Pituitary Adenylate Cyclase Activating Polypeptide ... - Semantic Scholar

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 6 Printed in U.S.A.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Vasoactive Intestinal Peptide (VIP) Stimulate Interleukin-6 Production through the Third Subtype of PACAP/VIP Receptor in Rat Bone Marrow-Derived Stromal Cells YIQIANG CAI*, XIAONAN XIN*, GIL-JIN SHIM, YOKO MOKUNO, HISANORI UEHARA, TAKAHISA YAMADA, TAKASHI AGUI, AND KOZO MATSUMOTO From Institute for Animal Experimentation (Y.C., X.X., T.Y., K.M.), and Second Department of Pathology (H.U.), University of Tokushima School of Medicine, Tokushima 770, and Institute for Experimental Animal Science (G.-J.S., Y.M., T.A.), Nagoya City University Medical School, Nagoya 467, Japan ABSTRACT Regulation of Interleukin-6 (IL-6) production in bone marrow (BM)-derived stromal cells by neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), was examined. Both forms of PACAP, PACAP-27 and PACAP-38, as well as VIP significantly increased IL-6 production by rat BM-derived stromal cells at physiological concentrations ranging from 10210–1028 M. The three related peptides (PACAP-27, -38, and VIP) stimulated the production of both cAMP and inositol 1,4,5trisphosphate (IP3) in rat BM-derived stromal cells with similar 50% effective concentrations. The stimulatory potency of the three related peptides for the production of IL-6, cAMP, and IP3 was almost con-

sistent, suggesting that the dual signaling transduction pathways may be involved in PACAP/VIP-induced IL-6 production in rat BMderived stromal cells. The messenger RNA (mRNA) for the third subtype of PACAP receptor (PVR3) was found to be abundantly expressed in both BM-derived stromal cells and the BM tissue, whereas little of the mRNA for type 1 (PVR1) nor type 2 (PVR2) was detected. Furthermore, the mRNAs for PACAP and VIP were detected in the BM tissue, suggesting that both PACAP/VIP and PVR3 are synthesized in vivo in the BM. The results shown in this paper suggest that PACAP/VIP and their receptor play an important role in the IL-6 production and perhaps in the hematopoiesis in the BM. (Endocrinology 138: 2515–2520, 1997)

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Pituitary adenylate cyclase activating polypeptide (PACAP), recently isolated from the ovine hypothalamus, is a neuropeptide that belongs to the vasoactive intestinal peptide (VIP)/glucagon/secretin family (8, 9). Its mRNA was shown to be abundantly expressed in the central nervous tissue (10, 11). Both forms of PACAP, PACAP-27 and PACAP-38, exert their diverse biological effects by stimulating the release of various hormones (8, 9, 12–14). The complementary DNAs (cDNAs) of three subtypes of PACAP/VIP receptor have been cloned (15–19). PVR1 binds both PACAP-27 and PACAP-38 with similar affinity, but VIP with 1000-fold less affinity than PACAPs, whereas both PVR2 and PVR3 show similar affinity for the three related peptides (15–20). In view of the important role of IL-6 in hematopoiesis (21, 22) and the existence of PACAP-positive cells in the BM (23), we investigated here a possible role of PACAP in the BM by determining the effect of PACAP/VIP on the production of IL-6 by rat BM-derived stromal cells in vitro. We found that the PVR3, but not the PVR1 nor PVR2, is expressed in BMderived stromal cells and functionally coupled to the induction of IL-6 production via dual intracellular signaling pathways. We discuss here a possible role of PACAP/VIP and PVR3 in the IL-6 production and hematopoiesis in the BM.

