| Retinal pigment epithelial cells (RPE) form a highly specialized monolayer between the neural retina and choroid layers of the eye. These cells carry out several functions critical for the maintenance of the photoreceptor cells of the retina. RPE cells are also thought to play an important role in the disease proliferative vitreoretinopathy (PVR). During the early stages of PVR, RPE cells undergo a transformation from their normal quiescent epithelial phenotype into a fibroblastic invasive proliferative phenotype. This change is known as an epithelial mesenchymal transformation (EMT). A similar EMT of RPE cells occurs in vitro when the cells are exposed to vitreous. Real time PCR (qPCR), western blotting, and immunocytochemistry were used to determine the mRNA and protein levels of the stress responsive protein HO-1 in RPE cells exposed to vitreous; qPCR was also used to determine the levels of metallothionein-1a, 2a, and 3 (MT-1a, MT-2a and MT-3). Various inhibitors of TGF-beta signaling, mitogen activated protein kinase (MAPK) signaling, and reactive oxygen species (ROS) were used to investigate the control of the stress responsive proteins HO-1, MT-1a, and MT-2a at the mRNA level. We determined that mRNA levels for HO-1, MT-1a, and MT-2a were increased in RPE cells exposed to vitreous while MT-3 mRNA levels were decreased. HO-1 protein also increased with the exposure of RPE cells to vitreous. It was found that the exposure of RPE cells to TGF-beta caused a significant increase in HO-1 mRNA and protein and that TGF-beta signaling was partly responsible for the vitreous mediated rise of HO-1 mRNA. ROS also contributed to the vitreous mediated increase in HO-1, as did the MAPK pathways p38 and JNK. None of these contributors to increased HO-1 mRNA levels had effects on MT-1a or MT-2a mRNA levels. We also show that inhibition of cyclooxygenase-2 activity did not affect the rise in HO-1, and that the hyper expression of HO-1 did not affect COX-2 mRNA levels with the exposure of RPE cells to vitreous. |
