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

No rAd.A20 treated mice developed liver carcinomas during the 6 months monitoring period.

“no rAd.A20 treated mice developed liver carcinomas during the 6 months monitoring period (14–16).”

• cites [14] Arvelo MB Cooper JT Longo C Daniel S Grey ST Mahiou J Czismadia E A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis Hepatology 2002 35 535 543 11870365 sourceDisputed

  The section “Expression of A20 in Hepatocytes Protects from Death Associated With FHF” says surviving mice were killed 2 to 4 weeks later, contrary to the claimed six-month monitoring period. The paper does not report whether those mice developed liver carcinomas. (LLM, conf=0.85)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [15] Longo CR Patel VI Shrikhande GV Scali ST Csizmadia E Daniel S Sun DW A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism Hepatology 2005 42 156 164 15962316 sourcePartly supported

  The section “Mice Engineered to Express A20 Are Protected From Lethality Following Extended and Radical Hepatectomy” reports no liver tumors among surviving mice, including those treated with rAd.A20. However, it says they were monitored for 8 weeks, not 6 months; the longer monitoring period is unsupported. (LLM, conf=0.91)

  Experimental — accuracy not yet measured
• cites [16] Ramsey HE Da Silva CG Longo CR Csizmadia E Studer P Patel VI Damrauer SM A20 protects mice from lethal liver ischemia/reperfusion injury by increasing peroxisome proliferator-activated receptor-alpha expression Liver Transpl 2009 15 1613 1621 19877201 PMC2976064 sourceSupported

Successful liver regeneration depends on an intact TNF-NFκB-IL-6-STAT3 pathway.

“Successful LR depends on an intact TNF-NFκB-IL-6-STAT3 pathway.”

• cites [5] Cressman DE Greenbaum LE DeAngelis RA Ciliberto G Furth EE Poli V Taub R Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice Science 1996 274 1379 1383 8910279 sourceNot checked — no full text of the source
• cites [27] Li W Liang X Kellendonk C Poli V Taub R STAT3 contributes to the mitogenic response of hepatocytes during liver regeneration J Biol Chem 2002 277 28411 28417 12032149 sourceDisputed

  The Abstract reports that hepatocyte DNA synthesis was impaired without STAT3, but liver regeneration still occurred. The Discussion likewise says liver mass was restored despite deficient DNA synthesis. These findings contradict the claim that successful regeneration depends on an intact pathway through STAT3. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [28] Yamada Y Kirilova I Peschon JJ Fausto N Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor Proc Natl Acad Sci USA 1997 94 1441 1446 9037072 PMC19810 sourceNot checked — no full text of the source
• cites [29] Moh A Iwamoto Y Chai GX Zhang SS Kano A Yang DD Zhang W Role of STAT3 in liver regeneration: survival, DNA synthesis, inflammatory reaction and liver mass recovery Lab Invest 2007 87 1018 1028 17660847 sourceDisputed

  The Abstract reports that surviving mice lacking liver STAT3 completely recovered liver mass after partial hepatectomy. The Conclusion (page not provided) says STAT3 is required for survival in the acute stage but plays a limited role in subsequent DNA synthesis and liver-mass recovery. Thus, the paper does not support an intact STAT3 pathway as a requirement for successful liver regeneration. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Hepatocyte-specific SOCS3 knockout mice show improved liver regeneration following hepatectomy.

“Accordingly, hepatocyte-specific SOCS3 knockout (hKO SOCS3) mice show improved LR following hepatectomy (11).”

• cites [11] Riehle KJ Campbell JS McMahan RS Johnson MM Beyer RP Bammler TK Fausto N Regulation of liver regeneration and hepatocarcinogenesis by suppressor of cytokine signaling 3 J Exp Med 2008 205 91 103 18158318 PMC2234364 sourceSupported

Short-term overexpression of A20 does not carry a significant carcinogenic risk.

“Our previous studies however indicate that short-term overexpression of A20 does not carry a significant carcinogenic risk. Indeed, no rAd.A20 treated mice developed liver carcinomas during the 6 months monitoring period (14–16).”

