Int J Stem Cells.  2025 Feb;18(1):87-98. 10.15283/ijsc24107.

Probiotic-Derived P8 Protein: Promoting Proliferation and Migration in Stem Cells and Keratinocytes

• Jang SB
• Kim Y
• Yeo HC
• Kang GH
• An BC
• Ryu Y
• Chung MJ
• Cho SG

• KMID: 2565199
• DOI: http://doi.org/10.15283/ijsc24107

Abstract

Probiotics exert various effects on the body and provide different health benefits. Previous reports have demonstrated that the P8 protein (P8), isolated from Lactobacillus rhamnosus, has anticancer properties. However, its efficacy in stem cells and normal cells has not been reported. In this study, the effect of P8 on cell proliferation and wound healing was evaluated, investigating its underlying mechanism. Based on scratch assay results, we demonstrated that P8 treatment significantly increases wound healing by activating the cell cycle and promoting stem cell stemness. Cellular mechanisms were further investigated by culturing stem cells in a medium containing Lactobacillus-derived P8 protein, revealing its promotion of cell proliferation and migration. Also, it is found that P8 enhances the expression of stemness markers, such as OCT4 and SOX2, along with activation of the mitogen-activated protein kinase (MAPK) signaling and Hippo pathways. These results indicate that P8 can promote cell growth by increasing stem cell proliferation, migration, and stemness in a manner associated with MAPK and Hippo signaling, which could contribute to the increased wound healing after P8 treatment. Furthermore, P8 could promote wound healing in keratinocytes by activating the MAPK signaling pathways. These results suggest that P8 might be a promising candidate to enhance stem cell culture efficiency by activating cell proliferation, and enhance therapeutic effects in skin diseases.

Keyword

Lactobacillus rhamnosus; Probiotics; Mesenchymal stem cells; Proliferation; Wound healing

Figure

• 

  Fig. 1 Effect of P8 on mesenchymal stem cell (MSC) proliferation. (A) 3D structure of the P8 protein, with its size, height and width shown. (B) Heatmap representing the predicted alignment error (PAE) from AlphaFold structure prediction. This map indicates the expected position error in Angstroms between residues in the predicted structure. (C) The viability of human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) was measured using CellRix after P8 treatment concentrations of 0, 10, 20, 40, and 60 μM for 24, 48, and 72 hours. Data are presented mean±SD. Statistical significance was determined using statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparison tests. *p<0.05 vs. control group for each time. n=3 per sample. (D) Morphology of hWJ-MSCs treated with 0, 20, and 40 μM of P8 (scale bar=90 μm). (E) The proliferation of hWJ-MSCs was measured using CellRix after P8 treatment (20 and 40 μM) for 24, 48, and 72 hours. Data are presented mean±SD. Statistical significance was determined using statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparison tests. **p<0.01, ****p<0.0001 vs. control group for each time. n=3 per sample.

• 

  Fig. 2 Effect of P8 on migration and wound healing in mesenchymal stem cells (MSCs) via activating MAPK and Hippo signaling pathways. (A) Confluent monolayer cultures of control and P8-treated human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) were scratched using a sterile pipette tip at 0 hour. Representative images of migration are shown for the control and P8 treatment groups at 4, 6, 8, 10, and 12 hours after scratching (scale bar=500 μm). (B) Graph showing the fold-change in the wound-closure rate for hWJ-MSCs using T Scratch software. Data are presented mean±SD. Statistical significance was determined using Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparison tests ***p<0.001, ****p<0.0001 vs. control group for each time. n=3 per sample. (C) Graph showing comparative mRNA expression levels of collagen type I, II, and III in hWJ-MSCs after P8 protein treatment (20 and 40 μM) for 24 hours based on real-time quantitative polymerase chain reaction (RT-qPCR). The relative gene expression levels were analyzed in triplicate and normalized to endogenous GAPDH. Data are presented mean±SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. *p<0.05, **p<0.01, ***p<0.001 vs. control group. n=3 per sample. (D) Protein expression levels of p-p38, p-ERK, p-Akt, p-mTOR, YAP, cyclinD1 and β-actin. Protein expression was detected via western blot analysis.

