| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 19, 2004 as doi:10.1096/fj.04-1737fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,1
* Institute of Vascular Medicine, Peking University Third Hospital;
Department of Physiology,
Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Health Science Center, Peking University, Beijing, P. R. of China
1 Correspondence: Institute of Vascular Medicine, Peking University Third Hospital, Beijing 100083, P. R. of China. E-mail: xwang{at}bjmu.edu.cn
SPECIFIC AIMS
Neuropeptide calcitonin gene-related peptide (CGRP) is found in cultured rat type II alveolar epithelial cells (AEII). The present study investigated proinflammatory factor IL-1ß-induced CGRP secretion from a non-neural source, A549 human AEII cells, and explored the mediating mechanism and its functional significance.
PRINCIPAL FINDINGS
1. IL-1ß induced the increased CGRP release in A549 cells
Exposure of A549 cells to IL-1ß (1 ng/mL) resulted in a time-dependent release of CGRP (Fig. 1
A). CGRP-like immunoreactive levels were significantly increased after 8 h incubation and further elevated after 36 h. Treatment with IL-1ß (0.001–50 ng/mL, for 24 h) caused a concentration-dependent increase in CGRP release (Fig. 1B
). IL-1ß-specific Ab (0.1 and 1 µg/mL) significantly attenuated the IL-1ß-induced CGRP release, which indicates that IL-1ß-stimulated CGRP release depends on IL-1ß immune activity. Pretreating the cells with cyclohexamide (CHA: 10 µM) or actinomycin D (Act D: 1 µM) for 30 min markedly decreased the IL-1ß-induced release of CGRP by 70.3% and 100%, respectively (Fig. 1C
), suggesting that the enhanced release of CGRP by IL-1ß depends on de novo transcription and translation elevation. RT-PCR data showed that A549 cells expressed only ß-type (not 
-type) CGRP. RNase protection assay was performed to test the change in mRNA level; the relative ratio showed that the level of ß-CGRP mRNA was significantly increased 0.5 h to 4 h after IL-1ß administration in vitro (Fig. 1D
).
|
2. PKC mediated IL-1ß-induced CGRP release
Although A549 cells can express PKC-, -
, -
, -
, -
, -
, and -µ, only PKC- is activated after IL-1ß stimulation. Exposure of the cells to IL-1ß (1 ng/mL) for 10, 30, and 60 min caused translocation of PKC- isoforms from the cytosol to the membrane fraction. In addition, PKC activator phorbol myristate acetate (PMA: 5–50 nM, 24 h) directly triggered CGRP release increase 91.1–146.5% in A549 cells. To determine further whether PKC activation was involved in IL-1ß-induced CGRP release, cells were pretreated with PKC inhibitors Ro 31-8220 (Ro: 500 and 1000 nM) or calphostin C (Cal: 100 and 500 nM) for 30 min, which significantly attenuated the IL-1ß-induced CGRP release by 28.4–46.6%. After the addition of 500 nM PMA to the cells for 20 h to exhaust PKC, IL-1ß-evoked CGRP release was completely inhibited.
3. Involvement of p38 MAPK in IL-1ß-evoked CGRP release
After IL-1ß stimulation, phosphorylation of p38 MAPK was significantly increased at 10 min and maximized at 20–30 min whereas the phosphorylated p44/42 MAPK level was unchanged. SB 203580, the inhibitor of p38 MAPK, completely blocked IL-1ß-induced CGRP release whereas the inhibitor of p44/42 MAPK, PD 98059 (2–20 µM), had no such effect. To further determine whether p38 MAPK was the downstream signal molecule of PKC, cells were pretreated with SB (6 µM) or Ro (1 µM) for 30 min before IL-1ß (1 ng/mL) stimulation for 20 min; the phosphorylation of p38 MAPK was significantly attenuated. After PKC was depleted by 500 nM PMA for 20 h, IL-1ß-induced p38 MAPK activity was inhibited as well.
4. NF-
B participated in IL-1ß-evoked CGRP release
EMSA showed that IL-1ß-induced NF-B was activated as early as 20 min and peaked at 1 h. To study whether NF-B was involved in the signal transduction pathway leading to IL-1ß-stimulated CGRP release, cells pretreated with NF-B inhibitors pyrrolidine dithiocarbamate (PDTC, 10–30 µM) and MG 132 (10 and 30 µM) for 30 min before the addition of 1 ng/mL IL-1ß showed significantly attenuated IL-1ß-induced CGRP release by 32.4–52.3%. To analyze whether the PKC-p38-MAPK pathway was upstream of NF-B, cells pretreated with the inhibitors Ro (1 µM), SB (6 µM) or PDTC (30 µM) before 1 ng/mL IL-1ß stimulation for 1 h showed attenuated activation of NF-B. One NF-Bp65 binding motif 1768 bp upstream of the transcription start site of ß-CGRP was determined by the TRANSFAC system.