• cites [14] Arvelo MB Cooper JT Longo C Daniel S Grey ST Mahiou J Czismadia E A20 protects mice from D-galactosamine/lipopolysaccharide acute toxic lethal hepatitis Hepatology 2002 35 535 543 11870365 sourceCould not verify

  The Results examine rAd.A20-treated mice over hours and days, and the Discussion addresses liver protection and adenoviral toxicity, but neither assesses carcinogenic risk. Searches for carcinoma, tumors, and six-month monitoring found no such follow-up in our copy. (LLM, conf=0.75)

  Experimental — accuracy not yet measured
• cites [15] Longo CR Patel VI Shrikhande GV Scali ST Csizmadia E Daniel S Sun DW A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism Hepatology 2005 42 156 164 15962316 sourcePartly supported

  The Discussion says limited rAd-mediated A20 expression may avoid tumor formation and reports no liver neoplasia in treated mice during long-term follow-up. The Results section specifies that surviving mice were monitored for eight weeks, not the six months stated in the citing sentence; this paper therefore does not support that duration. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [16] Ramsey HE Da Silva CG Longo CR Csizmadia E Studer P Patel VI Damrauer SM A20 protects mice from lethal liver ischemia/reperfusion injury by increasing peroxisome proliferator-activated receptor-alpha expression Liver Transpl 2009 15 1613 1621 19877201 PMC2976064 sourceSupported

• AssumptionRests on an assumption: The 6-month monitoring period without liver carcinomas is sufficient to conclude that short-term A20 overexpression does not carry significant carcinogenic risk.

A20 is induced in mice and humans as part of the hepatocyte's regenerative response following hepatectomy.

“A20, a newly identified player in LR, is induced in mice and humans as part of the hepatocyte’s regenerative response following hepatectomy (15, 20).”

• cites [15] Longo CR Patel VI Shrikhande GV Scali ST Csizmadia E Daniel S Sun DW A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism Hepatology 2005 42 156 164 15962316 sourcePartly supported

  The Results section reports a 13-fold rise in A20 mRNA in mouse liver 24 hours after hepatectomy, and the Discussion identifies A20 as part of the hepatocyte regenerative response. The paper does not show A20 induction after hepatectomy in humans; its reference to human A20 mRNA concerns a gene introduced into mouse livers. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [20] de Jonge J Kurian S Shaked A Reddy KR Hancock W Salomon DR Olthoff KM Unique early gene expression patterns in human adult-to-adult living donor liver grafts compared to deceased donor grafts Am J Transplant 2009 9 758 772 19353763 PMC2734955 sourceCould not verify

  The Discussion describes regenerative gene-expression patterns in human liver grafts, but does not identify A20 as an induced gene. A search for A20 found it only in the References, in the title of a mouse hepatectomy study; our copy does not state that A20 is induced in mice and humans following hepatectomy. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

IL-6 is the central trigger of liver regeneration via phosphorylation/activation of STAT3.

“IL-6, produced following hepatectomy by Kupffer cells and hepatocytes (25), is the central trigger of LR via phosphorylation/activation of STAT3 (27).”

• cites [25] Panesar N Tolman K Mazuski JE Endotoxin stimulates hepatocyte interleukin-6 production J Surg Res 1999 85 251 258 10423326 sourceNot checked — no full text of the source
• cites [27] Li W Liang X Kellendonk C Poli V Taub R STAT3 contributes to the mitogenic response of hepatocytes during liver regeneration J Biol Chem 2002 277 28411 28417 12032149 sourcePartly supported

  The Abstract calls IL-6 one of the most important initiators of liver regeneration and says STAT3 is induced in an IL-6-dependent fashion. It also reports that regeneration still occurred without STAT3 and that some IL-6 effects are mediated by other pathways. The paper supports IL-6–STAT3 signaling as an important contributor, but not the stronger claim that it is the central trigger of regeneration via STAT3 alone. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

A20 expression levels increase in deceased and living donor liver grafts hours following reperfusion.

“its expression levels increase in deceased and living donor liver grafts hours following reperfusion (20).”

• cites [20] de Jonge J Kurian S Shaked A Reddy KR Hancock W Salomon DR Olthoff KM Unique early gene expression patterns in human adult-to-adult living donor liver grafts compared to deceased donor grafts Am J Transplant 2009 9 758 772 19353763 PMC2734955 sourcePartly supported

  The Results section on deceased-donor grafts (page unavailable) names TNFAIP3/A20 among genes up-regulated after reperfusion. The living-donor Results section reports up-regulation of inflammatory genes but does not identify A20 as one of them; the Methods say POST biopsies were taken after reperfusion but do not establish the claimed interval of hours. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The rAd. dose and route of administration achieves maximal transgene expression in 30% of hepatocytes, 5 days after injection.

“This dose and route of administration achieves maximal transgene expression in 30% of hepatocytes, 5 days after injection (15).”

• cites [15] Longo CR Patel VI Shrikhande GV Scali ST Csizmadia E Daniel S Sun DW A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism Hepatology 2005 42 156 164 15962316 sourceSupported

A20 KO and HT hepatocytes have higher basal P-STAT3 levels than WT.

“Increased basal IL-6 levels in A20 KO and HT hepatocytes were paralleled by higher basal P-STAT3 levels, indicating a chronic state of IL-6-mediated activation of these hepatocytes (Fig. S3; n=2).”

At baseline, A20 KO livers have more P-STAT3/HNF4α positive hepatocytes than WT and HT livers.

