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Introduction
Lactobacillus is a genus that comprises an extremely diverse range of species and is widely distributed in nature. These gram-positive bacilli are classified as safe microorganisms. Moreover, Lactobacillus secretes various metabolites, including lactic acid, to promote digestion and absorption, lowers blood cholesterol, and suppresses harmful bacteria in the intestine (1). Recently, functional studies of Lactobacillus rhamnosus-derived proteins have been conducted. The protein derived from L. rhamnosus GG, which is most widely studied for their ability to survive and proliferate in gastric environment (2). It has been demonstrated that this protein is beneficial to the gut by enhancing the survival of intestinal crypts, reducing apoptosis of the intestinal epithelium and regulating intestinal inflammation (3, 4). For example, L. rhamnosus GG-derived protein p40 promotes epidermal growth factor receptor (EGFR)-dependent intestinal development when provided to fetus (5). Additionally, HM0539, another L. rhamnosus GG-derived protein, exhibits inhibitory effects on inflammatory bowel diseases by regulating NF-κB activation (6). Another protein, P8 derived from L. rhamnosus KCTC 12202BP, was identified by Cellbiotech Corp. while screening probiotic strains for new therapeutic proteins for colorectal cancer, and is known to suppress the growth of colorectal cancer cells (7, 8). However, the effects of P8 on normal or stem cells have not yet been evaluated.
Mesenchymal stem cells (MSCs) or multipotent stem cells, can be divided into two categories: one is derived from adult tissues, such as bone marrow, fat, and cartilage, and the other one is derived from fetal and maternal tissues such as umbilical cord, placenta and amniotic membrane (9). Among these, MSCs derived from fetal tissues exhibit proliferation and differentiation potential and express the specific markers of embryonic stem cells such as OCT4, SOX2, KLF4 genes and SSEA-4 surface antigen (10). Additionally, human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) are multipotent stem cells that can differentiate into various cell types such as chondrocytes, adipocytes, neurons, cardiomyocytes and osteocytes (11). Several growth factors such as brain-derived nerve growth factor, insulin-like growth factor, epidermal growth factor, and fibroblast growth factor are present in the conditioned medium from hWJ-MSCs (12). Signaling pathways involved in stem cell activation and proliferation include the Wnt pathway, characterized by high β-catenin activity (13) and the EGFR, which induces stem cell proliferation via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling (14). It can affect cell proliferation efficiency by regulating the expression of Cyclin D1, which is involved in cell cycle regulation, through the MAPK signaling pathway that regulates cell proliferation and the Hippo signaling pathway including YAP/TAZ, which is also involved in cell cycle regulation (15, 16). Based on these properties, hWJ-MSCs are used for cell therapy and cosmeceutical research related to regenerative medicine (17). However, mass production of MSC in vitro is required to achieve the desired cell number for clinical therapy.
Given that the gastrointestinal tract is related to skin homeostasis, its maintenance, functional integrity and associated microorganisms are important for the homeostasis of both organs (18). Additionally, in vitro studies have confirmed the skin-healing properties of probiotics, mediated by anti-inflammatory effects and cell regeneration, in HaCaT cells (19). Moreover, in some cases, intestinal microbe-derived probiotics have been used for the treatment of skin diseases, such as acne, burns, and atopic dermatitis (20).
In this study, we investigated the efficacy of the probiotic-derived protein P8 in promoting hWJ-MSC proliferation and stemness and identified the mechanisms underlying such effects. Our results suggest that P8 might be a promising candidate to improve stem cell growth by stimulating cell proliferation, migration, and stemness-related pathways. Additionally, after identifying the effects of P8 on migration and wound healing using HaCaT keratinocytes, we propose its potential therapeutic effects for skin therapies, such as skin regeneration, burns, atopic dermatitis, and psoriasis.
Materials and Methods
Preparation of P8 protein
The P8 protein, originally derived from L. rhamnosus KCTC 12202BP, isolated from human feces, was provided as a gift by Cell Biotech Co., Ltd.. As previously reported, this protein was isolated via 6x His-tag binding. The 6x His-tag was then removed using TEV protease with 1 mM DTT. Finally, the P8 protein was confirmed through size exclusion chromatography (HiLoad 26/60 Superdex 200 pg [GE Healthcare] equilibrated with 20 mM HEPES [pH 7.5]/150 mM NaCl) (7). We generated the P8 structure using the AlphaFold2 method, known for its high accuracy in protein structure prediction, to improve conformational sampling (21).
