Overexpression of HIC1 plays a protective effect on renal cell injury caused by lipopolysaccharide by inhibiting IL-6/STAT3 pathway

Sepsis is a life-threatening condition that can even occur due to an infection. Systemic inflammatory response syndrome acts a pivotal role in acute kidney injury (AKI). Although great advancements have been achieved for treating sepsis-induced AKI, its prognosis and pathophysiology remain unclear. In order to gain insights into the relevant role of hypermethylated in cancer 1 (HIC1) in AKI, a cellular model of AKI caused by sepsis was performed in lipopolysaccharide (LPS)-treated human kidney 2 (HK-2) cell line. The overexpression vector pcDNA3.1-HIC1 was transfected into HK-2 cell line to examine the effects of HIC1 on LPS-treated HK-2 cell line. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot and enzyme-linked immunosorbent assay (ELISA) assays were performed to examine the alterations in the expression levels of HIC1, cell apoptosis, or inflammation-related biomarkers. The apoptotic rate of HK-2 cell line was measured by flow cytometry. This study suggested that LPS treatment downregulated HIC1 and inhibited HK-2 cell viability, whereas HIC1 overexpressing reversed these effects. Importantly, HIC1 has a protective effect on LPS-induced cellular apoptosis and inflammatory response. Moreover, overexpression of HIC1 suppressed the LPS-induced activation of IL-6/STAT3 signaling pathway in HK-2 cell line. HIC1 protects HK-2 cell line against LPS-induced damage, which was partly through the inhibition of IL-6/STAT3 signaling pathways.

Sepsis; Renal cell injury; HIC1 cell line; IL-6/STAT3; LPS

[1] Arina P, Singer M. Pathophysiology of sepsis. Current Opinion in Anaesthesiology. 2021; 34: 77–84.

[2] Schlapbach LJ, Trück J, Roger T. The immunology of sepsis—understanding host susceptibility, pathogenesis of disease, and avenues for future treatment. Frontiers in Immunology. 2020; 11: 1263.

[3] Mayeux PR, MacMillan-Crow LA. Pharmacological targets in the renal peritubular microenvironment: implications for therapy for sepsis-induced acute kidney injury. Pharmacology & Therapeutics. 2012; 134: 139–155.

[4] Gonsalez SR, Cortês AL, Silva RCD, Lowe J, Prieto MC, Silva Lara LD. Acute kidney injury overview: from basic findings to new prevention and therapy strategies. Pharmacology & Therapeutics. 2019; 200: 1–12.

[5] Zarjou A, Agarwal A. Sepsis and acute kidney injury. Journal of the American Society of Nephrology. 2011; 22: 999–1006.

[6] White LE, Hassoun HT. Inflammatory mechanisms of organ crosstalk during ischemic acute kidney injury. International Journal of Nephrology. 2012; 2012: 505197.

[7] Tan RZ, Wang C, Deng C, Zhong X, Yan Y, Luo Y, et al. Quercetin protects against cisplatin‐induced acute kidney injury by inhibiting Mincle/Syk/NF-κB signaling maintained macrophage inflammation. Phytotherapy Research. 2020; 34: 139–152.

[8] Inagi R. Endoplasmic reticulum stress as a progression factor for kidney injury. Current Opinion in Pharmacology. 2010; 10: 156–165.

[9] Gomez H, Ince C, De Backer D, Pickkers P, Payen D, Hotchkiss J, et al. A unified theory of sepsis-induced acute kidney injury inflammation, microcirculatory dysfunction, bioenergetics, and the tubular cell adaptation to injury. Shock. 2014; 41: 3–11.

[10] Fleuriel C, Touka M, Boulay G, Guérardel C, Rood BR, Leprince D. HIC1 (Hypermethylated in Cancer 1) epigenetic silencing in tumors. The International Journal of Biochemistry & Cell Biology. 2009; 41: 26–33.

[11] Briones VR, Chen S, Riegel AT, Lechleider RJ. Mechanism of fibroblast growth factor-binding protein 1 repression by TGF-beta. Biochemical and Biophysical Research Communications. 2006; 345: 595–601.

[12] Tseng RC, Lee CC, Hsu HS, Tzao C, Wang YC. Distinct HIC1-SIRT1-p53 loop deregulation in lung squamous carcinoma and adenocarcinoma patients. Neoplasia. 2009; 11: 763–770.

[13] Kim MK, Jeon BN, Koh DI, Kim KS, Park SY, Yun CO, et al. Regulation of the cyclin-dependent kinase inhibitor 1A Gene (CDKN1A) by the repressor BOZF1 through inhibition of p53 acetylation and transcription factor Sp1 binding. Journal of Biological Chemistry. 2013; 288: 7053–7064.

[14] Hu B, Zhang K, Li S, Li H, Yan Z, Huang L, et al. HIC1 attenuates invasion and metastasis by inhibiting the IL-6/STAT3 signalling pathway in human pancreatic cancer. Cancer Letters. 2016; 376: 387–398.

[15] Zeng S, Wu X, Chen X, Xu H, Zhang T, Xu Y. Hypermethylated in cancer 1 (HIC1) mediates high glucose induced ROS accumulation in renal tubular epithelial cells by epigenetically repressing SIRT1 transcription. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms. 2018; 1861: 917–927.

[16] Shuai K. Modulation of STAT signaling by STAT-interacting proteins. Oncogene. 2000; 19: 2638–2644.

