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ARS Home » Northeast Area » Boston, Massachusetts » Jean Mayer Human Nutrition Research Center On Aging » Research » Publications at this Location » Publication #147191
Title: INDUCTION OF THE UBIQUITIN-PROTEASONE PATHWAY DURING THE KERATOCYTE TRANSITION TO THE REPAIR FIBROBLAST PHENOTYPE

Author
item STRAMER, B - TUFTS
item COOK, J - TUFTS
item FINI, M - TUFTS
item TAYLOR, A - HNRCA
item OBIN, MARTIN - HNRCA

Submitted to: Investigative Ophthalmology and Visual Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/2001
Publication Date: 7/1/2001
Citation: N/A

Interpretive Summary: Injury compromises the optical clarity of the corneal, thereby limiting vision. The present work demonstrates that injury induces the ubiquitin proteasome pathway (UPP) in the cornea, and that the upregulated UPP alters levels and activities of key proteins involved in the response to injury. These inlcude the transcription factor NF-kappaB, which is a key mediator of the post-wound inflammatory response, and the thiamine-dependent redox protein, tissue transketolase (TKT), which constitutes more than 20% of soluble protein in the cornea. In particular, the dramatic loss of TKT that occurs in the injured cornea is believed to contribute to pathologic light-scattering and compromised vision. In the present work, we demonstrate that the loss of TKT is due to degradation by the UPP. Because TKT plays an important role in maintaining cellular redox balance, loss of TKT may contribute to corneal pathogenesis via an increase in free radicals. These results raise the possibility that antioxidant intervention may preserve (some) corneal clarity following injury.

Technical Abstract: An established cell culture model was used in which freshly isolated rabbit corneal stromal cells acquire a repair fibroblast phenotype, thereby mimicking injury-induced stromal cell activation. Transition to the repair fibroblast phenotype during the 72 hours after initial plating was coincident with progressive UPP induction. Levels of Ub, Ub-conjugated proteins, ubiquitinylating enzymes E1 and E2-25K, and 26 S proteasome increased two- to fivefold in activated stromal cells. These increases were associated with enhanced (>10-fold) capacity for Ub-dependent proteolysis of (125)I-labeled H2A and with progressive (>6-fold) increases in the UPP substrate, inhibitor of kappaBalpha (IkappaBalpha). This finding suggests that rates of constitutive NF-kappaB activation, and thus IkappaBalpha degradation, are elevated in activated stromal cells. Both freshly isolated and activated stromal cells degraded IkappaBalpha in response to IL-1alpha; yet, only activated stromal cells maintained autocrine IL-1alpha expression after 24 hours. UPP induction was coincident with a more than 90% loss of tissue transketolase (TKT) and aldehyde dehydrogenase (ALDH) class 1. TKT was stabilized during the repair phenotype transition by proteasome inhibition and was degraded (>30%/h) by the UPP in cell-free assays. CONCLUSIONS. Coordinate induction of the UPP during stromal cell activation alters levels of IkappaBalpha and TKT, two UPP substrates that are implicated in the loss of tissue stasis and corneal clarity after injury.