help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kiriyama, Y.
Right arrow Articles by Tokumitsu, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiriyama, Y.
Right arrow Articles by Tokumitsu, Y.
Endocrinology Vol. 142, No. 8 3563-3569
Copyright © 2001 by The Endocrine Society

ARTICLES

Calcitonin Induces IL-6 Production via Both PKA and PKC Pathways in the Pituitary Folliculo-Stellate Cell Line

Yoshimitsu Kiriyama, Hiroyuki Tsuchiya, Takeshi Murakami, Kumi Satoh and Yukiko Tokumitsu

Department of Physiological Chemistry (Y.K.) and Laboratory of Molecular Design of Pharmaceutics (H.T.), Graduate School of Pharmaceutical Sciences, Hokkaido University; and Department of Laboratory Medicine, Hokkaido University Graduate School of Medicine (T.M.), Sapporo 060-0812, Japan; Department of Pharmacology, Hokkaido College of Pharmacy (K.S.), Otaru 047-0264, Japan; and Health Sciences University of Hokkaido (Y.T.), Ishikari-Tobetsu 061-0293, Japan

Address all correspondence and requests for reprints to: Yukiko Tokumitsu, Ph.D., Health Sciences University of Hokkaido, IshikariTobetsu 061-0293, Japan. E-mail: tyukiko{at}hoku-iryo-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been demonstrated that calcitonin-binding sites are present in a variety of tissue types, including in the pituitary gland. Interleukin-6 (IL-6) is also produced in the pituitary and it regulates the secretion of various hormones. In this study, we examined the expression of the calcitonin receptor and the mechanism of IL-6 production induced by calcitonin in the pituitary folliculo-stellate cell line (TtT/GF). The mRNA of calcitonin receptor subtype C1a, but not that of C1b, was detected by RT-PCR in TtT/GF cells and in the normal mouse pituitary. Calcitonin increased cAMP accumulation and IL-6 production in a concentration-dependent manner in TtT/GF cells. As calcitonin activates the PKA and PKC pathways, we investigated the contributions of PKA and PKC to IL-6 production. IL-6 production was only slightly increased by either 8-bromo-cAMP (1 mM) or phorbol 12-myristate 13-acetate (100 nM) alone. However, IL-6 was synergistically induced in the presence of both 8-bromo-cAMP (1 mM) and phorbol 12myristate 13-acetate (100 nM). Furthermore, calcitonin-induced IL-6 production was completely suppressed by H-89 (PKA inhibitor) or GF109203X (PKC inhibitor), indicating that the activation of both PKA and PKC is necessary for calcitonin-induced IL-6 production. On the other hand, pertussis toxin (Gi/Go signaling inhibitor) treatment achieved an approximately 9-fold increase in calcitonin-induced IL-6 production. These results show that calcitonin-stimulated IL-6 production is mediated via both PKA- and PKC-signaling pathways, whereas calcitonin also suppresses IL-6 production by activating Gi/Go proteins in folliculo-stellate cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN (CT) IS a 32-amino acid peptide hormone that regulates blood Ca2+ concentrations by inhibiting bone reabsorption. CT is mainly synthesized in the parafollicular cells of the thyroid gland and is secreted into blood (1). Binding sites for CT are distributed among many cell and tissue types, including osteoclasts, kidney, brain, and pituitary (2, 3, 4, 5, 6). The CT receptor (CTR) belongs to a subclass of the G protein-coupled receptor family that includes PTH/PTHrP, secretin, VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), CRF, etc. (7, 8, 9). Two isoforms of rodent CTRs, C1a and C1b, have been identified to date, and they have been shown to differ according to the absence or presence of a 37-amino acid insert in the second extracellular domain (10, 11). The CTR is able to couple to Gs, Gq, and Gi proteins (12, 13, 14) and activates adenylyl cyclase, PLC (15, 16), and PLD (17). PKA, PKC, and MAPK are all involved in the CTR signaling pathways (18, 19).

IL-6 controls the secretion of ACTH, PRL, GH, and LH from the anterior pituitary (20, 21). Moreover, IL-6 mRNA and IL-6 have been detected in the pituitary (22, 23) and are localized to folliculo-stellate (FS) cells (24, 25). IL-6 production is regulated via various signaling pathways, including both the PKA and PKC pathways (26, 27, 28).

FS cells of the anterior pituitary gland are of the agranular cell type and have a stellate morphology and surrounding follicular cavities (29). About 7.5% of the cells in the mouse pituitary are FS cells (25). FS cells have some features in common with glial cells, e.g. expression of GFAP, S100, and vimentin (30). The TtT/GF cell line, which was established by Inoue et al. from a mouse thyrotropic pituitary tumor, has been shown to possess many features of normal FS cells (31). TtT/GF cells produce IL-6 in response to VIP, PACAP, and TNF{alpha} (32, 33). Moreover, CT, like VIP and PACAP, stimulated cAMP accumulation and subsequent release of IL-6 production from glioma cells (34). These findings suggest the presence of the CTR and also imply that CT induces IL-6 production in FS cells.

