Low MBOAT7 expression, a genetic risk for MASH, promotes a profibrotic pathway involving hepatocyte TAZ upregulation - PubMed (original) (raw)

. 2025 Feb 1;81(2):576-590.

doi: 10.1097/HEP.0000000000000933. Epub 2024 May 22.

Xiaobo Wang 1, John Paul Kennelly 2, Hongxue Shi 1, Yuki Ishino 3, Kuniyuki Kano 3, Junken Aoki 3, Alessandro Cherubini 4, Luisa Ronzoni 4, Xiuqing Guo 5, Naga P Chalasani 6, Shareef Khalid 1 7, Danish Saleheen 1 7, Matthew A Mitsche 8, Jerome I Rotter 5, Katherine P Yates 9, Luca Valenti 4 10, Nozomu Kono 3, Peter Tontonoz 2, Ira Tabas 1 11 12 13

Affiliations

Low MBOAT7 expression, a genetic risk for MASH, promotes a profibrotic pathway involving hepatocyte TAZ upregulation

Mary P Moore et al. Hepatology. 2025.

Abstract

Background and aims: The common genetic variant rs641738 C>T is a risk factor for metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis (MASH), including liver fibrosis, and is associated with decreased expression of the phospholipid-remodeling enzyme MBOAT7 (LPIAT1). However, whether restoring MBOAT7 expression in established metabolic dysfunction-associated steatotic liver disease dampens the progression to liver fibrosis and, importantly, the mechanism through which decreased MBOAT7 expression exacerbates MASH fibrosis remain unclear.

Approach and results: We first showed that hepatocyte MBOAT7 restoration in mice with diet-induced steatohepatitis slows the progression to liver fibrosis. Conversely, when hepatocyte-MBOAT7 was silenced in mice with established hepatosteatosis, liver fibrosis but not hepatosteatosis was exacerbated. Mechanistic studies revealed that hepatocyte-MBOAT7 restoration in MASH mice lowered hepatocyte-TAZ (WWTR1), which is known to promote MASH fibrosis. Conversely, hepatocyte-MBOAT7 silencing enhanced TAZ upregulation in MASH. Finally, we discovered that changes in hepatocyte phospholipids due to MBOAT7 loss-of-function promote a cholesterol trafficking pathway that upregulates TAZ and the TAZ-induced profibrotic factor Indian hedgehog (IHH). As evidence for relevance in humans, we found that the livers of individuals with MASH carrying the rs641738-T allele had higher hepatocyte nuclear TAZ, indicating higher TAZ activity and increased IHH mRNA.

Conclusions: This study provides evidence for a novel mechanism linking MBOAT7-LoF to MASH fibrosis, adds new insight into an established genetic locus for MASH, and, given the druggability of hepatocyte TAZ for MASH fibrosis, suggests a personalized medicine approach for subjects at increased risk for MASH fibrosis due to inheritance of variants that lower MBOAT7.

Copyright © 2024 American Association for the Study of Liver Diseases.

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Conflict of interest statement

CONFLICTS OF INTEREST

Luca Valenti consults for Novo Nordisk and Pfizer. He is on the speakers’ bureau for Viatris. He received grants from Gilead. The remaining authors have no conflicts to report.

Figures

FIGURE 1

FIGURE 1. Hepatocyte MBOAT7 restoration after the development of hepatosteatosis reduces liver fibrosis and hepatic stellate cell (HSC) activation in MASH mice.

Mice were fed the FPC for 8 wks to induce hepatosteatosis and then injected with AAV8-TBG-Mboat7 or AAV8-TBG-GFP control (Ctrl) virus. The mice were continued on the FPC diet for an additional 8 wks. Livers were assayed for (A) MBOAT7 by IFM (scale bar = 50 μm); (B) Picrosirius red (scale bar = 200 μm); (C) αSMA+ by IFM (scale bar = 50 μm); (D-E) COL1A1 and OPN by IHC (scale bar = 100 μm); (F) mRNAs associated with HSC activation, expressed relative to the to the MASH control value; (G) Lipid-droplet area (scale bar, 100 μm); and (H) F4/80+ area (scale bar = 100 μm). The arrows in B-E show examples of positively stained cells. The values for all graphs are means ± SEM. In (A), the data are expressed as MBOAT7+ area in hepatocytes, normalized to the chow-control value, and in B-E, G, and H, the data are expressed as percent area. n = 6-7 mice/group, and values are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

FIGURE 2. Hepatocyte MBOAT7 silencing after the development of hepatosteatosis accelerates liver fibrosis and hepatic stellate cell activation in MASH mice.

