Upregulation of TGF-beta-induced tissue transglutaminase expression by PI3K-Akt pathway activation in human subconjunctival fibroblasts.
OBJECTIVE
Excessive scarring in subconjunctival tissues after filtering surgery seems to be characterized by aberrant extracellular matrix (ECM) production, and tissue transglutaminase (tTgase) plays an important role in this process. In the present study, the effects of transforming growth factor (TGF)-beta2 on the expression of tTgase, its activity in subconjunctival fibroblasts and whether the effects of TGF-beta are mediated by prosurvival signaling pathways were examined.


METHODS
Primary subconjunctival fibroblasts treated with TGF-beta2 were examined for the expression of tTgase with Western blot analysis. The modulation of extracellular tTgase activity by TGF-beta2 was measured by both the formation of fibronectin polymers and the ECM protein incorporation of fluorescein cadaverine. The expression of tTgase was analyzed by immunofluorescence staining and Western blot analysis of subconjunctival fibroblasts that were transiently transfected with an Akt dominant negative mutant gene or were treated with an Akt inhibitor or tTgase siRNA.


RESULTS
Treatment of subconjunctival fibroblasts with TGF-beta2 caused an increase in activation and expression of tTgase. The effects of TGF-beta stimulation of subconjunctival fibroblasts were twofold, causing both rapid activation of the ERK pathway within minutes of treatment and a more delayed activation of the phosphatidylinositol3-kinase-protein kinase B (PKB)/Akt pathway; however, only Akt activation was necessary for TGF-beta-induced tTgase expression. Transient transfection of subconjunctival fibroblasts with an Akt dominant negative mutant gene, or treatment with an Akt inhibitor (but not with an ERK inhibitor) or tTgase siRNA led to decreased activation and expression of tTgase.


CONCLUSIONS
TGF-beta2 activated the PI3K-Akt pathway, and this activation was essential for the expression and activity of tTgase in subconjunctival fibroblasts. The results indicate a novel biological function of the PI3K-Akt pathway in subconjunctival fibroblasts. Elevated expression and activity of tTgase may play an important role in the pathogenesis of diseases related to wound healing and fibrogenic reactions in subconjunctival fibroblasts.
Jung
Sun-Ah
SA
Myunggok Eye Research Institute at Kim's Eye Hospital, Konyang University College of Medicine, Nonsan, Korea.
Lee
Hyung Keun
HK
Yoon
Jin Sook
JS
Kim
Sung-Joo
SJ
Kim
Chan Yoon
CY
Song
Heesang
H
Hwang
Ki-Chul
KC
Lee
Jong Bok
JB
Lee
Joon H
JH
D015153:Blotting, Western; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D011503:Transglutaminases / Q000378:metabolism*; D015854:Up-Regulation
D005353:Fibronectins; D019204:GTP-Binding Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D051057:Proto-Oncogene Proteins c-akt; D053781:Transforming Growth Factor beta2; D011503:Transglutaminases