MANF induced p44/42MAPK phosphorylation in retinal Müller cells

doi:10.3877/cma.j.issn.2095-2007.2019.06.002

Abstract

Abstract:

Objective

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a potent neurotrophic factor for photoreceptors and retinal ganglion cells. But the mechanism underlying its neuroprotective effects is not well understood. Our present work investigate potential signaling mechanism and pathway underlying these action effects.

Methods

25 Sprague-Dawley male rats (weight 0.2 to 0.25 kg) were selected. They were randomly divided into untreated group and 4 treated groups with different treating time. There were 5 rats in each group. In treated group, the left eyes of SD rats were injected intravitreally with 10 μg MANF (3 μl), and the right eyes were injected with 3 μl PBS (phosphate buffered saline) as negative controls. Retinas were collected at 0.5 h, 1 h, 2 h, 4 h after injection. Müller cells were isolated from rat retina and cultured in DMEM + 10% fetal bovine serum. Cells were treated with MANF (100 ng/ml) and collected at given time points after addition of MANF. The levels of phospho-p44/42MAPK wereassessed by Western blot analysis. Immunohistochemical analysis using frozen section was employed to localize the MANF-induced phospho-p44/42MAPK. Images were analyzed using Image J analysis system. Relative density ratio of Western blot bands and immune fluorescence intensity were represented as means±standard errors(±s). Independent sample t test was used to compare MANF treated and control groups.

Results

A dramatic significant increase in phospho-p44/42MAPK was detected 30 min after MANF injection into the vitreous, and increase to the peak at 1 hour (t=21.18, 4.41; P<0.05), then declined to normal level at 4 hours. Immunostaining for phospho-p44/42MAPK detected a group of positive cells in the inner nuclear layer, with characteristics of Müller cells. The immune fluorescence intensity was significantly stronger than control group (t=11.41, P<0.05). The MANF-induced phospho-p44/42MAPK was confirmed in cultured Müller cells. Addition of MANF (100 ng/ml) to culture medium induced a significant increase in phospho-p44/42MAPK as soon as 5 min (t=4.48, 6.43; P<0.05). The increase in phospho-p44/42MAPK reached a peak at 15 min after treatment (t=19.57, 9.18; P<0.05) and declined to normal level by 30 min.

Conclusions

This data strongly suggests that the p44/42MAPK pathway is involved in mediating extracellular neurotrophic activity of MANF. Experiments with cultured Müller cells could provide the evidence that MANF directly had the interaction with Müller cells.

Key words: MANF, p44/42MAPK pathway, Retina, Müllercells

Xueting Pei, Jianmin Lu, Yiwen Li, Rong Wen. MANF induced p44/42MAPK phosphorylation in retinal Müller cells[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(06): 328-334.

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References 44

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分组		鼠数	磷酸盐缓冲液处理		中脑星形胶质细胞源性神经营养因子处理
分组		鼠数	磷酸化p44MAPK/p44MAPK	磷酸化p42MAPK/p42MAPK	磷酸化p44MAPK/p44MAPK	磷酸化p42MAPK/p42MAPK
未处理组		5	0.61±0.039	0.54±0.090	0.61±0.039	0.54±0.090
处理组
	0.5 h组	5	0.55±0.064	0.43±0.038	1.32±0.130^*	0.95±0.026*
	1 h组	5	0.88±0.032	0.89±0.058	2.61±0.087^*	1.24±0.075^*
	2 h组	5	0.49±0.104	0.53±0.075	0.37±0.069	0.62±0.054
	4 h组	5	0.42±0.057	0.43±0.026	0.35±0.041	0.38±0.022

分组		鼠数	磷酸盐缓冲液处理		中脑星形胶质细胞源性神经营养因子处理
分组		鼠数	磷酸化p44MAPK/p44MAPK	磷酸化p42MAPK/p42MAPK	磷酸化p44MAPK/p44MAPK	磷酸化p42MAPK/p42MAPK
未处理组		5	0.61±0.039	0.54±0.090	0.61±0.039	0.54±0.090
处理组
	0.5 h组	5	0.55±0.064	0.43±0.038	1.32±0.130^*	0.95±0.026*
	1 h组	5	0.88±0.032	0.89±0.058	2.61±0.087^*	1.24±0.075^*
	2 h组	5	0.49±0.104	0.53±0.075	0.37±0.069	0.62±0.054
	4 h组	5	0.42±0.057	0.43±0.026	0.35±0.041	0.38±0.022

分组		鼠数	中脑星形胶质细胞源性神经营养因子处理
分组		鼠数	磷酸化p44MAPK/p44MAPK	磷酸化p42MAPK/p42MAPK
未处理组		5	0.28±0.032	0.37±0.018
处理组
	5 min组	5	0.65±0.092^*	0.81±0.045^*
	15 min组	5	1.46±0.074^*	1.12±0.025^*
	30 min组	5	0.20±0.034	0.24±0.065
	60 min组	5	0.15±0.016	0.18±0.103

分组		鼠数	中脑星形胶质细胞源性神经营养因子处理
分组		鼠数	磷酸化p44MAPK/p44MAPK	磷酸化p42MAPK/p42MAPK
未处理组		5	0.28±0.032	0.37±0.018
处理组
	5 min组	5	0.65±0.092^*	0.81±0.045^*
	15 min组	5	1.46±0.074^*	1.12±0.025^*
	30 min组	5	0.20±0.034	0.24±0.065
	60 min组	5	0.15±0.016	0.18±0.103

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