In the present study we examined the effects of CT on IL-6 production in FS cells using H-89, a PKA inhibitor (35); GF109203X, a PKC inhibitor (36); PD98059, a MAPK kinase-1 inhibitor (37); U73122, a PLC/PLD inhibitor (38); D609, a phosphatidylcholine (PC)-PLC inhibitor (39); propranolol, a phosphatidic, phosphohydrolase (PPH) inhibitor (40); and pertussis toxin (PTX), a Gi/Go protein activation inhibitor (41). The results show that CT activates both PKA and PKC, resulting in the induction of IL-6 production, whereas CT also suppresses IL-6 production in part by activating PTX-sensitive G proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All peptides were purchased from Peninsula Laboratories, Inc. (San Carlos, CA). The ELISA kit for measuring IL-6 was purchased from R & D Systems (Minneapolis, MN). H-89 and U73122 were obtained from Calbiochem (La Jolla, CA), GF109203X and propranolol were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). D609, PD98059, phorbol 12-myristate 13-acetate (PMA), and the Wizard PCR Preps DNA Purification System were purchased from Alexis (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), Sigma (St. Louis, MO), and Promega Corp. (Madison, WI), respectively. SuperScript II reverse transcriptase, oligo(deoxythymidine)12–18 primer, and RNASEOUT Recombinant Ribonuclease Inhibitor were purchased from Life Technologies, Inc. (Gaithersburg, MD). 8-Bromo-cAMP and the Expand High Fidelity PCR System were obtained from Roche Molecular Biochemicals (Mannheim, Germany). The cAMP assay kit and PTX were provided by Yamasa Shoyu (Chiba, Japan) and Kaken Pharmaceutical Co. Ltd. (Kyoto, Japan), respectively. All peptides, 8-bromo-cAMP, and propranolol were prepared in sterile distilled water. PTX was prepared in sterile 2 M urea/0.1 M phosphate buffer (pH 6.5). The other drugs were prepared in dimethylsulfoxide.

Cells and cell culture
The mouse FS cell line (TtT/GF cell) was obtained from RIKEN Cell Bank (Saitama, Japan). TtT/GF cells were cultured in DMEM supplemented with penicillin G potassium (50 µg/ml), streptomycin sulfate (100 µg/ml), and heat-inactivated FCS (10%) at 37 C in 5% CO2/95% air. Confluent cells were routinely passaged using 0.25% trypsin and 0.05% EDTA in PBS (pH 7.4).

Animals
Adult male BALB/c mice (6–7 wk old) were acclimated to standard laboratory conditions (12-h light, 12-h dark cycle at 22-24 C) and were fed laboratory chow and water ad libitum. All of the experimental procedures were approved by the animal care and use committee at Hokkaido University. Four mouse pituitaries were used for RNA extraction.

RT-PCR
Total RNA of TtT/GF cells or the mouse pituitary homogenates were isolated by AGPC methods (42). The integrity of total RNA was verified by ethidium bromide staining. Reverse transcriptase reactions were performed on 1 µg RNA using 200 U SuperScript II reverse transcriptase in the presence of 0.5 µg oligo(deoxythymidine)12–18 primer, 500 µM of each deoxynucleotide triphosphate, 20 U RNASEOUT Recombinant Ribonuclease Inhibitor, 50 mM Tris-HCl, 10 mM dithiothreitol, 75 mM KCl, and 3 mM MgCl2 in a 20-µl volume at 42 C for 1 h. The enzyme was inactivated by heating the samples to 95 C for 5 min. The sense (5'-gttgaggttgtgcccaatgga-3') and antisense (5'-ccctggaaatgaatcagagag-3') primers for the CTR amplify bands of 656 bp for C1b and 545 bp for C1a. The sense and antisense primers for the CTR are equivalent to the positions of 1114–1134 and 1749–1769, respectively, of mouse C1b sequence (GenBank MMU18542). The sense (5'-gcagacaacgtgggctccaag-3') and antisense (5'-gatgttcagcatgttcagcag-3') primers for 36B4 amplify a band of 447 bp. 36B4 was used as a control for the quality of the cDNA for each PCR reaction. One twentieth of the resulting cDNAs was subjected to PCR reactions in a 50-µl reaction mixture containing 300 nM of both sense and antisense primers, 2.6 U enzyme mixture (thermostable Taq and Pwo DNA polymerases), 1.5 mM MgCl2, 200 µM of each deoxyribonucleotide triphosphate, and 1 x kit buffer. PCR amplification was performed after 5 min of denaturation at 94 C. The cycle consisted of denaturation (45 sec at 94 C), annealing (45 sec at 58 C for the CTR and at 60 C for 36B4), extension (1 min at 72 C), and 7 min of final extension at 72 C after amplification. The CTR was amplified by 35 cycles; 36B4 was amplified by 25 cycles. The PCR products were analyzed on 1.5% agarose gels in the presence of 0.5 mg/ml ethidium bromide. In addition, control PCR reactions were performed with each primer pair on RNA samples that had been incubated in the absence of reverse transcriptase. The PCR products were purified from gels using the Wizard PCR preps DNA Purification System. The identity of the PCR products migrating at the predicted size was verified by direct sequencing using the same primers as those used for the PCR reaction in an ABI 373 DNA sequencer.