[17] Yang J, Stark GR. Roles of unphosphorylated STATs in signaling. Cell Research. 2008; 18: 443–451.

[18] Morris R, Kershaw NJ, Babon JJ. The molecular details of cytokine signaling via the JAK/STAT pathway. Protein Science. 2018; 27: 1984–2009.

[19] Sirkisoon SR, Carpenter RL, Rimkus T, Anderson A, Harrison A, Lange AM, et al. Interaction between STAT3 and GLI1/tGLI1 oncogenic transcription factors promotes the aggressiveness of triple-negative breast cancers and HER2-enriched breast cancer. Oncogene. 2018; 37: 2502–2514.

[20] Unver N, McAllister F. IL-6 family cytokines: key inflammatory mediators as biomarkers and potential therapeutic targets. Cytokine & Growth Factor Reviews. 2018; 41: 10–17.

[21] Wang L, Cao L, Wang H, Liu B, Zhang Q, Meng Z, et al. Cancer-associated fibroblasts enhance metastatic potential of lung cancer cells through IL-6/STAT3 signaling pathway. Oncotarget. 2017; 8: 76116–76128.

[22] Ning F, Zheng H, Tian H, Wang T, Hao D, Han S, et al. Research on effect of adiponectin on sepsis-induced lung injury in rats through IL-6/STAT3 signaling pathway. Panminerva Medica. 2020; 62: 184–186.

[23] Li Y, Yao M, Wu T, Zhang L, Wang Y, Chen L, et al. Loss of hypermethylated in cancer 1 (HIC1) promotes lung cancer progression. Cellular Signalling. 2019; 53: 162–169.

[24] Pianta TJ, Gobe GC, Owens EP, Endre ZH. Overview of pathophysiology of acute kidney injury: human evidence, mechanisms, pathological correlations and biomarkers and animal models. In: Waikar S., Murray P., Singh A. (eds) Core Concepts in Acute Kidney Injury (pp. 45–67). 1st edn. Springer: New York, NY. 2018.

[25] Chen WY, Wang DH, Yen RC, Luo J, Gu W, Baylin SB. Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage responses. Cell. 2005; 123: 437–448.

[26] Liao Y, Guo S, Chen Y, Cao D, Xu H, Yang C, et al. VSIG4 expression on macrophages facilitates lung cancer development. Laboratory Investi-gation. 2014; 94: 706–715.

[27] Lin YM, Wang CM, Jeng JC, Leprince D, Shih HM. HIC1 interacts with and modulates the activity of STAT3. Cell Cycle. 2013; 12: 2266–2276.

[28] Wu W, Zhang L, Lin J, Huang H, Shi B, Lin X, et al. Hypermethylation of the HIC1 promoter and aberrant expression of HIC1/SIRT1 contribute to the development of thyroid papillary carcinoma. Oncotarget. 2016; 7: 84416–84427.

[29] Kuwabara T, Imajoh-Ohmi S. LPS-induced apoptosis is dependent upon mitochondrial dysfunction. Apoptosis. 2004; 9: 467–474.

[30] Liu S, Gallo DJ, Green AM, Williams DL, Gong X, Shapiro RA, et al. Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide. Infection and Immunity. 2002; 70: 3433–3442.

[31] Lin M, Rikihisa Y. Ehrlichia chaffeensisdownregulates surface Toll-like receptors 2/4, CD14 and transcription factors PU.1 and inhibits lipopolysaccharide activation of NF-kappa B, ERK 1/2 and p38 MAPK in host monocytes. Cellular Microbiology. 2004; 6: 175–186.

[32] Li N, Grivennikov SI, Karin M. The unholy trinity: inflammation, cytokines, and STAT3 shape the cancer microenvironment. Cancer Cell. 2011; 19: 429–431.

[33] Gupta S, Jain A, Syed SN, Snodgrass RG, Pflüger-Müller B, Leisegang MS, et al. IL-6 augments IL-4-induced polarization of primary human macrophages through synergy of STAT3, STAT6 and BATF transcription factors. Oncoimmunology. 2018; 7: e1494110.

[34] Ranganathan P, Jayakumar C, Ramesh G. Proximal tubule-specific overexpression of netrin-1 suppresses acute kidney injury-induced interstitial fibrosis and glomerulosclerosis through suppression of IL-6/STAT3 signaling. American Journal of Physiology-Renal Physiology. 2013; 304: F1054–F1065.

[35] Xiao Y, Yang N, Zhang Q, Wang Y, Yang S, Liu Z. Pentraxin 3 inhibits acute renal injury-induced interstitial fibrosis through suppression of IL-6/Stat3 pathway. Inflammation. 2014; 37: 1895–1901.

[36] Laudisi F, Cherubini F, Monteleone G, Stolfi C. STAT3 interactors as potential therapeutic targets for cancer treatment. International Journal of Molecular Sciences. 2018; 19: 1787.

[37] Wang X, Wang Y, Xiao G, Wang J, Zu L, Hao M, et al. Hypermethylated in cancer 1(HIC1) suppresses non-small cell lung cancer progression by targeting interleukin-6/Stat3 pathway. Oncotarget. 2016; 7: 30350–30364.

[38] Sun X, Qu Q, Lao Y, Zhang M, Yin X, Zhu H, et al. Tumor suppressor HIC1 is synergistically compromised by cancer-associated fibroblasts and tumor cells through the IL-6/pSTAT3 axis in breast cancer. BMC Cancer. 2019; 19: 1180.