Background & Aims

Glycogen synthase kinase 3 (Gsk3) a and b are ubiquitously expressed, constitutively active serine/threonine kinases, which play complex roles in cell activation, proliferation and death. The major regulatory mechanism of Gsk3s is mediated by inhibitory phosphorylations at serine 21 (a) or 9 (b) upon stimulation. Although active Gsk3s has been shown to promote the development of liver IRI, the significance of their inhibitory phosphorylation in the disease process is not clear.

Methods

In a murine liver partial warm ischemia model (90m in cephalad lobes), we analyzed Gsk3ab phosphorylation profiles during IR. The functions of serine 21 (a)/9 (b) inhibitory phosphorylation in liver IRI were determined by using dual Gsk3aS21A and Gsk3bS9A knock-in (KI) mutant mice. To differentiate roles in liver immune activation and hepatocellular damages against IR, we studied bone marrow chimeras in vivo, and macrophages and hepatocytes in vitro.

Results

Liver IR triggered transient activation of both Gsk3 isoforms, followed by their inactivation via increases and decreases of S21 or S9 phosphorylation. Gsk3 KI mutant mice developed significantly less severe liver IRI at both 6h and 24h post reperfusion (lower sALT levels, better preserved liver histology with lower Suzuki scores), despite elevated liver inflammatory immune activation, as compared with WT mice. Increased liver autophagy induction was observed in Gsk3 KI mutant livers in response to IR. In vitro analysis of bone marrow-derived macrophages and Kupffer cells confirmed that Gsk3 KI mutant cells increased TNF-a, but decreased IL-10 productions upon TLR stimulations. However, mutant hepatocytes became more resistant to TNF-a induced cell death, which was associated with increased levels of Bcl-2 and Bcl-xL expressions, and abrogated by Gsk3 inhibition. In bone marrow chimeric mice, WT recipients of Gsk3 KI mutant bone marrow suffered significantly more severe, while Gsk3 KI mutant recipients of WT bone marrow were protected from, liver IRI.

Conclusions

IR-induced Gsk3 inhibitory phosphorylation downregulates liver immune response, but promotes hepatocellular injury against IR. Thus, cell-type specific targeting of Gsk3 is necessary to achieve better and safer therapeutic efficacy.

CITATION INFORMATION: Zhou H, Zhu J, Yue S, Busuttil R, Kupiec-weglinski J, Lu L, Zhai Y. Distinctive Roles of Gsk3 Inhibitory Phosphorylation in Liver Inflammatory Immune Response and Hepatocellular Injury Against Ischemia Reperfusion. Am J Transplant. 2016;16 (suppl 3).

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