Mice were fed the FPC for 8 wks to induce hepatosteatosis and then injected with AAV8-H1-shMboat7 or AAV8-H1-shControl (shCtrl) virus. The mice were continued on the FPC diet for an additional 8 wks. Livers were assayed for (A) MBOAT7 by IFM (scale bar = 50 μm); (B) Picrosirius red (scale bar = 200 μm); (C) αSMA+ by IFM (scale bar = 50 μm); (D-E) COL1A1 and OPN by IHC (scale bar = 100 μm); (F) mRNAs associated with HSC activation, expressed relative to the to the MASH control value ; (G) Lipid-droplet area (scale bar, 100 μm); and (H) F4/80+ area (scale bar = 100 μm). The arrows in B-E show examples of positively stained cells. The values for all graphs are means ± SEM. In (A), the data are expressed as MBOAT7+ area per hepatocyte area, normalized to the MASH-shCtrl value, and in B-E, G, and H, the data are expressed as percent area. n = 7-8 mice/group, and values are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001****, p < 0.0001.

FIGURE 3

FIGURE 3. Evidence in hepatic cells linking MBOAT7 LoF to elevated TAZ/IHH and hepatic stellate cell (HSC) activation.

(A-B) Immunoblots of MBOAT7, TAZ, and IHH in human and mouse primary hepatocytes treated with siMboat7 or siCtrl, with densitometric quantification relative to β-actin (n = 3 technical replicates/group). (C) Immunoblots of MBOAT7, TAZ, and IHH in AML12 hepatocytes transfected with plasmids encoding MBOAT7 or GFP control (Ctrl), with densitometric quantification (n = 3 technical replicates/group). (D) Immunoblots of MBOAT7, TAZ, and IHH in wildtype versus _Mboat7_−/− mouse primary hepatocytes treated for 6 h with 0.4 mM oleic acid, with densitometric quantification (n = 4 technical replicates/group). (E) Primary HSCs from wildtype mice were incubated for 48 h with conditioned medium (CM) obtained from oleic acid-treated wildtype or _Mboat7_−/− mouse primary hepatocytes or with medium not exposed to hepatocytes (Basal), with or without a Smothened (Smo) inhibitor to block hedgehog signaling in the HSCs. The HSCs were then assayed for Opn and Timp1 mRNA (n = 4 technical replicates/group). (F-G) Human primary hepatic 3D spheroids were treated with siMBOAT7 or siCtrl and assayed for the indicated mRNAs. Values are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

FIGURE 4. Evidence in experimental MASH and human liver linking MBOAT7 LoF to elevated TAZ.

(A) Immunoblots of TAZ, IHH, and p-S89-TAZ in the livers of mice given AAV8-TBG-GFP (Ctrl) or AAV8-TBG-Mboat7 from Figure 1, with densitometric quantification (n = 6-7/group). (B) Immunoblots of MBOAT7, TAZ, and IHH in the livers of mice given AAV8-H1-shCtrl or AAV8-H1-shMboat7 from Figure 2, with densitometric quantification (n = 7-8/group). (C) The percent nuclear TAZ was assayed by IFM in liver sections from subjects with MASH and the indicated genotypes affecting MBOAT7 (n = 30 CC, 50 TC, 25 TT), PNPLA3 (n = 33 CC, 50 CG, 30 GG), and HSD17B13 (n = 63 TT, 36 T/TA, 6 TA/TA). A one-way analysis of covariance (ANCOVA) was performed with sex, age, BMI, and fasting blood glucose as covariates for MBOAT7 rs641738, PNPLA3 rs738409, and HSD17B13 rs72613567. (D) Liver specimens from obese individuals with the indicated genotypes affecting MBOAT7 (n = 46 CC, 47 TC, 29 TT), PNPLA3 (n = 60 CC, 56 CG, 9 GG), and HSD17B13 (n = 82 TT, 40 T/TA, 3 TA/TA) were assayed for IHH mRNA, expressed relative to the first group for each locus. The data were log-transformed, and a one-way analysis of covariance (ANCOVA) was performed with sex, age, fasting blood glucose, LDL-cholesterol, and HDL-cholesterol as covariates for MBOAT7 rs641738 and PNPLA3 rs738409; and sex, age, and body mass index as covariates for HSD17B13 rs72613567. Values are means ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 5

FIGURE 5. MBOAT7 LoF in hepatocytes enhances the cholesterol trafficking-activated TAZ pathway.

