Inhibition of sphingosine kinase 2 attenuates hypertrophic scar formation via upregulation of Smad7 in human hypertrophic scar fibroblasts

Zeng,Jian; Jiang,Bin; Xiao,Xia; Zhang,Rou

doi:10.3892/mmr.2020.11313

Inhibition of sphingosine kinase 2 attenuates hypertrophic scar formation via upregulation of Smad7 in human hypertrophic scar fibroblasts

Authors:
- Jian Zeng
- Bin Jiang
- Xia Xiao
- Rou Zhang
View Affiliations

Affiliations: Department of Medical Cosmetology, The Second Affiliated Hospital, University of South China, Hengyang, Hunan 421001, P.R. China
Published online on: July 9, 2020 https://doi.org/10.3892/mmr.2020.11313
Pages: 2573-2582
Copyright: © Zeng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aims of the present study were to investigate the role of sphingosine kinase 2 (Sphk2) in hypertrophic scar (HS) formation and its underlying mechanisms. The expression levels of Sphk2 and Smad7 in HS tissues and healthy skin tissues of patients undergoing plastic surgery were determined using immunohistochemical staining. Subsequently, the expression levels of Sphk2 and collagen I in human embryonic skin fibroblasts (control) and human HS fibroblasts (HSF) were detected using western blot analysis and immunofluorescence assay, respectively. Following Sphk2 silencing, Smad7 overexpression or both Sphk2 and Smad7 silencing, HSF proliferative ability was assessed using Cell Counting Kit‑8 assay and proliferation‑associated proteins were evaluated using western blot analysis. In addition, the level of apoptosis in HSF was assessed using flow cytometry and expression levels of apoptotic‑associated proteins were determined using western blotting. Furthermore, the expression levels of collagen I and proteins in the TGF‑β1/Smad signaling pathway were detected using western blot analysis. The results indicated that the expression of Sphk2 was significantly increased, while Smad7 expression was decreased in HS tissue. Moreover, the upregulation of Sphk2 and collagen I expression levels was identified in HSF. The present results also indicated that Sphk2 silencing or Smad7 overexpression inhibited proliferation, but promoted apoptosis of HSF, coupled with changes in the expression levels of proliferation‑associated proteins, with an increase in p21 and a decrease in cyclin D1 expression levels, and apoptosis‑associated proteins, with an increase in Bax and cleaved caspase‑3, and a decrease in Bcl‑2, which were reversed following transfection with both Sphk2 and Smad7 using small interfering RNA in HSF. In addition, the expression levels of transforming growth factor‑β1, phosphorylated (p)‑Smad2, p‑Smad3 and collagen I were reduced following Sphk2 silencing or Smad7 overexpression, which were abolished by silencing both Sphk2 and Smad7. Collectively, the present results indicated that inhibition of Sphk2 attenuated HS formation via upregulation of Smad7 expression, thus Sphk2 may serve as a potential therapeutic target for the treatment of HS.