HE HEMATOPOIETIC and immune systems are reported to be modulated by various neuropeptides (1–7). Neuropeptides, such as substance P and substance K, were reported to induce the release of interleukin (IL)-1, tumor necrosis factor-a, and IL-6 from human blood monocytes (3). The DW/J dwarf mice, which lack acidophilic anterior pituitary cells and are deficient in GH and other anterior pituitary-derived hormones, were found to exhibit a decrease in the number of peripheral blood cells including erythroid, myeloid, and lymphoid lineages (4, 5). Recently, it was reported that the high level of messenger RNA (mRNA) for neuropeptide Y was expressed in B cell precursor lymphoblasts from acute lymphoblastic leukemia patients, implying a role of neuropeptide Y in B cell development and/or pathologic disorders of B cells (6). However, the precise roles of individual neuropeptides in regulating hematopoietic system are not yet elucidated. Received September 9, 1996. Address all correspondence and requests for reprints to: Kozo Matsumoto, Institute for Animal Experimentation, University of Tokushima School of Medicine, Tokushima 770, Japan. * Present address: Department of Medicine and Molecular Genetics (Y.C.) and Department of Molecular Pharmacology (X.X.), Albert Einstein School of Medicine, Bronx, New York 10467.

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INCREASE IN IL-6 PRODUCTION BY PACAP/VIP IN BONE MARROW Materials and Methods

Materials Experimental reagents were obtained from the following sources: FCS, penicillin, streptomycin, and gentamicin, Life Technologies (Grand Island, NY); PACAP-27, PACAP-38, and VIP, Peptide Institute, Inc. (Osaka, Japan); DMEM, Nissui Pharmaceutical Co. Ltd., (Tokyo, Japan); oligo deoxythymidine (dT), Pharmacia, (Uppsala, Sweden); 3-isobutyl-1-methylxanthine (IBMX) and MEM nonessential amino acid, Sigma Chemical Co. (St. Louis, MO); Moloney-murine leukemia virus (M-MLV) reverse transcriptase, Bethesda Research Laboratories (Gaithersburg, MD); Taq DNA polymerase, acetylthiocholine iodide, mouse recombinant (r) IL-3 (WAKO, Osaka, Japan); restriction enzymes Mbo I, Takara (Kyoto, Japan); [125I] cAMP assay kit and [3H]inositol 1,4,5-trisphosphate (IP3) assay kit, Amersham International, (Buckinghamshire, UK); 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein (Dil-Ac-LDL), Biomedical Technologies Inc. (Stoughton, MA).

Preparation of rat BM cells A femoral bone was removed from a 3-month-old male F344/Tj rat. The BM cells were isolated by flushing the bone with DMEM supplemented with 5% FCS, 10 mm HEPES, 5.5 mm glutamine, 0.16 mm lasparagine, 0.55 mm l-arginine-HCl, 13 MEM nonessential amino acid, 50 mm 2-mercaptoethanol amine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 mg/ml gentamicin [modified DMEM (M-DMEM)] using a needle and syringe. BM cells were dispersed by repeated pipetting, washed twice with M-DMEM, and then cultured with 25 ml of MDMEM in an 80-cm2 flask (Nunc, Kamstrup, Denmark). After 24 h, unattached cells were harvested and recultured for the megakaryocyte development assay. Attached cells were continuously cultured by adding 25 ml of fresh medium. At 3-day intervals, the old medium and unattached cells were removed and fresh medium was added. After 7 days in culture, stromal cells formed a confluent monolayer. The stromal cell population was determined by counting macrophages, endothelial cells, and adipocytes as described previously (24). Macrophages and endothelial cells were identified by the avidity of Dil-Ac-LDL uptake. Both cell types were distinguished morphologically in fluorescence-positive cells. Adipocytes were identified by staining with Sudan III. In three independent primary cultures, contents of macrophages, endothelial cells, and adipocytes were 10.2 6 0.3% (mean 6 sem), 12.4 6 1.5% (mean 6 sem), and ,1%, respectively. In this way, other remaining cells were determined to be fibroblastic stromal cells. For passaging, the cells were removed with a rubber scraper, dispersed by repeated pipetting, and then one tenth were seeded into another flask. Primary culture cells were used for the IL-6 production assay, and passaged culture cells were used for the detection of the PACAP and PACAPR mRNA and cAMP production assay.