cAMP assay
Confluent TtT/GF cells in six-well plates were washed twice and incubated with serum-free DMEM for 14 h. Cells were washed twice and incubated with various agents for 10 min in a final volume of 1 ml modified Tyrode’s HEPES buffer [137 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgCl2, and 20 mM HEPES (pH 7.4)] containing 0.1% BSA and 0.3 mM isobutylmethylxanthine at 37 C. The reaction was terminated by the addition of 100 µl 1 N HCl. The cAMP content in the supernatant was measured using the cAMP assay kit.

Protein assay
The protein concentration of each sample was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s methodology using BSA as the standard.

IL-6 production
TtT/GF cells were plated at 1 x 105 cells/ml into six-well plates and were incubated for 72 h. Cells were washed twice and incubated with serum-free DMEM for 14 h. Unless otherwise indicated, cells were treated with various agents for 6 h after serum-free DMEM incubation. Supernatants obtained by centrifugation (2000 x g, 15 min) were stored at -80 C and assayed for immunoreactive IL-6 using ELISA kits.

Statistical analysis
The data are expressed as the mean ± SEM of at least three independent experiments. Multiple comparisons were examined by ANOVA, followed by the Bonferroni’s multiple range test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CTR mRNA expression in TtT/GF cells and the mouse pituitary
RT-PCR was performed using total RNA from TtT/GF cells and the normal mouse pituitary. The two receptors are distinguished by the 111-bp insert present in the C1b receptor, but not in the C1a receptor. As shown in Fig. 1Go, the predicted 545-bp product corresponding to the C1a receptor was expressed in both TtT/GF cells and mouse pituitary. In contrast, the predicted 656-bp product corresponding to the C1b receptor was not detected in either TtT/GF cells or mouse pituitary even at 40 cycles (data not shown). Sequencing confirmed that the 545-bp PCR product from TtT/GF cells and mouse pituitary was the C1a isoform of the mouse CTR.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 1. RT-PCR analysis of CTR mRNA expression in TtT/GF cells and mouse pituitary. RT (-), Negative control for each sample without reverse transcriptase. 36B4 was used as an internal control.

 
cAMP accumulation induced by CT
CT, calcitonin gene-related peptide (CGRP) or amylin increased cAMP accumulation in a similar concentrationdependent manner (Fig. 2AGo). CGRP and amylin are 37-amino acid peptides and are structurally related to CT. It has been shown that CGRP and amylin cross-react with the CTR (43, 44). To examine the mechanism of how CT stimulates its own receptor, we observed the inhibitory effects of the following substances on CT-induced cAMP accumulation: CT antagonist, salmon CT (sCT)-(8–32), CGRP antagonist, rat CGRP (rCGRP)-(8–37), and amylin antagonist, rat amylin-(8–37). As shown in Fig. 2BGo, 100 nM CT-induced cAMP accumulation was reduced to 18 ± 2% by 100 nM sCT-(8–32) and to 78 ± 6% by 10 µM rCGRP-(8–37), which reduced rCGRP-induced cAMP accumulation to 14 ± 3% (Fig. 2CGo). However, 10 µM rat amylin-(8–37), which reduced rat amylin-induced cAMP accumulation to 68 ± 3% (Fig. 2DGo), had no effect on rCT-induced cAMP accumulation. sCT-(8–32) (100 nM), 10 µM rCGRP-(8–37), or 10 µM rat amylin-(8–37) alone did not affect basal cAMP accumulation (data not shown).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Effects of rCT, rCGRP, or rat amylin on cAMP accumulation in TtT/GF cells. A, Cells were stimulated with the indicated concentrations of rCT, rCGRP, and rat amylin. B, Cells were stimulated with 100 nM rCT in the presence or absence of 100 nM sCT-(8–32), 10 µM rCGRP-(8–37), or 10 µM rat amylin-(8–37). C, Cells were stimulated with 100 nM rCGRP in the presence or absence of 10 µM rCGRP-(8–37). D, Cells were stimulated with 100 nM rat amylin in the presence or absence of 10 µM rat amylin-(8–37). cAMP accumulation was determined as described in Materials and Methods. Data are the mean ± SEM of at least three independent experiments. *, P < 0.05 compared with control.