View Figures

View References

1	Miletta NR, Donelan MB and Hivnor CM: Management of trauma and burn scars: The dermatologist's role in expanding patient access to care. Cutis. 100:18–20. 2017.PubMed/NCBI
2	van der Veer WM, Bloemen MC, Ulrich MM, Molema G, van Zuijlen PP, Middelkoop E and Niessen FB: Potential cellular and molecular causes of hypertrophic scar formation. Burns. 35:15–29. 2009. View Article : Google Scholar : PubMed/NCBI
3	Sideek MA, Teia A, Kopecki Z, Cowin AJ and Gibson MA: Co-localization of LTBP-2 with FGF-2 in fibrotic human keloid and hypertrophic scar. J Mol Histol. 47:35–45. 2016. View Article : Google Scholar : PubMed/NCBI
4	Zuccaro J, Ziolkowski N and Fish J: A systematic review of the effectiveness of laser therapy for hypertrophic burn scars. Clin Plast Surg. 44:767–779. 2017. View Article : Google Scholar : PubMed/NCBI
5	Willows BM, Ilyas M and Sharma A: Laser in the management of burn scars. Burns. 43:1379–1389. 2017. View Article : Google Scholar : PubMed/NCBI
6	Gras C, Ratuszny D, Hadamitzky C, Zhang H, Blasczyk R and Figueiredo C: miR-145 contributes to hypertrophic scarring of the skin by inducing myofibroblast activity. Mol Med. 21:296–304. 2015. View Article : Google Scholar : PubMed/NCBI
7	Wang XQ, Song F and Liu YK: Hypertrophic scar regression is linked to the occurrence of endothelial dysfunction. PLoS One. 12:e01766812017. View Article : Google Scholar : PubMed/NCBI
8	Liu B, Guo Z and Gao W: miR-181b-5p promotes proliferation and inhibits apoptosis of hypertrophic scar fibroblasts through regulating the MEK/ERK/p21 pathway. Exp Ther Med. 17:1537–1544. 2019.PubMed/NCBI
9	Pyne NJ, Dubois G and Pyne S: Role of sphingosine 1-phosphate and lysophosphatidic acid in fibrosis. Biochim Biophys Acta. 1831:228–238. 2013. View Article : Google Scholar : PubMed/NCBI
10	Schwalm S, Pfeilschifter J and Huwiler A: Sphingosine-1- phosphate: A Janus-faced mediator of fibrotic diseases. Biochim Biophys Acta. 1831:239–250. 2013. View Article : Google Scholar : PubMed/NCBI
11	Ravichandran S, Finlin BS, Kern PA and Özcan S: Sphk2(−/-) mice are protected from obesity and insulin resistance. Biochim Biophys Acta Mol Basis Dis. 1865:570–576. 2019. View Article : Google Scholar : PubMed/NCBI
12	Schwalm S, Timcheva TM, Filipenko I, Ebadi M, Hofmann LP, Zangemeister-Wittke U, Pfeilschifter J and Huwiler A: Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts. Biol Chem. 396:813–825. 2015. View Article : Google Scholar : PubMed/NCBI
13	Schwalm S, Beyer S, Frey H, Haceni R, Grammatikos G, Thomas D, Geisslinger G, Schaefer L, Huwiler A and Pfeilschifter J: Sphingosine kinase-2 deficiency ameliorates kidney fibrosis by up-regulating Smad7 in a mouse model of unilateral ureteral obstruction. Am J Pathol. 187:2413–2429. 2017. View Article : Google Scholar : PubMed/NCBI
14	Zhou J, Chen J and Yu H: Targeting sphingosine kinase 2 by ABC294640 inhibits human skin squamous cell carcinoma cell growth. Biochem Biophys Res Commun. 497:535–542. 2018. View Article : Google Scholar : PubMed/NCBI
15	Shin SH, Cho KA, Hahn S, Lee Y, Kim YH, Woo SY, Ryu KH, Park WJ and Park JW: Inhibiting sphingosine kinase 2 derived-sphingosine-1-phosphate ameliorates psoriasis-like skin disease via blocking Th17 differentiation of naive CD4 T lymphocytes in mice. Acta Derm Venereol. 99:594–601. 2019. View Article : Google Scholar : PubMed/NCBI
16	Shi J, Li J, Guan H, Cai W, Bai X, Fang X, Hu X, Wang Y, Wang H, Zheng Z, et al: Anti-fibrotic actions of interleukin-10 against hypertrophic scarring by activation of PI3K/AKT and STAT3 signaling pathways in scar-forming fibroblasts. PLoS One. 9:e982282014. View Article : Google Scholar : PubMed/NCBI
17	Zuo J, Chen Z, Zhong X, Lan W, Kuang Y and Huang D: FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis, and collagen expression. Connect Tissue Res. 59:120–128. 2018. View Article : Google Scholar : PubMed/NCBI
18	Chen H, Xu Y, Yang G, Zhang Q, Huang X, Yu L and Dong X: Mast cell chymase promotes hypertrophic scar fibroblast proliferation and collagen synthesis by activating TGF-β1/Smads signaling pathway. Exp Ther Med. 14:4438–4442. 2017.PubMed/NCBI
19	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
20	Xiao Y, Xu D, Song H, Shu F, Wei P, Yang X, Zhong C, Wang X, Müller WE, Zheng Y, et al: Cuprous oxide nanoparticles reduces hypertrophic scarring by inducing fibroblast apoptosis. Int J Nanomedicine. 14:5989–6000. 2019. View Article : Google Scholar : PubMed/NCBI
21	Zhou Y, Zhao Y, Du H, Suo Y, Chen H, Li H, Liang X, Li Q and Huang X: Downregulation of CFTR is involved in the formation of hypertrophic scars. Biomed Res Int. 2020:95262892020.PubMed/NCBI