Cell culture
The experimental protocol for hWJ-MSCs followed our recent publication and was approved by the Konkuk University Ethics Committee (7001355-202, 010-BR-407) (22). hWJ-MSCs were cultured in an MSC medium composed of α-minimum essential medium (MEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (P/S; Thermo Fisher Scientific) at 37℃ in a humidified atmosphere of 5% CO2. Spontaneously immortalized human keratinocyte HaCaT cells were cultured in Dulbecco’s modified Eagle medium (DMEM)-High (Thermo Fisher Scientific) supplemented with 10% FBS and 1% P/S (Thermo Fisher Scientific) at 5% CO2 and 37℃ in a humidified incubator.
RNA isolation and real-time quantitative polymerase chain reaction analysis
LaboPassTM Labozol reagent (Cosmo Genetech) was used to isolate total RNA from hWJ-MSCs, which was reverse- transcribed into complementary DNA using the LaboPassTM M-MuLV Reverse Transcriptase kit (CMRT010; Cosmo Genetech). The quantitative polymerase chain reaction (qPCR) mixture was prepared using EzAmpTM qPCR 2X MasterMix (EBT-1802; ELPIS-BIOTECH) and run on a QuantStudioTM 3 Real-Time PCR System (Thermo Fisher Scientific). The respective methods were performed in accordance with the manufacturer’s protocol. The relative expression results were normalized based on GAPDH expression, an internal control, and the fold-change in gene expression was calculated using the comparative 2-ΔΔCt method (23). The primers used in this study are listed in Table 1.
Western blotting
RIPA buffer (LPS Solution Inc.) containing a protease inhibitor (Thermo Fisher Scientific) was used to prepare protein lysates of control cells and cells treated with different concentrations of the P8 protein from all experimental groups. The protein concentration of each cell line was quantified using a Microplate BCA Protein Assay Kit (Invitrogen), and 10 μg of each protein sample was separated using the BoltTM 4%∼12% Bis-Tris Plus Gels 12-well (Thermo Fisher Scientific) and transferred onto iBoltTM 2 transfer stacks (Thermo Fisher Scientific). The membrane was blocked in 5% Skim milk in TBST (0 mM Tris-HCL, 150 mM NaCl, 0.1% Tween-20) for an hour and incubated with primary antibodies (1：1,000), such as anti-phospho-ERK1/2 (CSB-PA000749; Cusabio Technology), anti-Phospho-p38 (sc-101758; Santa Cruz), anti-β-actin conjugated with horseradish peroxidase (HRP) (sc-47778; Santa Cruz), anti-phospho-Akt (sc-293125; Santa Cruz), anti-phospho-mTOR (sc-293133; Santa Cruz), anti-YAP (sc-15407; Santa Cruz), anti-Cyclin D1 (sc-717; Santa Cruz) and anti-phospho-JNK (sc-6254; Santa Cruz) overnight at 4℃ followed by incubation. The blots were then washed three times with 1x TBST, and the proteins were incubated with secondary antibodies (1：1,000) for a day, followed by three washes with 1x TBST. The secondary antibodies used were HRP-conjugated anti-rabbit IgG (#7074; Cell Signaling Technology) and HRP-conjugated anti-mouse IgG (#7076; Cell Signaling Technology). The proteins, processed using Clarity Western ECL substrate (Bio-Rad Laboratories), were visualized using an InvitrogenTM iBrightTM Imagers CL-1000 (Thermo Fisher Scientific).
Cell viability and proliferation assays
To determine the effect of P8 treatment on cell viability and proliferation, hWJ-MSCs were seeded at a density of 5×103 cells/well in 96-well plates. Culture medium was used for cells measuring cell viability and serum-free culture medium was used for cells measuring cell proliferation. After incubation for 24 hours, various concentrations of P8 (10, 20, 40, and 60 μM) were added to each well, and cells were incubated for an additional 24, 48, and 72 hours. The medium was then replaced with fresh medium, and cell viability rates were determined using the Cellrix Viability Assay Kit (B1007-500; Medifab) according to the manufacturer’s recommended protocol. Absorbance was measured at 450 nm using a Bio-RAD x-MarkTM spectrophotometer (Bio-Rad Laboratories).
