells
Next, we investigated the modulation of IL-6 mRNA levels by forskolin and PACAP. The maximal effect of forskolin on IL-6 mRNA levels occurred at 2 h after experiment, with an approximate tenfold increase when compared with the basal level (Fig. 6A). Significant stimulatory effects of forskolin were observed at 1 and 10 μM.
We also examined time- and dose-dependent changes in IL-6 mRNA levels in the presence of PACAP (Fig. 6B). Maximal stimulation by PACAP was observed at 2 h after experiment, with an approximate ninefold increase of IL-6 mRNA levels. PACAP stimulated IL-6 mRNA levels in a dose-dependent manner with significant stimulation at 10 nM to 1 μM.
Effects of PACAP on IL-6 production in 4B cells
To examine whether PACAP induced a functional response in the 4B cells, we examined the effects of PACAP on IL-6 output levels or production (Fig. 7). Incubation with 0.01 nM to 10 μM forskolin or 0.1 to 100 nM PACAP increased IL-6 output levels in a dose-dependent manner (ANOVA; P<0.0001). PACAP stimulation was significant at 100 nM.
Effects of IL-6 on CRF and AVP 5′-promoter activities in 4B cells
IL-6 is a candidate modulator of CRF and AVP gene expression. Therefore, we examined the time- and dose-dependency of the IL-6 effects on both CRF and AVP 5′-promoter activities. To examine CRF 5′-promoter activity, 4B cells transfected with CRF-907luc were incubated with IL-6 (Fig. 8A). The time course revealed a gradual increase in CRF 5′-promoter following exposure to 5 nM IL-6. Maximal stimulation by IL-6 occurred after 24 h, with an approximate twofold increase when compared with the basal level. IL-6 stimulation of CRF 5′-promoter was dose dependent with significant stimulatory effects observed at 500 pM.
Next, 4B cells transfected with AVP-803luc were incubated with IL-6 to examine changes in AVP 5′-promoter activity (Fig. 8B). There were gradual increases in AVP 5′-promoter activity in the presence of 5 nM IL-6. The maximal effect of IL-6 was observed after 24 h, with an approximate twofold increase when compared with the basal level. IL-6 stimulated AVP 5′-promoter activity in a dose-dependent manner, with 500 pM inducing significant stimulation.
Effects of anti-IL-6 Ab on PACAP-induced CRF or AVP 5′-promoter activity in 4B cells
Finally, to determine whether endogenous IL-6 production is involved in the PACAP-induced CRF or AVP 5′-promoter activity, 4B cells were pre-incubated for 30 min with anti-IL-6 MAB, and then incubated for 2 h with 10 or 100 nM PACAP. As shown in Fig. 9B, the stimulatory effects of 100 nM or a smaller dose, 10 nM, PACAP on both activities were significantly, but not completely, inhibited by treatment with the anti-IL-6 MAB.
In this study, we first found that PACAP stimulated activity of both CRF and AVP promoter in hypothalamic 4B cells. Although PACAP has been shown to modulate hypothalamic CRF gene expression in vivo (Grinevich et al. 1997), it was difficult to study the mechanism regulating CRF transcription in parvocellular neurons because of the lack of availability of a representative cell line. We used a homologous hypothalamic 4B cell, which shows the characteristics of the parvocellular cells of the PVN, because the cells were shown to express CRF, AVP, and CRF1 and glucocorticoid receptors (Kasckow et al. 2003b). In this and other recent studies (Liu et al. 2006), basal levels of both CRF and AVP mRNA were very low or undetectable in the 4B cells, presumably caused by changes in the cell condition through current passages of the cells, while PACAP stimulated both CRF and AVP mRNA levels in 4B cells. PACAP induced intracellular cAMP production in hypothalamic 4B cells and stimulated CRF gene transcription via the cAMP-dependent pathway. Indeed, nerve fibers containing PACAP connect to CRF neurons (Hannibal et al. 1995, Legradi et al. 1998). Other studies also suggest that PACAP stimulates CRF gene via the cAMP/PKA signaling pathway (Agarwal et al. 2005). In hypothalamic 4B cells, the cAMP–PKA pathway was involved in PACAP-induced CRF stimulation of gene transcription. Activation of the PKA pathway, causing phosphorylation of CREB, acts on the CRF promoter (Seasholtz et al. 1988, Spengler et al. 1992, Itoi et al. 1996). A functional CRE in the 5′-promoter region takes a part in regulating CRF gene expression (Seasholtz et al. 1988, Spengler et al. 1992). H89, a PKA inhibitor, completely blocked a smaller dose of PACAP-induced CRF stimulation of gene transcription in hypothalamic 4B cells. However, the inhibitor did not completely block a larger dose of PACAP-induced CRF stimulation, suggesting the limitation of the inhibitor. Otherwise, it is possible that an additional pathway may be involved in this regulation. The difference between CRF/AVP promoter activity and cAMP response to PACAP also may suggest that the effect of PACAP is mediated by other signals in addition to cAMP.
