The Expression of Bone Morphogenetic Protein 2 and Matrix Metalloproteinase 2 through Retinoic Acid Receptor Beta Induced by All-Trans Retinoic Acid in Cultured ARPE-19 Cells

Abstract

Purpose: All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells.

Methods: The effects of ATRA (concentrations from 10-9 to 10-5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10-9 to 10-5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ.

Results: RARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10-9 to 10-5 mol/l) with a maximum effect observed at 10-6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10-6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135.

Conclusion: ATRA induced upregulation of RARβ in ARPE-19 cells and stimulated these cells to secrete BMP-2 and MMP-2.

Fig 1. The Effect of ATRA on survival in ARPE-19 cells.

ATRA caused a low level of apoptosis (<10%) at concentrations from 10⁻⁹ to 10⁻⁶ mol/l, but the cell morphology remained normal. The cells were inhibited at the highest concentration of ATRA treatment 10⁻⁵ mol/l (Fig 1A). At both 24 h and 48 h, the cell proliferation was inhibited by ATRA in a dose-dependent manner. A small reduction (<10%) in the number of cells also occurred with a lower ATRA concentration of 10⁻⁶ mol/l, but these changes were not statistically significant (P>0.05). When the ATRA concentration was 10⁻⁵ mol/l, the cell survival rate was below 50% at both time points (Fig 1B).

Fig 2. RARs and RXRs expressions in ARPE-19 cells.

RARs mRNA was strongly expressed in ARPE-19 cells, whereas RXRs expression was not detected (Fig 2A and 2B). RARβ mRNA levels was increased after ATRA treatment when compared with controls. Expression of RARβ mRNA was dose dependent with a maximal effect observed with 10⁻⁵ mol/l ATRA. In contrast, there were no significant changes in the mRNA levels of RARα and RARγ (P>0.05) (Fig 2C and Fig 2D).

Fig 3. Time-related changes in expression of RARβ mRNA and protein.

A significant increase in the RARβ band intensity was detected for both mRNA (Fig 3A and 3B) and protein (Fig 3A and 3C) at 24 h and 48 h after treatment with 10⁻⁶ mol/l ATRA (P<0.05).

Fig 4. BMP-2 and MMP-2 expression in ARPE-19 cells at different times points following ATRA treatment.

ATRA at 10⁻⁶ mol/l upregulated the expression of BMP-2 mRNA in ARPE-19 cells, and this effect was time dependent. There were no significant changes in BMP-2 mRNA in ARPE-19 cells after incubation with ATRA for 12 h, but the mRNA levels of BMP-2 were significantly increased after treatment with 10⁻⁶ mol/l ATRA for 48 h and 72 h (p<0.001) (Fig 4A). However, the marked expression of MMP-2 mRNA was upregulated in ARPE-19 cells after incubation with ATRA at 10⁻⁶ mol/l for 6 h, peaked at 12 h and 24 h (P<0.001), but was then down regulated to normal levels at 72 h (P>0.05) (Fig 4B). The expression levels of BMP-2 and MMP-2 protein were similar to the levels of those mRNA in ARPE-19 cells (Fig 4A and 4B). Cell supernatant from the cells treated with ATRA concentrations of 10⁻⁶ mol/l were analyzed by ELISA and compared with controls (medium with 0.1% DMSO). ATRA also upregulated the BMP-2 protein level in a time dependent manner. BMP-2 protein expression was significantly increased after the cells were treated with 10⁻⁶ mol/l ATRA at both 48 h and 72 h (p<0.05) (Fig 4C). The MMP-2 protein level was similar to its mRNA level and was significantly increased at 12 h (P<0.05) (Fig 4D).

Fig 5. LE135 could block the pathway of RARβ in ARPE-19 cells.

Compared to the control, LE135 (10^-6mol/l) could block the ATRA-induced increase in RARβ expression at either the mRNA (P<0.05) (Fig 5A) or the protein level (P<0.001) (Fig 5B and 5C) at 48 h.

Fig 6. ATRA-induced BMP-2 and MMP-2 expression were inhibited by RARβ.

The increased expression of BMP-2 and MMP-2 induced by ATRA were almost completely blocked by pre-treatment of the ARPE19 cells with LE135. ATRA at 10⁻⁶ mol/l upregulated the expression of BMP-2 and MMP-2 on both mRNA and protein levels in ARPE-19 cells (P<0.05), but there were no significant changes after incubation with LE135 (10^-6mol/l) or ATRA (10^-6mol/l) added to LE135 (10^-6mol/l) (Fig 6A, 6B and 6C).