Cell migration assay
hWJ-MSCs and HaCaT cells grown to 95% confluence in 6-well plates (SPL Lifesciences) were pretreated with 10 μg/mL mitomycin C (Sigma-Aldrich) for 2 hours at 37℃ in a 5% CO2 incubator. A wound was created using a sterile 1 mL pipette tip, the cells were gently rinsed using PBS (Gibco), and complete medium (α-MEM and DMEM-High, respectively) with 1% P/S was added. Migration into the wound was monitored at the indicated time points through imaging at ×40 magnification. The images were analyzed using T-scratch software, and the fold-change difference in the wound closure percentage was determined.
Immunocytochemistry
The cells were fixed with 4% paraformaldehyde (Biosesang), permeabilized with 1X Phosphate-Buffered Saline, 0.1% Tween 20 (PBST) (0.2% [vol/vol] Triton X-100 in PBS), and blocked with 10% bovine serum albumin (vol/vol) in PBST composed of 0.1% Triton X-100 (vol/vol, Sigma-Aldrich) in PBS (Gibco). The cells were then incubated overnight at 4℃ with anti-OCT-3/4 (sc-9081; Santa Cruz), anti-SOX-2 (sc-20088; Santa Cruz), and anti-SSEA-4 (sc-21704; Santa Cruz) primary antibodies in PBST containing 1% bovine serum albumin. Next, the cells were washed three times for 10 minutes each with PBST and incubated with secondary antibodies, specifically Alexa Fluor goat-anti-mouse 488 (#A-11001; Thermo Fisher Scientific) and Alexa Fluor goat-anti-rabbit 546 (#A-11035; Thermo Fisher Scientific), for 2 hours at room temperature, and washed three times for 10 minutes each with PBST. Samples were visualized using a fluorescence microscope (LSM 900 with Airyscan 2; Zeiss) for imaging.
Statistical analysis
All statistical analyses were performed using GraphPad Prism software (GraphPad Software). Most experiments were replicated three times. The results were reported as the mean±SD. Adjusted p-values were calculated using one-way ANOVA and two-way ANOVA to determine statistical significance. In all figures, the following designations are used: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Results
Probiotic-derived protein P8 increases cell proliferation
The P8 protein, derived from L. rhamnosus CBT-LR5 (KCTC 12202BP), is 8 kDa protein with a height of 53 Å and a width of 33 Å. The matrix also provides another representation of the data, allowing the accuracy of the forecasts to be assessed by identifying segments of solid entities, shown as dark green squares, and areas of inherent disorder, shown as dark green lines (Fig. 1A, 1B). We investigated the effect of P8 on the proliferation of hWJ-MSCs. Initially, to determine the optimal concentration of the P8 protein, screening was performed to evaluate its effects on hWJ-MSCs. For this, 5×103 of hWJ-MSCs were seeded, stabilized, and treated with various concentrations of P8 (0, 10, 20, 40, and 60 μM) for 3 days. An increase in cell viability was observed from 48 to 72 hours, with 20 and 40 μM of P8 being most effective (Fig. 1C). As such, these concentrations were selected for further analysis of P8 efficacy. Based on changes in hWJ-MSC confluency from 48 hours to 72 hours, it was shown that 20 and 40 μM of P8 concentration is the most effective, as with the previous cell viability results (Fig. 1D, 1E). These results indicate that the probiotic-derived P8 protein promotes hWJ-MSC proliferation.