IL-6 bioassay and estimation of relative amounts of the IL-6 mRNA Rat BM-derived stromal cells were seeded onto a 96-well microplate at a density of 5,000/well in 200 ml of M-DMEM and cultured overnight. On the following day, the culture medium was aspirated, fresh medium was added, and the cells were then stimulated with various reagents. After incubation for various periods, the conditioned medium was removed and kept at 280 C until use. The amount of IL-6 in the conditioned medium was estimated using an IL-6-dependent B cell hybridoma cell line, MH60.BSF2, which was a generous gift from Dr. K. Himeno (University of Tokushima School of Medicine) with the permission of Dr. T. Hirano (Osaka University Medical School). MH60.BSF2 cells were cultured for 3 d in 100 ml M-DMEM in the presence of 10 ml stromal-cell-conditioned medium or various concentrations of human rIL-6 (a gift from Dr. T. Hirano) as the standard. Cell growth was estimated colorimetrically using a method described previously (25). For estimation of relative amounts of the IL-6 mRNA, cells were stimulated by 1027 m each peptide for 12 h, and the total RNA was prepared by a method described previously (26). RT-PCR was then performed as described previously (27).

Determination of cAMP accumulation BM-derived stromal cells were harvested and 0.1 million cells were incubated with PACAP-27, PACAP-38, or VIP in a volume of 0.5 ml of

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M-DMEM containing 1 mm IBMX. After incubation for 30 min at 37 C, the reaction was terminated by adding 10 ml of 5% NP40 solution and boiling samples for 3 min. The reaction mixture was centrifuged at 2,000 3 g for 15 min at 4 C, and the supernatants were removed to be used for RIAs. RIA was performed according to the procedure supplied with the cAMP assay kit.

IP3 production assay Cultured rat BM-derived stromal cells were suspended in M-Krebs solution. Cells (0.1 million) were preincubated at 37 C for 5 min and then incubated with various concentrations of PACAP-27, PACAP-38, or VIP for various times at 37 C. The total volume of the reaction mixture was 250 ml. The reaction was terminated by adding 250 ml of ice-cold 15% trichloroacetic acid solution. After the tubes had been kept on ice for 20 min, samples were centrifuged at 2,000 3 g for 20 min, and the supernatants were removed. The supernatants were washed three times with 10 vol of H2O-saturated diethyl ether, and the pH value was adjusted to 7.5 with a saturated NaHCO3 solution. The amount of IP3 was determined with an IP3 assay kit.