5. IL-1ß-induced CGRP reduced IL-1ß-evoked MCP-1 and IL-8 secretion
Results from a number of studies have shown that pulmonary epithelial cells, especially type II cells, help recruit of inflammatory cells to the lungs by generating chemokines IL-8 and MCP-1 in response to TNF- and IL-1ß. RT-PCR data showed that A549 cells expressed the CGRP-1 receptor (CRLR), a functional receptor of CGRP. To investigate the effect of IL-1ß-induced CGRP on IL-1ß-induced chemokines, we used hCGRP8-37, a CGRP-1 receptor antagonist, simultaneously with IL-1ß after incubation for 24 h. The supernatants were collected and IL-8 or MCP-1 levels were measured by ELISA (Fig. 2
A). hCGRP8-37 at a concentration of 0.1 nM significantly enhanced the IL-1ß-induced IL-8 and MCP-1 secretion. Cells were also treated with IL-1ß in combination with exogenous human CGRP (0.1–10 nM) for 24 h. ELISA results showed that exogenous CGRP reduced IL-1ß-induced IL-8 and MCP-1 levels in a concentration-dependent manner (Fig. 2B
). Thus, CGRP might play a role as a neuroendocrine immune modulator in inhibiting inflammation progress in the lung.
|
It is been well known that CGRP acts on its receptor, which is coupled to adenylyl cyclase, leading to an elevation of intracellular cAMP and its protein kinase (PK). To further investigate whether cAMP-PK mediated the CGRP effect, cells pretreated with Rp-cAMPs (11 µM), a cAMP-PK inhibitor, significantly attenuated the inhibitory effect of CGRP (Fig. 2C
).
CONCLUSIONS AND SIGNIFICANCE
We demonstrated for the first time that A549 human AEII cells expressed CGRP and that proinflammatory cytokine IL-1ß in vitro increased ß-CGRP synthesis and release in a time- and concentration-dependent manner via the PKC-p38-MAPK-NF-B pathway. Investigating the CGRP-1 receptor antagonist hCGRP8-37 and exogenous hCGRP, we showed that AEII cell-derived ß-CGRP could suppress inflammatory chemokine IL-8 and MCP-1 secretion induced by IL-1ß (Fig. 3
).
|
CGRP is a highly conservative peptide. In humans, two isoforms, - and ß-CGRP, differ only in three amino positions, and their functions are similar. In the lung, CGRP-like immunoreactivity is localized in the nerve fibers of the airway mucosa and around vascular smooth muscle; it is also found in pulmonary neuroepithelial cells/NEBs, including Clara cells. CGRP is predicted to be important in controlling lung circulation and airway hyper-responsiveness. Hastings and Hua demonstrated that only ß-CGRP is expressed in cultured rat AEII cells and that CGRP secretion is stimulated by the PKC activator PMA. In light of their report, our results showed that only ß-CGRP was expressed in A549 human AEII cells and that IL-1ß induction of CGRP release was PKC dependent.
The p44/42 and p38 MAPKs, which are conserved eukaryotic signaling pathways mediating the effects of extracellular stimuli, are candidate targets of PKC. They are distinguished by activating signals, substrate specificity, and cellular responses. In the present study, after PKC was exhausted or blocked, IL-1ß-induced p38 MAPK was significantly inhibited, indicating that PKC is a potent upstream molecule of p38 MAPK involved in IL-1ß-induced CGRP release. The activated p38 MAPK can directly activate IB kinase ß (IKKß), which then phosphorylates the inhibitor of NF-B, IB. After the degradation of IB, NF-B is set free, and it translocates to nuclear binding the relative motifs to regulate the transcription of target genes. However, p38 MAPK activity was not completely inhibited by PKC exhaustion in our study, which suggests the presence of other PKC-independent signaling mechanisms in mediating its activation.
As "defenders of the alveolus," AEII cells play important roles in normal pulmonary function and in the response of the lung to toxic compounds. AEII cells express neuropeptides other than CGRP, such as neuropeptide FF and adrenomedullin. Thus, AEII cells may act as an important immune tissue, taking part in immunomodulation in lung inflammatory disease.
Many inflammatory diseases are accompanied by an increase of CGRP release and synthesis. The CGRP effect on immunomodulation has long been discussed; some investigators consider the effect an inflammatory mediator. Rat -CGRP has a chemotactic active fragment and can induce eosinophil migration in rat airways. CGRP has also been demonstrated to magnify IL-1ß-induced edema in vivo. Lymphocyte-derived CGRP can inhibit Con A-induced proliferation and IL-2 production in rat thymocytes in an autocrine/paracrine mode. In the present study, a very low concentration of hCGRP8-37 (0.1 nM), 
10-fold more than that of endogenous CGRP, significantly magnified the IL-1ß-induced IL-8 and MCP-1 secretion (Fig. 2A
). Exogenous CGRP reduced IL-1ß-induced chemokine levels (Fig. 2B
), indicating that endogenous CGRP may play an inhibitory role in the inflammatory progress. In lung or airway inflammatory diseases, CGRP released from terminals of primary sensory afferents and neuroendocrine cells leads to a high local concentration of CGRP; CGRP not only inhibits the immunoreactivity of lung epithelium toward immune cells but also enhances the phagocytosis of the peripheral macrophages to attenuate the inflammation.
In the present study, our findings provide novel evidence that AEII cell-derived CGRP may act as anti-inflammatory factor in an autocrine/paracrine mode and play an important inhibitory role in the local area in lung inflammatory disease.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1737fje;
This article has been cited by other articles:
|
|
 |
|
  M. Chen, W. Li, N. Wang, Y. Zhu, and X. Wang ROS and NF-{kappa}B but not LXR mediate IL-1beta signaling for the downregulation of ATP-binding cassette transporter A1 Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1493 - C1501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Li, T. Wang, C. Ma, T. Xiong, Y. Zhu, and X. Wang Calcitonin gene-related peptide inhibits interleukin-1beta-induced endogenous monocyte chemoattractant protein-1 secretion in type II alveolar epithelial cells Am J Physiol Cell Physiol, September 1, 2006; 291(3): C456 - C465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wang, L. Jia, T. Wang, W. Sun, S. Wu, and X. Wang Endogenous Calcitonin Gene-related Peptide Protects Human Alveolar Epithelial Cells through Protein Kinase C{epsilon} and Heat Shock Protein J. Biol. Chem., May 27, 2005; 280(21): 20325 - 20330. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