 
IL-6 production induced by CT
To study the time course of the effects of CT on IL-6 production, TtT/GF cells were treated with 1 µM CT, and supernatants were collected at various times from 1–24 h after CT treatment. IL-6 production induced by CT in TtT/GF cells was detectable as early as 1 h (17 ± 4.8 pg/ml) and reached a maximum at 6 h (360.4 ± 21.7 pg/ml). IL-6 production then gradually decreased after 6 h (Fig. 3AGo). The concentration-dependent effects of CT on IL-6 production were examined 6 h after CT stimulation. The amount of IL-6 production was detectable at concentrations as low as 30 nM CT (9.8 ± 1.5 pg/ml) and rose steadily with 1 µM CT (346.3 ± 25.4 pg/ml; Fig. 3BGo).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 3. Time course (A) and concentration-response curve (B) of IL-6 production induced by CT in TtT/GF cells. A, Cells were incubated with 1 µM CT for the indicated durations. B, Cells were incubated for 6 h with the indicated concentrations of CT. The amount of IL-6 released into the medium was assayed by ELISA. Data are the mean ± SEM of at least three independent experiments. *, P < 0.05 compared with control.

 
CT-induced IL-6 production requires both PKA and PKC pathways
CT activates the PKA and PKC pathways via the accumulation of cAMP, diacylglycerol (DG), and intracellular Ca2+ (15, 16). 8-Bromo-cAMP and PMA were used to examine whether PKA, PKC, or both kinases are involved in the IL-6 production pathways in TtT/GF cells. IL-6 production was slightly increased by 1 mM 8-bromo-cAMP, but was not increased by 100 nM PMA. However, induction of IL-6 production was significant in the presence of both 1 mM 8bromo-cAMP and 100 nM PMA (Table 1Go). The selective PKA inhibitor, H-89 (35), was used to study the role of PKA in IL-6 production induced by CT. IL-6 production was not affected by 20 µM H-89 and was completely inhibited by 50 µM H-89 (Fig. 4AGo). Moreover, the specific inhibitor of both conventional and novel PKC isoforms, GF109203X (36), was used to study the role of PKC on CT-induced IL-6 production. GF109203X caused a concentration-dependent inhibition of IL-6 production stimulated by CT, and inhibition was complete in the presence of 50 µM GF109203X (Fig. 4BGo). IL-6 was not detected in the presence of 50 µM H-89 or 50 µM GF109203X alone (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Effects of various agents on IL-6 production in TtT/GF cells

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Effects of PKA and PKC inhibitors on CT-induced IL-6 production in TtT/GF cells. Cells were preincubated for 3 h with the indicated concentrations of H-89 (A) or GF10932X (B), and then were incubated with 1 µM CT for 6 h. The amount of IL-6 released into the medium was assayed by ELISA. Data are the mean ± SEM of at least three independent experiments. *, P < 0.05 compared with control.

 
Effects of signaling inhibitors on CT-induced IL-6 production
It has been reported that CT activates G proteins, MAPK, PLC, and PLD in addition to activating PKA and PKC (12, 13, 14, 15, 16, 17, 18, 19). MAPK kinase-1 inhibitor, PD98059 (37) (10 µM), reduced CT-induced IL-6 production to 67 ± 3% and the inhibitory effect of PD98059 was consistent up to 50 µM (46 ± 3%; Fig. 5AGo). Moreover, we examined the effects of PLC/PLD inhibitor, U73122 (38), on CT-induced IL-6 production. U73122 reduced CT-induced IL-6 production in a concentrationdependent manner, and inhibition by 50 µM U73122 was 16 ± 5% (Fig. 5BGo). D609 is a PC-PLC inhibitor (39), and propranolol, a ß-adrenergic receptor antagonist, is a well established PPH inhibitor (40). PLD catalyzes the hydrolysis of PC and generates phosphatidic acid, which is dephosphorylated by PPH to produce DG (45). Thus, propranolol inhibits DG production via the PLD pathway. D609 and propranolol failed to suppress CT-induced IL-6 production in TtT/GF cells (Fig. 6Go). IL-6 was not detected in the presence of 50 µM PD98059, 50 µM U73122, 50 µM D609, or 300 µM propranolol alone (data not shown).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Effects of MAPK and PLC/PLD inhibitors on CT-induced IL-6 production in TtT/GF cells. Cells were preincubated for 3 h with the indicated concentration of PD98059 (A) or U73122 (B), and then were incubated with 1 µM CT for 6 h. The amount of IL-6 released into the medium was assayed by ELISA. Data are the mean ± SEM of at least three independent experiments. *, P < 0.05 compared with control.

 


View larger version (46K):
[in this window]
[in a new window]
  		Figure 6. Effects of PC-PLC and PPH inhibitors on CT-induced IL-6 production in TtT/GF cells. Cells were preincubated for 3 h with the indicated concentrations of D609 or propranolol, and then were incubated with 1 µM CT for 6 h. The amount of IL-6 released into the medium was assayed by ELISA. Data are the mean ± SEM of at least three independent experiments.

