Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models

  1. Komal Ramani  Is a corresponding author
  2. Nirmala Mavila  Is a corresponding author
  3. Aushinie Abeynayake
  4. Maria Lauda Tomasi  Is a corresponding author
  5. Jiaohong Wang
  6. Michitaka Matsuda
  7. Eki Seki
  1. Karsh Division of Gastroenterology and Hepatology, Cedars-Sinai Medical Center, United States
  2. Applied Cell Biology Division, Department of Biomedical Sciences, Cedars-Sinai Medical Center, United States
7 figures, 2 tables and 6 additional files

Figures

Figure 1 with 1 supplement
Expression, phosphorylation, and scaffolding activity of AKAP12 is altered in CCl4-treated mouse liver and human liver fibrosis.

Mice were administered CCl4 or mineral oil (control) as in methods. (A) Total protein (left panel) was immunoblotted with AKAP12, α-SMA, or GAPDH (control) antibody and blots were quantified by …

Figure 1—figure supplement 1
Complete human tissue arrays of 11 normal livers and 16 liver fibrosis tissues stained with PLA probes to detect AKAP12-HSP47 interaction and Alexa fluor probes to detect HSP47 (green) or AKAP12 (red) as described under Materials and methods.

Magnification at 60×; scale bar=25 µm. PLA, proximity ligation assay.

Figure 2 with 1 supplement
CRISPR-directed editing of AKAP12’s activation-responsive phospho-sites enhances AKAP12’s scaffolding activity and inhibits HSC activation.

(A) Cell extracts from human HSCs cultured for 0, 3, or 6 days (see Materials and methods) were processed for co-immunoprecipitation of AKAP12 and HSP47 or for α-SMA western blotting. Data …

Figure 2—figure supplement 1
CRISPR-directed editing of AKAP12’s activation-responsive phospho-sites enhances AKAP12’s HSP47 scaffolding activity in mouse HSCs.

Activated mouse HSCs were transfected with CRISPR reagents and GFAP-Cas9 vector to cause CRISPR-directed HDR as described under Materials and methods. Untransfected (WT) or cells with Cas9 alone …

PKCα phosphorylates AKAP12 and inhibits its interaction with HSP47.

(A) AKAP12 is phosphorylated by PKCα at its activation-responsive phospho-sites. Recombinant WT or AKAP12 phospho-mutants were in vitro translated from their vectors and subjected to in vitro kinase …

Figure 4 with 1 supplement
In vivo gene editing of the Akap12 region corresponding to its activation-responsive phospho-sites in the CCL4 mouse model using GFAP-SaCas9.

(A) Schematic diagram of the mouse Akap12 locus showing the exon 3 region containing AKAP12’s activation-responsive phospho-site regions and two SaCas9 target sgRNAs (1 and 2). The PDEL mutation …

Figure 4—figure supplement 1
In vivo gene editing of the Akap12 region corresponding to its activation-responsive phospho-sites in the CCl4 mouse model using LRAT-Cas9.

(A) Specificity of PDEL CRISPR for HSCs using LRAT-SaCas9 was evaluated by multiplex PCR amplification of genomic DNA from HSCs using a PDEL-specific primer and two primers around the PDEL primer …

Figure 5 with 1 supplement
Phospho-editing of AKAP12 regulates liver injury and fibrosis in the CCL4 mouse model.

Gross changes in mouse body weight (A) and liver/body weight ratio (B) after PDEL or PMUT GFAP-SaCas9-mediated CRISPR editing of AKAP12’s phospho-sites under oil or CCL4 treatment conditions. …

Figure 5—figure supplement 1
Phospho-editing of AKAP12 using LRAT-Cas9 regulates liver fibrosis in the CCl4 mouse model.

(A) Gross changes in mouse body weight (left panel) and liver/body weight ratio (right panel) after PDEL LRAT-Cas9-mediated CRISPR editing of AKAP12’s phospho-sites under oil or CCl4 treatment …

Phospho-editing of AKAP12 regulates AKAP12’s HSP47-scaffolding activity, HSC activation, and HSP47’s collagen-chaperoning activity in the CCl4 mouse model.

(A) AKAP12-HSP47 co-immunoprecipitation, AKAP12, HSP47, and α-SMA western blotting from liver protein of CR-PDEL experiment. Data represented as GAPDH normalized densitometry are mean±SE from six …

Figure 7 with 2 supplements
HSC-specific phospho-editing of AKAP12 regulates the ER stress response.

(A) Heat map of total liver and HSCs ER stress/UPR signaling components in four groups, oil+EV, oil+CR, CCl4+EV and CCL4+CR. Proteomics data utilized to prepare the heatmap are presented in Supplemen…

Figure 7—figure supplement 1
Densitometric quantification of blots from Figure 7B–E.
Figure 7—figure supplement 2
Interaction between IRE1α and HSP47 in desmin-positive HSCs of CRISPR-PDEL model by PLA staining.

