Abstract
Purpose: :
Retinal pigment epithelium (RPE)-derived blebs have been observed in eyes with age-related macular degeneration (AMD). Cumulative oxidative injury caused by cigarette smoke to the RPE may play a role in AMD. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, induces actin rearrangement and membrane blebbing in RPE cells, as well as sub-RPE deposits in mice. Heat-shock protein 25/27 (Hsp25/27) is a key regulator of actin filaments dynamics regulated by oxidative stress. The objective of the present study was to further characterize the role of phosphorylated Hsp25/27 in regulating actin cytoskeleton dynamics and blebbing during non lethal HQ-induced oxidative stress as well as the involvement of p38 and ERK pathways in vitro and in vivo.
Methods: :
Human donor eyes with AMD were used for immunohistochemistry. Confluent ARPE-19 cells were treated with HQ 100µM for various periods of time with or without either p38 inhibitor SB203580, or okadaic acid or ERK inhibitor PD98059. After treatment, cells were harvested for RNA and protein extraction. siRNA technology was used to knock-down Hsp27 expression. Western blot, real-time PCR and immunofluorescence staining were performed. We also used the experimental model for sub-RPE deposits of mice chronically exposed to oral HQ for 7 months. RPE-choroid complexes were dissected at the time of sacrifice and protein and RNA were subsequently extracted.
Results: :
Immunohistochemistry revealed that phosphorylated Hsp27 was upregulated in RPE from AMD patients. Also, exposure to HQ led to Hsp27mRNA upregulation, dimers formation and Hsp27 phosphorylation in ARPE-19 cells. A crosstalk between p38 and ERK pathways mediated HQ-induced Hsp27 phosphorylation and actin aggregates formation. SB203580 decreased HQ-induced actin rearrangement and blebs formation whereas okadaic acid increased these effects. Hsp27 siRNA and PD98059 robustly decreased HQ-induced Hsp27 phosphorylation and actin rearrangement. Also, RPE/choroid from mice chronically exposed to HQ showed increased Hsp25, p38 and ERK phosphorylation, while Hsp25 expression was dramatically decreased.
Conclusions: :
Our study suggests that phosphorylated Hsp25/27 may be a major contributor to RPE-derived blebs formation and HQ-induced actin reorganization which may play a key role in sub-RPE deposits formation relevant to the pathogenesis of AMD in smokers.
Keywords: retinal pigment epithelium • cytoskeleton • signal transduction