P75 neurotrophin receptor activates epidermal stem cell differentiation via ERK, JNK and AKT pathways

As reported, p75 neurotrophin receptor (p75NTR) is involved in activation and differentiation of epidermal stem cells (ESCs). However, the possible signaling pathways of p75NTR in ESCs remain unknown. In this study, p75NTRvo ESCs were constructed to identify the role of p75NTR in cell differentiation. Here we found that P75NTR activated and promoted the differentiation of ESCs. Functionally, P75NTR significantly promoted cell proliferation and migration, and inhibited cell apoptosis. Mechanically, P75NTR stimulated the secretion of NGF in ESCs, and activated ERK, JNK and AKT signaling pathway. Finally in-vivo assay demonstrated that P75NTR effectively promoted wound healing in mice model. In conclusion, P75NTR activates differentiation of epidermal stem cells via ERK, JNK and AKT signaling pathways, and promotes the secretion of NGF and wound healing. Thus, P75NTR may favor skin wound healing in the clinical practice. www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2018


INTRODUCTION
Skin serves as a protective organ for mammals, which prevents pathogens, radiation etc [1,2].The dysfunction of skin homeostasis usually result in dire consequences, involving acute, chronic inflammation or infected wounds [3,4].And skin wound healing is an intrinsic self-saving chain reaction, which is crucial to facilitate the replacement of damaged or lost tissues.It is reported that epidermal stem cells are responsible for maintaining skin homeostasis [5].However, the mechanisms of the differentiation of epidermal stem cells were not clear.
p75 neurotrophin receptor (p75NTR), together with the tyrosine kinase receptor tropomyosin-receptor kinase (Trk), is reported to mediate neurotrophin (NT) functions [6][7][8].p75NTR serves as a low-affnity receptor to mediate diverse functions [9].Generally, p75NTR is proteolytically cleaved by γ-secretase to generate intracellular domain after ligand activation [10,11].It is reported that p75NTR interacts with a variety of proteins [12] that in turn induce different pathways [13].This interaction allows p75NTR to play an important role in the regulation of some processes, such as proliferation, migration and differentiation [14].However the potential signaling pathways that p75NTR participated in ESCs remain unknown.
In the present study, firstly, we examined the expression of stem cell bio-makers and p75NTR using western blot.And then, we investigated the related signalling pathways, involving ERK, JNK and AKT.Finally, wound healing assay was performed in mice model.Our findings indicated that P75NTR may favor skin wound healing in the clinical practice.

Isolation of ESCs and transfection of p75NTR
To elucidate the role of p75NTR in the progression of ESCs, we firstly isolated ESCs from newborn mice.And then, p75NTR vo ESCs were constructed and subjected to western blot.We found that p75NTR expression was markedly increased in p75NTR vo ESCs compared with that in empty vector (EV) ESCs (Figure 1A).( * p < 0.05).Furthermore, we examined the expression of stem cell biomakers following p75NTR vo ESCs construction.As shown in Figure 1B-1D, compared with empty vector, intergrin β1 expression in p75NTR vo ESCs was negative, while CK10 and CK19 expression p75NTR vo ESCs was positive, indicating that p75NTR may affect the progression of ESCs.

P75NTR promotes secretion of NGF in ESCs
Firstly, we used ELISA to detect the expression of NGF in two groups.In the present study, we found that the expression level of NGF was 103.28 ± 0.88 in p75NTR vo ESCs, and 90.42 ± 0.77 in EV ESCs, indicating that P75NTR expression in p75NTR vo ESCs stimulated NGF secretion.In addition, during the wound-healing process among the three groups, at post-operation 14 days, we examined the levels of the NGF proteins, and demonstrated that the p75NTR vo ESCs group was able to express more NGF (122.55 ± 2.76) compared with that (95.12 ± 0.98) in EV ESCs group ( * p < 0.05).

P75NTR mediates the proliferation and apoptosis in ESCs
To evaluate the role of p75NTR expression in ESCs proliferation and apoptosis, P75NTR vo ESCs were subjected to flow cytometry and CCK-8 assays.Meanwhile, freshly isolated normal ESCs were used as control.We observed that P75NTR vo ESCs showed a significantly lower apoptosis rate (0.003 ± 0.001) than EV ESCs (0.063 ± 0.011) (Figure 2A, p < 0.05).As expected, P75NTR vo ESCs also had a significantly higher proliferative capacity (1.23 ± 0.13) than EV ESCs (0.72 ± 0.09) (Figure 2B, p < 0.05).These results indicated that P75NTR mediates the proliferation and apoptosis in ESCs.

P75NTR promotes the migration of ESCs
To evaluate the effect of p75NTR on ESCs migration, P75NTR vo ESCs were subjected to transwell invasion assay.And then freshly isolated ESCs were used as control.As shown in Figure 2C, P75NTR vo ESCs showed significantly higher cell migration ability (251 ± 32) compared with EV ESCs (157 ± 63) (p < 0.05).These data indicated that P75NTR mediates the migration in ESCs.

ERK, JNK and AKT signaling mediates P75NTR pathway
To clarify which signalling pathways are involved in p75NTR mediated the biology of ESCs, we further investigated the expression of ERK, JNK and AKT.In this study, there are no animals to be sick or dead.We found that the expression of the phosphorylated ERK, JNK and AKT was significantly increased in p75NTR vo ESCs at 14 day when compared with EV ESCs (Figure 3).

P75NTR promotes wound healing in mice model
In this work, from 1st to 3rd group, all mice were injected with 500 μL of ESCs medium, 500 μL of EV ESCs medium, and 500 μL of p75NTR vo ESCs medium, respectively.The groups were injected on 0, 3 day and the area of the wound was measured by tracing the wound on 0, 3, 7 and 14 day post-injury.P75NTR mediated ESCs promoted wound healing.The lesion area of the three groups at day 3, 7, 14 was compared with each other, as shown in Figure 4A.We found that compared with the group injected with EV ESCs, the group injected with p75NTR vo ESCs increased wound healing area by about 14.86% at day 3, 32.53% at day 7 and 6.4% at day 14.However, there was no significant differences between normal and EV ESCs group (p > 0.05).As shown in Figure 4B, we used HE staining to identify that P75NTR promotes wound healing in mice model.Finally, as shown in Figure 4C, western blot further revealed that P75NTR obviously increased the expression of CK10, indicating that P75NTR indeed activate the differentiation of ESCs to promotes wound healing.

