Abstract
Purpose: :
The Unfolded Protein Response (UPR) is a cellular stress pathway activated upon accumulation of unfolded proteins in the Endoplasmic reticulum (ER). We have shown that UPR is highly activated by retention of extracellular matrix proteins in the lens resulting in cataract. However, UPR activation is also required for professional secretory cell development to drive the necessary ER expansion. Since lens fiber cell differentiation requires the synthesis of many new membrane proteins, we tested the hypothesis that UPR is activated during lens fiber cell differentiation.
Methods: :
Lenses were extracted from embryonic and newborn FVB/N mice. The molecular markers of UPR were evaluated by immunohistochemistry, rt-PCR and Western blotting.
Results: :
Normal lenses show activation of all three UPR pathways during development. IRE1 is an ER-localized transmembrane protein that, when activated, performs a site-specific cleavage of Xbp1 mRNA to produce a transcription factor. In normal lenses we detected the splicing of Xbp1 mRNA and the production of the XBP1(S) protein in E15.5 and E16.5 lenses. The second UPR pathway is mediated by ATF6, a transmembrane protein that, upon ER stress, undergoes proteolytic cleavage enabling its cytosolic domain to translocate into the nucleus as a transcription factor. In normal lenses, ATF6 was first detected in the nucleus of lens cells undergoing primary fiber cell elongation at E11.5. The third arm of the UPR is mediated by PERK which, upon activation, phosphorylates eIF2α causing a general attenuation in protein translation. The active form of PERK was detected at the apical tips of lens fiber cells at E12.5 and the phosphorylation of eIF2α was present at high levels in E14.5, E15.5 and E16.5 embryonic lenses. Phosphorylation of eIF2α also induces selective translation of some mRNAs such as ATF4 which is a transcription factor regulating many stress related genes. ATF4 expression was first detected in the nucleus of E11.5 lens cells undergoing primary fiber cell elongation.
Conclusions: :
Overall, these data show that UPR is activated during normal lens development and support the hypothesis that UPR may be important in the development and differentiation of the normal lens.
Keywords: development • differentiation • signal transduction: pharmacology/physiology