Progranulin increases phagocytosis by retinal pigment epithelial cells in culture

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Retinal pigment epithelium (RPE) cells have been generally known to reside in the outer retina between the photoreceptors and Bruch's membrane. The RPE maintains the homeostasis of the retina and choroid (Ablonczy et al., 2014). Major functions of the RPE include the regeneration of rhodopsin, transportation of glucose and nutrients into the retina, and phagocytosis of the shed photoreceptor outer segments (POS) (Redmond et al., 1998; Curcio and Millican, 1999; Jablonski et al., 2000; Sparrow et al., 2010; Ablonczy et al., 2014). Keeping up the normal physiology of the photoreceptors, the POS undergo a constant renewal process every day (Young, 1976; Nguyen‐Legros and Hicks, 2000). Previous studies have shown that the turnover rate for an entire POS is 10 days (Young, 1971), and each RPE contacts and digests 250 to 300 POS in rat retina (Gao and Hollyfield, 1992; Kim et al., 2005).
RPE phagocytosis is composed of three distinct steps: first, recognition/binding; second, internalization; and third, digestion (Kevany and Palczewski, 2010). RPE membrane receptor integrin alpha‐v and beta‐5, Mer tyrosine kinase (MerTK), and CD36 participate in the outer segment recognition and internalization step (Finnemann et al., 1997; Finnemann and Rodriguez‐Boulan, 1999; D'Cruz et al., 2000; Shelby et al., 2015). Incomplete phagocytic function has been shown to accelerate RPE degeneration, and then turn out toxic vice‐generative production, lipofuscin. The excessive lipofuscin could contribute to abnormal phagocytosis (Delori et al., 2001). Excessive lipofuscin accumulation is characteristic of various blinding diseases such as age‐related macular degeneration (AMD) and retinitis pigmentosa in the human eye (Wang et al., 2001; Katz, 2002; Duncan et al., 2003; Hunter et al., 2012; Li, 2013). Mutations of the MerTK gene in Royal College of Surgeons rats result in the synthesis of nonfunctional MerTK proteins (Charbel Issa et al., 2009). This phenomenon causes a failure in the uptake of shed POS by the RPE cells, and then the accumulation of the shed POS leads to photoreceptor degeneration (Tzameret et al., 2015; Liu et al., 2016). A mutation of the MerTK gene in humans causes an autosomal recessive form of retinitis pigmentosa (Edwards and Szamier, 1977; Thompson et al., 2002).
Progranulin has been classified into two isoforms according to its glycosylation status: the glycosylated isoform (58–68 kDa) and the fully glycosylated mature secretory isoform (∼88 kDa) (Shankaran et al., 2008). Progranulin is released by a variety of cells (Bateman et al., 1990). Progranulin contributes to development, tumor proliferation, and ischemic brain injury (He and Bateman, 2003). Previous research has demonstrated that progranulin protected the vascular damage against focal cerebral ischemia via suppression of blood‐brain barrier disruption (Egashira et al., 2013), and recombinant progranulin could be used as a novel neurovascular protective drug with anti‐inflammatory effects after delayed t‐PA treatment (Kanazawa et al., 2015). Progranulin is expressed predominantly in neurons and microglia (Hrabal et al., 1996). Another study showed that progranulin is upregulated in activated microglia cells characteristic for actively phagocytizing engulfed materials in the brain (Diaz‐Cueto et al., 2000; Baker et al., 2006). Moreover, our previous report suggested that progranulin expressed in primary retinal cell culture which was picked out from postnatal day 8 mice retina (Kuse et al., 2016). Considering these reports, progranulin and retinal phagocytosis may be closely related.
Thus, the purpose of this study was to determine whether progranulin has any effect on shed POS phagocytosis using our previous quantification method (Murase et al., 2015). In this study, we exposed primary human RPE (hRPE) cells to progranulin and performed phagocytosis assays, Western blot analysis, and immunohistochemistry.
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