Data representative of the PLA/fluorescence count is mean±SE from six experiments (200× magnification, scale bar=40 µm). *p<0.01 versus oil+EV, #p<0.01 versus CCl4+EV. EV, empty vector; HSC, hepatic …

Tables

Table 1
Phospho-peptide mapping of human HSCs, mouse HSCs, and mouse hepatocytes.
Cell typeObserved precursor massNeutral loss of phosphate massPhospho-peptide sequencePeptide modification
Day 0 human HSC1988.78121890.0297KRKVDTSVSWEALICVGSSKKPhospho (ST)[16]
Day 7 human HSC2148.97581854.9KRKVDTSVSWEALICVGSSKPhospho (ST)[16,17],
Day 7 human HSC2148.99321855.3141, 1854.9KRKVDTSVSWEALICVGSSKKPhospho (ST)[4,6,16]
Day 0 mouse HSCNDNDKRKVDTSVSWEALICVGSSKKND
Day 7 mouse HSC1998.131801.7952KRKVDTSVSWEALICVGSSKPhospho (ST)[3,6]
Day 7 mouse HSC2054.221857.9027KRKVDTSVSWEALICVGSSKKPhospho (ST)[14,15]
Mouse hepatocytesNDNDKRKVDTSVSWEALICVGSSKND
  1. S=Serine, T=Threonine, ND=not detected.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Human)AKAP12GenBankAccession ID: NM_005100.4
Gene (Mus musculus)Akap12GenBankAccession ID: NM_031185.3
Transfected construct (Human)Negative control siRNAThermo Fisher ScientificCat# 4404021silencerselect siRNA
Transfected construct (Human)Prkca siRNA-AThermo Fisher ScientificCat# s11092silencerselect siRNA
Transfected construct (Human)Prkca siRNA-BThermo Fisher ScientificCat# s11094silencerselect siRNA
Sequence-based reagentHuman PDEL region forward primer-653 bp ampliconThis paperPCR primerAGCTACTTCCGATGGAGAGA
Sequence-based reagentHuman PDEL region reverse primer-653 bp ampliconThis paperPCR primerCAGGAATAAACTTCTTGATTGAGACC
Sequence-based reagentHuman PDEL-specific primerThis paperPCR primerGACCCTCTCCTTGCTCTTTTCTTATC
Sequence-based reagentMouse PDEL region forward primer-780 bp ampliconThis paperPCR primerGATGAAGAGCCAGGAGAATACC
Sequence-based reagentMouse PDEL region reverse primer-780 bp ampliconThis paperPCR primerGGAAACCCAAGATTCCTCTCTAC
Sequence-based reagentMouse PDEL region amplicon sequencing forward primer-298 bp ampliconThis paperPCR primerACAAGGAAGAAGAGCTGGATAAG
Sequence-based reagentMouse PDEL region amplicon sequencing reverse primer-298 bp ampliconThis paperPCR primerCTGGCAGGAAGAGCATCTG
Sequence-based reagentMouse PDEL -specific primerThis paperPCR primerGCCTTCCTCGCTCTCTTCTTATC
Sequence-based reagentHuman guide sequenceThis paperCRISPR guide RNA sequenceGGAAGAACCAAAGCGCAAGGTG
Sequence-based reagentMouse guide sequence #1This paperCRISPR guide RNA sequenceGTCAGAGGAGCCAAAGCGCAGG
Sequence-based reagentMouse guide sequence #2This paperCRISPR guide RNA sequenceGGCCCTCCTTCATCATCTGAA
Sequence-based reagentHuman PDEL HDR donorThis paperCRISPR donor RNA sequenceGCCAAAGCCGGAAGAACCAAAGCGCAAGGTCGATAAGAAAAGAGCAAGGAGAGGGTCCTCTTCT
Sequence-based reagentMouse PDEL HDR donorThis paperCRISPR donor RNA sequenceGAGGAGCAAAGGTCAGAGGAGCCAAAGCGCCGGGTGGATAAGAAGAGAGCGAGGAAGGCATCCTCTTCA
Sequence-based reagentMouse pMUT HDR donorThis paperCRISPR donor RNA sequenceAGGTCAGAGGAGCCAAAGCGCAGGGTGGATGCTGCAGTGGCTTGGGAGGCGTTGATTTGTGTCGGAGCGGCCAAGAAGAGAGCGAGGAAGGCATCCTCTTCA
Recombinant DNA reagentOmicsLink expression clone of human AKAP12 in pRECEIVER-WG16 vectorGenecopoeia, MDEX-H3212-WG16Vector for in vitro translation of AKAP12 controlled by T7 promoter
Recombinant DNA reagentAAV-GFAP-Sacas9Vector Biolabs, PACat #7125HSC-specific gene editing AAV vector
Recombinant DNA reagentAAV-LRAT-Sacas9Vector Builder cloning serviceHSC-specific gene editing AAV vector
Peptide, recombinant proteinPKCα protein, activeMilliporeSigma, MA14-484In vitro kinase assay
Peptide, recombinant proteinHSP47 recombinant, humanProspec NJHSP-047Recombinant binding assay
Chemical compound, drugCarbon tetrachloride (CCl4)Sigma-AldrichCat #270652HPLC grade
OtherLipofectamine RNAiMAXThermo Fisher ScientificCat #13778075Transfection of siRNA
OtherDharmafect Duo reagentDharmacon, COCat #T-2010-02Transfection of CRISPR components
Commercial assay or kitQuikChange II site-directed mutagenesis KitAgilent Technologies, CACat #200521Mutagenesis of AKAP12 plasmid
Commercial assay or kitNon-radioactive TNT Coupled Transcription/Translation systemPromega, WICat #L4610In vitro translation
Commercial assay or kitHydroxyproline Assay KitCell Biolabs Inc, CACat #STA-675Hydroxyproline measurement in liver
Commercial assay or kitALT colorimetric activity assay kitCayman Chemical, MACat #700260ALT measurement in plasma
Commercial assay or kitAST colorimetric activity assay kitsCayman Chemical, MACat #701640ALT measurement in plasma
Biological sample (Homo sapiens)Primary human hepatic stellate cellsScienCell IncorporationCat #5300
Biological sample (H. Sapiens)Human tissue arrayHuman tissue biorepository, US Biolabs Inc MDXLiv086-01
AntibodyAnti-AKAP12 antibody (JP74 clone, mouse monoclonal)Abcamab49849Immunoprecipitation: (1 µg/500 µg) extract; western: (1:2000) in 5% milk/TBS-Tween-20; PLA: (1:250) dilution in PLA buffer
AntibodyAnti-Phosphoserine antibody (rabbit polyclonal)Abcamab9332PLA: (1:250) dilution
AntibodyAnti-α-SMA antibody (rabbit polyclonal)Abcamab5694Western: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-PKCα antibody (rabbit polyclonal)GenetexGTX130453Western: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-Collagen I alpha (Friedman, 2008) antibody (COL-1 clone, mouse monoclonal)Novus BiologicalsNB600-450Western: (1:1000) in 5% milk/TBS-Tween-20; PLA: (1:250) dilution in PLA buffer
AntibodyAnti-HSP47 antibody (clone # 950806, mouse monoclonal)Novus BiologicalsMAB9166-100Western: (1:2000) in 5% milk/TBS-Tween; PLA: (1:250) dilution in PLA buffer
AntibodyAnti-Biotin antibody (rabbit polyclonal IgG)Abcamab53494Western: (1:1000) in 5% milk/TBS-Tween-20
AntibodyAnti-GAPDH antibody (rabbit polyclonal IgG)Proteintech10494-1-APWestern: (1:2000) in 5% milk/TBS-Tween
AntibodyAnti-SaCas9 antibody (Clone 11C12, mouse monoclonal)GenetexA01951Western: (1:2000) in 5% milk/TBS-Tween
AntibodyAnti-desmin antibody (rabbit polyclonal IgG)Proteintech16520-1-APImmunostaining: (1:250) dilution in PLA buffer
AntibodyAnti-albumin antibody (rabbit polyclonal IgG)Proteintech16475-1-APImmunostaining: (1:250) dilution in PLA buffer
AntibodyAnti-IRE1α antibody (rabbit polyclonal IgG)Proteintech27528-1-APWestern: (1:1000) in 5% milk/TBS-Tween-20; PLA: (1:250) dilution in PLA buffer
AntibodyAnti-Phospho-IRE1α (S724) antibody (rabbit polyclonal IgG)Abcamab124945Western: (1:1000) in 5% BSA/TBS-Tween-20
AntibodyAnti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (rabbit polyclonal IgG)Cell Signaling Technology8828Western: (1:2000) in 5% BSA/TBS-Tween-20
AntibodyAnti-SMAD2 antibody (rabbit polyclonal IgG)Proteintech12570-1-APWestern: (1:2000) in 5% BSA/TBS-Tween-20
AntibodyAnti-SMAD3 antibody (rabbit polyclonal IgG)Proteintech25494-1-APWestern: (1:2000) in 5% BSA/TBS-Tween-20
AntibodyPhospho-p38 MAPK (Thr180/Tyr182) Antibody (rabbit polyclonal IgG)Cell Signaling Technology9211Western: (1:2000) in 5% BSA/TBS-Tween-20
AntibodyP38 MAPK Antibody (rabbit polyclonal IgG)Cell Signaling Technology9212Western: (1:2000) in 5% BSA/TBS-Tween-20
AntibodyAnti-BIP/GRP78 antibody (rabbit polyclonal IgG)Proteintech11587-1-APWestern: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-IL1β antibody (rabbit polyclonal IgG)Proteintech26048-1-APWestern: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-IL6 antibody (rabbit polyclonal IgG)Proteintech21865-1-APWestern: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-IL17 antibody (Clone 1B3D5, mouse monoclonal)Proteintech66148-1-IgWestern: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-IL10 antibody (rabbit polyclonal IgG)Proteintech20850-1-APWestern: (1:2000) in 5% milk/TBS-Tween-20
AntibodyAnti-calreticulin antibody (clone EPR3924, rabbit monoclonal)Abcamab92516Immunostaining: (1:250) dilution in PLA buffer
AntibodyClean-Blot IP Detection (HRP) (secondary antibody)Life Technologies21230Detection: co-immunoprecipitation-immunoblot: (1:1000) in 5% milk/TBS-Tween-20
AntibodyStreptavidin-HRP (secondary antibody)Cell Signaling Technology3999Detection: Biotin western blots: (1:5000) in 5% milk/TBS-Tween-20
AntibodyGoat anti rabbit IgG H&L (Alexa Fluor 488 green) (secondary antibody)Abcamab150077Detection: immunoflorescence: (1:1000) in PLA buffer
AntibodyGoat Anti-Mouse IgG H&L (Alexa Fluor 488 green) (secondary antibody)Abcamab150113Detection: immunoflorescence: (1:1000) in PLA buffer
AntibodyGoat Anti-Mouse IgG H&L (Alexa Fluor 647 far red) (secondary antibody)Abcamab150115Detection: immunoflorescence: (1:1000) in PLA buffer
AntibodyGoat Anti-Rabbit IgG H&L (Alexa Fluor 647 far red) (secondary antibody)Abcamab150079Detection: immunoflorescence: (1:1000) in PLA buffer
AntibodyDuolink In Situ PLA Probe Anti-Mouse PLUS (secondary antibody)MilliporeSigmaDUO92001Detection: PLA: (1:600) in PLA buffer
AntibodyDuolink In Situ PLA Probe Anti-Mouse MINUS (secondary antibody)MilliporeSigmaDUO92004Detection: PLA: (1:600) in PLA buffer

