Induction of the ubiquitin-proteasome pathway during the keratocyte transition to the repair fibroblast phenotype.
OBJECTIVE
To examine dynamics and function of the ubiquitin (Ub)-proteasome pathway (UPP) during corneal stromal cell acquisition of the repair fibroblast phenotype.


METHODS
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.


RESULTS
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). Because IkappaBalpha expression is induced by nuclear factor (NF)-kappaB, 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.
Stramer
B M
BM
Vision Research Laboratories, New England Eye Center, Tufts University School of Medicine, Tufts University, Boston, MA 02111, USA.
Cook
J R
JR
Fini
M E
ME
Taylor
A
A
Obin
M
M
D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D015151:Immunoblotting; D007375:Interleukin-1 / Q000378:metabolism; D008025:Ligases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000096:biosynthesis*; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D015398:Signal Transduction; D014174:Transketolase / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000096:biosynthesis*; D015854:Up-Regulation; D014945:Wound Healing
D020672:I-kappa B Proteins; D007375:Interleukin-1; D009097:Multienzyme Complexes; D014452:Ubiquitins; D000444:Aldehyde Dehydrogenase; D014174:Transketolase; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases