Abstract
Purpose: :
Corneal stromal cells undergo a phenotypic transition into repair cells as a response to injury (in vivo) or serum (in cell culture). A hallmark of this transition is loss of corneal crystallins. We recently showed that up–regulation of ubiquitin (Ub)–proteasome pathway (UPP) activity is correlated with loss of the corneal crystallin transketolase (TKT). Treatment with the serum cytokine platelet–derived growth factor–BB (PDGF) stimulated TKT loss on a per cell basis, however transforming growth factor–beta2 (TGF–beta2) did not have this effect. The goal of the current study was to define further the mechanisms controlling TKT levels.
Methods: :
Rabbit corneal stromal cells were harvested by collagenase digestion and plated in serum–free medium. Cells were treated with or without PDGF or TGF–beta2 and clasto–lactacystin (beta)–lactone, a proteasome inhibitor. Intracellular TKT protein and mRNA levels were quantified by Westerns and quantitative RT–PCR, respectively.
Results: :
Cells treated with PDGF became elongated, but their overall size remained the same. The TKT protein level was reduced as previously observed, both on a per cell basis (1.9 +/– 0.1 versus 1.2 +/– 0.3) and relative to beta–actin (1.3 +/– 0.1 versus 0.8 +/– 0.1). UPP inhibition at the same time as PDGF treatment prevented this TKT loss. In contrast, treatment with PDGF did not affect the TKT mRNA level. Treatment with TGF–beta2 produced a much different result. Cells became larger with well–developed stress fibers. The TKT protein level remained unchanged as previously observed on a per cell basis (1.9 +/– 0.1 versus 1.8 +/– 0.2), but was reduced slightly relative to beta–actin (1.3 +/– 0.1 versus 1.1 +/– 0.1). There was a more substantial reduction in the TKT mRNA level (2.43 +/– 0.07 fold). UPP inhibition at the same time as TGF–beta2 treatment did not affect the TKT level.
Conclusions: :
The results indicate that serum cytokines target different mechanisms to control TKT levels. PDGF stimulates TKT loss by increasing the rate of UPP–mediated protein degradation, however it does not affect the TKT mRNA level. Therefore, cells retain the potential to reaccumulate TKT. In contrast TGF–beta2 does not stimulate TKT degradation. The amount of TKT per cell remains constant, although the level is decreased slightly relative to total protein, which correlates with increased cell size. Nevertheless, the TKT mRNA level is reduced. Over a prolonged time period, this may lead to a reduction in the TKT protein level as the existing TKT pool turns over.
Keywords: cornea: stroma and keratocytes • growth factors/growth factor receptors • cornea: basic science