 
Inhibition of CT-induced IL-6 production by Gi/Go proteins
PTX was used to investigate whether CT mediates Gi/Go proteins. PTX covalently modifies the {alpha}-subunits of Gi and Go proteins, resulting in the prevention of Gi/Go protein activation (41). The effects of 1 µg/ml PTX on cAMP accumulation were examined (Table 2Go). Although PTX alone did not affect cAMP accumulation, PTX increased CT-induced cAMP accumulation approximately 2.5-fold. Moreover, 0.5 and 1 µg/ml PTX treatment increased CT-induced IL-6 production to 871 ± 135% and 806 ± 52%, respectively (Fig. 7Go). IL-6 was not detected in the presence of 0.5 µg/ml PTX alone and was induced slightly in the presence of 1 µg/ml PTX alone (44.4 ± 17.7 pg/ml).


View this table:
[in this window]
[in a new window]
  		Table 2. Effects of PTX on cAMP accumulation in TtT/GF cells

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 7. Effects of Gi/Go protein activation inhibitor on CT-induced IL-6 production in TtT/GF cells. Cells were preincubated for 16 h with the indicated concentrations of PTX and then were incubated with 1 µM CT for 6 h. The amount of IL-6 released into the medium was assayed by ELISA. Data are the mean ± SEM of at least three independent experiments. *, P < 0.05 compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The C1a subtype of CTR mRNA was expressed in the mouse pituitary and TtT/GF cells. As some secretory anterior pituitary cells, such as thyrotrophs, also express CTR (46), the C1a mRNA in the mouse pituitary is derived from not only FS cells, but also other anterior pituitary cells. On the other hand, the mRNA of the C1b subtype was not detected in either mouse pituitary or TtT/GF cells. C1b is considered to be a receptor for endogenous sCT-like peptides (47, 48, 49). The physiological plasma level of CT in rats is approximately 60 pg/ml (0.02 nM) to 480 pg/ml (0.1 nM) (50). Although these levels of CT are able to increase cAMP accumulation, these concentrations of CT are not able to increase IL-6 production from FS cells. On the other hand, the pituitary also synthesizes and secretes CT-like peptides (47, 48). Therefore, it is speculated that CT alone in the pituitary or in combination with plasma CT may induce IL-6 production from FS cells.

The receptor activity-modifying proteins (RAMPs) are a family of three single transmembrane proteins, RAMP1, -2, and -3, that modify the glycosylation of CTR-like receptor (CRLR) and are coexpressed with CRLR. RAMP1 is necessary for CRLR to act as CGRP receptor; RAMP2 or RAMP3 must be present for CRLR to act as an adrenomedullin receptor (51). It has been demonstrated that high affinity amylin receptor-like phenotypes consist of CTR and RAMPs (52, 53), but it remains unclear which subtype of RAMP is necessary for CT activation. Although CT, CGRP, and amylin activate their own receptors, high concentrations of CGRP or amylin interact with CTR (35, 36). We examined the inhibitory effects of CT, CGRP, and amylin antagonists on CT-induced cAMP accumulation. CT-induced cAMP accumulation was inhibited strongly by sCT-(8–32) and weakly by CGRP-(8–37). However, amylin-(8–37) had no effect on CT-induced cAMP accumulation. We confirmed that CGRP and CT cross-react at high concentrations, but we also demonstrated that CT will primarily activate its own receptor.

The FS cell is a unique type of cell that forms follicles or ductules into which their own apical cell surfaces can project microvilli and cilia. Because of their localization and appearance, it seems that FS cells regulate hormone secretion and/or the metabolism of granulated pituitary cells. It has been shown that FS cells produce growth factors, including IL-6 (32, 33, 54, 55, 56, 57). IL-6 production is positively and negatively regulated by a variety of signals, namely lipopolysaccharide, cytokines, cAMP, and DG (26, 27, 28). Both cAMP and PMA were necessary for increasing IL-6 production in TtT/GF cells. Moreover, CT-induced IL-6 production was also inhibited by both the PKA-selective inhibitor, H-89, and the PKC-selective inhibitor, GF109203X. These findings indicate that both PKA and PKC are necessary for CT-induced IL-6 production. In addition to PKA and PKC, MAPK, PLC, and PLD are also involved in the CT signaling pathways (15, 16, 17, 19). In TtT/GF cells, a MAPK-selective inhibitor, PD98059 partially inhibited CT-induced IL-6 production. U73122 is a PLC/PLD inhibitor that was previously thought to be a specific phosphatidylinositol-PLC inhibitor (38). U73122 inhibited IL-6 production stimulated by CT. However, the PC-PLC inhibitor, D609, and the PLD inhibitor, propranolol, failed to suppress CT-induced IL-6 production. These findings imply that CT induces DG by PI-PLC, but not by PC-PLC or PLD, in TtT/GF cells. Recent studies have shown that Gs, Gq, and Gi/Go proteins are all involved in the CTR signaling pathways (12, 13, 14). In the present study we demonstrated that a Gi/Go inhibitor, PTX, increased CT-stimulated cAMP and IL-6 production. On the other hand, PTX alone produced no significant or only a slight increase in cAMP accumulation and IL-6 production. These findings indicate that CT also activates Gi/Go proteins, which suppress cAMP accumulation and IL-6 production. Recently, it was reported that RAMPs decrease CT-induced cAMP accumulation in COS-7 cells expressing levels of human CTR (53).

