Role of the proteasome in TGF-beta signaling in lens epithelial cells.
OBJECTIVE
The durability of the ubiquitin proteasome pathway in the mammalian lens makes this enzyme system a potential contributor to certain cataracts and posterior capsular opacification (PCO). The present study addresses proteasome involvement in TGF-beta induced, cataract-associated gene activation in human lens cells.


METHODS
HLE B-3 cells were treated with TGF-beta, in combination with the proteasome inhibitors MG-132 or lactacystin. TGF-beta target gene expression was measured by semiquantitative RT-PCR. Annexin-FITC staining and flow cytometry were used to assess apoptosis levels. Western blot analyses were performed with anti-SnoN and anti-Smad2 antibodies.


RESULTS
TGF-beta induced the expression of alpha-smooth muscle actin, fibronectin, and TGF-beta-inducible gene mRNA in HLE B-3 cells and primary cultured human lens cells from donor tissues. TGF-beta also induced a time-dependent decrease in the level of the Smad repressor SnoN. Gamma-glutamyl-cysteine synthetase (gamma-GCS) mRNA levels decreased in the presence of TGF-beta. Proteasome inhibitor cotreatment blocked the induction of alpha-SMA mRNA, the loss of SnoN protein, the decrease in gamma-GCS mRNA, and TGF-beta-induced apoptosis.


CONCLUSIONS
The HLE B-3 cell line and primary cultured human lens cells respond similarly to TGF-beta treatments by activating cataract-related gene expression. This response in both of these model systems is blocked by inhibiting the proteasome. This suggests that the proteasome can mediate cataract and PCO-associated changes and therefore is a novel target of medical therapy.
Hosler
Matthew R
MR
Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07101-1709, USA.
Wang-Su
Shuh-Tuan
ST
Wagner
B J
BJ
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000199:Actins / Q000235:genetics; D017302:Annexins / Q000378:metabolism; D015153:Blotting, Western; D002386:Cataract / Q000235:genetics; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D051899:Smad2 Protein / Q000378:metabolism; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000199:Actins; D017302:Annexins; D015853:Cysteine Proteinase Inhibitors; D005353:Fibronectins; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C060615:SKIL protein, human; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; D005721:Glutamate-Cysteine Ligase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine