Bidirectional, optical sign-dependent regulation of BMP2 gene expression in chick retinal pigment epithelium

Abstract

Purpose: We explored the role of bone morphogenic protein 2 (BMP2) in defocus-induced ocular growth using gene expression changes in RPE as a surrogate.

Methods: Young White-Leghorn chickens were used in this study. Normal gene expression of BMP2 and its receptors was examined in retina, RPE, and choroid, and BMP2 protein expression assessed in the same tissues using Western blots and immunohistochemistry. Quantitative PCR (qPCR) was used to assess the effects of short-term exposure (2 or 48 hours) to monocular +10 and -10 diopter (D) lenses, on RPE gene expression of BMP2 and its receptors. Ocular growth was assessed using A-scan ultrasonography.

Results: In the eyes of untreated chickens, BMP2 mRNA was expressed more highly in RPE compared to retina and choroid and all three tissues expressed BMP2 protein. The gene expression for all three receptors also was detected in these tissues, with BMPR2 showing highest and BMPR1B lowest expression. BMP2 was up-regulated in the RPE from eyes wearing +10 D lenses, which exhibited shorter than normal vitreous chambers (VCDs) and thickened choroids, while BMP2 was down-regulated in the RPE from eyes wearing -10 D lenses, which developed enlarged VCDs. These treatments did not induce differential expression of BMP receptors in RPE.

Conclusions: That mRNA expression of BMP2 in chick RPE shows bidirectional, defocus sign-dependent changes is suggestive of a role for BMP2 in eye growth regulation, although the diffuse ocular expression of BMP2 and its receptors suggests complex growth-modulatory signal pathways.

Figure 1.

Results of electrophoresis using a 1.2% agarose gel and ethidium bromide staining, for 8 RPE RNA samples checked for RNA integrity. Lane M, marker; lanes 1 to 8, RNA samples.

Figure 2.

mRNA expression of BMP2, and BMP types I and II receptors (BMPR1A, BMPR1B, BMPR2) in normal untreated chick retina (A), RPE (B), and choroid (C); GAPDH used as the housekeeping gene. Data are expressed as mean MNE ± SEM.

Figure 3.

Western blots showing protein expression of BMP2 for non-reducing (A) and reducing (B) conditions. In cases, lane M was loaded with marker, lane 1 with mouse brain lysates (positive control), and lanes 2 to 4, with chick retina, RPE, and choroid, respectively, while lane 5 was loaded with commercial BMP2 protein. Differences in BMP2 expression, between tissues and between conditions, were evident, with the choroid showing the highest expression and multiple forms. Molecular weights of main mature and proprotein forms of BMP2 are 13.0 and 40.3 kDa, respectively. Negative control using BMP2 peptide preabsorbed primary antibody was included (C). Primary antibody concentration 1:500.

Figure 4.

Labeled section from the wall of the posterior eyecup, either double-labeled for BMP2 (red) and DAPI (blue, A), with BMP2 alone (B), with DAPI alone (C), with light microscopy image overlaid (D), or isotype control (E). CHO, choroid; SCL-C, sclera cartilaginous layer; SCL-F, sclera fibrous layer. *Basal side of RPE. ▾Inner boundary between choroid and sclera. ↓Border between cartilaginous and fibrous layers of sclera. Scale bar = 200 μm.

Figure 5.

Effects of +10 D (A) and −10 D (B) lens treatments on axial length (AL), vitreous chamber depth (VCD), and choroidal thickness (CT) following 2 hours (n = 6, 11 respectively) and 48 hours (n = 6, 18 respectively) of lens wear, shown as interocular differences (treated-control eyes, mean ± SEM). Asterisks are placed on top of data when ocular dimensions after treatment were compared with before treatment. ***P < 0.001.

Figure 6.

Differential expression of BMP2 mRNA in RPE after 2 and 48 hours of imposed defocus (+10 D, A; −10 D, B). Ratios of values for treated and fellow eyes expressed as mean ± SEM. **P < 0.01. ***P < 0.001.

Figure 7.

BMP2 mRNA expression in RPE after +10 D (A) and −10 D (B) lens treatments. Expression relative to GAPDH plotted as mean MNE ± SEM. Differences in gene expression between lens-treated and fellow eyes reached statistical significance for all four groups. Expression in −10 D fellow eyes of −10 D lens-treated eyes decreased relative to eyes from untreated birds but did not reach statistical significance; no such yoking is evident in the +10 D lens data.

Figure 8.

BMP receptor mRNA expression in RPE after +10 and −10 D lens treatments and in eyes of untreated birds. No differences in gene expression between lens-treated and fellow eyes or between right and left eyes of untreated birds were observed (A, P > 0.05). mRNA expression of BMPR2 was significantly down-regulated in treated and fellow eyes compared to untreated eyes after −10 D lens treatment for 2 and 48 hours (P < 0.01). **P < 0.01.

Figure 9.

Expression of GAPDH in RPE normalized to total RNA (μg); data plotted as the ratio of expression levels in treated and fellow eyes for treated birds, and for untreated birds, the ratio of levels in right and left eyes. Dotted line indicates a ratio of 1.0.