Abstract
The lamina cribrosa (LC) is a key site of fibrotic damage in glaucomatous optic neuropathy and the precise mechanisms of LC change remain unclear. Elevated Ca2+ is a major driver of fibrosis, and therefore intracellular Ca2+ signaling pathways are relevant glaucoma-related mechanisms that need to be studied. Protein kinase C (PKC), mitogen-activated MAPK kinases (p38 and p42/44-MAPK), and the PI3K/mTOR axis are key Ca2+ signal transducers in fibrosis and we therefore investigated their expression and activity in normal and glaucoma cultured LC cells. We show, using Western immune-blotting, that hyposmotic-induced cellular swelling activates PKCα, p42/p44, and p38 MAPKs, the activity is transient and biphasic as it peaks between 2 min and 10 min. The expression and activity of PKCα, p38 and p42/p44-MAPKs are significantly (p < 0.05) increased in glaucoma LC cells at basal level, and at different time-points after hyposmotic stretch. We also found elevated mRNA expression of mRNA expression of PI3K, IP3R, mTOR, and CaMKII in glaucoma LC cells. This study has identified abnormalities in multiple calcium signaling pathways (PKCα, MAPK, PI3K) in glaucoma LC cells, which might have significant functional and therapeutic implications in optic nerve head (ONH) fibrosis and cupping in glaucoma.
Highlights
Glaucoma is an optic neuropathy characterized by optic nerve head (ONH) cupping and pallor and loss of retinal ganglion cells leading to blindness [1]
The induction of PKCα expression and activity using hyposmotic-induced cellular swelling to model glaucoma has not been reported in lamina cribrosa (LC) cells
We performed a timeswelling to model glaucoma has not been reported in LC cells
Summary
Glaucoma is an optic neuropathy characterized by optic nerve head (ONH) cupping and pallor and loss of retinal ganglion cells leading to blindness [1]. The ECM remodeling includes increased deposition of collagens [4], loss of elastin structure [5], and higher transforming growth factor-β2 (TGF-β2) and matrix metalloproteinase-2 (MMP-2) protein levels in human glaucoma LC tissue [6]. Fibrosis is the result of tissue stiffening caused by the excess deposition of ECM proteins in response to chronic inflammation, provoked by different stimuli such as hypoxia, oxidative stress, mechanical stretch, and growth factors (e.g., TGFβ). These stimuli can promote fibroblasts to proliferate, migrate, and acquire an activated phonotype change into myofibroblasts contributing to further tissue remodeling and subsequently fibrosis [10]. It has been shown in cancer cells that the mechanism behind Ca2+ -induced cell proliferation is caused by the opening of Ca2+ channels allowing Ca2+ entry, followed by downstream activation of the MAPKs pathway promoting cell proliferation [14]
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