Effects of changes in SHP2 expression on the activation of human hepatic stellate cell LX-2 based on ERK1/2 signaling pathway

Abstract

Abstract: Objective To explore the effects of changes in protein tyrosine phosphatase 2 (SHP2) expression on activation and extracellular signal regulated kinase 1/2 (ERK1/2) in human hepatic stellate cell LX-2. Methods Adenovirus Ad-SHP2 with wild-type SHP2 gene and Ad-shRNA/SHP2 with shRNA targeting SHP2 were introduced into LX-2 cells cultured in vitro, respectively. Application of real-time fluorescence quantitative PCR and Western blot to analyze expression of SHP2 and ERK1/2 in LX-2 cells. Expression of phosphorylated ERK1/2 (p-ERK1/2) in LX-2 cells was analyzed by Western blot analysis. The expression of α-smooth muscle actin (α-SMA), an activated marker of hepatic stellate cells was detected immunocytochemical staining and Western blot. DMEM was used instead of adenovirus to transfect LX-2 cells in control group, the LX-2 cells was transfected with empty virus Ad-GFP in Ad-GFP group. Ad-SHP2 was transfected into LX-2 cells in Ad-shRNA/SHP2 group. Results Wild-type SHP2 and shRNA targeting SHP2 significantly increased and decreased the expression of SHP2 in LX-2 cells (P<0.05). Immunocytochemical staining showed that compared with the positive expression integral optical density (IOD) of α-SMA protein in LX-2 cells in control group and Ad-GFP group (0.073±0.003, 0.074±0.004), that in Ad-shRNA/SHP2 group (0.048±0.003) showed a significant (P<0.05) decrease and in Ad-SHP2 group (0.127±0.004) remarkably (P<0.05) increased. Expression levels of α-SMA and p-ERK1 protein in LX-2 cells of Ad-shRNA/SHP2 group (0.154±0.033, 0.134±0.023) significantly (P<0.05) lowered and in Ad-SHP2 group (0.517±0.036, 0.522±0.053) exhibited a significant (P<0.05) increase compared with those in control group (0.342±0.043, 0.302±0.035) and Ad-GFP group (0.308±0.038, 0.315±0.037). There was no significant difference in ERK1 mRNA and protein expression of LX-2 cells among 4 groups (P<0.05). Conclusion Overexpressed SHP2 can promote activation of LX-2 cells, while low expression of SHP2 can suppress activation of LX-2 cells, and the ERK1/2 signaling pathway mediates the effects of SHP2 on activation of LX-2 cells.

Key words: Human hepatic stellate cell LX-2, SHP2, Hepatic stellate cell activation, ERK1/2

HAO Li-sen, GAO Ying-ying, ZHAN Zong-yuan, LIAN Jun, LIU Tian, WANG Jin-mei, YANG Dong-jie, MAO Jia-qi. Effects of changes in SHP2 expression on the activation of human hepatic stellate cell LX-2 based on ERK1/2 signaling pathway[J]. Chinese Hepatolgy, 2025, 30(12): 1674-1677.

