Chordin-like 2 influences the differentiation fate of retinal pigment epithelium cells by dynamically regulating BMP pathway

INTRODUCTION

E pithelial-mesenchymal transition (EMT) plays many important roles in embryonic development and tissue repair. In embryonic development, reversible EMT can drive tissue growth and organ formations. However, disordered EMT in tissue repair can also cause fibrosis and even support tumor metastasis

. Therefore, sequential and controlled EMT is indispensable to keep normal pathophysiologic process. In retinal pigment epithelium (RPE), EMT is regarded as one of the vital pathological processes in both of neovascular and non-neovascular age-related macular degeneration (AMD) andproliferation vitreoretinopathy (PVR)

. Fibrotic RPE cells also decrease the therapeutic effect of anti-vascular endothelial growth factor (VEGF) in wet AMD

.

, sub-confluent RPE cells have two directions to differentiation, one is normal RPE cells, and another is EMT. Recently, transplantation of multiple-derived RPE cells is becoming a potential therapeutic method to treat dry AMD, but excessive EMT is a tough problem for them to differentiate into normal RPE in subretinal cavity

. Therefore, inhibiting irreversible EMT and controlling it in a proper level is an urgent affair.

Bone morphogenetic proteins (BMPs), such as BMP2, BMP4,BMP5, BMP6 and BMP7, are series of secreted cytokines which have many different functions. For example, BMP4 had inhibitory effects on EMT, and in chick optical cup, it induced RPE differentiation

, but BMP4 also mediated the programed cell death in the embryo

. Similarly, the negative effect of BMP on cell proliferation existed in many cells, such as pulmonary artery smooth muscle cells, pancreatic α-cells and breast cancer

. Therefore, the accurate regulation of BMP pathway is crucial to correct function of BMP.

The regulation mechanism of BMP pathway is very complex.Because BMP pathway not only has many crosstalk with other signaling pathway, such as Wnt and Notch signaling pathway, but also has many endogenous inhibitors, especially a series of endogenous inhibitors of BMP pathway is a unique feature

. It has many kinds of endogenous inhibitors like Noggin, Dan family, Chordin-like 1 and Chordin-like 2

. In among that, Gremlin-l and Noggin already got more research compared with Chordin-like 1, and Chordin-like 2. However,the mutation of Chordin-like 1, encoded by

, was founded in X-linked megalocornea, and in the research, BMP4 was downregulated after knocking down

, that was an interesting appearance, besides, in developing retina of human,

also had a gradual upregulation

. Therefore,in this article, we explored the functions of another strange endogenous BMP inhibitor, Chordin-like 2, which is encoded by

and contains highly conserved cysteine-rich domain. It inhibits BMP signaling by binding BMPs, especially BMP2, BMP4, BMP5, BMP6, and BMP7

. In zebrafish embryo, Chordin-like 2 has an exact function in dorsoventral formation

. Besides, Chordin-like 2 also plays a role in regenerating osteoarthritic cartilage, and is expressed in uterus and colon, connective tissues, osteoblasts, epithelial cells of reproductive organs and bladder

. In RPE, the functions of Chordin-like 2 are still uncertain. Therefore, human fetal RPE cells were used to investigate its exact functions.

The protein levels of BMP4 in culture media were qualified by ELISA kit (abcam,Shanghai) according to the manufacturer’s protocol. The culture media was extracted after transfection. Standard curve was used to calculate the exact concentration of the protein.To avoid error, the number of RPE cells from each well had no significant differences, and the culture media in each well kept the same volume.

MATERIALS AND METHODS

The cells were isolated from aborted fetus acquired from the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital), and the research was registered in ClinicalTrials.gov (NCT02868424) and Chinese Clinical Trial Registry (ChiCTR-OPC-15006757). At last,Medical Ethics was signed.

The common protocol was referred to isolate and culture cells

, the components of medium were showed in Table 1, seeding density of cells was 10 000/cm

. In normal conditions, fetal RPE(fRPE) cells will occur EMT when the cells are passaged to passage (P4). Cells were digested by trypsin and were passaged repetitively to establish the model of EMT. Exogenous TGF-β pathway inhibitor, SB431542 [Sigma, 10 μmol/L, dissolved in dimethyl-sulfoxide (DMSO)], was used to alleviate the cells which undergone EMT. Exogenous BMP pathway inhibitor,LDN193189, was used to inhibit BMP pathway.