• 

  Fig. 3 P8 improves stemness of mesenchymal stem cells (MSCs). (A) RT-qPCR was performed to measure changes in the expression of stemness-related genes (OCT4, SOX2, and KLF4). The difference in stemness-related gene expression levels between control and 20 and 40 μM P8-treated human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) was assessed. The relative gene expression levels were analyzed in triplicate and normalized to endogenous GAPDH. Data are presented mean±SD. Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparison tests. *p<0.05, ***p<0.0001 vs. control group for each time. n=3 per sample. (B-D) The expression of stemness-associated proteins (OCT4, SOX2, and SSEA-4) was visualized using immunofluorescence staining. Samples were captured using a confocal microscope (scale bar=20 μm). The intensity of OCT4, SOX2, and SSEA-4 fluorescence was determined based on the mean fluorescence intensity using ImageJ. Data are presented mean±SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. **p<0.01, ***p<0.001, ****p<0.0001 vs. control group. n=3 per sample.

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  Fig. 4 Effect of P8 on migration and wound healing in HaCaT cells. (A) Confluent monolayer cultures of control and P8-treated HaCaT cells were scratched using a sterile pipette tip at 0 hour. Representative images of migration at 24, 48, and 72 hours after scratching (scale bar=500 μm). (B) Graph showing the HaCaT cell wound-closure rate, obtained using T Scratch software. Data are presented mean±SD. Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparison tests. *p<0.05, ****p<0.0001 vs. control group for each time. n=3 per sample. (C) Control and P8 protein-treated HaCaT cells were cultured under the indicated conditions for 72 hours. Cell lysates were subjected to western blot analysis using antibodies against p-ERK, p-JNK, and β-actin.

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  Fig. 5 MAPK signaling pathway in stem cells. (A) Hypothetical model of signaling pathways in human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) cultured under P8-treatment conditions. P8 administration to hWJ-MSCs resulted in increased phosphorylation of ERK, which increased the expression of YAP and stimulated the expression of Cyclin D1. (B) Summary of effects of P8-treatment in stem cells. P8 protein can increase cell proliferation, stemness, migration and cell cycle when treated to stem cells.

Reference

References

1. Dempsey E, Corr SC. 2022; Lactobacillus spp. for gastrointestinal health: current and future perspectives. Front Immunol. 13:840245. DOI: 10.3389/fimmu.2022.840245. PMID: 35464397. PMCID: PMC9019120.

2. Capurso L. 2019; Thirty years of Lactobacillus rhamnosus GG: a review. J Clin Gastroenterol. 53 Suppl 1:S1–S41. DOI: 10.1097/MCG.0000000000001170. PMID: 30741841.

3. Yan F, Polk DB. 2012; Characterization of a probiotic-derived soluble protein which reveals a mechanism of preventive and treatment effects of probiotics on intestinal inflammatory diseases. Gut Microbes. 3:25–28. DOI: 10.4161/gmic.19245. PMID: 22356855. PMCID: PMC3337122.

4. Fong FLY, Kirjavainen PV, El-Nezami H. 2016; Immunomodulation of Lactobacillus rhamnosus GG (LGG)-derived soluble factors on antigen-presenting cells of healthy blood donors. Sci Rep. 6:22845. DOI: 10.1038/srep22845. PMID: 26961406. PMCID: PMC4785377.

5. Shen X, Liu L, Peek RM, et al. 2018; Supplementation of p40, a Lactobacillus rhamnosus GG-derived protein, in early life promotes epidermal growth factor receptor-dependent intestinal development and long-term health outcomes. Mucosal Immunol. 11:1316–1328. DOI: 10.1038/s41385-018-0034-3. PMID: 29875401. PMCID: PMC6162144.

6. Li Y, Yang S, Lun J, et al. 2020; Inhibitory effects of the Lactobacillus rhamnosus GG effector protein HM0539 on inflammatory response through the TLR4/MyD88/NF-кB axis. Front Immunol. 11:551449. DOI: 10.3389/fimmu.2020.551449. PMID: 33123130. PMCID: PMC7573360.

7. An BC, Hong S, Park HJ, et al. 2019; Anti-colorectal cancer effects of probiotic-derived p8 protein. Genes (Basel). 10:624. DOI: 10.3390/genes10080624. PMID: 31430963. PMCID: PMC6723380.

8. An BC, Ahn JY, Kwon D, et al. 2023; Anti-cancer roles of probiotic-derived P8 protein in colorectal cancer cell line DLD-1. Int J Mol Sci. 24:9857. DOI: 10.3390/ijms24129857. PMID: 37373005. PMCID: PMC10298382.

9. Berebichez-Fridman R, Montero-Olvera PR. 2018; Sources and clinical applications of mesenchymal stem cells: state-of-the-art review. Sultan Qaboos Univ Med J. 18:e264–e277. DOI: 10.18295/squmj.2018.18.03.002. PMID: 30607265. PMCID: PMC6307657.

10. Musiał-Wysocka A, Kot M, Sułkowski M, Badyra B, Majka M. 2019; Molecular and functional verification of Wharton's jelly mesenchymal stem cells (WJ-MSCs) pluripotency. Int J Mol Sci. 20:1807. DOI: 10.3390/ijms20081807. PMID: 31013696. PMCID: PMC6515095.

11. Marino L, Castaldi MA, Rosamilio R, et al. 2019; Mesenchymal stem cells from the Wharton's jelly of the human umbilical cord: biological properties and therapeutic potential. Int J Stem Cells. 12:218–226. DOI: 10.15283/ijsc18034. PMID: 31022994. PMCID: PMC6657936.

12. Drobiova H, Sindhu S, Ahmad R, Haddad D, Al-Mulla F, Al Madhoun A. 2023; Wharton's jelly mesenchymal stem cells: a concise review of their secretome and prospective clinical applications. Front Cell Dev Biol. 11:1211217. DOI: 10.3389/fcell.2023.1211217. PMID: 37440921. PMCID: PMC10333601.

13. Yu M, Qin K, Fan J, et al. 2023; The evolving roles of Wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities. Genes Dis. 11:101026. DOI: 10.1016/j.gendis.2023.04.042. PMID: 38292186. PMCID: PMC10825312.

14. Liang X, Ding Y, Lin F, et al. 2019; Overexpression of ERBB4 rejuvenates aged mesenchymal stem cells and enhances angiogenesis via PI3K/AKT and MAPK/ERK pathways. FASEB J. 33:4559–4570. DOI: 10.1096/fj.201801690R. PMID: 30566395.

15. Ou WB, Lundberg MZ, Zhu S, et al. 2021; YWHAE-NUTM2 oncoprotein regulates proliferation and cyclin D1 via RAF/MAPK and Hippo pathways. Oncogenesis. 10:37. DOI: 10.1038/s41389-021-00327-w. PMID: 33947829. PMCID: PMC8097009.

16. Ohgushi M, Minaguchi M, Sasai Y. 2015; Rho-signaling-directed YAP/TAZ activity underlies the long-term survival and expansion of human embryonic stem cells. Cell Stem Cell. 17:448–461. DOI: 10.1016/j.stem.2015.07.009. PMID: 26321201.

17. Abbaszadeh H, Ghorbani F, Derakhshani M, et al. 2020; Regenerative potential of Wharton's jelly-derived mesenchymal stem cells: a new horizon of stem cell therapy. J Cell Physiol. 235:9230–9240. DOI: 10.1002/jcp.29810. PMID: 32557631.

18. De Pessemier B, Grine L, Debaere M, Maes A, Paetzold B, Callewaert C. 2021; Gut-skin axis: current knowledge of the interrelationship between microbial dysbiosis and skin conditions. Microorganisms. 9:353. DOI: 10.3390/microorganisms9020353. PMID: 33670115. PMCID: PMC7916842.

19. Brandi J, Cheri S, Manfredi M, et al. 2020; Exploring the wound healing, anti-inflammatory, anti-pathogenic and proteomic effects of lactic acid bacteria on keratinocytes. Sci Rep. 10:11572. DOI: 10.1038/s41598-020-68483-4. PMID: 32665600. PMCID: PMC7360600.

20. Gao T, Wang X, Li Y, Ren F. 2023; Jul. 13. The role of probiotics in skin health and related gut-skin axis: a review. Nutrients. 15(14):3123. DOI: 10.3390/nu15143123. PMID: 37513540. PMCID: PMC10385652.

21. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. 2022; ColabFold: making protein folding accessible to all. Nat Methods. 19:679–682. DOI: 10.1038/s41592-022-01488-1. PMID: 35637307. PMCID: PMC9184281.

22. Lim KM, Dayem AA, Choi Y, et al. 2021; High therapeutic and esthetic properties of extracellular vesicles produced from the stem cells and their spheroids cultured from ocular surgery-derived waste orbicularis oculi muscle tissues. Antioxidants (Basel). 10:1292. DOI: 10.3390/antiox10081292. PMID: 34439540. PMCID: PMC8389225.

23. Livak KJ, Schmittgen TD. 2001; Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. DOI: 10.1006/meth.2001.1262. PMID: 11846609.

24. Han X, Lee A, Huang S, Gao J, Spence JR, Owyang C. 2019; Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids. Gut Microbes. 10:59–76. DOI: 10.1080/19490976.2018.1479625. PMID: 30040527. PMCID: PMC6363076.