P8 increases MSC migration and wound healing via MAPK and Hippo signaling pathways
To determine the effect of P8 on hWJ-MSC migration, we performed a wound healing assay by conducting a scratch assay. The cells were scratched in the middle of the plates using a 1 mL sterile pipette tip, and the wound sites of the control and P8-treatment groups of hWJ-MSCs were measured 0, 4, 6, 8, 10, and 12 hours later (Fig. 2A). As shown in Fig. 2A, the degree of wound closure in the P8-treatment group was significantly greater after 10 and 12 hours compared to that in the control group (Fig. 2B). We further observed an increase in collagen type I, II, and III mRNA expression levels in the 20 and 40 μM of P8 treatment group after 24 hours (Fig. 2C). These results indicate that the probiotic-derived P8 protein increases wound-healing in hWJ-MSCs. To investigate the effect of P8 on MSC proliferation, we examined the MAPK signaling pathway including Akt/mTOR signaling and Hippos signaling pathway over time via western blotting (Fig. 2D). The expression of phospho-ERK and phospho-p38 in hWJ-MSCs was upregulated in the P8-treatment groups compared to that in the control group at 48 and 72 hours through MAPK signaling pathway. Furthermore, we confirmed the expression levels of YAP and Cyclin D1 proteins, crucial in cell cycle regulation, in hWJ-MSCs after P8 treatment. We observed an increase in their levels in the P8-treatment group compared to levels in the control group at 24 hours, but by 72 hours their levels resembled those of the control group. Similar to that of YAP, the expression of Cyclin D1 was also slightly increased in the P8-treatment group compared to that in the control group at 24 hours, with levels similar to those in the control group at 48 and 72 hours. These results show that the MAPK signaling pathway in MSCs is activated by P8, leading to increased cell proliferation through the upregulation of YAP and Cyclin D1 expression.
P8 improves MSC stemness
MSCs, comprising a type of multipotent stem cell, have multi-directional differentiation potential. To assess the effect of P8 on MSC stemness, we analyzed stemness-associated gene expression in these cells after treatment with different concentrations of P8. Specifically, RT-qPCR analysis was performed to identify the expression of stemness-related genes in hWJ-MSCs. The expression levels of OCT4, SOX2, and KLF4 were increased in MSCs treated with P8 for 48 hours (Fig. 3A). The expression of OCT4, SOX2, and SSEA-4 in hWJ-MSCs was also confirmed via immunofluorescence staining, and it was significantly increased in MSCs treated with 40 μM of P8, compared with that in control and 20 μM P8-treated MSCs (Fig. 3B-3D). The upregulation of stemness marker expression indicated that P8 might enhance MSC stemness to improve cell self-renewal.
P8 promotes wound healing in HaCaT cells by activating MAPK signaling pathways
We further investigated whether P8 could induce migration and wound healing effects in HaCaT keratinocytes. As done previously with hWJ-MSCs, we assessed the migration-inducing effects of P8 by performing a wound healing assay. Because of differences in cell properties, we observed the wound closure of the control and P8-treatment groups of HaCaT cells at 24, 48, and 72 hours after creating the scratch (Fig. 4A). The extent of wound closure was significantly increased after 48 hours depending on the concentration of P8, with the wound sites almost completely closed in the 40 μM P8-treatment group after 72 hours (Fig. 4B). Then, we measured the expression level of MAPK proteins, specifically p-ERK and p-JNK, after treating HaCaT cells with P8 for 72 hours (Fig. 4C). The data showed increased p-ERK protein expression in a time-dependent manner. However, the expression levels of p-JNK was similar compared to those of control group. Taken together, we showed that treating HaCaT cells with P8 can promote migration and wound healing by activating the MAPK signaling pathways. These results further show that P8 can increase the proliferation of keratinocytes.
Discussion
L. rhamnosus has been shown to provide a variety of benefits to the body, including helping to build and repair the gut barrier and modulating inflammatory responses in the gut, but there are few reports of L. rhamnosus-derived proteins (24, 25). In a previous study, Cellbiotech Corp. reported that an 8 kDa protein (P8) isolated from L. rhamnosus KCTC 12202BP inhibits the growth of colon cancer cells (7, 8). However, the function of P8 had not been evaluated in normal cells or stem cells. According to other reports, functional factors of probiotic proteins derived from L. rhamnosus directly affect not only intestine-related cells but also dendritic and epithelial cells (26, 27). Additionally, these proteins have a role in maintaining tissue homeostasis by activating macrophages via cytokines to activate functional proteins, such as IL-10 (28). Therefore, we hypothesized that P8 would have a positive effect on stem cells or normal cells. In this study, we investigated the mechanism underlying the effects of P8 on stem cells and normal cells in vitro, as well as its effect on cell proliferation and wound healing.