DISCUSSION
As known to all, the epidermis, a kind of selfrenewal tissue, was constructed by multiple compartment layers of keratinocytes.The basal layer is composed of proliferating epidermal stem cells; the upper layers includes viable and differentiation cells; the horny layer comprises terminally differentiated cells [15].Epidermal homeostasis and stabilization relay on the status of epidermal stem cells situated in the basal layer, which is able to generate newly differentiated cells to replace old cells, such as apoptotic, injured and neucrotic cells.In recent decades, related molecular mechanisms underlying cell differentiation of epidermal stem cells have been extensively investigated [16].According to our previous study, we assumed that the nerve growth factor may be involved into these processes.
The nerve growth factor contains two kinds of structurally different receptors, p75NTR and the tropomyosin-related kinase A (TrkA).NGF is reported to mediate a wide range of physiological and pathological processes in humans by binding to their receptors.p75NTR, a member of tumor necrosis factor receptor superfamily, comprises three domains, including an extracellular domain, a transmembrane domain, and an intracellular domain.Of three domains, the extracellular domain has four cysteine rich repeats, and is required for interaction of NGF-p75NTR [17][18][19].Once NGF-p75NTR compounds occur, p75NTR can regulate cell apoptosis, proliferation and differentiation.In the present study, we firstly succeeded to isolate and culture ESCs, and then constructed p75NTR vo ESCs from newborn mice.We demonstrated that p75NTR expression decreased the expression of intergrin β1, but increased the expression of CK10 and CK19 compared with those in EV ESCs.Furthermore, we simulated similar environments to the wounded skin tissues using in-vitro p75NTR vo ESCs.We found that P75NTR obviously promoted cell proliferation and migration, and inhibited cell apoptosis.At the same time, we also demonstrated that P75NTR indeed stimulated the secretion of NGF in ESCs.
Mechanically, ERK signalling pathway plays an important role in regulating the migration of epithelial cells during wound healing [20][21][22].When ERK was activated, ERK translocated into the nucleus to regulate cellular proliferation [23,24].RhoA-JNK signalling pathway can promote epithelial wound healing by regulating cell survival and protein synthesis [25,26].Dahai Hu et al. showed that HAESCs accelerated keratinocyte proliferation and migration to promote wound healing via the ERK, JNK, and AKT signal pathways [27].To clarify which signalling pathways are involved in the differentiation of ESCs, we investigated the specific role of ERK, JNK and AKT signaling pathways in p75NTR vo ESCs.Our data revealed that the expression of p-ERK, p-JNK and p-AKT significantly increased in p75NTR vo ESCs groups, indicating that ERK, JNK and AKT was involved in p75NTR-mediated ESCs during wound healing.In addition, it has been reported that ERK and AKT signalling can enhance the synthesis and secretion of certain factors, (VEGF, MMP9, et al.) by modulating related transcription factor [28,29].Thus it is plausible that activated ERK and AKT signalling may promote NGF synthesis and secretion in p75NTR vo ESCs.
In conclusion, we confirmed that p75NTR-mediated ESCs accelerated the wound-healing process and upregulats NGF expression through the pERK/ERK, pJNK/ JNK and pAKTK/AKT signalling pathways.Thus, P75NTR may favor skin wound healing in the clinical practice.

Cell isolation, culture and transfection
We isolated ESCs using enzymatic digestion and Col IV rapid adhesion.Briefly, five newborn mice were purchased from the Shandong University Laboratory Animal Centre (Jinan, China).The newborn mice were anesthetized with 25% pentobarbital sodium (35 mg/kg), shaved backs and wiped with 75% alcohol.1cm square fresh skin pieces were collected and digested in 0.25% Dispase (Gibco) at 4° C, separate the epidermis to digested in 0.25% protease (Sigma) at 4° C overnight.Cells were collected by filtration and centrifuged, and then seeded to a pre-spread with Col IV dish.The keratinocyte serum free medium (K-SFM, Gibco) was added and replaced every other day.
Primary ESCs were separately infected with nerve growth factor (NGFR-p75NTR) lentivirus particles, NGFR-p75NTR-shRNA lentivirus particles and Puro lentivirus particles.Lentivirus particles were obtained from the Genome Ditech Company (Shanghai, China).After the ESCs (1 × 10 4 /well) were seeded in 96-well plate for 24 hours, cells were co-cultured with 10 µL of virus from the three different gradients and polybrene for 8h, and then the medium should be replaced.Next, cells were observed using an inverted fluorescence microscope (Nikon, Japan) after 3 or 4 days transfection to calculate the transfection efficiency and the multiplicity of infection.
ESCs (1 × 10 6 cells/ml) were cultured for 3 days.To obtain culture medium from ESCs culture, the medium was changed without FBS and the cells were cultured for 2 days.

Flow cytometry of the cells surface markers
The Annexin-V-FITC/PE kit (Becton, Dickinson and Company (BD), America) was employed in flow cytometry to probe cellular apoptosis according to the manufacturer's protocol.The cells were collected, washed three times with pre-chilled PBS, and then resuspended in 1×binding buffer.Cells were stained at room temperature in the dark with 5 µL of PE-7-AAD and Annexin-V-FITC for 15 min.Then, another 400 µL of 1×binding buffer was added to each tube before testing.The data was collected and analyzed using FCM (BD LSR Fortessa).

Enzyme-linked immunosorbent assay (ELISA)
Proteins in ESCs medium were analyzed using ELISA kits (R&D Systems, USA) to detect the expression of NGF.According to the manufacturer's protocol, NGF in culture medium was collected and measured from ESCs cultures.

