The PNPLA3 I148M variant promotes lipid-induced hepatocyte secretion of CXC chemokines establishing a tumorigenic milieu
The I148M variant of the Patatin-like phospholipase domain-containing 3 (PNPLA3) protein is associated with an increased risk for liver inflammation and hepatocellular carcinoma (HCC), but the underlying mechanism is unknown. We hypothesized that enhanced CXC chemokine secretion mediates hepatic inflammation that accelerates development of HCC. Expandable primary human (upcyte®) hepatocytes and human PLC/PRF/5 hepatoma cells were lentivirally transduced with both PNPLA3 I148M variants and stimulated with lipids. Cytokine levels in culture supernatant and patient sera (n = 80) were analyzed by ELISA. Supernatants were assessed in transmigration experiments, tube formation, and proliferation assays. In vitro, lipid stimulation of transduced hepatocytes dose-dependently induced the production of interleukin-8 and CXCL1 in hepatocytes carrying the PNPLA3 148M variant. In line, sera from PNPLA3 148M-positive patients with alcoholic liver cirrhosis contained higher levels of interleukin-8 and CXCL1 than patients with wild-type PNPLA3. Supernatants from lipid-stimulated hepatocytes with the PNPLA3 148M variant induced enhanced migration of white blood cells, angiogenesis, and cell proliferation in comparison with supernatants from wild-type hepatocytes via CXC receptors 1 and 2. Increased production of interleukin-8 and CXCL1 by hepatocytes carrying the PNPLA3 148M variant contributes to a pro-inflammatory and tumorigenic milieu in patients with alcoholic liver disease.
The PNPLA3 148M variant is associated with cirrhosis and hepatocellular carcinoma.
Lipid stimulation of hepatocytes with this variant induces IL-8 and CXCL1.
Supernatants from hepatocytes with this variant promote migration and angiogenesis.
Sera from patients with this variant contained enhanced levels of IL-8 and CXCL1.
The PNPLA3 148M variant contributes to a tumorigenic milieu via IL-8 and CXCL1.
KeywordsPNPLA3 148M IL-8 CXCL1 cirrhosis HCC alcoholic liver disease rs738409
We thank Jennifer Söhne, Gudrun Hack, Lisa Behnke, and Saskia Schmitz for their perfect technical assistance.
This study was funded by the Deutsche Krebshilfe (70112169) to HDN and US. FS was funded by grant SNF 310030_169196 from the Swiss National Fund. JN received funding by the German Research Foundation (DFG SFB/TRR 57). The funding organizations had no role in the design of the study, and collection, analysis, and interpretation of the data, or writing of the manuscript.
Compliance with ethical standards
The study protocol was approved by the ethics committee of the University of Bonn (number 351/15) and complied with the ethical guidelines given by the Declaration of Helsinki. Written informed consent was given from all patients.
Conflict of interest
The authors declare that they have no competing interests.
- 9.Stickel F, Buch S, Nischalke HD, Weiss KH, Gotthardt D, Fischer J, Rosendahl J, Marot A, Elamly M, Casper M, Lammert F, McQuillin A, Zopf S, Spengler U, Marhenke S, Kirstein MM, Vogel A, Eyer F, Felden J, Wege H, Buch T, Schafmayer C, Braun F, Deltenre P, Berg T, Morgan MY, Hampe J (2018) Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis. Am J Gastroenterol 113:1475–1483PubMedGoogle Scholar
- 12.BasuRay S, Wang Y, Smagris E et al (2019) Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis. Proc Natl Acad Sci 116:9521–6Google Scholar
- 17.Smagris E, BasuRay S, Li J et al (2015) Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis. Hepatol Baltim Md 61:108–118Google Scholar
- 19.Speliotes EK, Butler JL, Palmer CD et al (2010) PNPLA3 variants specifically confer increased risk for histologic nonalcoholic fatty liver disease but not metabolic disease. Hepatol Baltim Md 52:904–912Google Scholar
- 23.McCoy JP Jr (2001) Handling, storage, and preparation of human blood cells. In: Robinson JP, Darzynkiewicz Z, Dobrucki J et al (eds) Current protocols in cytometry. John Wiley & Sons, Inc., HobokenGoogle Scholar
- 34.Hassan MM, Kaseb A, Etzel CJ et al (2013) Genetic variation in the PNPLA3 gene and hepatocellular carcinoma in USA: risk and prognosis prediction. Mol Carcinog 52:139–147Google Scholar
- 44.Cui X, Li Z, Gao J et al (2016) Elevated CXCL1 increases hepatocellular carcinoma aggressiveness and is inhibited by miRNA-200a. Oncotarget 7:65052–65066Google Scholar
- 45.Cao Z, Fu B, Deng B et al (2014) Overexpression of chemokine (C-X-C) ligand 1 (CXCL1) associated with tumor progression and poor prognosis in hepatocellular carcinoma. Cancer Cell Int 14:86Google Scholar