Detection of the mRNA for PACAP and PACAPR Total RNA was prepared from cultured rat BM-derived stromal cells or BM tissue according to the method described previously (26). Two micrograms of RNA were reversely transcribed with 200 U of M-MLV reverse transcriptase using 2 mg of oligo dT as the primer in 40 ml reaction mixture and the synthesized cDNA was used as a template for PCR. Sense and antisense primers for PACAP were 59-CTGTTGGTCTACGGGATAAT-39 (nucleotide 603–622 of the rat PACAP cDNA) and 59-CTACAAGTACGCTATTCGGC-39 (nucleotide 1081–1100 of the rat PACAP cDNA), respectively (28). For VIP, sense and antisense primers were 59-GTCTCTTTAAAAGCAGACTC-39 (nucleotide 132–151 of the rat VIP cDNA) and 59TATGAAATTATAAGCCTTTC-39 (nucleotide 710–729 of the rat VIP cDNA), respectively (29). For PVR1, sense and antisense primers were 59-GCCGATAGTAATTCCTTGGA-39 (nucleotide 328–347 of the rat PVR1 cDNA) and 59-TGAATGACAGGGCATCGAGT-39 (nucleotide 1066 –1085 of the rat PVR1 cDNA), respectively (15). For PVR2, sense and antisense primers were 59-TGAATGACAGGGCATCGAGT-39 (nucleotide 413– 432 of the rat PVR2 cDNA) and 59-TTTGGAGCTCCAGCCCAGGA-39 (nucleotide 1299–1318 of the rat PVR2 cDNA), respectively (17). For PVR3, sense and antisense primers were 59-CCCGAGGATGAGAGTAAGAT-39 (nucleotide 405–424 of the rat PVR3 cDNA) and 59-AACAGGATCTGGTACGTGGA-39 (nucleotide 1122–1141 of the rat PVR3 cDNA), respectively (18). The PCR reaction mixture (100 ml) contained 100 pmol of each primer, 200 mm each of deoxynucleotides, 50 mm KCl, 10 mm Tris-HCl (pH 8.8 at room temperature), 1.5 mm MgCl2, 0.1% Triton X-100, 5.0 U Taq DNA polymerase, and 5 ml of the above RT product. PCR was performed with a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT) using 35 cycles (94 C, 1 min; 62 C, 1.5 min; 72 C, 1.5 min) for PACAP and PVR3, 35 cycles (94 C, 45 sec; 52 C, 45 sec; 72 C, 45 sec) for VIP, 35 cycles (94 C, 1 min; 60 C, 1.5 min; 72 C, 1.5 min) for PVR1, and 35 cycles (94 C, 1 min; 63 C, 1.5 min; 72 C, 1.5 min) for PVR2. After amplification, 20 ml of the PCR products were digested by MboI in the case for checking if correct products were amplified. PCR products were electrophoresed in a 1.2% agarose gel for undigested PCR products or a 4% NuSieve agarose gel for digested PCR products. After running, gels were soaked with 0.5 mg/ml ethidium bromide solution and photographed with Polaroid film (Polaroid Corp., Cambridge, MA) under the UV light.

Results PACAP/VIP-induced IL-6 production by rat BM-derived stromal cells

In order to examine whether the effect of PACAP/VIP on the IL-6 production in the BM, we stimulated rat BM-derived stromal cells by PACAP-27, PACAP-38, and VIP. As shown in Fig. 1, three peptides all augmented IL-6 production by rat BM-derived stromal cells with similar time course. Three peptides, however, had no effect on the cell number in culture for 3 days. Figure 2 shows that the potency of the three

INCREASE IN IL-6 PRODUCTION BY PACAP/VIP IN BONE MARROW

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respectively. In order to show that IL-6 was certainly produced by PACAP-stimulated BM-derived stromal cells, an increase in the steady-state level of the IL-6 mRNA was examined in the stimulated cells. As shown in Fig. 3, the level of IL-6 mRNA was higher in cells stimulated by three kinds of peptides than in control cells. Furthermore, conditioned medium of PACAP-38-stimulated stromal cells supported megakaryocyte development in cooperation with IL-3 (data not shown), indicating that IL-6 produced by stromal cells are also biologically active on other cells than MH60.BSF-2 cells. PACAP was reported to augment the IL-6 production by rat anterior pituitary cells at the concentration of 10210 m, whereas VIP did not even at 1026 m (13). We presumed that the discrepancy in the capability of VIP in induction of IL-6 production in the two cell types may be due to the different receptor subtypes expressed. In order to clarify it, we examined second messengers in PACAP/VIP-stimulated BM-derived stromal cells. FIG. 1. Stimulation of IL-6 production by PACAP-27, PACAP-38, and VIP in rat BM-derived stromal cells. Cells were seeded onto wells of a microtiter plate at a density of 5,000/well. After overnight culture, the medium was aspirated, fresh medium was added, and then cells were stimulated by 1027 M PACAP-27 (E), PACAP-38 (■), or VIP (Ç) for the indicated times. The conditioned medium was removed and assayed IL-6 concentrations using an IL-6-dependent B cell hybridoma, MH60.BSF2, with human rIL-6 as the standard. Closed circles represent the basal production of IL-6. Data are means 6 SEM of triplicate observations and are representative of three similar results.