In this regard, it is of interest whether RAMPs can modify the ability of Gi/Go proteins to couple to the CTR. In conclusion, we demonstrated that CT stimulates IL-6 production via both PKA and PKC pathways. However, CT also activates Gi/Go proteins and negatively regulates IL-6 production in TtT/GF cells.


   Footnotes
 
Abbreviations: CT, Calcitonin; CGRP, calcitonin gene-related peptide; CRLR, CTR-like receptor; CTR, CT receptor; DG, diacylglycerol; FS, folliculo-stellate; PC, phosphatidylcholine; PMA, phorbol 12-myristate 13-acetate; PPH, phosphohydrolase; PTX, pertussis toxin; RAMP, receptor activity-modifying protein; rCGRP, rat CGRP; sCT, salmon CT.

Received December 6, 2000.

Accepted for publication April 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Plata-Salaman CR 1996 Anorexia during acute and chronic disease. Nutrition 12:69–78[CrossRef][Medline]
  2. Tisdale MJ 1997 Biology of cachexia. J Natl Cancer Inst 89:1763–1773[Abstract/Free Full Text]
  3. Dewys WD, Begg C, Lavin PT, et al. 1980 Prognostic effect of weight loss prior to chemotherapy in cancer patients. Eastern Cooperative Oncology Group. Am J Med 69:491–497[CrossRef][Medline]
  4. Bruera E 1997 ABC of palliative care. Anorexia, cachexia, and nutrition. Br Med J 315:1219–1222[Free Full Text]
  5. Bruera E 1998 Pharmacological treatment of cachexia: any progress? Support Care Cancer 6:109–113[CrossRef][Medline]
  6. Plata-Salaman CR 2000 Central nervous system mechanisms contributing to the cachexia-anorexia syndrome. Nutrition 16:1009–1012[CrossRef][Medline]
  7. Inui A 1999 Cancer anorexia-cachexia syndrome: are neuropeptides the key? Cancer Res 59:4493–4501[Abstract/Free Full Text]
  8. Maltoni M, Fabbri L, Nanni O, et al. 1997 Serum levels of tumour necrosis factor {alpha} and other cytokines do not correlate with weight loss and anorexia in cancer patients. Support Care Cancer 5:130–135[Medline]
  9. Noguchi Y, Yoshikawa T, Matsumoto A, Svaninger G, Gelin J 1996 Are cytokines possible mediators of cancer cachexia? Surg Today 26:467–475[CrossRef][Medline]
  10. Meguid MM, Fetissov SO, Varma M, et al. 2000 Hypothalamic dopamine and serotonin in the regulation of food intake. Nutrition 16:843–857[CrossRef][Medline]
  11. Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG 2000 Central nervous system control of food intake. Nature 404:661–671[Medline]
  12. Cummings DE, Schwartz MW 2000 Melanocortins and body weight: a tale of two receptors. Nat Genet 26:8–9[CrossRef][Medline]
  13. Hagan MM, Rushing PA, Schwartz MW, et al. 1999 Role of the CNS melanocortin system in the response to overfeeding. J Neurosci 19:2362–2367[Abstract/Free Full Text]
  14. Lopez-Soriano J, Carbo N, Tessitore L, Lopez-Soriano FJ, Argiles JM 1999 Leptin and tumor growth in rats. Int J Cancer 81:726–729[CrossRef][Medline]
  15. Chance WT, Balasubramaniam A, Fischer JE 1995 Neuropeptide Y and the development of cancer anorexia. Ann Surg 221:579–87[Medline]
  16. Chance WT, Balasubramaniam A, Thompson H, Mohapatra B, Ramo J, Fischer JE 1996 Assessment of feeding response of tumor-bearing rats to hypothalamic injection and infusion of neuropeptide Y. Peptides 17:797–801[CrossRef][Medline]
  17. Asakawa A IA, Kaga T, Yuzuriha H, et al. 2001 Ghrelin is an appetite-stimulating signal from stomach with structural resemblance to motilin. Gastroenterology 120:337–345[CrossRef][Medline]
  18. Tschop M, Smiley DL, Heiman ML 2000 Ghrelin induces adiposity in rodents. Nature 407:908–913[CrossRef][Medline]
  19. Wren AM, Small CJ, Ward HL, et al. 2000 The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 141:4325–4328[Abstract/Free Full Text]
  20. Ghigo E, Arvat E, Camanni F 1998 Orally active growth hormone secretagogues: state of the art and clinical perspectives. Ann Med 30:159–168[Medline]
  21. Emrich LJ, Priore RL, Murphy GP, Brady MF 1985 Prognostic factors in patients with advanced stage prostate cancer. Cancer Res 45:5173–5179[Abstract/Free Full Text]