hWJ-MSCs are known to exhibit multipotency and can differentiate into various cell types such as chondrocytes, adipocytes, neurons, cardiomyocytes, and osteocytes (11). They are also used for the treatment of various diseases, owing to their robust proliferative capacity and potent wound-healing properties (17, 29). When lactic acid bacteria-derived proteins are administered to skin keratinocytes, skin regeneration ability is increased (19). hWJ-MSCs are reported to have a therapeutic effect on atopic dermatitis (30, 31). Therefore, in this study, we plated hWJ-MSCs cells, stabilized them for 1 day, then treated them with P8 for 3 days to observe the time-dependent effect on cell proliferation. The results showed that hWJ-MSC proliferation increased in a time-dependent manner after P8 treatment. There was almost no difference or slightly decrease in proliferation rates between the 20 and 40 μM treatment groups at 24, 48, and 72 hours after P8 treatment, indicating that P8 did not have a concentration-dependent effect on the prolieration of WJ-MSCs.
The mammalian MAPK signaling pathway is shared by several distinct cascades, namely ERK1/2, Jun amino-terminal kinases (JNK1/2/3), and p38-MAPK. Further, the MAPK/ERK pathway is associated with cell proliferation, differentiation, migration, senescence, and apoptosis (32, 33). As such, we identified the signaling pathway associated with the increase in hWJ-MSC and HaCaT cell wound healing and proliferation after P8 treatment. Wound closure occurred faster in hWJ-MSCs treated with P8 compared to that in the control group, in both a time- and concentration-dependent manner. Therefore, we analyzed the genes and proteins involved in cell proliferation and wound closure. Collagen, a principal component of the extracellular matrix, performs pivotal functions in the regulation of the phases of wound healing, whether in its native, fibrillar conformation or as soluble components within the wound milieu (34). Type I and type III collagens are indispensable for wound healing and tissue regeneration, with an increase in type III collagen synthesis during the early phases and an increase in Type I collagen synthesis during the late stages (35). As collagen type I, III mRNA expression levels increased in the P8 treated group, it is confirmed that migration and wound healing effects of P8 protein on WJ-MSCs by increasing collagen synthesis. In addition, a concentration- and time-dependent increase in the protein expression of phospho-ERK and phospho-p38 was observed. These results suggested that P8 increases wound healing by inducing cell proliferation.
Next, we examined the expression of proteins associated with the cell cycle. YAP/TAZ signaling regulates several signaling pathways and is a key regulator of the Hippo pathway, which is involved in cell proliferation (36). Moreover, in MSCs, YAP/TAZ signaling interacts with other pathways, such as Wnt/β-catenin, Notch, and MAPK, to regulate cell functions. After translocation into the nuclear, YAP binds to TEAD as a transcription factor and is involved in cell proliferation, cell migration, lineage function, and maintenance of differentiation. In the case of cell proliferation, YAP can regulate the expression of Cyclin D1 (37, 38). Cyclin D1 controls the transition from the G1 to S phase of the cell cycle, and its regulation via ERK1/2 occurs at the transcriptional level and is mediated by the direct binding of ERK1/2 to multiple sites in the Cyclin D1 promoter (39, 40). In our study, when hWJ-MSCs were treated with P8, it was shown that the protein levels of Cyclin D1 and YAP were increased. Also, expression levels of proteins related to MAPK pathway including ERK, p38 were increased too. It was suggested that P8 activates both the MAPK signaling pathway and the cell cycle-related Hippo signaling pathway therefore hWJ-MSCs to induce an increase in cell proliferation and wound healing by allowing translocation of YAP and expression of cell cycle related Cyclin D1 (Fig. 5A). Based on the effect of P8 on hWJ-MSCs, we further assessed the potential wound-healing effect on HaCaT cells. We found that phospho-ERK was significantly increased in HaCaT cells treated with P8 in a time- and concentration-dependent manner, compared to levels in the control group.
In summary, we analyzed the pathways involved in the effects of L. rhamnosus-derived P8 protein on hWJ-MSCs and HaCaT cells. The results showed that P8 stimulates the MAPK signaling pathway in both cell types, resulting in increased cell proliferation and wound healing. In addition, the self-renewal capacity and pluripotency of hWJ-MSCs treated with P8 increased, suggesting that this protein has a potential to be used in mass cell culture studies and as a basis for cell therapeutics to contribute to increase wound healing (Fig. 5B). These findings provide insights into the enhancing effect of P8 on cell proliferation and wound healing, which could be ultimately used to increase the efficiency of stem cell or normal cell culture, suggesting the potential of probiotics that can be used as therapeutic candidates for skin diseases.
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