FIG. 2. Dose response of PACAP-27, PACAP-38, and VIP in stimulation of IL-6 production by rat BM-derived stromal cells. Experimental conditions were the same as in Fig. 1, except that cells were stimulated for 3 days by various concentrations of PACAP-27 (E), PACAP-38 (■), or VIP (Ç). Data are means 6 SEM of triplicate observations and are representative of three similar results.

peptides are similar and that they exert their effects at physiological concentrations ranging from 10210–1028 m. The 50% effective concentrations (EC50) for PACAP-27, PACAP-38 and VIP were 2.0 3 1029, 6.3 3 10210, and 8.0 3 10210 m,

Second messengers of PACAP in rat BM-derived stromal cells

It has been demonstrated that the three subtypes of PACAPRs are coupled to the activation of adenylate cyclase (15–19). Therefore, the accumulation of cAMP by BM-derived stromal cells were assayed after stimulation of PACAP-27, PACAP-38, and VIP. As shown in Fig. 4, the three peptides evoked a significant and dose-dependent elevation of cAMP levels. The EC50 were approximately 4.4 3 1029, 6.6 3 10210, and 3.3 3 1029 m for PACAP-27, PACAP-38, and VIP, respectively. The three peptides were all effective to the augmentation of cAMP production, although their potency was slightly different; PACAP-38 was 5- to 6-fold more potent than VIP. These results suggest that PACAPR on rat BM-derived stromal cells are either PVR2 or PVR3, because PVR1 was reported to bind VIP with 1,000-fold less affinity than two forms of PACAP (15, 16). Because it has been shown that the third subtype of PACAPR recently discovered, PVR3, is coupled to the phospholipase C (PLC) activation (19) but PVR2 is unknown (17), we further examined whether PACAP and VIP are capable of stimulating

FIG. 3. Comparison of relative amounts of the IL-6 mRNA in control and stimulated stromal cells. Cells were stimulated by 1027 M PACAP-27, PACAP-38, or VIP for 12 h, and then total RNA was prepared. Amounts of the IL-6 mRNA were compared between control and stimulated stromal cells by performing RT-PCR using various amplification cycles with respect to both the IL-6 and b-actin mRNAs. This is representative of three similar results.

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FIG. 4. Dose-dependent stimulation of cAMP accumulation in BMderived stromal cells by PACAPs and VIP. Cells (0.1 million) were incubated with various concentrations of PACAP-27 (E), PACAP-38 (■), or VIP (Ç) at 37 C for 30 min and assayed accumulated cAMP. Data are means 6 SEM of triplicate observations and are representative of two similar independent results.

PLC in BM-derived stromal cells. As shown in Fig. 5A, IP3 was rapidly produced by the stimulation of PACAP-27, PACAP-38, and VIP with a peak at 15–30 sec after addition of each peptide. The rapid increase in IP3 production within 30 sec is consistent with the previous report (30). The potency of the three peptides on the stimulation of IP3 production was similar each other (Fig. 5B). The EC50 were 4.8 3 10210, 1.2 3 10210, and 3.0 3 10210 m for PACAP-27, PACAP-38, and VIP, respectively. It is, therefore, proposed that PACAPR on rat BM-derived stromal cells seems to be the third type of PACAPR. In order to clarify it, the PACAPR mRNA expressed in rat BM-derived stromal cells are examined by RT-PCR using each PACAPR-specific primers. Expression of the PVR3 mRNA in BM-derived stromal cells and the BM tissue

We examined expression of PACAPRs by RT-PCR with three pairs of primers, which were unique to the rat PVR1, PVR2, and PVR3. Primers were synthesized according to information of the rat cDNA sequences (15, 17, 18). We clearly detected the abundant expression of mRNA for PVR3 in cultured BM-derived stromal cells comparable to that of the brain (Fig. 6). In contrast to PVR3, little expression of PVR1 nor PVR2 was detected in the BM-derived stromal cells, although expression of them in the brain was well observed. That the PCR certainly amplified respective PACAPR mRNAs was corroborated by digestion analysis with restriction enzyme, MboI (data not shown). In order to test whether the PVR3 is expressed in vivo, we isolated the total RNA from the rat BM tissue and used it for the RT-PCR and MboI digestion experiments. The data showed the same result as in cultured cells (data not shown), suggesting that PVR3 is only expressed in the rat BM in vivo.