References

[1] Tsuchida T, Friedman S L. Mechanisms of hepatic stellate cell activation [J]. Nat Rev Gastroenterol Hepatol, 2017, 14(7):397-411.
[2] Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression[J]. Nat Rev Gastroenterol Hepatol, 2021, 18(3): 151-166.
[3] Higashi T, Friedman S L, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis [J]. Adv Drug Deliv Rev, 2017, 121: 27-42.
[4] Asmamaw M D, Shi X J, Zhang L R, et al. A comprehensive review of SHP2 and its role in cancer [J]. Cell Oncol (Dordr), 2022, 45(5):729-753.
[5] Zhang J, Zhang F, Niu R. Functions of Shp2 in cancer [J]. J Cell Mol Med, 2015, 19(9): 2075-2083.
[6] Tibaldi E, Zonta F, Bordin L, et al. The tyrosine phosphatase SHP-1 inhibits proliferation of activated hepatic stellate cells by impairing PDGF receptor signaling[J]. Biochim Biophys Acta, 2014, 1843(2): 288-298.
[7] 郝礼森, 宋洁, 吴荣鹏, 等. 蛋白酪氨酸磷酸酶SHP2在四氯化碳诱导的肝纤维化大鼠肝组织中的动态表达[J]. 中华肝脏病杂志, 2021,29(09):844-848.
[8] 郝礼森, 潘恩亮, 季景秀, 等. 含SH2结构域蛋白酪氨酸磷酸酶2表达变化对肝纤维化大鼠肝组织中细胞外信号调节激酶1/2活性的影响[J].陕西医学杂志, 2023,52(12):1642-1647.
[9] Arocho A, Chen B, Ladanyi M, et al. Validation of the 2- DeltaDeltaCt calculation as an alternate method of data analysis for quantitative PCR of BCR- ABL P210 transcripts[J]. Diagn Mol Pathol, 2006, 15(1): 56-61.
[10] 郝礼森, 张晓岚, 李玉林, 等. 大鼠纤维化肝组织中PTEN的动态表达及其与肝星状细胞增殖和活化的关系[J]. 中华肝脏病杂志, 2008, 16(10): 743-747.
[11] 张朋垒, 张明婷, 郝礼森, 等. 四氯化碳诱导的大鼠肝纤维化肝组织中SHP2表达与在体肝星状细胞活化及增殖的关系[J]. 肝脏, 2023,28(05): 549-553.
[12] Xie H, Huang S, Li W, et al. Upregulation of Src homology phosphotyrosyl phosphatase 2 (Shp2) expression in oral cancer and knockdown of Shp2 expression inhibit tumor cell viability and invasion in vitro [J]. Oral Surg Oral Med Oral Pathol Oral Radiol, 2014, 117: 234-242.
[13] Cai Z, Hao X Y, Liu F X. MicroRNA-186 serves as a tumor suppressor in oral squamous cell carcinoma by negatively regulating the protein tyrosine phosphatase SHP2 expression [J]. Arch Oral Biol, 2018, 89: 20-25.
[14] Yuan Y, Fan Y, Gao Z, et al. SHP2 promotes proliferation of breast cancer cells through regulating Cyclin D1 stability via the PI3K/AKT/GSK3β signaling pathway [J]. Cancer Biol Med, 2020, 17(3):707-725.
[15] Hu Z, Fang H, Wang X, et al. Overexpression of SHP2 tyrosine phosphatase promotes the tumorigenesis of breast carcinoma [J]. Oncol Rep, 2014, 32(1): 205-212.
[16] Han T, Xiang D M, Sun W, et al. PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients [J]. J Hepatol, 2015, 63(3): 651-660.
[17] 郝礼森, 展宗媛, 宋洁, 等. 腺病毒介导的shRNA下调SHP2表达对人肝星状细胞LX-2凋亡的影响[J]. 中华肝脏病杂志, 2023, 31(12): 1313-1317.
[18] 郝礼森, 苗笑佳, 宋洁, 等. SHP2过表达抑制人肝星状细胞LX-2凋亡[J]. 中国现代医学杂志, 2024,34(5):32-36.
[19] Guo Y J, Pan W W, Liu S B, et al. ERK/MAPK signalling pathway and tumorigenesis [J]. Exp Ther Med, 2020, 19(3): 1997-2007.
[20] Song Y Y, Liang D, Liu D K, et al. The role of the ERK signaling pathway in promoting angiogenesis for treating ischemic diseases [J]. Front Cell Dev Biol, 2023, 11:1164166.
[21] Naim A, Baig M S. Matrix metalloproteinase-8 (MMP-8) regulates the activation of hepatic stellate cells (HSCs) through the ERK-mediated pathway[J]. Mol Cell Biochem, 2020, 467(1-2): 107-116.
[22] Lucas R M, Luo L, Stow J L. ERK1/2 in immune signalling [J]. Biochem Soc Trans, 2022, 50(5): 1341-1352.