Cell counting kit-8
CCK-8 (Dojindo, Japan) was employed to test cell proliferation.Both normal and stable-transfected cells (approximately 2 × 10 3 ) were seeded into 96-well plates and cultured for 24 h, 48 h, 72 h, 96 h, respectively.When reached the detected time, 10ul of CCK-8 were added into each well and incubated for 1h in incubator.The absorbance was measured at the wavelength of 450 nm used the Multimode Reader (Molecular Devices, MD, American).

In vitro cell migration and metastasis assay
The non-coated and pre-coated with diluted Matrigel transwell membrane (8.0um pore size, Corning, USA) were employed to test cell invasion ability.Cells (approximately 5 × 10 5 ) were suspended in 200 ul epidermal growth factor (EGF)-free medium and seeded on the upper chamber while 700 ul medium contains EGF was added to the lower chamber as an chemo-attract.Incubated 24 h for migration and 36 h for metastasis assay.Cells can't invade the membrane were removed by the cotton swab, the membrane was fixed by methanol and

In vivo experiments in mice
C57BL/6 mice (6 weeks old, male, 20-25 g) were purchased from the Shandong University Laboratory Animal Centre (Jinan, China).The mice were randomly divided into three groups (n = 6) and housed in a pathogenfree barrier facility.After mice were anesthetized with 25% pentobarbital sodium (35 mg/kg), their backs were shaved and wiped with 75% alcohol.Full-thick skins measuring 1cm × 1cm were cut to create the wound model.The mice  were revived by the mice by intraperitoneal injection of 5 mL lactated Ringer's liquid immediately.From 1st to 3rd group, the mice were respectively injected with 500 μL of ESCs medium, 500 μL of EV ESCs medium and 500 μL of p75NTRvo ESCs medium.The groups were injected on 0, 3 day and the area of the wound was measured by tracing the wound on 0, 3, 7 and 14 day post-injury.The wound area of the seven groups on 3, 7, 14 day was compared with which of their own on 0 day by a metric ruler measure.(Wound area rate of (3, 7, 14) D) % = (the wound area of (3, 7, 14) D)/(the wound area of 0 D).Use a metric ruler to measure the area of the wound.

Statistical analysis
The SPSS 17.0 software was used to perform the statistical analyses and the data were expressed as the mean ± standard deviation (SD).Student's t test was applied to perform the intergroup comparison and analysis of variance (ANOVA) was used to compare multiple groups.P ˂ 0.05 was considered statistically significant.

CONFLICTS OF INTEREST
None.

Figure 1 :
Figure 1: The effect of p75NTR on the expression of ESCs bio-markers.We firstly isolated ESCs from newborn mice.And then, p75NTR vo ESCs were constructed (A) and subjected to western blot.Compared with empty vector, intergrin β1 expression (B) in p75NTR vo ESCs was negative, while CK10 (C) and CK19 (D) expression p75NTR vo ESCs was positive.* p < 0.05, v.s.EV ESCs.

Figure 2 :
Figure 2: The effects of p75NTR on cell activities of ESCs.(A) The flow cytometry results and data analysis of p75NTR on cell apoptosis.* p < 0.05, v.s.EV ESCs.(B) The effects of p75NTR on cell proliferation of ESCs.* p < 0.05, v.s.EV ESCs.(C) The effects of p75NTR on cell migration ability of ESCs.* p < 0.05, v.s.EV ESCs.

Figure 4 :
Figure 4: Wound closure status in normal ESCs, empty vector (EV) ESCs and p75NTRvo ESCs in mice model.(A) Representative photographs of skin full-thick excision wounds in mice treated with ESCs, PBS-treated control and p75NTR vo ESCs for 0, 3, 7 and 14 days.* p < 0.05, compared to EV ESCs groups; n = 5. (B) The HE staining in normal ESCs, EV ESCs and p75NTR vo ESCs at day 14.(C) The expressions of CK10 were detected using western blot in normal ESCs, EV ESCs and p75NTR vo ESCs on day 14.* p < 0.05, v.s.EV ESCs.