25. Khailova L, Baird CH, Rush AA, Barnes C, Wischmeyer PE. 2017; Lactobacillus rhamnosus GG treatment improves intestinal permeability and modulates inflammatory response and homeostasis of spleen and colon in experimental model of Pseudomonas aeruginosa pneumonia. Clin Nutr. 36:1549–1557. DOI: 10.1016/j.clnu.2016.09.025. PMID: 27745813. PMCID: PMC5641477.

26. Cai S, Kandasamy M, Rahmat JN, et al. 2016; Lactobacillus rhamnosus GG activation of dendritic cells and neutrophils depends on the dose and time of exposure. J Immunol Res. 2016:7402760. DOI: 10.1155/2016/7402760. PMID: 27525288. PMCID: PMC4971325.

27. Yan F, Polk DB. 2020; Probiotics and probiotic-derived functional factors-mechanistic insights into applications for intestinal homeostasis. Front Immunol. 11:1428. DOI: 10.3389/fimmu.2020.01428. PMID: 32719681. PMCID: PMC7348054.

28. Mikulic J, Longet S, Favre L, Benyacoub J, Corthesy B. 2017; Secretory IgA in complex with Lactobacillus rhamnosus potentiates mucosal dendritic cell-mediated Treg cell differentiation via TLR regulatory proteins, RALDH2 and secretion of IL-10 and TGF-β. Cell Mol Immunol. 14:546–556. DOI: 10.1038/cmi.2015.110. PMID: 26972771. PMCID: PMC5518813.

29. Liau LL, Ruszymah BHI, Ng MH, Law JX. 2020; Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells. Curr Res Transl Med. 68:5–16. DOI: 10.1016/j.retram.2019.09.001. PMID: 31543433.

30. Daltro SRT, Meira CS, Santos IP, Ribeiro Dos Santos R, Soares MBP. 2020; Mesenchymal stem cells and atopic dermatitis: a review. Front Cell Dev Biol. 8:326. DOI: 10.3389/fcell.2020.00326. PMID: 32478072. PMCID: PMC7240073.

31. Yang J, Xiao M, Ma K, et al. 2023; Therapeutic effects of mesenchymal stem cells and their derivatives in common skin inflammatory diseases: atopic dermatitis and psoriasis. Front Immunol. 14:1092668. DOI: 10.3389/fimmu.2023.1092668. PMID: 36891306. PMCID: PMC9986293.

32. Zhang W, Liu HT. 2002; MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12:9–18. DOI: 10.1038/sj.cr.7290105. PMID: 11942415.

33. Liu D, Yi C, Fong CC, et al. 2014; Activation of multiple signaling pathways during the differentiation of mesenchymal stem cells cultured in a silicon nanowire microenvironment. Nanomedicine. 10:1153–1163. DOI: 10.1016/j.nano.2014.02.003. PMID: 24566272.

34. Mathew-Steiner SS, Roy S, Sen CK. 2021; Collagen in wound healing. Bioengineering (Basel). 8:63. DOI: 10.3390/bioengineering8050063. PMID: 34064689. PMCID: PMC8151502.

35. Singh D, Rai V, Agrawal DK. 2023; Regulation of collagen I and collagen III in tissue injury and regeneration. Cardiol Cardiovasc Med. 7:5–16. DOI: 10.26502/fccm.92920302. PMID: 36776717. PMCID: PMC9912297.

36. Park S, Choe M, Yeo H, et al. 2018; Yes-associated protein mediates human embryonic stem cell-derived cardiomyocyte proliferation: involvement of epidermal growth factor receptor signaling. J Cell Physiol. 233:7016–7025. DOI: 10.1002/jcp.26625. PMID: 29693249.

37. Yang K, Wu Y, Cheng P, et al. 2016; YAP and ERK mediated mechanical strain-induced cell cycle progression through RhoA and cytoskeletal dynamics in rat growth plate chondrocytes. J Orthop Res. 34:1121–1129. DOI: 10.1002/jor.23138. PMID: 26694636.

38. Pan C, Hao X, Deng X, et al. 2024; The roles of Hippo/YAP signaling pathway in physical therapy. Cell Death Discov. 10:197. DOI: 10.1038/s41420-024-01972-x. PMID: 38670949. PMCID: PMC11053014.

39. Tchakarska G, Sola B. 2020; The double dealing of cyclin D1. Cell Cycle. 19:163–178. DOI: 10.1080/15384101.2019.1706903. PMID: 31885322. PMCID: PMC6961668.

40. Wang Z. 2021; Regulation of cell cycle progression by growth factor-induced cell signaling. Cells. 10:3327. DOI: 10.3390/cells10123327. PMID: 34943835. PMCID: PMC8699227.