“the number of P-STAT3/HNF4α positive hepatocytes, at baseline, was higher in A20 KO than in WT and HT livers (Fig. 4D and 4E; n=3–4; p<0.01, and <0.05, respectively).”

miR203 levels are lower in A20 KO and HT livers than WT.

“we checked by qPCR for miR203 levels and found them significantly lower in KO (p<0.01) and HT (p<0.05) vs. WT livers (Fig. 4F; n=5–7).”

• Also stated in: § Discussion · plain

  “We discovered that A20 knockdown (KO and HT) significantly decreased hepatic levels of miR-203.”

A20 overexpression enhances STAT3 phosphorylation at baseline, 6h, and 24h after LPS/TNF treatment in HepG2 cells.

“STAT3 phosphorylation, downstream of IL-6, was enhanced at baseline (~150 fold; p<0.001), 6h (p<0.001) and 24h (p<0.05) after LPS/TNF treatment, when compared to C and rAd.βgal-transduced cells (Fig. 2B; n=3–4).”

Overexpression of A20 increases hepatocyte proliferation in NMuLi cells.

“Overexpression of A20 increased by 1.6 fold cell counts/well when compared to C and rAd.βgal transduced cells, 24h after addition of 10% FBS, (Fig. 1A, n=4–6; p<0.05).”

Total loss of A20 increases basal and TNF-induced IL-6 production by mouse primary hepatocytes.

“Total loss of A20 significantly increased basal (p<0.05) and TNF-induced (p<0.001) IL-6 production by MPH, when compared to HT and WT (Fig. 3B; n=4).”

Greater hepatocyte proliferation in rAd.A20 vs rAd.βgal livers occurs despite higher basal hepatic IL-6 levels in rAd.βgal livers.

“greater hepatocyte proliferation in rAd.A20 vs. rAd.βgal livers occurred despite significantly higher basal hepatic IL-6 levels in the latter (Fig. S5; n=4–5; p<0.001).”

Decreased STAT3 phosphorylation in A20 KO mouse primary hepatocytes correlates with higher basal and IL-6-induced SOCS3 mRNA levels compared to WT.

“Decreased STAT3 phosphorylation in A20 KO MPH correlated with significantly higher basal (p<0.05) and IL-6 induced (p<0.01 at 1h, p<0.05 at 3h) SOCS3 mRNA levels, compared to WT (Fig. 3D; n= 4–5).”

The Nter and 7Zn domains of A20 independently increase hepatocyte proliferation in NMuLi cells.

“Similarly, rAd.Nter and rAd.7Zn-transduced cells showed a 1.8 and 1.9 fold increase in cell counts/well (Fig. 1A; n=3–4; p<0.05 vs. C and p<0.01 vs. rAd.βgal). This indicated that independent overexpression of Nter or 7Zn increases proliferation in NMuLi cells.”

The number of Ki67 proliferating hepatocytes is not significantly different among A20 KO, HT, and WT livers.

“Despite increased P-STAT3 levels in A20 KO livers, the number of Ki67 proliferating hepatocytes was not significantly different among groups (Fig. S4C and S4D; n=3–4).”

A20 overexpression inhibits EH-induced hepatic upregulation of SOCS3 mRNA and protein in mice.

“Overexpression of A20 inhibited EH-induced hepatic upregulation of SOCS3 mRNA, when compared to C or rAd.βgal treated livers, 24h following surgery (Fig. 5A; n=3–6). We confirmed these results by showing decreased SOCS3 immunostaining in A20 overexpressing livers, 36h following EH (Fig. 5B; n=5–6).”

A20 overexpression decreases TNF/LPS-induced IL-6 secretion in HepG2 cells.

“IL-6 levels were significantly lower in supernatants of A20 overexpressing HepG2 compared to all other groups (Fig. 2A; n=4–7; p<0.01 vs. C; and p<0.001 vs. rAd.Nter, rAd.7Zn, and rAd.βgal).”

A20 overexpression decreases EH-induced upregulation of FGG and SAA1 in mouse livers.

“Overexpression of A20 significantly decreased EH-induced upregulation of FGG (Fig. 7A and 7B; n=6; p< 0.01), and reduced baseline and EH-induced upregulation of SAA1.”

A20 overexpression enhances hepatocyte proliferation after extended hepatectomy in mice.

“hepatocyte proliferation was significantly enhanced, as shown by increased number of Ki67/HNF4α positive hepatocytes, in rAd.A20 vs C and rAd.βgal treated livers (Fig. 5E; n=5–6; p<0.001).”

A20 HT livers express higher levels of SAA1 and FGG following EH.

“Conversely, A20 HT livers expressed higher levels of SAA1 (Fig 7C; n=5–6; p<0.01), and FGG (Fig. 7D; n=6) following EH.”