To explore the exact effects of

on fRPE cells, siRNA was used to knock down

.Cells were divided into 3 groups at P2: negative control group (NC group),

group and BMP+

group. BMP4 was used to investigate the effects without Chordin-like 2 regulations. After transfecting, the efficiency of transfection was detected by qPCR (Figure 3A). Four days after transfection, the morphology was observed by phase contrast microscope and had no significant difference (Figure 3B). And we collected the culture medium and analyzed the concentration of BMP4 in the medium by ELISA. Interestingly,BMP4 secretion in

group was decreased compared with NC group (Figure 3C). And in

group, the cells tended to occur EMT because of a higher expression of

and

and a lower expression of

and

Inversely, the cells in NC group and BMP+

expressed higher RPE-related genes and lower EMT-related genes, besides, with BMP4 treatment,

was downregulated even more in BMP+

group (Figure 3D).

Phase contrast microscope (Nikon TS-100) was used to observe the morphology of cells. The appearance of cell pigments is the sign of cells which has normal functions, and they were analyzed by phase contrast microscope in bright field.

近年來，隨著“精益”被國內醫療機構逐漸認知，如何快速習得一套便實可用的精益醫療管理體系，為醫院精益管理的院內實踐落地作戰略指導，并引領管理不斷推進，是每位精益學習院長期待的。

Total RNAs in cells were extracted by TRIzol (Invitrogen Life Technologies, Shanghai, China). Revert aid first strand cDNA synthesis kit (Thermo, Shanghai, China) was used to synthesize cDNA and cDNA was amplified by FastStart Universal SYBR Green Master (Roche, Shanghai, China; Table 2). The method of ΔΔCT was calculated to analyze target gene expression,and the results showed the mRNA transcription level of target genes.

was regarded as internal reference.

Because of upregulating gradually along with normal differentiation of fetal RPE cells,

is likely to play a potential role in RPE differentiation. To upregulate the expression of

, plasmid-

(p

) was established. Cells which should undergo EMT at P4 were divided into two groups: negative control group (NC group)and p

group. Four days after transfecting, the cells in two groups were observed and harvested to detect the transfection efficiency (Figure 4A). The cells showed an epithelial-like shape in the p

group (Figure 4B).Meanwhile, the secretion of BMP4 in culture medium was upregulated correspondingly (Figure 4C). In p

group,

and

had higher expression than NC group, however,

and

was downregulated (Figure 4D). Meanwhile, about the results of Western blot, both phosphorylated-SMAD2 and especially phosphorylated-SMAD1 were upregulated in the p

group, which means the BMP pathway and TGF-β pathway were activated at the same time. Besides, the expression level of total SMAD1 and SMAD2 was also analyzed by Western blot, the results showed that overexpression of

could make SMAD1 upregulate, however, the expression of SMAD2 had no significant difference (Figure 4E).

Conventional method was used to extract total proteins by radio immunoprecipitation assay (RIPA)and protease inhibitor. The protein samples were separated and transferred by SDS-PAGE and polyvinylidene fluoride(PVDF) membrane (Millipore, Shanghai, China). Blocker of the membranes was 5% nonfat dry milk for 1.5h. The membranes with proteins were hybridized with primary antibodies: phosphorylated-SMAD2 (BIOSS, Beijing, China 1:1000), phosphorylated-SMAD1 (Ruiying Biology, Suzhou,China 1:1000) overnight at 4°C. At last, the membranes were incubated with secondary antibodies (Servicebio, Wuhan,China) for 2h. Exposing the bands by chemiluminescence imaging system (Sysgene). β-actin was served as an internal reference.

