Abstract
MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.
Highlights
The retinal pigment epithelium (RPE) is formed by a monolayer of highly specialized cells, with the basolateral side facing Bruch’s membrane and the choroid, and the apical side in contact with the outer segments of photoreceptor cells [1]
Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; we evaluated ADAM9 expression, sMer, and photoreceptor outer segments (POS) phagocytosis
We demonstrate that HG compromises Mer Tyrosine Kinase (MerTK) expression and activation in ARPE-19 cells
Summary
The retinal pigment epithelium (RPE) is formed by a monolayer of highly specialized cells, with the basolateral side facing Bruch’s membrane and the choroid, and the apical side in contact with the outer segments of photoreceptor cells [1]. The extracellular region of MerTK may be cleaved by Human A Disintegrin And Metalloproteases (ADAMs) and released as a soluble form (sMer) [7,8]. It has been demonstrated that sMer acts as a decoy receptor of MerTK ligands, inhibiting MerTK activation and, phagocytosis. Law et al showed that the presence of sMer in the extracellular space decreased POS binding to the RPE surface, whereas inhibition of MerTK cleavage increased it [9]. This study demonstrated that ADAM9 is responsible for MerTK shedding, and that miR-126 regulates ADAM9 expression. HG exposure decreased miR-126 levels, with a corresponding increment in ADAM9 expression. Overexpression of miR-126 can attenuate ADAM9 expression [11]
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