Perturbed liver gene zonation in a mouse model of non-alcoholic steatohepatitis.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

IL-6-GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models.

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model.

Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.

Monomeric/dimeric forms of Fgf15/FGF19 show differential activity in hepatocyte proliferation and metabolic function.

Human Fibroblast Growth Factor 19 (FGF19) and mouse ortholog Fgf15 play similar roles in liver regeneration and metabolism via the activation of Fgfr4/b-klotho (Klb). Monomeric FGF19 and dimeric Fgf15 are both necessary for liver regeneration and proper bile acid (BA) metabolism. FGF19 elicits stronger effects than Fgf15 on glucose and fatty acid metabolism and only FGF19 induces hepatocellular carcinoma (HCC). However, inhibiting FGF19/FGFR4 signaling in HCC patients is associated with toxicity due to elevated BA levels. Here, we examine the structure/function relationship in Fgf15/FGF19 to better understand the molecular basis for their distinct functions. We demonstrate that FGF19 is a more effective activator of Fgfr4 and of downstream signaling (Erk, Plcg1) than Fgf15. Furthermore, we use site-directed mutagenesis to show that the presence or absence of an unpaired cysteine in Fgf15/19 modulates ligand structure and determines the ability of these molecules to induce hepatocyte proliferation, with monomers being more potent activators. Consistent with these findings, an engineered dimeric variant of FGF19 is less effective than wild-type FGF19 at inducing liver growth in cooperation with the Wnt-enhancer RSPO3. In contrast to effects on proliferation, monomeric and dimeric ligands equally inhibited the expression of Cyp7a1, the enzyme catalyzing the rate limiting step in BA production. Thus, structure and function of Fgf15/FGF19 are intricately linked, explaining why FGF19, but not Fgf15, induces liver tumorigenesis. Our data provide insight into FGF19/FGFR4 signaling and may inform strategies to target this pathway while limiting on-target toxicity due to dysregulation of BA production or induction of hepatocyte proliferation.

A SIRT2-Selective Inhibitor Promotes c-Myc Oncoprotein Degradation and Exhibits Broad Anticancer Activity.

Targeting sirtuins for cancer treatment has been a topic of debate due to conflicting reports and lack of potent and specific inhibitors. We have developed a thiomyristoyl lysine compound, TM, as a potent SIRT2-specific inhibitor with a broad anticancer effect in various human cancer cells and mouse models of breast cancer. Mechanistically, SIRT2 inhibition promotes c-Myc ubiquitination and degradation. The anticancer effect of TM correlates with its ability to decrease c-Myc level. TM had limited effects on non-cancerous cells and tumor-free mice, suggesting that cancer cells have an increased dependency on SIRT2 that can be exploited for therapeutic benefit. Our studies demonstrate that SIRT2-selective inhibitors are promising anticancer agents and may represent a general strategy to target certain c-Myc-driven cancers.

Microtubules regulate hypoxia-inducible factor-1α protein trafficking and activity: implications for taxane therapy.

Disruption of the microtubule cytoskeleton impairs tumor angiogenesis by inhibiting the hypoxia-inducible factor (HIF-1α) pathway. However, the signaling cascade linking microtubule disruption to HIF-1α inactivation has not been elucidated. Here, we show that microtubule-targeting drug (MTD) treatment impaired HIF-1α protein nuclear translocation, which significantly down-regulated HIF transcriptional activity. We provide strong evidence that HIF-1α protein associates with polymerized microtubules and traffics to the nucleus, with the aid of the dynein motor protein. Together, these data suggest that microtubules are critically involved in the nuclear trafficking and transcriptional activity of HIF-1α. We also show that the connection between the microtubule cytoskeleton and HIF-1α regulation is lost in renal cell carcinoma (RCC), where HIF-1α is overexpressed because of mutations in the von Hippel Lindau (VHL) tumor suppressor protein. Specifically, we show that MTD treatment of RCC cells did not impair HIF-1α nuclear accumulation or transcriptional activity, and had no effect on the polysome association profile of HIF-1α. Interestingly, we found that HIF-1α protein did not bind microtubules in RCC. Moreover, restoration of VHL function failed to restore the ability of MTDs to inhibit HIF-1α, suggesting that VHL does not contribute to this phenotype. Together, these results suggest that HIF-1α regulation is microtubule-independent, and likely contributes to the chemoresistant nature of RCCs. Further understanding of the microtubule-dependent HIF-1α regulation, and lack thereof in RCC, is essential given the importance of HIF-1α in tumor biology, and the widespread use of MTDs in clinical oncology.

Microtubule disruption targets HIF-1alpha mRNA to cytoplasmic P-bodies for translational repression.

The hypoxia inducible factor 1α (HIF-1α) is overexpressed in solid tumors, driving tumor angiogenesis and survival. However, the mechanisms regulating HIF-1α expression in solid tumors are not fully understood. In this study, we find that microtubule integrity and dynamics are intricately involved in orchestrating HIF-1α translation. HIF-1α messenger RNA (mRNA) traffics on dynamic microtubules when it is actively translated. Microtubule perturbation by taxol (TX) and other microtubule-targeting drugs stalls HIF-1α mRNA transport and releases it from polysomes, suppressing its translation. Immunoprecipitation of the P-body component Argonaute 2 (Ago2) after microtubule disruption shows significant enrichment of HIF-1α mRNAs and HIF-targeting microRNAs (miRNAs). Inhibition of HIF-repressing miRNAs or Ago2 knockdown abrogates TX's ability to suppress HIF-1α translation. Interestingly, microtubule repolymerization after nocodazole washout allows HIF-1α mRNA to reenter active translation, suggesting that microtubule dynamics exert tight yet reversible control over HIF-1α translation. Collectively, we provide evidence for a new mechanism of microtubule-dependent HIF-1α translation with important implications for cell biology.