22	Song Y, Guo B, Ma S, Chang P and Tao K: Naringin suppresses the growth and motility of hypertrophic scar fibroblasts by inhibiting the kinase activity of Akt. Biomed Pharmacother. 105:1291–1298. 2018. View Article : Google Scholar : PubMed/NCBI
23	Zhou X, Xie Y, Xiao H, Deng X, Wang Y, Jiang L, Liu C and Zhou R: MicroRNA-519d inhibits proliferation and induces apoptosis of human hypertrophic scar fibroblasts through targeting Sirtuin 7. Biomed Pharmacother. 100:184–190. 2018. View Article : Google Scholar : PubMed/NCBI
24	Qiu W, Yang Z, Fan Y and Zheng Q: MicroRNA-613 inhibits cell growth, migration and invasion of papillary thyroid carcinoma by regulating SphK2. Oncotarget. 7:39907–39915. 2016. View Article : Google Scholar : PubMed/NCBI
25	Zhang L, Liu X, Zuo Z, Hao C and Ma Y: Sphingosine kinase 2 promotes colorectal cancer cell proliferation and invasion by enhancing MYC expression. Tumor Biol. 37:8455–8460. 2016. View Article : Google Scholar
26	Leili H, Nasser S, Nadereh R, Siavoush D and Pouran K: Sphingosine kinase-2 inhibitor ABC294640 enhances doxorubicin-induced apoptosis of NSCLC cells via altering survivin expression. Drug Res (Stuttg). 68:45–53. 2018. View Article : Google Scholar : PubMed/NCBI
27	Xiao Y, Liu R, Li X, Gurley EC, Hylemon PB, Lu Y, Zhou H and Cai W: Long noncoding RNA H19 contributes to cholangiocyte proliferation and cholestatic liver fibrosis in biliary atresia. Hepatology. 70:1658–1673. 2019. View Article : Google Scholar : PubMed/NCBI
28	Wang XC, Wang T, Zhang Y, Wang LL, Zhao RY and Tan W: Tacrolimus inhibits proliferation and induces apoptosis by decreasing survivin in scar fibroblasts after glaucoma surgery. Eur Rev Med Pharmacol Sci. 22:2934–2940. 2018.PubMed/NCBI
29	Li XY, Li T, Li XJ, Wang JN and Chen Z: TSG-6 induces apoptosis of human hypertrophic scar fibroblasts via activation of the Fas/FasL signalling pathway. Folia Biol (Praha). 64:173–181. 2018.PubMed/NCBI
30	Zhang G, Zheng H, Zhang G, Cheng R, Lu C, Guo Y and Zhao G: MicroRNA-338-3p suppresses cell proliferation and induces apoptosis of non-small-cell lung cancer by targeting sphingosine kinase 2. Cancer Cell Int. 17:462017. View Article : Google Scholar : PubMed/NCBI
31	Fan X, Yuan J, Xie J, Pan Z, Yao X, Sun X, Zhang P and Zhang L: Long non-protein coding RNA DANCR functions as a competing endogenous RNA to regulate osteoarthritis progression via miR-577/SphK2 axis. Biochem Biophys Res Commun. 500:658–664. 2018. View Article : Google Scholar : PubMed/NCBI
32	Ma L, Li LY and Zhao TL: Anti-inflammatory effects of ginsenoside Rg3 on the hypertrophic scar formation via the NF-κB/IκB signaling pathway in rabbit ears. Pharmazie. 75:102–106. 2020.PubMed/NCBI
33	Volk SW, Wang Y, Mauldin EA, Liechty KW and Adams SL: Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing. Cells Tissues Organs. 194:25–37. 2011. View Article : Google Scholar : PubMed/NCBI
34	Zhou R, Zhang Q, Zhang Y, Fu S and Wang C: Aberrant miR-21 and miR-200b expression and its pro-fibrotic potential in hypertrophic scars. Exp Cell Res. 339:360–366. 2015. View Article : Google Scholar : PubMed/NCBI
35	Nicholas SE, Rowsey TG, Priyadarsini S, Mandal NA and Karamichos D: Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma. PLoS One. 12:e01823902017. View Article : Google Scholar : PubMed/NCBI
36	Zhang Y, Shan S, Wang J, Cheng X, Yi B, Zhou J and Li Q: Galangin inhibits hypertrophic scar formation via ALK5/Smad2/3 signaling pathway. Mol Cell Biochem. 413:109–118. 2016. View Article : Google Scholar : PubMed/NCBI
37	Zhao JC, Zhang BR, Shi K, Wang J, Yu QH and Yu JA: Lower energy radial shock wave therapy improves characteristics of hypertrophic scar in a rabbit ear model. Exp Ther Med. 15:933–939. 2018.PubMed/NCBI
38	Chen H, Xu Y, Yang G, Zhang Q, Huang X, Yu L and Dong X: Mast cell chymase promotes hypertrophic scar fibroblast proliferation and collagen synthesis by activating TGF-β1/Smads signaling pathway. Exp Ther Med. 14:4438–4442. 2017.PubMed/NCBI
39	Seki N, Toh U, Kawaguchi K, Ninomiya M, Koketsu M, Watanabe K, Aoki M, Fujii T, Nakamura A, Akagi Y, et al: Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways. J Cell Biochem. 113:2346–2355. 2012. View Article : Google Scholar : PubMed/NCBI
40	Wu SP, Yang Z, Li FR, Liu XD, Chen HT and Su DN: Smad7-overexpressing rat BMSCs inhibit the fibrosis of hepatic stellate cells by regulating the TGF-β1/Smad signaling pathway. Exp Ther Med. 14:2568–2576. 2017. View Article : Google Scholar : PubMed/NCBI
41	Tang B, Zhu B, Liang Y, Bi L, Hu Z, Chen B, Zhang K and Zhu J: Asiaticoside suppresses collagen expression and TGF-β/Smad signaling through inducing Smad7 and inhibiting TGF-βRI and TGF-βRII in keloid fibroblasts. Arch Dermatol Res. 303:563–572. 2011. View Article : Google Scholar : PubMed/NCBI