FIG. 5. IP3 production stimulated by PACAPs and VIP in BM-derived stromal cells. (A) Cells (0.1 million) were stimulated by 1027 M PACAP-27 (E), PACAP-38 (■), or VIP (Ç) for the indicated times and assayed IP3 accumulation. B, Cells (0.1 million) were stimulated by various concentrations of PACAP-27 (E), PACAP-38 (■), or VIP (Ç) for 30 s and assayed IP3 accumulation. Data are means 6 SEM of triplicate observations and are representative of two similar results.

Expression of the mRNA for PACAP and VIP in the BM tissue

The fact that the physiological concentrations (1029 m) of PACAPs and VIP up-regulate the production of IL-6 by BMderived stromal cells raised a question as whether there exists the endogenous ligands in the BM. To examine this possibility, we attempted to detect the mRNAs for PACAP and VIP by RT-PCR. Figure 7 shows that the mRNAs for PACAP and VIP are expressed in the BM tissue. That the resulting PCR products were specific for PACAP and VIP was confirmed by MboI digestion (data not shown). These


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FIG. 7. Detection of the mRNA for PACAP and VIP in the rat BM tissue. Lanes 1, rat BM tissue; 2, rat brain. This is representative of two similar results. FIG. 6. Detection of the mRNA for PVR3 in rat BM-derived stromal cells. Total RNA was prepared from the BM-derived stromal cells and rat whole brain. RT-PCR was performed using PCR primers unique to three subtypes of PACAPRs. Lanes 1, rat BM-derived stromal cells; 2, rat brain. This is representative of four similar results.

results support the previous report showing the existence of PACAP-positive cells in rat BM tissue (23) and suggest that the PACAP/VIP/PVR3 ligand-receptor system plays a physiological role in producing IL-6 in the BM. Discussion

The role of IL-6 on hematopoiesis has been well investigated (21, 22). It is proposed that IL-6 may be one of the factors capable of inducing entry of primitive stem cells into cell cycle and that IL-6 may act as a competence factor that is required to confer responsiveness to the classical hematopoietic growth factors. The regulation of IL-6 production and secretion by stromal cells in the BM has not been completely elucidated. IL-1, transforming growth factor-b, and tumor necrosis factor-a were reported to be inducers of IL-6 production by a murine BM-derived stromal cell clone (31). Recently, hormonal and/or neuronal regulation of cytokine production has been noted in other immune tissues. However, little is known about hormonal or neuronal regulation of cytokine production in the BM. We report here that neuropeptides PACAPs and VIP, which were already reported to be secretagogues for IL-6 in anterior pituitary cells (13, 14, 32), also induce IL-6 production in rat BMderived stromal cells (Figs. 1–3). We also demonstrated the existence of receptors for PACAP/VIP, which are coupled to the activation of both adenylate cyclase and PLC (Figs. 4 and 5). Furthermore, we could show evidence that endogenous ligands PACAP and VIP were synthesized in the BM tissue (Fig. 7). These results suggest that the PACAP/VIP and their receptor system plays a physiological role in regulating IL-6 production in the BM. Recently, the calcitonin gene-related peptide was shown to increase dose-dependently cAMP and IL-6 production in a murine BM-derived stromal cell line (7). Furthermore, we recently showed that endothelin, a potent vasoactive polypeptide, also plays a role in the regulation of IL-6 production by rat BM-derived stromal cells (24). Taken together, it is thus raised the possibility that multiple neuro-hormonal factors may be involved in hematopoiesis by controlling the production of IL-6 in the BM. The cDNAs for three subtypes of PACAPR have been cloned so far. The type 1 receptor, PVR1, preferentially binds two forms of PACAP, PACAP-27 and PACAP-38, and binds VIP with 1,000-fold less affinity than PACAPs (15, 16). Furthermore, the PVR1 was reported to be coupled to the activation of both adenylate cyclase and PLC (33). The type 2 receptor, PVR2, was

