Functional proteomics of failed filtering blebs.
OBJECTIVE
To identify and determine the function of the proteins associated with failed filtering blebs following trabeculectomy.


METHODS
Tenon's tissues, obtained during surgery for failed filtering blebs or obtained during cataract surgery on normal eyes, were analyzed by proteomics. The proteins showing significant differences between the two tissues were selected for identification by mass spectrometry. The location and expression pattern of ribosomal S6 kinase 2 (RSK2), one of the altered proteins, were determined. The effect of basic fibroblast growth factor (bFGF) on the expression pattern and function of RSK2 in NIH3T3 fibroblast cells was then investigated by an RNA knockdown technique.


RESULTS
Eight proteins were found differentially expressed in failed filtering blebs; the identified proteins included those associated with intracellular signaling pathways. The expression of RSK2, one of the identified proteins, was found to be decreased compared with that of the control. RSK2 was located in Tenon's tissue using an immunohistochemical technique. In culture, the bFGF-induced cell proliferation was inhibited by the RNA knockdown of RSK2. The level of mRNA and protein expression of actin was increased by RSK2 RNA knockdown, but bFGF-induced protein expression of actin was not promoted by RSK2 RNA knockdown. Whereas RSK2 RNA knockdown increased the expression and activity of mitogen-activated protein kinase (MAPK), activation of MAPK induced by bFGF was not promoted by RSK2 knockdown.


CONCLUSIONS
The expression of eight proteins in the failed filtering blebs was significantly different from that in the Tenon's capsules used as a control. The effect of RSK2 expression on fibroblast cells suggests that RSK2 may be associated with wound healing in filtering blebs.
Kanamoto
Takashi
T
Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. tkana@hiroshima-u.ac.jp
Souchelnytskyi
Nazariy
N
Kiuchi
Yoshiaki
Y
D000199:Actins / Q000378:metabolism; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001768:Blister / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D015536:Down-Regulation / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D041681:NIH 3T3 Cells; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D014130:Trabeculectomy; D017211:Treatment Failure; D014945:Wound Healing
D000199:Actins; D015415:Biomarkers; D020543:Proteome; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D020928:Mitogen-Activated Protein Kinases