Down-regulation of collagen I biosynthesis in intestinal epithelial cells exposed to indomethacin: a comparative proteome analysis.
In contrast to accumulated knowledge about gastroduodenal injury associated with nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin, small intestinal mucosal injuries have been noticed only recently, and the precise mechanism remains to be elucidated. To clarify the mechanism, we performed 2-DE on IEC-6 rat normal intestinal cells that were treated with indomethacin (200μΜ, 24h) or a vehicle control and identified 18 up-regulated and 8 down-regulated proteins through MALDI-TOF/TOF mass spectrometry. Among these proteins, collagen I and proteins involved in collagen I biosynthesis and maturation, including prolyl 4-hydroxylase subunit α1, protein disulfide isomerase A3 (PDIA3), calreticulin, and endoplasmin, were all down-regulated by indomethacin. Immunohistochemical staining of the intestinal mucosa of indomethacin-administered rats showed a decrease of collagen I on the apical surface of intestinal cells. Cell death induced by indomethacin was prominently suppressed when IEC-6 cells were grown on collagen I-coated plates. cis-4-Hydroxy-l-proline, a proline analog that inhibits collagen synthesis, depressed IEC-6 cell viability in a concentration-dependent manner. Cell death was also induced by short interfering RNA knockdown of endogenous collagen I in IEC-6 cells. In conclusion, by comparative proteome analysis, we identified down-regulation of collagen I as an important mechanism in NSAID-induced intestinal injury.


CONCLUSIONS
Small intestinal lesions induced by NSAIDs are of great concern in clinical settings. Various hypotheses have been proposed for the origin of these inflammatory responses, such as reduction in the blood flow, intestinal hypermotility, abnormal intestinal mucosal permeability, mitochondrial dysfunction, and reactive oxygen species, many of which are related to the inhibition of prostaglandin synthesis. However, the precise mechanism is yet to be known. The cellular process of the lesions must involve up- and down-regulations of a large number of proteins and complex interactions between them. To elucidate it, global and systematic identification of the proteins in intestinal cells affected by NSAIDs is essential. We found that the proteins exhibiting reduced expression by indomethacin treatment are collagen I and the proteins involved in collagen I synthesis and maturation. Consistent with this, immunohistochemical analysis showed that the indomethacin-treated rat intestinal mucosal cells exhibits decreased collagen I expression on its apical surface. Furthermore, the cell-protective effect of collagen on intestinal mucosal cells was demonstrated by the use of a collagen-synthesis inhibitor, short interfering RNA (siRNA) knockdown of endogenous collagen I, and cell cultivation on collagen I-coated plates versus uncoated plates. These results give important information on the role of the collagen synthesis in intestinal mucosa in the mechanism of NSAID-induced small intestinal lesions.
2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan. Electronic address: in2119@poh.osaka-med.ac.jp.
Shoko
S
Edogawa
Department of Chemistry, Osaka Medical College, Osaka, Japan.
Akiko
A
Sakai
2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan.
Takuya
T
Inoue
2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan.
Satoshi
S
Harada
2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan.
Toshihisa
T
Takeuchi
2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan.
Eiji
E
Umegaki
Department of Chemistry, Osaka Medical College, Osaka, Japan.
Hideyuki
H
Hayashi
2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan.
Kazuhide
K
Higuchi
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D024042:Collagen Type I / Q000096:biosynthesis* / Q000494:pharmacology; D015536:Down-Regulation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006909:Hydroxyproline / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D008297:Male; D020543:Proteome / Q000378:metabolism; D017207:Rats, Sprague-Dawley
D000894:Anti-Inflammatory Agents, Non-Steroidal; D024042:Collagen Type I; D020543:Proteome; D006909:Hydroxyproline; D007213:Indomethacin
Collagen I; Intestine; NSAID; Proteome analysis