PACAP also stimulated both AVP gene transcription and mRNA expression via the cAMP–PKA pathway in hypo-thalamic 4B cells. Gene transcription of AVP is stimulated by Fos/Jun family member proteins through an activation protein 1 (AP1) site in neuroblastoma cells (Yoshida et al. 2006). This effect is achieved via both PKA and protein kinase C signaling pathways. Therefore, our results are consistent with those of the prior study showing that intracellular cAMP production is involved in the stimulation of AVP gene transcription via the PKA pathway in hypothalamic 4B cells. PACAP is detected in nerve terminals that innervate AVP-containing neurons in the rat hypothalamus (Shioda et al. 1997b), and its receptor mRNA is highly expressed in AVP-containing neurons (Shioda et al. 1997b). Another report demonstrated that PAC1 receptors were expressed in the parvocellular and magnocellular subdivisions of the PVN (Nomura et al. 1996). The 4B cells also expressed PAC1 and VPAC receptors. Therefore, activation of PAC1 or VPAC receptors by PACAP induces production of intracellular cAMP, activating the transcription of AVP gene in hypothalamic cells. Taken together, we propose that PACAP-induced intracellular cAMP production is involved in stimulating the transcription of both CRF and AVP genes at least via the PKA pathway in hypothalamic 4B cells.
The role of PACAP in the PVN of the hypothalamus has not been clearly determined, although PACAP might contribute to stress responses through CRF and AVP stimulation. The extended amygdala and the bed nuclei of the stria terminalis are identified as innervation sites of PACAP neurons, suggesting an important role in stress responses (Piggins et al. 1996, Kozicz & Arimura 2002). PACAP also stimulates cAMP production in the AP. PACAP, therefore, could regulate pituitary corticotrophs directly (Boutillier et al. 1994). CRF and AVP neurons in the parvocellular region of PVN project to the external zone of the ME (Seasholtz et al. 1988, Gonzalez-Hernandez et al. 2006). Furthermore, AVP and CRF in parvocellular PVN neurons exert synergistic effects on ACTH secretion from the AP (Gillies et al. 1982, Mouri et al. 1993). Therefore, PACAP may contribute to stress responses through stimulation of CRF and AVP neurons of the hypothalamus, causing ACTH secretion.
The treatment with both PACAP and forskolin showed no additive effect on IL-6 5′-promoter activity. An adenylate cyclase inhibitor significantly inhibited PACAP-induced IL-6 5′-promoter activity. Therefore, PACAP may stimulate IL-6 production through intracellular cAMP pathway. On the other hand, it is possible that the differences in the activation of IL-6 promoter between PACAP and forskolin may suggest the differences in signal pathway, in addition to cAMP, in the IL-6 regulation.
Cytokines affect the HPA axis at multiple levels (Dunn 2004). Silverman et al. (2004). demonstrated that IL-6 was able to activate the HPA axis directly by actions on the hypothalamus, the pituitary, and the adrenal cortex. IL-6 and its receptor mRNA levels in the hypothalamus are modulated by an acute stress or changes in the HPA axis (Komori et al. 2003). Therefore, IL-6 in the hypothalamus is also a candidate for stimulation of CRF and AVP gene expression. PACAP could participate in the regulation of CRF and AVP genes via IL-6 production. For example, both forskolin and PACAP stimulated IL-6 promoter activity, IL-6 mRNA, and protein release. Therefore, PACAP stimulates IL-6 synthesis and secretion. PACAP increased IL-6 output levels to 70 pg/ml which would correspond to about 3.5 pM in the medium, while significant stimulatory effects on both CRF and AVP promoter activities required the treatment with at least 500 pM IL-6 in our study. This result suggests that IL-6 may not be the only stimulus in response to PACAP. However, we found that the stimulatory effects of PACAP on both activities were significantly inhibited by treatment with the anti-IL-6 MAB. Therefore, we propose that endogenous IL-6 production is involved in the PACAP-induced CRF and AVP 5′-promoter activities, and IL-6 stimulates transcription of both genes in an autocrine manner in hypothalamic 4B cells. Considering the delayed response to IL-6, and the partial inhibition of PACAP effects by IL-6 Ab, IL-6 may be important to sustain the activity of CRF and AVP promoter. Finally, note that subpopulations of parvocellular cells coexpress CRF, AVP, and IL-6, and project to the external layer of the ME (Gonzalez-Hernandez et al. 2006). Thus, it is possible that IL-6, produced in the hypothalamus, both directly and indirectly through CRF and AVP, stimulates ACTH secretion in pituitary corticotrophs.
