Lee et al. Experimental & Molecular Medicine (2018) 50:48 DOI 10.1038/s12276-018-0074-5 Experimental & Molecular Medicine ARTICLE Open Access Altered AKAP12 expression in portal fibroblasts and liver sinusoids mediates transition from hepatic fibrogenesis to fibrosis resolution Hye Shin Lee1, Jinhyeok Choi1,TaekwonSon1,Hee-JunWee1,Sung-JinBae1,JiHaeSeo2,JiHyunPark1, Soo Hyung Ryu3, Danbi Lee4,MyoungKukJang5,EunsilYu6, Young-Hwa Chung4 and Kyu-Won Kim1,7 Abstract Liver fibrosis can be reversed by removing its causative injuries; however, the molecular mechanisms mediating the resolution of liver fibrogenesis are poorly understood. We investigate the role of a scaffold protein, A-Kinase Anchoring Protein 12 (AKAP12), during liver fibrosis onset, and resolution. Biliary fibrogenesis and fibrosis resolution was induced in wild-type (WT) or AKAP12-deficient C57BL/6 mice through different feeding regimens with 0.1% 3,5- diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing chow. AKAP12 expression in portal fibroblasts (PFs) and liver sinusoidal endothelial cells (LSECs) gradually decreased as fibrosis progressed but was restored after cessation of the fibrotic challenge. Histological analysis of human liver specimens with varying degrees of fibrosis of different etiologies revealed that AKAP12 expression diminishes in hepatic fibrosis from its early stages onward. AKAP12 KO mice displayed reduced fibrosis resolution in a DDC-induced biliary fibrosis model, which was accompanied by impaired 1234567890():,; 1234567890():,; normalization of myofibroblasts and capillarized sinusoids. RNA sequencing of the liver transcriptome revealed that genes related to ECM accumulation and vascular remodeling were mostly elevated in AKAP12 KO samples. Gene ontology (GO) and bioinformatic pathway analyses identified that the differentially expressed genes were significantly enriched in GO categories and pathways, such as the adenosine 3′,5′-cyclic monophosphate (cAMP) pathway. Knockdown of the AKAP12 gene in cultured primary PFs revealed that AKAP12 inhibited PF activation in association with the adenosine 3′,5′-cyclic monophosphate (cAMP) pathway. Moreover, AKAP12 knockdown in LSECs led to enhanced angiogenesis, endothelin-1 expression and alterations in laminin composition. Collectively, this study demonstrates that AKAP12-mediated regulation of PFs and LSECs has a central role in resolving hepatic fibrosis. Introduction liver fibrosis—liver malfunction with severe complications Liver fibrosis is the final outcome of chronic hepatic arises due to the distortion of liver architecture caused by damage caused by multiple etiologies, such as virus aberrant extracellular matrix (ECM) deposition. However, infections, alcohol over-consumption, and autoimmune an important characteristic of liver fibrosis that can pro- or metabolic diseases1,2. In cirrhosis—the end stage of vide new therapeutic strategies is its reversibility, even at an advanced stage, upon removal of the causative injury3,4. Correspondence: K-W. Kim ([email protected]) 1College of Pharmacy and Research, Institute of Pharmaceutical Sciences Seoul Hepatic non-parenchymal cells are involved not only in National University Seoul 08826, Korea fibrogenesis but also in fibrosis resolution. Hepatic stellate 2 Department of Biochemistry, School of Medicine, Keimyung University, Daegu cells (HSCs) and portal fibroblasts (PFs) are mesenchymal 42601, Korea Full list of author information is available at the end of the article cells capable of transforming into myofibroblasts that © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.Toviewacopyofthislicense,http:// creativecommons.org/licenses/by-nc-nd/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Lee et al. Experimental & Molecular Medicine (2018) 50:48 Page 2 of 13 aberrantly produce ECM proteins under fibrogenic con- (DDC)-supplemented diet; 0.1% DDC (Sigma) was mixed ditions2,5. These cells are removed by apoptosis6 or with normal mouse chow and fed to weight-matched 8- to senescence7 during fibrosis resolution; however, some 10-week-old male mice. The mice were kept on the DDC remain in a quiescent state8. The molecular mechanisms diet for 3 weeks, following which they were returned to a underlying the transition from fibrogenesis to fibrosis normal diet for the next 2 weeks. At the end of the resolution are not fully understood; however, shifting the experimental period, the mice were killed via deep anes- balance between pro-fibrotic pathways, such as TGF-β1 thesia and killed by cardiac perfusion. and PDGF signaling9,10, and anti-fibrotic ones, such as peroxisome proliferator-activated receptor gamma Human tissue specimens – (PPARγ) and cAMP pathways11 13, may mediate this Tissues from normal and fibrotic human livers were transition. obtained through biopsy specimens from patients who Under fibrotic conditions, liver sinusoids undergo vas- visited the Asan Medical Center, University of Ulsan, cular remodeling, which includes angiogenesis and College of Medicine, Seoul, Korea, for diagnosis. Informed capillarization characterized by the loss of fenestrae and consent was obtained from all patients. The patients’ the formation of basement membrane14. As differentiated clinical characteristics are summarized in supplementary liver sinusoidal endothelial cells (LSECs) function to Table 1. Fibrosis grades were determined by gross and maintain the quiescent status of HSCs, loss of LSEC dif- microscopic examination of the liver tissues. The study ferentiation under fibrosis contributes to HSC activa- was approved by the Institutional Review Board of the tion15,16. Forced re-differentiation of LSECs by a soluble Asan Medical Center, Seoul, Korea. guanylate cyclase activator promotes fibrosis regression in rodent models17, suggesting that the normalization of Histological analysis and immunohistochemistry LSECs to their differentiated state is a prerequisite for Immunofluorescence staining was performed on paraf- proper liver fibrosis resolution. fin sections or frozen sections. The paraffin sections were AKAP12 is a scaffold protein known to integrate de-paraffinized and stained with Picro-Sirius Red (Abcam, effector proteins such as protein kinase A, protein kinase Cambridge, MA, USA) according to the manufacturer’s C, and protein phosphatase 2B for the β-adrenergic instructions to visualize the deposition of ECM proteins. receptor pathway, through which it has a role in reg- To stain the paraffin sections, antigen retrieval was con- ulating cAMP compartmentalization and the re- ducted for 30 min at 95 °C in Tris or citrate buffer, sensitization of β-adrenergic receptors18,19. As AKAP12 depending on antibody compatibility. After blocking with is widely expressed by several cell types, it is reported to 5% normal donkey serum (Sigma-Aldrich) in PBS solu- have diverse physiological functions, such as inhibition of tion, the sections were incubated overnight at 4 °C with angiogenesis20, promotion of meningeal reconstruc- primary antibodies for AKAP12 (I. Gelman, Roswell Park tion21,22, and tumor suppression23. In connection to Cancer Institute), aSMA (Dako), laminin (Sigma), SE-1 chronic liver diseases, AKAP12 is reported to be down- (R&D Systems), CD31 (BD Biosciences), Thy-1 (BD regulated by an epigenetic mechanism in human hepa- Biosciences), collagen I (Southern Biotech), collagen III tocellular carcinoma24. However, little is known regarding (Southern Biotech), Desmin (R&D Systems), VEGFR2 its expression and function in normal and fibrotic livers. (Cell Signaling), F4/80 (Abcam), SE-1 (R&D Systems), and In this study, we aim to investigate the molecular collagen IV (Southern Biotech). After extensive washing mechanisms by which non-parenchymal cells—in parti- in PBS containing 0.1% Tween-20 solution, the sections cular, PFs and LSECs—modulate hepatic fibrosis resolu- were treated with Alexa 488- or 546-conjugated second- tion. By examining the expression profiles of AKAP12 in ary antibodies (1:750, Invitrogen, Carlsbad, CA, USA) for normal and fibrotic livers, we investigate the role of this 1 h at room temperature followed by counter staining protein in hepatic fibrogenesis and fibrosis resolution. with Hoechst (Sigma). Fluorescent images were taken under a confocal microscope (Carl Zeiss AG, Oberko- Materials and methods chen, Germany), and immuno-positive areas were quan- Animals and fibrosis induction tified by ImageJ software. Unless specified otherwise, 5–10 All mouse experiments were reviewed and approved by images from a single animal were quantified, and the the Committee for Care and Use of Laboratory Animals at mean value was considered to be a representative value Seoul National University according to the Guide