  22. Pollard M 1998 Lobund-Wistar rat model of prostate cancer in man. Prostate 37:1–4[CrossRef][Medline]
  23. Plata-Salaman CR, Ilyin SE, Gayle D 1998 Brain cytokine mRNAs in anorectic rats bearing prostate adenocarcinoma tumor cells. Am J Physiol 275:R566–R573
  24. Chavez M, Seeley RJ, Woods SC 1995 A comparison between effects of intraventricular insulin and intraperitoneal lithium chloride on three measures sensitive to emetic agents. Behav Neurosci 109:547–550[CrossRef][Medline]
  25. Schwartz MW, Seeley RJ, Woods SC, et al. 1997 Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119–2123[Abstract]
  26. Grill HJ, Ginsberg AB, Seeley RJ, Kaplan JM 1998 Brainstem application of melanocortin receptor ligands produces long-lasting effects on feeding and body weight. J Neurosci 18:10128–10135[Abstract/Free Full Text]
  27. Nijenhuis WA, Oosterom J, Adan RA 2001 AgRP(83–132) acts as an inverse agonist on the human-melanocortin-4 receptor. Mol Endocrinol 15:164–171[Abstract/Free Full Text]
  28. Plata-Salaman CR, Sonti G, Borkoski JP, Wilson CD, French-Mullen JM 1996 Anorexia induced by chronic central administration of cytokines at estimated pathophysiological concentrations. Physiol Behav 60:867–875[Medline]
  29. Lonnroth C, Moldawer LL, Gelin J, Kindblom L, Sherry B, Lundholm K 1990 Tumor necrosis factor-{alpha} and interleukin-1{alpha} production in cachectic, tumor-bearing mice. Int J Cancer 46:889–896[Medline]
  30. Torelli GF, Meguid MM, Moldawer LL, et al. 1999 Use of recombinant human soluble TNF receptor in anorectic tumor- bearing rats. Am J Physiol 277:R850–R855
  31. Smith BK, Kluger MJ 1993 Anti-TNF-{alpha} antibodies normalized body temperature and enhanced food intake in tumor-bearing rats. Am J Physiol 265:R615–R619
  32. Sherry BA, Gelin J, Fong Y, et al. 1989 Anticachectin/tumor necrosis factor-alpha antibodies attenuate development of cachexia in tumor models. FASEB J 3:1956–1962[Abstract]
  33. Leon LR, Kozak W, Peschon J, Kluger MJ 1997 Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice. Am J Physiol 272:R563–R569
  34. Fantuzzi G, Zheng H, Faggioni R, et al. 1996 Effect of endotoxin in IL-1ß-deficient mice. J Immunol 157:291–296[Abstract]
  35. Fattori E, Cappelletti M, Costa P, et al. 1994 Defective inflammatory response in interleukin 6-deficient mice. J Exp Med 180:1243–1250[Abstract/Free Full Text]
  36. Lipton JM, Catania A 1998 Mechanisms of antiinflammatory action of the neuroimmunomodulatory peptide alpha-MSH. Ann NY Acad Sci 840:373–380[Abstract/Free Full Text]
  37. Huang QH, Entwistle ML, Alvaro JD, Duman RS, Hruby VJ, Tatro JB 1997 Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever. J Neurosci 17:3343–3351[Abstract/Free Full Text]
  38. Rajora N, Boccoli G, Burns D, Sharma S, Catania AP, Lipton JM 1997 {alpha}-MSH modulates local and circulating tumor necrosis factor-{alpha} in experimental brain inflammation. J Neurosci 17:2181–2186[Abstract/Free Full Text]
  39. Huang QH, Hruby VJ, Tatro JB 1999 Role of central melanocortins in endotoxin-induced anorexia. Am J Physiol 276:R864–R871
  40. Gayle D, Ilyin SE, Plata-Salaman CR 1999 Feeding status and bacterial LPS-induced cytokine and neuropeptide gene expression in hypothalamus. Am J Physiol 277:R1188–R1195
  41. Chance WT, Sheriff S, Kasckow JW, Regmi A, Balasubramaniam A 1998 NPY messenger RNA is increased in medial hypothalamus of anorectic tumor-bearing rats. Regul Pept 75–76:347–353
  42. Benoit SC, Schwartz MW, Lachey JL, et al. 2000 A novel selective melanocortin-4 receptor agonist reduces food intake in rats and mice without producing aversive consequences. J Neurosci 20:3442–3448[Abstract/Free Full Text]
  43. Bernstein IL, Sigmundi RA 1980 Tumor anorexia: a learned food aversion? Science 209:416–418[Abstract/Free Full Text]
  44. Tisdale MJ 1999 Wasting in cancer. J Nutr 129:243S–246S
