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The Sumoylation Modulated Tumor Suppressor p53 Regulates Cell Cycle Checking Genes to Mediate Lens Differentiation.

PURPOSE
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Furthermore, we have recently demonstrated that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. In addition, we have also shown that p53 controls both transcription factors, C-Maf and Prox-1 as well as lens crystallin genes, αA, β- and γ-crystallins. Here, we have examined whether p53 also regulates other known target genes during its modulation of lens differentiation. The human and mouse lens epithelial cells, FHL124 and αTN4-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.


METHODS
Mice used in this study were handled in compliance with the "Protocol for the Care and Use of Laboratory Animals" (Sun Yat-sen University). Adult mice were used for the collection of lens cells. These samples were used for extraction of total proteins. A total of 32 embryonic mice {8 at 14.5 ED, 8 at 17.5 ED and 8 newborns for wild type} were used for immunohistochemistry, which were used for co-localization study. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J.


RESULTS
Immunohistochemistry revealed that both the cell cycle checking genes, p21 and Gadd45α and the apoptotic genes, Bcl-2 and PUMA, display developmental changes associated with p53 during mouse lens development. Knockdown of p53 in the mouse lens epithelial cells caused inhibition of lens differentiation. Associated with this inhibition, the cell cycle genes displayed significant downreglation, the apoptotic genes was also attenuated but to a much less degree. In addition, we found that bFGF can induce dose-dependent upregulation of the upstream kinases, CHK1/2 and ERK1/2, both known to phosphorylate p53 and activate the later. Furthermore, We showed that in both developing lens and human lens epithelial cells, p53 can be co-localized with the catalytic subunit of the protein phoshphatase-1 (PP-1), suggesting that PP-1 regulates p53 phosphorylation status both in vivo and in vitro.


CONCLUSION
Taken together, our results suggest that during mouse lens development, p53 activity is regulated by ERK and CHK kinases-mediated activation, and by PP-1-mediated inactivation. p53 can regulate multiple groups of genes to mediate lens differentiation.