All-trans retinoic acid stimulates the secretion of TGF-β2 via the phospholipase C but not the adenylyl cyclase signaling pathway in retinal pigment epithelium cells
- 137 Downloads
By investigating that (i) all-trans retinoic acid (ATRA) affects human retinal pigment epithelium (RPE) in expressing and secreting transforming growth factor (TGF)-β2 and (ii) U73122 (phospholipase C inhibitor) and SQ22536 (adenylyl cyclase inhibitor) regulate the ATRA-induced secretion of TGF-β2 in human RPE, we sought to interpret the signaling pathway of ATRA in promoting the development of myopia.
The RPE cell line (D407) was treated with (i) ATRA (10 μM), (ii) U73122 (5–40 μM) and ATRA (10 μM), or (iii) SQ22536 (5–40 μM) and ATRA (10 μM). The control group was no-treated. After stimulated at 2, 4, 8, 16, 24, and 48 h, The expression and secretion of TGF-β2 was detected.
TGF-β2 in the cytoplasm was time-dependent increased by ATRA (p < 0.001). A time-dependent increase in the TGF-β2 protein of the supernatant was induced by ATRA (p < 0.001). U73122 (in the range of 5 to 40 μM) could suppress the secretion of TGF-β2 induced by ATRA (p < 0.001), and 40 μM U73122 could completely inhibit the up-regulated effect of 10 μM ATRA. However, SQ22536 (in the range of 5 to 40 μM) had no impact on the secretion of TGF-β2 induced by ATRA (p > 0.05).
In RPE cells, ATRA stimulates the secretion of TGF-β2 via the phospholipase C signaling pathway but not the adenylyl cyclase signaling pathway. U73122 may inhibit the promotion of ATRA in the development of myopia.
KeywordsTransforming growth factor-β2 U73122 SQ22536 Retinal pigment epithelium cells All-trans retinoic acid
All-trans retinoic acid
Dulbecco’s modified Eagle’s medium
Enzyme-linked immunosorbent assay
Phosphate buffer saline
Protein kinase A
Protein kinase C
Retinoic acid receptor
Retinal pigment epithelium
Retinoic X receptor
Transforming growth factor
Retinoic acid (RA) is a biological activity meditator with many functions including proliferation, cell differentiation and apoptosis [1, 2]. It has been reported that RA may be a retinal signal involved in inhibiting the remodeling of scleral extracellular matrix, resulting in slower ocular growth. Yuko Sekoa found that retinal RA increased within 5 days after form-deprivation in the chicken eyes. McFadden SA found that retinal RA accelerates the elongation of the eyes and increases in the myopic eyes of guinea pig. And conversely, it becomes lower in the inhibited elongation eyes [3, 4, 5, 6, 7, 8]. Previous studies have reported that all-trans retinoic acid (ATRA) can not only up-regulate the expression of tight junction-associated protein in the retinal pigment epithelium (RPE)-choroid complex and also promotes the epithelial barrier function of the RPE monolayer during the development of myopia [9, 10]. All of these results indicate that RPE plays an important role in the retinal RA signal controlling the scleral remodeling of myopia. It was reported that several cytokines synthesized and secreted by RPE cells have been involved in the progression of myopia [11, 12, 13]. Of these factors, it has been proved that transforming growth factor (TGF)-β2 affects the production of collagen and proliferation of scleral fibroblast cells and control the development of myopia . TGFβ is both a critical mediator of collagen, MMP-2, and proteoglycan production in the extracellular matrix. It implies that TGFβ may have potential influence in controlling scleral remodeling, which induces myopia development [15, 16, 17, 18, 19, 20]. According to these studies, we infer that retinal RA regulates the TGF-β2 level of RPE in the development of myopia, which controls the scleral remodeling. However, the mechanism by which ATRA could be transported from the choroid to the sclera has not been clarified. In the present study, we sought the effect of ATRA on the expression and secretion of human RPE in vitro and inhibited the signaling pathways of RPE with U73122 (phospholipase C and A2 inhibitor) and SQ22536 (adenylyl cyclase inhibitor) in order to survey which signaling pathway inhibitor can suppress the changes of TGF-β2 induced by ATRA in RPE.
Materials and reagents
In this study, human RPE cell line (D407) was purchased from the Retinal Cell Biology Laboratory, University of South Carolina (Columbia, South Carolina, USA). All-trans retinoic acid (ATRA), U73122, SQ22536 and DMSO were purchased from Sigma-Aldrich (Poole, UK). The human TGF-β2 ELISA kit was purchased from Bender MedSystems (Austria). Antibodies of TGF-β2 and β-actin were purchased from Santa Cruz (Santa Cruz, CA, USA). Complete protease inhibitor cocktail tablets were obtained from Roche Diagnostics (Roche, Switzerland). The protein marker was purchased from NEB (New England Biolabs, China), and the ECL western blot kit was purchased from Pierce (Lockford, IL, USA). All-trans retinoic acid (ATRA), U73122 and SQ22536 were dissolved in DMSO for subsequent coculture with cells.
Cell culture and treatment
D407 cells, cultured as previously described . There were 4 groups, Group A: Cells were treated with 10 μM ATRA for 2, 4, 8, 16, 24 and 48 h; Group B: Cells were treated with U73122 (B1, 5 μM; B2, 10 μM; B3, 20 μM; B4, 40 μM) for 30 min, then treated with 10 μM ATRA for 24 h; Group C: Cells were treated with SQ22536 (C1, 5 μM; C2, 10 μM; C3, 20 μM; C4, 40 μM) for 30 min, then treated with 10 μM ATRA for 24 h; Group D: Cells treated only with equivalent volume of DMSO served as blank controls.