免疫熒光原位雜交檢測結果判定：熒光顯微鏡的藍色通道下觀察DAPI染色，藍色熒光者表明為有核細胞；紅色通道下觀察CEP-8信號，紅色亮點數目即為8號染色體數目，CTC的 8號染色體呈多倍體，即CEP8信號點≥3個，血源性白細胞8號染色體呈二倍體，即CEP8信號點≤2個；另外，CD45染色為紅色，在紅色通道下觀察細胞是否表達CD45，CTC不表達CD45而細胞周圍無紅色熒光。結合三色通道下的疊加圖像，CTC陽性判讀標準：CEP-8信號點≥3個且DAPI＋CD45-，胃癌患者外周血中CTC的細胞核經DAPI染色顯示為藍色熒光，細胞核內可見有3個或3個以上紅色信號點時，認為是循環胃癌細胞（圖1）。

Cell cycle was analyzed by flow cytometry. Cell cycle kit (Keygen Biotech, Nanjing, China)was used to detect the cell proliferation. According to the kit protocol, cells were harvested by trypsin and were fixed by 75% ethyl alcohol for 18-24h. After fixed, the cells were washed by 4℃ phosphate buffer saline (PBS) and was centrifuged in 2000 rpm for 5min. The 20 μL RNase (50 μg/mL)was used to resuspend cells and digesting cells in 37℃ for 30min. After adding propidium iodide (PI) and filtrating the cells in the dark environment, the samples were analyzed by flow cytometry. The results were analyzed by FlowJo_V10.

Primary fRPE cells in lower passage will regain some stem cell properties and have stable proliferation capacity

. Therefore, these cells are suitable to investigate RPE differentiation and damage repairment. Normal fRPE cells appear a cobblestone-like shape with abundant pigments and express RPE specific genes,such as

,

,

,

,

and

. Generally,in lower passage, the subconfluent cells can re-differentiate into normal morphology and proliferate stably, however, with serial passage to P3 or P4, the cells gradually have a disorder redifferentiation with a mesenchymal-like appearance, for example, EMT cells have fusiform appearance and the size of cell body become longer, besides, EMT-RPE cells also have poor proliferation until to the complete death. This process is similar as the repetitive RPE injury

. In our research,in P2, fRPE cells still had the capacity to re-differentiate into normal RPE, but in P4, fRPE cells transdifferentiated into mesenchymal-like and lost pigments (Figure 1A). Meanwhile,

, a marker of EMT, was upregulated (Figure 1B), but the specific RPE genes were decreased (Figure 1C). Chordinlike 2 (

) also was downregulated in P4 (Figure 1D).Therefore, Chordin-like 2 was expressed synchronously with normal RPE redifferentiation

.

RESULTS

All experiments were repeated over three times. The data were presented as mean±standard error(mean±SEM) and GraphPad Prism 6 was used to analyze the data. Differences between the data were analyzed by

-test,paired

-test, besides, the comparison of different markers was occurred between two groups was analyzed by multiple

-tests.

＜0.05 was considered statistically significant.

For further verifying the expression pattern of Chordin-like 2 and its relationship with BMP4 and other target genes along with the process of cell differentiation. The P4 cells which should turn into EMT were divided into two groups: Control group and SB431542 group. Cells in SB431542 group were daily treated with SB431542 (Sigma, 10 μmol/L). SB431542 is a synthetic TGF-β inhibitor, and it has been confirmed to inhibit EMT and to promote RPE differentiation

. After seeding, cells were harvested at 2

, 5

, 10

, 15

, 30

day and meanwhile, the expression of

,

,

,

was detected by qPCR.

is the key transcription factor and the trigger for EMT occurrence and

is the marker in normal RPE cells. After treated by SB431542,the cells differentiated into RPE-like appearance over time,but in control group, the cells showed a fusiform shape and failed to redifferentiation successfully (Figure 2A). In the differentiation process,

,

,

, and

had a completely different expression process in two groups.In control group,

and

had an initial higher expression than SB431542 group, but after transitorily upregulated, these two genes were downregulated and kept a lower expression, and at the same time,

also kept a lower expression. On the contrary, although

had a similar expression process as

and

, their expression level was higher than SB431542 group all the time.However, in SB431542 group,

had a higher final expression and

was upregulated stably, but BMP4 kept relatively lower expression at the first five days and upregulated dramatically at the follow days.

had a slight upregulation and decreased after the first 5d (Figure 2B).

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