  45. Loprinzi CL, Kugler JW, Sloan JA, et al. 1999 Randomized comparison of megestrol acetate versus dexamethasone versus fluoxymesterone for the treatment of cancer anorexia/cachexia. J Clin Oncol 17:3299–3306[Abstract/Free Full Text]
  46. McCarthy HD, Crowder RE, Dryden S, Williams G 1994 Megestrol acetate stimulates food and water intake in the rat: effects on regional hypothalamic neuropeptide Y concentrations. Eur J Pharmacol 265:99–102[CrossRef][Medline]
  47. Beck SA, Tisdale MJ 1990 Effect of megestrol acetate on weight loss induced by tumour necrosis factor alpha and a cachexia-inducing tumour (MAC16) in NMRI mice. Br J Cancer 62:420–424[Medline]
  48. Bozzetti F, Cozzaglio L, Gavazzi C, et al. 1998 Nutritional support in patients with cancer of the esophagus: impact on nutritional status, patient compliance to therapy, and survival. Tumori 84:681–686[Medline]
  49. Hilton JM, Mitchelhill KI, Pozvek G, Dowton M, Quiza M, Sexton PM 1998 Purification of calcitonin-like peptides from rat brain and pituitary. Endocrinology 139:982–992[Abstract/Free Full Text]
  50. Fox J, Lowe SH, Conklin RL, Petty BA, Nemeth EF 1999 Calcimimetic compound NPS R-568 stimulates calcitonin secretion but selectively targets parathyroid gland Ca2+ receptor in rats. J Pharmacol Exp Ther 290:480–486[Abstract/Free Full Text]
  51. McLatchie LM, Fraser NJ, Main MJ, et al. 1998 RAMPs regulate the transport and ligan specificity of the calcitonin-receptor-like receptor. Nature 393:333–339[CrossRef][Medline]
  52. Christopoulos G, Perry KJ, Morfis M, et al. 1999 Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol 56:235–242[Abstract/Free Full Text]
  53. Tilakaratne N, Christopoulos G, Zumpe ET, Foord SM, Sexton PM 2000 Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment. J Pharmacol Exp Ther 292: 61–72
  54. Gloddek J, Pagotto U, Paez Pereda M, Arzt E, Stalla GK, Renner U 1999 Pituitary adenylate cyclase-activating polypeptide, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells. J Endocrinol 160:483–490[Abstract]
  55. Hentges S, Boyadjieva N, Sarkar DK 2000 Transforming growth factor-ß3 stimulates lactotrope cell growth by increasing basic fibroblast growth factor from folliculo-stellate cells. Endocrinology 141:859–867[Abstract/Free Full Text]
  56. Jin L, Zhang S, Burguera BG, et al. 2000 Leptin and leptin receptor expression in rat and mouse pituitary cells. Endocrinology 141:333–339[Abstract/Free Full Text]
  57. Perez Castro C, Nagashima AC, Pereda MP, et al. 2000 The gp130 cytokines interleukin-11 and ciliary neurotropic factor regulate through specific receptors the function and growth of lactosomatotropic and folliculostellate pituitary cell lines. Endocrinology 141:1746–1753[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. Sun, F. Wu, F. Sun, and P. Huang
Adenosine Promotes IL-6 Release in Airway Epithelia
J. Immunol., March 15, 2008; 180(6): 4173 - 4181.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
G. Vlotides, M. Cruz-Soto, T. Rubinek, T. Eigler, C. J. Auernhammer, and S. Melmed
Mechanisms for Growth Factor-Induced Pituitary Tumor Transforming Gene-1 Expression in Pituitary Folliculostellate TtT/GF Cells
Mol. Endocrinol., December 1, 2006; 20(12): 3321 - 3335.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. M. Bilezikjian, A. M. O. Leal, A. L. Blount, A. Z. Corrigan, A. V. Turnbull, and W. W. Vale
Rat Anterior Pituitary Folliculostellate Cells Are Targets of Interleukin-1{beta} and a Major Source of Intrapituitary Follistatin
Endocrinology, February 1, 2003; 144(2): 732 - 740.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Y. Kiriyama, Y. Nomura, and Y. Tokumitsu
Calcitonin gene expression induced by lipopolysaccharide in the rat pituitary
Am J Physiol Endocrinol Metab, June 1, 2002; 282(6): E1380 - E1384.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kiriyama, Y.
Right arrow Articles by Tokumitsu, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiriyama, Y.
Right arrow Articles by Tokumitsu, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals