Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models
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Curated by eLife
Evaluation Summary:
The work is relevant to colleagues who study non-alcoholic fatty liver disease, the most common chronic disease in the world. It starts with steatosis (fat deposition) in the liver and progresses to very devastating stages of liver fibrosis. This study provides novel insight into mechanisms that result in liver inflammation and fibrosis and identifies a novel disease pathway, which is an attractive target for the treatment of liver fibrosis. The study can be improved, especially by refining the quality of microscopic images and techniques of protein chemistry.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12’s scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12’s scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12’s site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12’s phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47’s collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.
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Evaluation Summary:
The work is relevant to colleagues who study non-alcoholic fatty liver disease, the most common chronic disease in the world. It starts with steatosis (fat deposition) in the liver and progresses to very devastating stages of liver fibrosis. This study provides novel insight into mechanisms that result in liver inflammation and fibrosis and identifies a novel disease pathway, which is an attractive target for the treatment of liver fibrosis. The study can be improved, especially by refining the quality of microscopic images and techniques of protein chemistry.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #3 (Public Review):
This is a well-performed study by an experienced group that has identified AKAP12 as a scaffolding partner of HSP47 in normal HSCs that blocks HSP47's collagen chaperoning activity and its interaction with UPR signals in HSCs. Furthermore, site-specific phosphorylation of AKAP12 inhibits its interaction with HSP47. This induces HSP47's collagen chaperoning activity, collagen production, and HSP47's interaction with UPR signaling proteins upon pro-fibrogenic stimulation. Blocking AKAP12 phospho-modification inhibits HSC activation as well as overall liver injury possibly via modulation of the ER stress response and inhibition of ER stress-linked inflammatory signals.
The paper fills an important gap in understanding the interaction of AKAP12 and HSP47.
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Reviewer #2 (Public Review):
Liver fibrosis is a pathological process that accompanies chronic liver diseases and causes significant morbidity and mortality worldwide. Thus, the understanding of the mechanisms involved in this process with the goal to develop effective treatments is of high significance. In this manuscript, the authors identify AKAP12 as a player in the activation of hepatic stellate cells (HSCs), the main liver cell type involves in the production of extracellular matrix and inflammatory mediators during the process of liver fibrosis. Using a mouse model of liver fibrosis (CCl4 treatment) and in-vitro experiments with isolated HSC, they show that phosphorylation of AKAP12 mediated by PKCalpha diminishes AKAP12 scaffolding activity towards HSP47, a chaperone of collagen in the ER. AKAP12 phosphorylation is increased in …
Reviewer #2 (Public Review):
Liver fibrosis is a pathological process that accompanies chronic liver diseases and causes significant morbidity and mortality worldwide. Thus, the understanding of the mechanisms involved in this process with the goal to develop effective treatments is of high significance. In this manuscript, the authors identify AKAP12 as a player in the activation of hepatic stellate cells (HSCs), the main liver cell type involves in the production of extracellular matrix and inflammatory mediators during the process of liver fibrosis. Using a mouse model of liver fibrosis (CCl4 treatment) and in-vitro experiments with isolated HSC, they show that phosphorylation of AKAP12 mediated by PKCalpha diminishes AKAP12 scaffolding activity towards HSP47, a chaperone of collagen in the ER. AKAP12 phosphorylation is increased in activated HSCs and induces HSP47 chaperoning activity with enhanced production of collagen but also ER stress with production of inflammatory mediators. Interestingly, by using CRISPR-AVV6 vectors for gene editing to specifically target AKAP12's activation phospho-sites (both deletion and mutation of the sites) in HSCs they were able to effectively suppress the liver injury and fibrotic response in mice treated with CCl4. The therapeutic effect was both mediated by a decrease in collagen production and also by modulation of the ER-stress and the mediated inflammatory response both in HSCs and in hepatocytes. The study would have been strengthened by using other alternative experimental models of liver fibrosis (i.e DDC, MCD diet) to broaden the significance of the mechanism. Although the study is interesting and reports a novel mechanism for drug design to potentially target the process of liver fibrosis, several issues related to data presentation call for questions on the rigor of the study.
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Reviewer #1 (Public Review):
The manuscript by Ramani et al. investigates the involvement of targeting A-kinase anchoring protein 12 (AKAP12), a protein that is described to exhibit a plethora of functions, for its regulation in liver injury and wound healing. Using molecular biology and gene editing techniques, the researchers delineate AKAP12's interaction with a chaperone, heat-shock protein 47, to understand the pathophysiology of hepatic cells. They conclude this protein's communications as a potential therapeutic target.
The authors build on their previous observation that AKAP12 phosphorylation impacts hepatic stellate cell activation, a process that may lead to liver fibrosis. In this manuscript, a link is described, heat-shock protein 47, a chaperone that is inhibited by AKAP12's phosphorylation state and therefore is unable to …
Reviewer #1 (Public Review):
The manuscript by Ramani et al. investigates the involvement of targeting A-kinase anchoring protein 12 (AKAP12), a protein that is described to exhibit a plethora of functions, for its regulation in liver injury and wound healing. Using molecular biology and gene editing techniques, the researchers delineate AKAP12's interaction with a chaperone, heat-shock protein 47, to understand the pathophysiology of hepatic cells. They conclude this protein's communications as a potential therapeutic target.
The authors build on their previous observation that AKAP12 phosphorylation impacts hepatic stellate cell activation, a process that may lead to liver fibrosis. In this manuscript, a link is described, heat-shock protein 47, a chaperone that is inhibited by AKAP12's phosphorylation state and therefore is unable to mature collagen. The authors go through many motions to address their hypothesis (including tetrachloro carbon treatment, western blotting, microscopy, Crispr/Cas9 gene editing, proteomic profiling, etc.), which is a strength of the study. Since I am not an expert in liver (patho)physiology, I can only comment on the technical aspect of this manuscript, which is sound but would benefit from an improved presentation of the data. An impact on society can be envisioned, if the protein interactions can be pharmacologically addressed, for instance with a small molecule screen.
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