A20 promotes liver regeneration by decreasing SOCS3 expression to enhance IL-6/STAT3 proliferative signals

Abstract

Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent anti-inflammatory and NF-κB inhibitory protein, has established pro-proliferative properties in hepatocytes, in part through decreasing expression of the Cyclin Dependent Kinase Inhibitor, p21. Both C-terminal (7-Zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro-proliferative function of A20 in hepatocytes. We ascribed one of A20’s pro-proliferative mechanisms to increased and sustained IL-6 induced STAT3 phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL-6 signaling, SOCS3. This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL-6-induced STAT3 phosphorylation. Following liver resection in mice pro-proliferative targets downstream of IL-6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL-6/STAT3 pro-inflammatory targets were increased in A20 deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20-deficient livers. Altogether these results demonstrate that A20 enhances IL-6/STAT3 pro-proliferative signals in hepatocytes by down-regulating SOCS3, likely through a miR203-dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes and prompts the pursuit of A20-based therapies to promote liver regeneration and repair.

Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration

“…These results agree with our published data showing that hepatocytes retrieved from A20 HET mice produce higher levels of TNF and IL-6 in response to LPS, than WT hepatocytes. 12 Excessive and extended production of TNF and IL-6 in HET livers likely relates to their inability to mount an adequate post-PH surge of NF-κB inhibitory A20, a pre-requisite for secondary containment of these largely NF-κB-dependent cytokines. 13 However, even if reduced, levels of the antiapoptotic A20 9 in HET livers following PH were still sufficient to preclude higher rate of hepatocyte apoptosis.…”

“…36 However, excessive levels of IL-6 following PH halt hepatocyte proliferation 17 by increasing p21 transcription to block Cyclin/CDK complexes 37 and by binding STAT3 to inhibit its pro-regenerative signals. 38 Having reported that A20 overexpression decreases p21 mRNA and protein levels, 4,11,12 we were intrigued by the comparable levels of p21 mRNA in HET and WT livers post PH. This suggested that A20 was either not a physiologic regulator of p21 mRNA, or that homozygous (not heterozygous) KO of A20 (paralleled by even higher IL-6 levels) was required to increase p21 mRNA levels.…”

“…4 A20 confers this advantage by (i) limiting inflammation through inhibition of NF-κB activation, (ii) protecting hepatocytes from apoptosis, 9 (iii) reducing oxidative stress by increasing peroxisome proliferator-activated receptoralpha (PPARα) expression, 10 (iv) promoting hepatocyte proliferation by decreasing cyclin-dependent kinase inhibitor (CDKI) p21, enhancing interleukin-6/signal transducer and activator of transcription-3 (IL-6/STAT3) regenerative signals, and (v) optimizing energy production by improving fatty acid (FA) metabolism. 4,11,12 A20 homozygous KO mice are born cachectic, and die within 3-6 weeks of birth from systemic inflammation, predominantly in the liver. 13 This highlights A20's prominence in the liver's physiologic anti-inflammatory and anti-apoptotic defense mechanisms.…”

“…Significant upregulation of A20 in humans and mice livers following hepatectomy suggests that A20 is also an active player in the liver's physiologic regenerative response. 1,4,9,12 We explored this hypothesis by evaluating LR following two-third partial hepatectomy (PH) in female A20 heterozygous knockout (HET) mice that show no pathological phenotype at baseline, including in the liver. This procedure is impossible in shortlived cachectic A20 KO mice.…”

Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

“…Previous studies have indicated that the STAT3 signal transduction pathway is involved in the liver injury regeneration and apoptosis mechanisms (40)(41)(42). Lou et al (43) revealed that hepatocyte-specific STAT3-deficient mice suffered more severe liver damage in a warm ischemia/reperfusion rat model.…”