In conclusion, this study demonstrated that PACAP stimulates transcription of CRF and AVP genes via a cAMP-dependent PKA pathway in hypothalamic 4B cells. The PACAP-induced intracellular cAMP pathway was also involved in the regulation of IL-6 gene transcription and protein production in hypothalamic 4B cells.
We thank Dr Ken Itoh’s laboratory (Center for Advanced Medical Research) for technical assistance. K K is supported by a grant from the Funds for the Promotion of Aomori Medical Research. This work was also supported in part by Health and Labor Science Research Grants (Research on Measures for Intractable Diseases) from the Ministry of Health, Labor, and Welfare of Japan, and by a grant to T S from the Ministry of Education, Science and Culture of Japan (No. 18591014). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
AgarwalA Halvorson LM & Legradi G 2005 Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene. Brain Research. Molecular Brain Research13845–57.
BoutillierAL Monnier D Koch B & Loeffler JP 1994 Pituitary adenyl cyclase-activating peptide: a hypophysiotropic factor that stimulates proopiomelanocortin gene transcription and proopiomelanocortin-derived peptide secretion in corticotropic cells. Neuroendocrinology60493–502.
Gonzalez-HernandezT Afonso-Oramas D Cruz-Muros I Barroso-Chinea P Abreu P del Mar Perez-Delgado M Rancel-Torres N & del Carmen Gonzalez M 2006 Interleukin-6 and nitric oxide synthase expression in the vasopressin and corticotrophin-releasing factor systems of the rat hypothalamus. Journal of Histochemistry and Cytochemistry54427–441.
HannibalJ Mikkelsen JD Fahrenkrug J & Larsen PJ 1995 Pituitary adenylate cyclase-activating peptide gene expression in corticotropin-releasing factor-containing parvicellular neurons of the rat hypothalamic paraventricular nucleus is induced by colchicine but not by adrenalectomy acute osmotic ether or restraint stress. Endocrinology1364116–4124.
HarmarAJ Arimura A Gozes I Journot L Laburthe M Pisegna JR Rawlings SR Robberecht P Said SI Sreedharan SP et al.1998 International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacological Reviews50265–270.
KasckowJ Mulchahey JJ Aguilera G Pisarska M Nikodemova M Chen HC Herman JP Murphy EK Liu Y Rizvi TA et al.2003b Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line. Journal of Neuroendocrinology15521–529.
KomoriT Miyahara S Yamamoto M Matsumoto T Zhang K Nakagawa M Nomura S Motomura E Shiroyama T & Okazaki Y 2003 Effects of odorants on the hypothalamic–pituitary–adrenal axis and interleukin-6 (IL-6) and IL-6 receptor mRNA expression in rat hypothalamus after restraint stress. Chemical Senses28767–771.
KoziczT & Arimura A 2002 Dopamine- and cyclic AMP-regulated phosphoprotein-immunoreactive neurons activated by acute stress are innervated by fiber terminals immunopositive for pituitary adenylate cyclase-activating polypeptide in the extended amygdala in the rat. Regulatory Peptides10963–70.
MastorakosG Weber JS Magiakou MA Gunn H & Chrousos GP 1994 Hypothalamic–pituitary–adrenal axis activation and stimulation of systemic vasopressin secretion by recombinant interleukin-6 in humans: potential implications for the syndrome of inappropriate vasopressin secretion. Journal of Clinical Endocrinology and Metabolism79934–939.
MiyataA Jiang L Dahl RD Kitada C Kubo K Fujino M Minamino N & Arimura A 1990 Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). Biochemical and Biophysical Research Communications170643–648.
NeumannH Schmidt H Cavalie A Jenne D & Wekerle H 1997 Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Journal of Experimental Medicine185305–316.
NomuraM Ueta Y Serino R Yamamoto Y Shibuya I & Yamashita H 1999 Effects of centrally administered pituitary adenylate cyclase-activating polypeptide on c-fos gene expression and heteronuclear RNA for vasopressin in rat paraventricular and supraoptic nuclei. Neuroendocrinology69167–180.