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Yoshihito Oura, Motokazu Tsujikawa, Machiko Nakamura, Kohji Nishida; Overexpression of HTRA1 in rod photoreceptor cells induces apoptosis via TGF beta signaling.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4208.
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© ARVO (1962-2015); The Authors (2016-present)
AMD is characterized by degeneration of photoreceptor cell and retinal pigmental epitherium (RPE). The latest study showed that human HTRA1 transgenically expressed in mouse RPE caused PCV and occult CNV. HTRA1 is intimately connected with pathogenesis of AMD, but the physiological expression and function of HtrA1 in retina remains unclear. Here, we found that HtrA1 was mainly expressed in photoreceptor cells and induced the apoptosis of photoreceptor cells via TGF beta signaling.
To analyze localized expression of HTRA1 in retina, we demonstrated immunohistochemistry and immunoblotting for zebrafish from 5 days post-fertilization (dpf) to 3 years-old. And we investigated how SNP mutation of human HTRA1 promotor had an effect on expression of HTRA1 in vivo by Met-luc reporter assay.<br /> We generated a transgenic zebrafish line of human HTRA1 using the tol2 transposon system. We compared the cell viability between HTRA1 transgenic fish and wild-type fish with two methods, counting GFP-labelled rod photoreceptor cells and TUNEL assay to detect apoptosis. And we observed morphological changes in detail by electron microscopy. Moreover, We analyzed how overexpression of HTRA1 effected on TGF beta signaling with checking mRNA and protein expression of its downstream genes.
We detected that HTRA1 protein in retina expressed in 3 month old and after and increased with aging. HTRA1 localized on inner segment of photoreceptor cells and RPE. We also found that HTRA1 expressed in Y79 cells and ARPE-19 cells and was up-regulated by SNP mutation of HTRA1 promotor. Overexpressing human HTRA1, rod photoreceptor cells decreased at 5 dpf due to apoptosis, but other retinal layers were unchanged. On electron microscopy analysis, we found lipofustin-like deposits and macrophages between photoreceptor cell layer and RPE that were known as initial changes in early AMD. overexpression of HTRA1 induced changes of TGFbeta-Akt-Foxo3a signaling and downstream transcriptional genes connected with anti-oxidant and cell senescence.
Overexpressed HTRA1 in photoreceptor cells induced their own apoptosis and AMD-like changes in retina. Changes of TGFbeta-Akt-Foxo3a signaling contributed to apoptosis of rod photoreceptor cells.
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