Hepatocyte-Specific Deletion of Yes-Associated Protein Improves Recovery From Acetaminophen-Induced Acute Liver Injury.

Overdose of acetaminophen (APAP) is the major cause of acute liver failure (ALF) in the Western world with very limited treatment options. Previous studies from our groups and others have shown that timely activation of liver regeneration is a critical determinant of transplant-free survival of APAP-induced ALF patients. Here, we report that hepatocyte-specific deletion of Yes-associated protein (Yap), the downstream mediator of the Hippo Kinase signaling pathway results in faster recovery from APAP-induced acute liver injury. Initial studies performed with male C57BL/6J mice showed a rapid activation of Yap and its target genes within first 24 h after APAP administration. Treatment of hepatocyte-specific Yap knockout (Yap-KO) mice with 300 mg/kg APAP resulted in equal initial liver injury but a significantly accelerated recovery in Yap-KO mice. The recovery was accompanied by significantly rapid hepatocyte proliferation supported by faster activation of Wnt/ß-catenin pathway. Furthermore, Yap-KO mice had significantly earlier and higher pro-regenerative inflammatory response following APAP overdose. Global gene expression analysis indicated that Yap-KO mice had a robust activation of transcription factors involved in response to endoplasmic reticulum stress (XBP1) and maintaining hepatocyte differentiation (HNF4α). In conclusion, these data indicate that inhibition of Yap in hepatocytes results in rapid recovery from APAP overdose due to an earlier activation of liver regeneration.

Inhibition of Glycogen Synthase Kinase 3 Accelerated Liver Regeneration after Acetaminophen-Induced Hepatotoxicity in Mice.

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. Timely initiation of compensatory liver regeneration after APAP hepatotoxicity is critical for final recovery, but the mechanisms of liver regeneration after APAP-induced ALF have not been extensively explored yet. Previous studies from our laboratory have demonstrated that activation of ß-catenin signaling after APAP overdose is associated with timely liver regeneration. Herein, we investigated the role of glycogen synthase kinase 3 (GSK3) in liver regeneration after APAP hepatotoxicity using a pharmacological inhibition strategy in mice. Treatment with specific GSK3 inhibitor (L803-mts), starting from 4 hours after 600 mg/kg dose of APAP, resulted in early initiation of liver regeneration in a dose-dependent manner, without modifying the peak regenerative response. Acceleration of liver regeneration was not secondary to alteration of APAP-induced hepatotoxicity, which remained unchanged after GSK3 inhibition. Early cell cycle initiation in hepatocytes after GSK3 inhibition was because of rapid induction of cyclin D1 and phosphorylation of retinoblastoma protein. This was associated with increased activation of ß-catenin signaling after GSK3 inhibition. Taken together, our study has revealed a novel role of GSK3 in liver regeneration after APAP overdose and identified GSK3 as a potential therapeutic target to improve liver regeneration after APAP-induced ALF.

Bile acids promote diethylnitrosamine-induced hepatocellular carcinoma via increased inflammatory signaling.

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC, but the mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)-initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in threefold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3-, CD20-, and CD45-positive inflammatory cell aggregates. Microarray-based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-κB and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNF-α and IL-1ß mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IκBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2, and Oct4. Quantification of total and 20 specific bile acids in liver, and serum revealed a tumor-associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic, and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Rheostats and toggle switches for modulating protein function.

The millions of protein sequences generated by genomics are expected to transform protein engineering and personalized medicine. To achieve these goals, tools for predicting outcomes of amino acid changes must be improved. Currently, advances are hampered by insufficient experimental data about nonconserved amino acid positions. Since the property "nonconserved" is identified using a sequence alignment, we designed experiments to recapitulate that context: Mutagenesis and functional characterization was carried out in 15 LacI/GalR homologs (rows) at 12 nonconserved positions (columns). Multiple substitutions were made at each position, to reveal how various amino acids of a nonconserved column were tolerated in each protein row. Results showed that amino acid preferences of nonconserved positions were highly context-dependent, had few correlations with physico-chemical similarities, and were not predictable from their occurrence in natural LacI/GalR sequences. Further, unlike the "toggle switch" behaviors of conserved positions, substitutions at nonconserved positions could be rank-ordered to show a "rheostatic", progressive effect on function that spanned several orders of magnitude. Comparisons to various sequence analyses suggested that conserved and strongly co-evolving positions act as functional toggles, whereas other important, nonconserved positions serve as rheostats for modifying protein function. Both the presence of rheostat positions and the sequence analysis strategy appear to be generalizable to other protein families and should be considered when engineering protein modifications or predicting the impact of protein polymorphisms.

Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression.

LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as 'linkers' to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite 'mismatched' interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer-dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.