originally identified as the receptor for VIP, but was shown to bind two forms of PACAP as well as VIP. The PVR2 was shown to be coupled to the adenylate cyclase activation, but it is unknown if it is coupled to the PLC activation (17). Recently, the third type of PACAPR, PVR3, was identified, which also binds two forms of PACAP and VIP with similar affinity (18, 19). The PVR3 was also shown to be coupled to the adenylate cyclase activation. The coupling to the PLC activation of the PVR3 was indirectly demonstrated by eliciting a Ca21 influx by PACAPs and VIP in Xenopus oocytes expressed this receptor (19). With respect to the induction of IL-6 production by BM-derived stromal cells, two forms of PACAP and VIP were equipotent. Furthermore, the three peptides stimulated both adenylate cyclase and PLC in these cells. These results suggest that the PVR3 may be responsible to the induction of IL-6 production in these cells. To clarify it, expression of the three subtypes of PACAPR mRNA was examined by RT-PCR using primers unique to the respective PACAPR subtypes. The result shows that BM-derived stromal cells express the PVR3 only, indicating that the PVR3 is responsible to the induction of IL-6 by BM-derived stromal cells in response to PACAPs or VIP. With respect to the role of PACAP/VIP in regulating IL-6 production, it is very interesting to compare our results in the BM with those in the anterior pituitary gland. Firstly, in the anterior pituitary IL-6 was regarded to be a stimulator for the secretion of various anterior pituitary hormones (34). Secondly, it has been shown that IL-6 is produced in the anterior pituitary in situ in response to PACAP/VIP as well as inflammatory cytokines (13, 32, 35, 36). Finally, it has been evidenced that agranular hormone-nonsecreting folliculo-stellate (FS) cells produce IL-6 in the anterior pituitary (14, 37). A role of FS cells in the pituitary may be analogous to that of stromal cells in the BM. Both types of cells secrete IL-6 in response to various stimulations, including PACAP/VIP. IL-6 is regarded to play a particular role in both tissues; in stimulating the release of pituitary hormones in the pituitary and in supporting the growth and differentiation of hematopoietic cells in the BM. However, with respect to the potency of PACAPs and VIP, there exists discrepancy in the two tissues. In the pituitary, the effect of VIP is controversial. Thus, VIP was reported to be as potent as PACAPs in cAMP production but much less potent than PACAPs in IL-6 production (14). In other report, VIP is as potent as PACAPs in IL-6 production (32). This may be due to the existence of multiple PACAPR subtypes and multiple target cells for PACAP/VIP in the pituitary. In fact, cDNAs of both PVR1 and PVR3 were cloned from the same rat anterior pituitary mRNA pool (16, 18). In contrast, BM-derived stromal cells were shown to express the PVR3 only by very specific RT-PCR analysis. Cultured BM-

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derived stromal cells are heterogenous consisted by fibroblastic cells, macrophages, endothelial cells, and adipocytes. Nevertheless, the fact that stromal cells express the PVR3 only may suggest that the target of PACAP/VIP may be a single type of cells. PACAPR subtypes were reported to localize in various tissues. However, it is noteworthy to find that the BM tissue express the PVR3 only. It should be interesting that only the PVR3 plays a role in regulating hematopoiesis in the BM through regulating IL-6 production by BM stromal cells. Although we showed in this report that the ligands, PACAP and VIP were also synthesized in the BM, it should be necessary to clarify the cell type synthesizing the ligands in situ, in order to fully understand roles of PACAP/VIP in the BM.

15. 16. 17. 18. 19.

Acknowledgment We thank Drs. K. Himeno and T. Hirano for IL-6-dependent cell line, MH60.BSF2 and human rIL-6.

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