A20 overexpression rescues EH-induced upregulation of CCND1 and CCNA mRNA in mouse livers.

“overexpression of A20 rescued this outcome, allowing for a significant increase of both cyclins, 24h following EH (Fig. 5C & D; n=3–6).”

A20 KO livers have more SOCS3/albumin positive hepatocytes around portal and central veins than HT and WT livers.

“we noted significantly more SOCS3/albumin positive hepatocytes around the portal and central veins of A20 KO livers, as compared to HT and WT livers (Fig. 4B and 4C; n=3–4; p<0.01).”

Both Nter and 7Zn domains of A20 are required to inhibit NF-κB.

“Both C-terminal (7-Zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB.”

The effect of A20 on STAT3 phosphorylation maps to its 7Zn domain.

“These results indicate that this novel effect of A20 indeed maps to its 7Zn domain.”

A20 reducing p21 and SOCS3 establishes its pro-proliferative properties in hepatocytes.

“This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes”

A20 promotes hepatocyte proliferation through decreasing SOCS3 expression.

“Altogether, these results uncover a novel mechanism by which A20 (7Zn domain) promotes hepatocyte proliferation through decreasing SOCS3 expression.”

Increased SOCS3 levels in A20 HT livers are associated with impaired hepatocyte proliferation and delayed liver regeneration after EH.

“Increased SOCS3 levels in HT livers associated with impaired hepatocyte proliferation and delayed LR, as evidenced by lower number of Ki67/HNF4α positive hepatocytes in HT vs. WT livers, 36h after EH (Fig. 6C; n=2 (WT) and 5 (HT); p<0.001).”

A20 KO mice die within 4-5 weeks of birth mainly from unfettered liver inflammation.

“A20 KO mice are born cachectic and die within 4–5 weeks of birth mainly from unfettered liver inflammation, which shows A20’s high rank in the hierarchy of the liver physiologic anti-inflammatory armamentarium (21).”

• cites [21] Lee EG Boone DL Chai S Libby SL Chien M Lodolce JP Ma A Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice Science 2000 289 2350 2354 11009421 PMC3582399 sourceNot checked — no full text of the source

A20-mediated modulation of SOCS3 expression in hepatocytes is at least in part epigenetically regulated by A20's effect on miR-203.

“Since SOCS3 is an evolutionarily conserved target of miR-203 (26), A20-mediated modulation of SOCS3 expression in hepatocytes is, at least in part, epigenetically regulated by A20’s effect on miR-203.”

• cites [26] Wei T Orfanidis K Xu N Janson P Stahle M Pivarcsi A Sonkoly E The expression of microRNA-203 during human skin morphogenesis Exp Dermatol 2010 19 854 856 20698882 sourceNot checked — no full text of the source

SOCS3 is a negative regulator of IL-6 signaling.

“STAT3 inducible SOCS3 is part of a negative feedback loop that inhibits IL-6 signaling, i.e. STAT3 phosphorylation (30).”

• cites [30] Croker BA Krebs DL Zhang JG Wormald S Willson TA Stanley EG Robb L SOCS3 negatively regulates IL-6 signaling in vivo Nat Immunol 2003 4 540 545 12754505 sourceNot checked — no full text of the source

A20 knockdown significantly impairs and delays liver regeneration.

“In contrast, A20 knockdown significantly impairs and delays LR (Studer et al, in preparation).”

• cites Studer et al, in preparationNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

SOCS3 is an evolutionarily conserved target of miR-203.

“Since SOCS3 is an evolutionarily conserved target of miR-203 (26)”

• cites [26] Wei T Orfanidis K Xu N Janson P Stahle M Pivarcsi A Sonkoly E The expression of microRNA-203 during human skin morphogenesis Exp Dermatol 2010 19 854 856 20698882 sourceNot checked — no full text of the source

Inappropriately high IL-6 levels inhibit liver regeneration by increasing p21.

“Indeed, inappropriately high IL-6 levels rather inhibit LR by increasing expression of the Cyclin Dependent Kinase Inhibitor (CDKI), p21 (9).”

• cites [9] Wustefeld T Rakemann T Kubicka S Manns MP Trautwein C Hyperstimulation with interleukin 6 inhibits cell cycle progression after hepatectomy in mice Hepatology 2000 32 514 522 10960443 sourceNot checked — no full text of the source

A20's pro-proliferative function segregates with its 7Zn not Nter domain.

“This novel A20 function segregates with its 7Zn not Nter domain.”

High and sustained STAT3 phosphorylation in A20/7Zn overexpressing hepatocytes is key to their pro-proliferative advantage.

“We believe that high and sustained STAT3 phosphorylation in A20/7Zn overexpressing hepatocytes is key to their pro-proliferative advantage, regardless of whether these cells are treated with IL-6.”