Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells.
BACKGROUND
Alveolar epithelial cells are known as progenitor cells for the restoration from the damage in the lung. Calcitonin gene-related peptide (CGRP) has been reported to play an important role in the proliferation of various types of epithelial and endothelial cells. We investigated the effects of CGRP on the proliferation of alveolar epithelial cells in vitro and in vivo.


METHODS
A549 cells were cultured in Dulbecco Modified Eagle Medium with 5% fatal bovin serum for 24 hours, then CGRP was added in vitro. The proliferation of DNA synthesis was measured using 5-bromo-2-deoxyuridine, an analog of thymidine, by enzyme-linked immunosorbent assay.As one intracellular response to CGRP, we examined activation of p44/42- extracellular signal-regulated kinase (ERK) pathway by adding CGRP, using western blotting method.Recombinant adenovirus encoding nuclear-targeted-human beta-CGRP (rhCGRP) was administered into Male Wister rat (n = 5, 10 weeks old) lungs by intratracheal instillation in vivo. 7 days after the administration of CGRP, rat lungs were harvested and histological findings and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) were evaluated to examine cell proliferation.


RESULTS
In vitro study, CGRP increased the proliferation of A549 cells in a dose and time dependent manner. CGRP8-37 (inhibitor of CGRP receptor) decreased CGRP induced proliferation of DNA synthesis. Phosphorylation of ERK pathway was observed within 15 minutes and peaked in one hour. U0126 (inhibitor of ERK pathway) decreased CGRP induced proliferation of DNA synthesis.In vivo study, histological examination of the lung indicated proliferation of alveolar epithelial cells in the rhCGRP-treated group and the nuclei of alveolar epithelial cells were positive for PCNA immunostaining.


CONCLUSIONS
In this study, we conclude that CGRP stimulates proliferation of human alveolar epithelial cells in vivo and in vitro.
Kawanami
Yukiko
Y
Department of Respiratory Disease, University of Occupational and Environmental Health, Japan, Kitakyushu City, Fukuoka, Japan. yu-kawanami@y5.dion.ne.jp
Morimoto
Yasuo
Y
Kim
Heungnam
H
Nakamura
Takehiro
T
Machida
Kazuhiko
K
Kido
Takashi
T
Asonuma
Etsuko
E
Yatera
Kazuhiro
K
Yoshii
Chiharu
C
Kido
Masamitsu
M
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004261:DNA Replication; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D002070:Butadienes; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D009570:Nitriles; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D047428:Protein Kinase Inhibitors; D018015:Receptors, Calcitonin Gene-Related Peptide; D011994:Recombinant Proteins; C113580:U 0126; C058636:calcitonin gene-related peptide (8-37); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015740:Calcitonin Gene-Related Peptide