Imposed Optical Defocus Induces Isoform-Specific Up-Regulation of TGFβ Gene Expression in Chick Retinal Pigment Epithelium and Choroid but Not Neural Retina

Abstract

Purpose: This study investigated the gene expression of TGFβ isoforms and their receptors in chick retina, retinal pigment epithelium (RPE), and choroid and the effects of short-term imposed optical defocus.

Methods: The expression of TGFβ isoforms (TGF-β1, 2, 3) and TGFβ receptors (TGFBR1, 2, 3) was examined in the retina, RPE, and choroid of young White-Leghorn untreated chicks (19 days-old). The effects on the expression of the same genes of monocular +10 and -10 D defocusing lenses, worn for either 2 or 48 h by age-matched chicks, were also examined by comparing expression in treated and untreated fellow eyes. RNA was purified, characterized and then reverse transcribed to cDNA. Differential gene expression was quantified using real-time PCR.

Results: All 3 isoforms of TGFβ and all 3 receptor subtypes were found to be expressed in all 3 ocular tissues, with apparent tissue-dependent differences in expression profiles. Data are reported as mean normalized expression relative to GAPDH. Sign-dependent optical defocus effects were also observed. Optical defocus did not affect retinal gene expression but in the RPE, TGF-β2 expression was significantly up-regulated with +10 D lenses, worn for either 2 h (349% increase ± 88%, p < 0.01) or 48 h (752% increase ± 166%, p < 0.001), and in the choroid, the expression of TGF-β3 was up-regulated with -10 D lenses, worn for 48 h (147% increase ± 9%, p < 0.01).

Conclusions: The effects of short term exposure to optical defocus on TGFβ gene expression in the RPE and choroid, which were sign-dependent and isoform specific, provide further supporting evidence for important roles of members of the TGFβ family and these two tissues in local signal cascades regulating ocular growth.

Fig 1

Percent change of gene expression for TGFβ isoforms (A, B, C) and TGFβ receptors (D, E, F), in RPE (A, D), retina (B, E), and choroid (C, F) from treated relative to fellow eyes of chicks wearing monocular +10 D lenses for 2 or 48 h; * p < 0.05; *** p < 0.001.

Fig 2

mRNA expression levels normalized to GAPDH for TGFβ isoforms (A, B, C) and TGFβ receptors (D, E, F), in RPE (A, D), retina (B, E), and choroid (C, F) from treated and fellow control eyes of chicks wearing monocular +10 D lenses for 2 or 48 h. Data from right and left eyes of untreated chicks shown for comparison. * p < 0.05; *** p < 0.001.

Fig 3

Percent change of gene expression for TGFβ isoforms (A, B, C) and TGFβ receptors (D, E, F), in RPE (A, D), retina (B, E), and choroid (C, F) from treated relative to fellow eyes of chicks wearing monocular -10 D lenses for 2 or 48 h; ** p < 0.01.

Fig 4

mRNA expression levels normalized to GAPDH for TGFβ isoforms (A, B, C) and TGFβ receptors (D, E, F), in RPE (A, D), retina (B, E), and choroid (C, F) from treated and fellow control eyes of chicks wearing monocular -10 D lenses for 2 or 48 h. Data from right and left eyes of untreated chicks shown for comparison. ** p < 0.01.

Fig 5. Stability of GAPDH expression across different treatment conditions in retina and choroid assessed by comparing the expression of GAPDH/total RNA (μg) from treated, fellow, and untreated eyes.