Additional files

Supplementary file 1

Kinase-prediction for AKAP12’s activation-responsive phospho-sites.

https://cdn.elifesciences.org/articles/78430/elife-78430-supp1-v1.docx
Supplementary file 2

Datasets of next-generation amplicon sequencing (NGS) from PDEL CRISPR mouse model.

Genomic DNA of HSCs isolated from oil or CCl4 injected mice treated with AKAP12 PDEL CRISPR +GFAP-Cas9 or LRAT-Cas9 were submitted for NGS to Azenta Life Sciences as described under methods. Hepatocytes from PDEL CRISPR +GFAP-Cas9 were also processed as above for NGS. Representative raw reads of WT, deletion or base changes are shown for each data set and summarized in the first summary tab of the excel.

https://cdn.elifesciences.org/articles/78430/elife-78430-supp2-v1.xlsx
Supplementary file 3

Datasets of next-generation amplicon sequencing (NGS) from PMUT CRISPR mouse model.

Genomic DNA of HSCs or hepatocytes isolated from oil or CCl4 injected mice treated with AKAP12 PMUT CRISPR +GFAP-Cas9 were submitted for NGS to Azenta Life Sciences as described under methods. Representative raw reads of WT or base changes are shown for each data set and summarized in the first summary tab of the excel.

https://cdn.elifesciences.org/articles/78430/elife-78430-supp3-v1.xlsx
Supplementary file 4

Proteomics analysis of total liver and HSCs from CRISPR PDEL mouse model.

Total protein from the liver or HSCs of AKAP12 PDEL CRISPR +GFAP-Cas9 mice was subjected to mass spectrometry-based proteomics analysis as described under methods. Proteomics dataset of whole liver, ER stress/UPR components of the liver and HSCs is shown. The summary tab in the excel explains each dataset.

https://cdn.elifesciences.org/articles/78430/elife-78430-supp4-v1.xlsx
Supplementary file 5

Mouse sgRNA off-target analysis.

https://cdn.elifesciences.org/articles/78430/elife-78430-supp5-v1.docx
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