Hypoxia is a natural state in the lens. In order to prevent hypoxia-driven apoptosis the lens has two stress switch protein kinase C (PKC) isoforms, PKC3 and PKC5. Both of these PKC isoforms have C1B activation domains which respond to oxidative stress (PKC3) or hypoxia (PKC5). The long-term objective of this proposal is to determine how these stress switch PKC's protect the lens from cataract formation. The previous work from this lab has shown that PKC3 is oxidatively activated and that this activation results in connexin 43 (Cx43), connexin 46 (Cx46), and connexin 50 (Cx50) gap junction inhibition. This would prevent the delivery of apoptotic signals to adjacent cells, known as the """"""""Bystander Effect"""""""". PKC5 , in contrast, may act to prevent mitochondrial induced apoptosis through activation of cytochrome C oxidase IV. The central hypothesis of this proposal is: The lens, which thrives under natural hypoxia, contains these two stress-switch PKC isoforms, PKC3 and PKC5, to provide protection for the lens during differentiation, oxidative stress and from hypoxia. This proposal will determine the protective roles of these stress-switch PKC's in both lens epithelial cells in culture and in whole lens, using control and knockout models. The approach includes biochemical studies, mass spectrometry (MS) analyses, structural studies using electron microscopy, and gating/impedance measurements on both cells in culture and whole lens.
The Specific Aims are: 1. Determine effects of growth factors, oxidative stress and hypoxia on lens epithelial cell Cx43 and Cx50, using cells in culture. Identify PKC3 and PKC5 phosphorylation and degradation/hyperphosphorylation sites using MS. Determine the effects of failure to degrade Cx43 in the PKC3 knockout mouse lens. 2. Determine how PKC5 protects the lens during hypoxia by activation of mitochondrial cytochrome C oxidase IV. Determine the effects of loss of PKC5 on lens protection from hypoxia using the PKC5 knockout mouse lens. 3. Use whole lens (control and knockout) and N2A connexin-deficient cells in culture to determine the effects of PKC3 and/or PKC5 activation, by oxidative stress or hypoxia. Determine how phosphorylation by each of these PKC's controls gating/impedance properties of lens fiber cell Cx50 and Cx46 in response to oxidative stress or hypoxia.
. As the human life span increases diseases of aging will be an increasing health concern. Many of these diseases will result in both oxidative insult and ischemic (hypoxia, or a drop in oxygen levels) episodes. Both could cause severe damage to ocular tissues such as retina (i.e., diabetic retinopathy, stroke, or macular degeneration) or lens (cataracts) and this could result in blindness. This proposal will determine how an ocular tissue, the lens, protects itself from such stress using two stress-switch enzymes, PKC3 and PKC5. Activation of these enzymes, using targeted drugs, could prevent or delay ocular damage during oxidative insult or ischemia.
|Schiffman, E L; Velly, A M; Look, J O et al. (2014) Effects of four treatment strategies for temporomandibular joint closed lock. Int J Oral Maxillofac Surg 43:217-26|
|Banerjee, Debarshi; Das, Satyabrata; Molina, Samuel A et al. (2011) Investigation of the reciprocal relationship between the expression of two gap junction connexin proteins, connexin46 and connexin43. J Biol Chem 286:24519-33|
|Burr, Diana B; Molina, Samuel A; Banerjee, Debarshi et al. (2011) Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo. Exp Eye Res 92:251-9|
|Das, Satyabrata; Wang, Huan; Molina, Samuel A et al. (2011) PKC?, role in lens differentiation and gap junction coupling. Curr Eye Res 36:620-31|
|Lauer, Jason; Banerjee, Debarshi; Shanks, Denton et al. (2010) NMR structure/function relationships of peptides corresponding to the C1B1 Region of PKC gamma. Protein Pept Lett 17:1-10|
|Banerjee, Debarshi; Gakhar, Gunjan; Madgwick, Dan et al. (2010) A novel role of gap junction connexin46 protein to protect breast tumors from hypoxia. Int J Cancer 127:839-48|
|Zhang, Yunong; Snider, Adam; Willard, Lloyd et al. (2009) Loss of Purkinje cells in the PKCgamma H101Y transgenic mouse. Biochem Biophys Res Commun 378:524-8|
|Akoyev, Vladimir; Das, Satyabrata; Jena, Snehalata et al. (2009) Hypoxia-regulated activity of PKCepsilon in the lens. Invest Ophthalmol Vis Sci 50:1271-82|
|Das, Satyabrata; Lin, Dingbo; Jena, Snehalata et al. (2008) Protection of retinal cells from ischemia by a novel gap junction inhibitor. Biochem Biophys Res Commun 373:504-8|
|Barnett, Michael; Lin, Dingbo; Akoyev, Vladimir et al. (2008) Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia. Exp Eye Res 86:226-34|
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