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Folami Lamoke, Chaunte Stampley, Wan Jin Jahng, Babak Baban, Manuela Bartoli; Regulation Of Thioredoxin Interacting Protein (TXNIP) Activity By PKC Delta In The Diabetic Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5418.
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© ARVO (1962-2015); The Authors (2016-present)
TXNIP is known to regulate the bio-availability of the endogenous antioxidant thioredoxin -1 (Trx1) and to promote inflammasome formation through its interaction with NOD-like receptor 3 (NLRP3). TXNIP expression is increased in response to hyperglycemia and this effect has been shown to promote neurovascular injury in the diabetic retina. In the present study we investigated the regulation of TXNIP by post-translational modifications and identified PKC delta as a regulator of its activity.
Immunoprecipitation, protein purification and mass spectrometry analysis were performed to identify sites of TXNIP phosphorylation in extracts of post mortem retinas from diabetic and non diabetic control donors (from Georgia Eye Bank) and from retinas of streptozotocin-induced diabetic rats (STZ-rats at 4 weeks of hyperglycemia) and age-matched normoglycemic control rats. Bovine retinal endothelial cells (BRECs) were treated with the PKC agonist phorbol 12-myristate 13-acetate (PMA, 1 micro molar) for different times. Western blotting and immunopreciptation analyses were conducted to assess TXNIP interaction with NLRP3 and PKC delta.
We found that TXNIP undergoes serine phosphorylation in hyperglycemic conditions (human and rat diabetic retinas). Mass spectrometry identified several phosphorylation site including serine 169 and a cluster of serine between residue 303 and 312. These phosphorylation sites were conserved among species and located in a PKC-consensus sequence. Treatment of BRECs with 25mM D-glucose for 5 days promoted TXNIP serine phosphorylation. Same effect was achieved by treatments of the BRECs with PMA which also promoted TXNIP interaction with NLRP3. Immunoprecipitation studies revealed that TXNIP formed a complex with PKC delta in response to hyperglycemia (human and STZ-rats).
Our results show that enhanced pro-oxidant and pro-inflammatory activity of TXNIP in the hyperglycemic milieu correlate with its increased phosphorylation at specific serine residues located in putative PKC consensus sequence. TXNIP serine phosphorylation is associated with its ability to interact with NLRP3, thus is potentially involved in its pro-inflammatory activity. In addition, our studies suggest that TXNIP serine phosphorylation is a PKC delta-dependent providing a new clue on the role of PKC delta in promoting oxidative stress and inflammatory reaction in the diabetic retina.
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