(A) Immunoblots of MBOAT7 and TAZ in AML12 cells transfected with ASTER-B/C ASO or control and siMboat7 or control, with densitometric quantification relative to β-actin loading control (n = 3 technical replicates/group). (B) Left, AML12 cells transfected with siMboat7 or control siRNA were incubated for 30 min ± MβCD-cholesterol, incubated with His6-tagged ALOD4, fixed, incubated with anti-His6 and then Alexa Fluor 488-labeled secondary antibody (green), and counterstained with DAPI/nuclear (blue); arrows show increased His6-ALOD4 binding at the plasma membrane. Right, AML12 cells transfected with siMboat7 or control siRNA were incubated for 60 min ± MβCD-cholesterol, incubated with His6-ALOD4, and then immunoblotted using anti-His6, with densitometric quantification relative to calnexin loading control (n = 3 technical replicates/group). (C) AML12 cells were treated with siCtrl or siMboat7 and then incubated for 4 h ± MβCD-cholesterol. Cell extracts were assayed for markers of cholesterol biosynthesis via qPCR (n = 3 technical replicates/group). (D) RhoA activity in control and Mboat7-silenced AML12 cells (n = 6 technical replicates/group). (E) Phosphatidylserine content in control and Mboat7-silenced AML12 cells (n = 3 technical replicates/group). (F) Left, Immunoblot of TAZ in wildtype and Ptdss1-knockdown (KD) AML12 cells. Right, Immunoblots of TAZ and MBOAT7 in Ptdss1-KD AML12 cells treated with control or siMboat7, with densitometric quantification (n = 3 technical replicates/group). (G) Left, Immunoblot of TAZ in wildtype and Agpat1-knockdown (KD) AML12 cells. Right, Immunoblots of TAZ and MBOAT7 in Agpat1-KD AML12 cells treated with control or siMboat7, with densitometric quantification (n = 3 technical replicates/group). (H) Immunoblots of MBOAT7 and TAZ in AML2 cells in control and Mboat7-silenced AML12 cells that were also treated with control or siCds2, with densitometric quantification (n = 3 technical replicates/group). Values are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

FIGURE 6. Hepatocyte MBOAT7 silencing after the development of hepatosteatosis shows evidence of upregulation of the cholesterol trafficking-induced TAZ pathway.

Mice were fed the FPC diet for 8 wks to induce hepatosteatosis and then injected with AAV8-H1-shMboat7 or AAV8-H1-shControl (shCtrl) virus. The mice were continued on the FPC diet for an additional 10 days. Livers were assayed for (A) phospho-S89-TAZ and total TAZ, (B) uncleaved and cleaved SREBP2, (C) mRNAs of sterol-responsive genes; (D) phosphatidylserine content; (E) diacylglycerol content; and (F) phopsho-S133-CREB and total CRE. The values for all graphs are means ± SEM. n = 4 mice/group, and values are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (G) Proposed PL-TAZ pathway linking MBOAT7 LoF to liver fibrosis in MASH. Due to impaired acylation, MBOAT7 LoF favors the conversion of PI to LPI.[22] However, consistent with the idea that MBOAT7 LoF promotes PI turnover, there is eventual conversion back to PI through a pathway involving AGPAT1 to form LPA and CDS2 to form CDP-DAG and then conversion of CD-DAG to PI.[22] This pathway leads to DAG formation, which can promote PC synthesis. The enzyme PSS1 converts PC into PS, which promotes ASTER-B/C-mediated transport of plasma membrane cholesterol to the cell interior, thereby triggering a TAZ-stabilizing pathway involving PKA, calcium, RhoA, and inhibition of TAZ phosphorylation[28]. TAZ (with its co-factor TEAD) induces IHH, which is then secreted, leading to HSC activation and liver fibrosis[26]. Though not depicted here, TAZ/TEAD likely induce other genes that also contribute to MASH progression.

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