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First published online October 18, 2006
Journal of Experimental Biology 209, 4371-4378 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02524
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PKC{gamma} knockout mouse lenses are more susceptible to oxidative stress damage

Dingbo Lin1, Micheal Barnett1, Samuel Lobell1, Daniel Madgwick1, Denton Shanks1, Lloyd Willard2, Guido A. Zampighi3 and Dolores J. Takemoto1,*

1 Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA
2 Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506, USA
3 Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, Los Angeles, CA90095, USA


Figure 1
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  		Fig. 1 Deletion of PKC{gamma} results in damage in 6-week-old lenses. Lenses were dissected and fixed immediately after 2-day-old or 6-week-old control and PKC{gamma} knockout mice were killed. Sections (1 µm thick) were stained with Toluidine Blue, and viewed and photographed at 20{chi} magnification under a light microscope. Scale is identical in each panel.

 

Figure 2
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  		Fig. 2 Knockout lenses are more susceptible to opacification. Intact lenses were dissected from PKC{gamma} knockout and control mice aged between 6- and 16-weeks old. The lenses were further incubated in serum-free DMEM medium at 37°C for 24 h. Transparent lenses were then treated with H2O2 or PBS for an additional 24 h at the indicated concentration of H2O2 (µmol l-1). The extent of lens opacification was determined, photographs taken (A; 6-week-old representative lenses) and the data graphed (B). Six lenses from wild-type and PKC{gamma} knockout mice treated with 0, 1, 5 and 10 µmol l-1 H2O2 were photographed and digitized, and the transparency of the lenses is given as the contrast ratio ± s.e.m. (C). Data were analyzed using the paired Student's t-test. *P<0.01. Note: approximately equal, though mixed, ages were used for control and PKC{gamma} knockout samples. Mixing of age groups was necessary as mice only had an average of two pups per litter and survival was poor.

 

Figure 3
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  		Fig. 3. H2O2 activates endogenous PKC{gamma} enzyme activity in lenses from the control, but not the PKC{gamma} knockout mice. The supernatants from whole lens extracts of control and PKC{gamma} knockout (KO) mice were used to determine PKC{gamma} enzyme activity and protein levels by western blot. (A) Endogenous PKC{gamma} was immunoprecipitated using PKC{gamma} antisera. PKC{gamma} enzyme activity was measured as described in the Materials and methods. Enzyme activity was expressed as a percentage of untreated specific PKC{gamma} activity. Untreated specific PKC{gamma} activity was expressed as 100%. Values are means ± s.e.m. for three independent experiments. The asterisks indicate statistical significance (P≤0.05). (B) Proteins from the supernatants were resolved by SDS-PAGE and immunoblotted with anti-PKC{gamma}, Cx50, Cx43, aquaporin 0 (AQP0) and caveolin 1 (Cav-1). Results demonstrate that the knockout is specific for PKC{gamma}. KO, PKC{gamma} knockout; PBS, phosphate-buffered saline; IB, immunoblot.

 

Figure 4
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  		Fig. 4. Phosphorylation of Cx50 on serines and threonines stimulated by H2O2 or TPA in lenses from the control, but not the PKC{gamma} knockout mice. Cx50 were immunoprecipitated from the lens whole cell extracts from 12-O-tetradecanoylphorbol 13-acetate (TPA) or H2O2 treated or untreated control or PKC{gamma} knockout (KO) lenses, and the protein complexes were resolved by 4%-15% SDS gradient gels and immunoblotted with anti-phosphoserine (pS), or anti-phosphothreonine (pT), or anti-Cx50 antisera. Phosphorylation of Cx50 on Ser and Thr are shown. Cx50 was measured as a loading control. NS, non-specific IgG, is a negative antibody binding control. Results are representative of three experiments. KO, PKC{gamma} knockout; PBS, phosphate-buffered saline; IP, immunoprecipitation; IB, immunoblot.

 

Figure 5
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  		Fig. 5. PKC{gamma} knockout abolishes the effects of H2O2 on decrease in lens gap junction dye transfer. Lucifer Yellow and rhodamine-dextran were microinjected as described in the Materials and methods. Confocal microscopy was used to measure the depth of Lucifer Yellow dye transfer (in µm) from the point of injection in the equatorial region of lenses. The movement of the dye through the extracellular spaces was accounted for by subtracting the rhodamine-dextran fluorescence. Each experimental group contained eight 6-week-old lenses; values are means ± s.e.m. *Significant difference at P≤0.05. KO, PKC{gamma} knockout, PBS, phosphate-buffered saline.

 

Figure 6
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  		Fig. 6. Structural changes of gap junctions occur in PKC{gamma} knockout lens. The plasma membrane of fiber cells exhibited similar structural features in 6-week-old control and PKC{gamma} knockout mice. The density of intra-membrane particles was approximately the same in both the protoplasmic (P) face of the membrane and the gap junctions. The principal difference was that the lenses of KO animals exhibited an abundance of small gap junction plaques (red areas; B). Scale bar, 120 nm. (C,D) Higher magnification views of gap junction plaques. The regions representing the gap junctions were identified by the small fracture step separating the protoplasmic (P) from the external (E) fracture faces. The density of channels in the plaques was similar in both control and knockout mice (~4500 µm-2). Scale bar, 60 nm.

 

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© The Company of Biologists Ltd 2006