Sparcl1 promotes nonalcoholic steatohepatitis progression in mice through upregulation of CCL2
Bin Liu, … , Minghua Zheng, Yan Lu
Bin Liu, … , Minghua Zheng, Yan Lu
Published October 15, 2021
Citation Information: J Clin Invest. 2021;131(20):e144801. https://doi.org/10.1172/JCI144801.
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Research Article Hepatology Metabolism

Sparcl1 promotes nonalcoholic steatohepatitis progression in mice through upregulation of CCL2

Abstract

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of chronic liver disease ranging from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms of NASH progression remain incompletely understood. White adipose tissue (WAT) has emerged as an important endocrine organ and contributes not only to the initial stage of NAFLD, but also to its severity. In the current study, through transcriptomic analysis we identified increased expression of Sparcl1, a secreted glycoprotein, in the WAT from NASH mice. Plasma Sparcl1 levels were similarly elevated and positively correlated with hepatic pathological features in NASH patients. Functional studies showed that both chronic injection of recombinant Sparcl1 protein and overexpression of Sparcl1 exaggerated hepatic inflammation and liver injury in mice. In contrast, genetic ablation of Sparcl1, knockdown of Sparcl1 in WAT, and treatment with a Sparcl1-neutralizing antibody dramatically alleviated diet-induced NASH pathogenesis. Mechanistically, Sparcl1 promoted the expression of C-C motif chemokine ligand 2 (CCL2) in hepatocytes through binding to Toll-like receptor 4 (TLR4) and activation of the NF-κB/p65 signaling pathway. Genetically or pharmacologically blocking the CCL2/CCR2 pathway attenuated the hepatic inflammatory response evoked by Sparcl1. Thus, our results demonstrated an important role for Sparcl1 in NASH progression, suggesting a potential target for therapeutic intervention.

Authors

Bin Liu, Liping Xiang, Jing Ji, Wei Liu, Ying Chen, Mingfeng Xia, Yuejun Liu, Wenyue Liu, Peiwu Zhu, Yi Jin, Yu Han, Jieli Lu, Xiaoying Li, Minghua Zheng, Yan Lu

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Figure 5

Sparcl1 activates TLR4 to upregulate CCL2 expression.

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Sparcl1 activates TLR4 to upregulate CCL2 expression. Hepa1-6 cells were...
Hepa1-6 cells were transfected with adenovirus expressing Flag-tagged vector control or Flag-tagged Sparcl1 for 36 hours. Protein lysates were immunoprecipitated using anti-Flag M2–agarose. The immunoprecipitates were then eluted with Flag peptide and immunoblotted using anti-Flag and anti-TLR4 antibodies. The immunoprecipitation of Flag-Sparcl1 interacting protein was visualized by Coomassie blue staining. (B) Protein-protein interaction of Sparcl1 and TLR4. HEK293T cells were transfected with Flag-tagged Sparcl1 and HA-tagged TLR4 for 48 hours. Cell lysates were immunoprecipitated with either anti-Flag antibody or anti-HA antibody, and the immunocomplexes were probed with the indicated antibodies. (C–E) Relative luciferase activity (C), relative mRNA level of CCL2 (D), and NF-κB binding to the CCL2 promoter (E) in Hepa1-6 cells after incubation of Sparcl1, in the absence or presence of CLI-095 (1 μM) or JSH-23 (20 μM). (F–H) Relative luciferase activity (F), relative mRNA level of CCL2 (G), and NF-κB binding to the CCL2 promoter (H) in Hepa1-6 cells after incubation with Sparcl1, in the absence or presence of TLR4 siRNA. (I) TLR4 activation in terms of SEAP activity in TLR4-MD2–overexpressing HEK Blue hTLR4 cells in response to Sparcl1 in the presence or absence of CLI-095. (J) Western blots showing phosphorylated p65 (P-P65) and total (T-P65) in the livers of HFHC diet–fed mice treated with Sparcl1 or saline. (K) Western blot showing phosphorylated p65 and total p65 in Hepa1-6 cells treated with Sparcl1 or saline. (L) Subcellular distribution of endogenous p65 in Hepa1-6 cells treated with Sparcl1 or vehicle control for 2 hours. (C–I and K–L) Hepa1-6 cells were preincubated with palmitate (0.2 mM) for 12 hours. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 by 1-way ANOVA (C–I).