Western blot analysis and ELISA
Western blot assay and ELISA were performed according to our previous study .
Data and statistical analysis
SPSS software 18.0 (IBM Corp, NY, USA) was used to perform the statistical analysis in the present study. Data are presented as the mean ± SD. Student’s t-test (SNK) and One-way ANOVA were used to determine the level of significance; p < 0.05 was defined as statistically significant. All in vitro cell experiments were repeated three times.
ATRA stimulates the expression and secretion of TGF-β2 in D407 cells
The effects of U73122 on the ATRA-induced secretion of TGF-β2 in D407 cells
The effects of SQ22536 on the ATRA-induced secretion of TGF-β2 in D407 cells
The metabolism of Vitamin A and its derivative retinoid happen in the retina. As a product of Vitamin A metabolism, RA is a critical factor of maintaining the normal functions of the eye. It has been reported that the structure and functions of RPE cells is regulated by RA . RA causes growth arrest of RPE cells and induces hexagonal changes in their shapes, which plays a crucial role in maintaining the RPE’s monolayer structure . In addition, RA was reported to up-regulate TSP-1 and enhance the concentration of TGF-β1 in RPE cells . In our research, we attempted to investigate the effect of RA on TGF-β2, which is closely related to myopia. We found that ATRA could up-regulate the expression of TGF-β2 in the RPE cells and increase the secretion of TGF-β2 time-dependently. In the mice, guinea pig and mouse, TGF-β2 was proved to be expressed in the retina, RPE-choroid complex and sclera [25, 26]. In a recent study, the TGF-β2 concentration of aqueous in highly myopic patients was significantly up-regulated, and was positively related to axial length of myopic patients.. According to these results, we inferred that increased RA in the retina of patients with myopia might induce the up-regulation of TGF-β2 in the RPE, which leads to the chondrogenic change in the sclera during the development of myopia. Nonetheless, the signaling pathway by which RA regulates TGF-β2 in RPE was not clear until now.
In the RPE, RA is bound to the retinoic X receptor (RXR) and retinoic acid receptor (RAR) to regulate gene transcription. There are two signaling pathways in RPE: (i) phospholipase C (PLC), calcium(Ca2+) channels, and protein kinase C (PKC), and (ii) adenylyl cyclase, calcium (Ca2+) channels, and cyclic AMP (cAMP)-dependent protein kinase A (PKA) . In our study, we used two inhibitors (U73122 and SQ22536) to inhibit the signaling pathways. As the inhibitor of phospholipase C and A2, U73122 can reduce the free cytosolic Ca2+ via inhibiting the hydrolysis of PPI (phosphatidylinositol) to IP3 (inositol triphosphate). It also inhibits the activation of G-protein phospholipase C coupling while remaining unaffected by the production of cAMP . SQ22536 is a cell-permeable adenylyl cyclase inhibitor. SQ22536 reduces the activity of adenylate cyclase, when the concentrations of GTP increases with Mg++ and Mn++, SQ22536 can inhibit that the activating effect of Mg++ and Mn++ on adenylate cyclase in the presence of catecholamines, . We found that after treatment with U73122 + ATRA, the concentration of secreted TGF-β2 was significantly lower than that of the ATRA-treated group. When the concentration of U73122 reached to 40 μM, the concentration of secreted TGF-β2 was not significantly different from that of the control group. These results indicated that ATRA stimulated the secretion of TGF-β2 via the PKC signaling pathway in RPE. On the other hand, we found that in the SQ22536-treated groups, there was no significant difference between the secretion of TGF-β2 in the SQ22536 + ATRA-treated group and the ATRA-treated group, which indicated that the adenylyl cyclase signaling pathway was not involved in the effect of the stimulation of ATRA on the secretion of TGF-β2. We concluded that retinal RA might bind to the RA receptors of RPE and activate the PKC signaling pathway, stimulating the secretion of TGF-β2 in the RPE and inducing scleral remodeling in the myopia.
In further studies, our group will inject ATRA and U73122 into the sub-retina of mice, observe the effects of the suppression of U73122 on the changes in RPE induced by ATRA, and determine whether the growth of eyeball induced by ATRA can be suppressed by U73122. We will attempt to clarify the mechanism behind ATRA’s control of myopia.
In RPE cells, ATRA stimulates the secretion of TGF-β2 via the phospholipase C signaling pathway but not the adenylyl cyclase signaling pathway. U73122 may inhibit the secretion of TGF-β2 induced by the stimulation of ATRA, which slow down the scleral remodeling in the myopia.
The study is supported by Hunan Science and Technology Planning Project (2015DK3007) which affords funding only,but has not any other contribution to our research.
Availability of data and materials
The data have not been placed in any online data storage. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
All the authors make a huge contribution to this study, Conceived and designed the Experiments: DZ ZD and JT. Performed the experiments: DZ ZD JT SL and SH. Analyzed the data: HT SH and DZ. Contributed reagents materials/analysis tools: RT and HT. Wrote the paper: DZ ZD and JT. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 13.Rosenthal R, Malek G, Salomon N, Peill-Meininghaus M, Coeppicus L, Wohlleben H, Wimmers S, Bowes RC, Strauss O. The fibroblast growth factor receptors, FGFR-1 and FGFR-2, mediate two independent signalling pathways in human retinal pigment epithelial cells. Biochem Biophys Res Commun. 2005;337:241–7.CrossRefGoogle Scholar
- 29.Tao Y, Pan M, Liu S, Fang F, Lu R, Lu C, Zheng M, An J, Xu H, Zhao F, Chen JF, Qu J, Zhou X. cAMP level modulates scleral collagen remodeling, a critical step in the development of myopia. PLoS One. 2013. https://doi.org/10.1371/journal.pone.0071441.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.