The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

  1. Younis Hazari
  2. Hery Urra
  3. Valeria A. Garcia Lopez
  4. Javier Diaz
  5. Giovanni Tamburini
  6. Mateus Milani
  7. Philippe Pihan
  8. Sylvere Durand
  9. Fanny Aprahamia
  10. Reese Baxter
  11. Menghao Huang
  12. X Charlie Dong
  13. Helena Vihinen
  14. Ana Batista-Gonzalez
  15. Patricio Godoy
  16. Alfredo Criollo
  17. Vlad Ratziu
  18. Fabienne Foufelle
  19. Jan G. Hengstler
  20. Eija Jokitalo
  21. Beatrice Bailly-maitre
  22. Jessica L Maiers
  23. Lars Plate
  24. Guido Kroemer
  25. Claudio Hetz

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Abstract

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

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  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Hazari et al reports on an important new function of the UPR IRE1 in liver fibrosis. The results are clearly and logically described. It shows that the genetic ablation of IRE1 in mice prevents liver fibrosis through retention of collagen in the ER and its degradation hence preventing secretion and accumulation in liver parenchyma. This effect was reversed was P4HB (a multifunctional enzyme that belongs to the protein disulfide isomerase) showing that IRE1 control P4HB.

    As such the paper is both scientifically and medically relevant.

    There is in my opinion a conceptual issue the paper does not directly address nor resolve.

    A clear functional distinction between IRE1 and XBP1 is not made nor attempted. This comes across as an unresolved issue. In the Introduction the authors cite ref 29 that provided evidence for a role of RIDD in alleviating hepatic cytotoxicity. The paper is based on chronic liver toxicity by CCL4 to identify the protective role of IRE1 and acute CCL4 toxicity to identify P4HB. IRE1 KO demonstrates that IRE1 controls collagen metabolism (degradation vs. secretion). However, many considerations involve XBP1 (see Fig. 8 as a example). Yet the paper concludes by saying "we propose that pharmacological inhibition of IRE1 activation..." Granted that IRE1 inhibition would definitely cause an attenuation of XBP1 splicing, I see a clear distinction between IRE1 and XBP1 still necessary to back the conclusion that inhibition of IRE1 and not XBP1 is the therapeutic modality one should develop.

    It is clear that IRE1 controls XBP1, but it also controls RIDD. Both independently control the fate of multiple downstream genes and also miRNAs in the case of RIDD. Because RIDD may offer some advantages in attenuating liver pathology, forfeiting some benefits IRE1 can offer via RIDD in order to blunt XBP1 may not be the optimal solution. Therefore, I suggest to complement the present study with experiments that target XBP1 specifically bypassing IRE1. Experiments of deletion (siRNA or Cre) as well as specific activation of XBP1 for which there exist commercially available molecules (e.g., IAX4 is a direct activator of XBP1 without UPR that also does not induce XBP1s-independent IRE1 signaling such as RIDD or JNK phosphorylation) will permit to better differentiate the role of XBP1 from IRE1 in P4HB regulation and collagen degradation vs. secretion in hepatic stellate cells and deposition in liver.

    Throughout the paper the authors refer to XBP1 and even center the Discussion on XBP1 more than on IRE1. Since determining the precise mechanism in this particular instance is critical to future treatments to prevent liver fibrosis these additional experiments should be performed.

    Minor point

    Fig. 4D. The label must have been erroneously copied and pasted. There is not way to distinguish what is different in lane 1-2 from lane 3-4. The legend is not helpful.

    Fig. S4B. Same problem.

    Significance

    This is an excellent paper with profound new medical implications with conceptual advances for the treatment of NASH.

    The limitations have been underscored in comments to authors.

    The audience remains specialized for the time being.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The current study aimed to investigate the possible contribution of the unfolded protein response (UPR), the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum (ER), to collagen biogenesis and liver disease. The authors targeted the ER sensor, inositol requiring transmembrane kinase/endoribonuclease 1 alpha (IRE1), using the IRE1Lox/Lox, Mx1Cre/- mouse strain where Cre is induced with 3 poly I:C injections. After confirmation of depleting IRE1 in the liver, they challenged mice with a high dose of carbon tetrachloride (CCl4). Phenotype analysis revealed the deleterious role of IRE1 in the liver in acute liver damage. Then, the authors determined the biological consequence of IRE1 deletion on the progression of experimental liver fibrosis. Consistently, IRE1 plays a promotive role in the CCl4-induced liver fibrosis mouse model since the results suggest that IRE1 knockout mice have reduced collagen deposition and AST/ALT activity (two major liver damage enzymes). To determine the potential mechanisms underlying the protective effects of IRE1 deficiency, the authors did mass spectrometry-based quantitative proteomics of liver samples from the experimental mice. Through bioinformatic analysis, they determined that protein and mRNA expression levels of P4HB decreased in IRE1 deficiency mice. Besides, in vitro and ex vivo studies suggested that IRE1 deficiency downregulated collagen generation. They further investigated the impact of IRE1 deficiency in liver steatosis in a mouse model fed a high-fat diet (HFD). Similarly, IRE1 deficiency suppressed liver steatosis and fibrosis in this mouse model. To clarify how IRE1 modulates collagen expression, the authors generated IRE1 knockout cell (KO) lines. They found that IRE1 KO cells may cause misfolding and intracellular accumulation of collagen inside the cells, reducing its secretion. Finally, they suggested the correlation of IRE1 associated gene (XBP1) and PH4B but not PH4A1 in human patients with non-alcoholic steatohepatitis (NASH). Overall, the current study may provide a potential molecular linkage between IRE1 and collagen in chronic liver disease progression. However, the inconsistent data presentation, poor data quality, and lack of suitable animal models make the current manuscript's impact on the field of chronic liver disease low.

    Major concern:

    1. The authors suggested that P450 levels are not changed in the liver samples of IRE1 KO mice compared to the wildtype (WT) ones. It has been demonstrated that IRE1 activation reduced P450 expression levels (PMID: 22291093). Can the authors explain the inconsistent findings?
    2. Why did the authors challenge mice with CCl4 for 12 weeks? The CCl4-induced liver fibrosis model would have a severe fibrotic liver phenotype at 8 weeks. Did the author check if IRE1 deficient mice have less liver injury than wildtype (WT) mice at early time points? Also, could you check the liver fibrosis phenotype using another typical liver fibrosis mouse model? The HFD-feeding mouse model would induce liver fibrosis after challenging mice for 50 weeks (PMID: 37270060).
    3. What are the mouse numbers, age, and sex in the CCl4 studies? In Fig. 2, several figures have inconsistent mouse numbers for the data presentation.
    4. There are many downregulated and upregulated target genes. What is the rationale for focusing on downregulated P4HB in IRE1 deficient mice, given that IRE1 has an RNase domain functioning in posttranscriptional regulation? In this case, how does IRE1 depletion upregulate P4HB?
    5. The author suggested that TGFb1 would promote collagen generation, but targeting IRE1 would reduce P4HB and the TGFb1-mediated collagen generation. The Fig. 4D does not support the statement. However, the authors' data demonstrated that TGFb1 cannot promote IRE1 and P4HB expression (Fig. 4G). If TGFb1 cannot promote collagen generation through activating IRE1 and P4HB, how can targeting IRE1 reduce TGFb1-induced collagen (Fig. 4H)?
    6. The authors stated that hepatic Ern1 deficiency suppresses the progression of liver steatosis. In fact, it has been reproducibly demonstrated that hepatic IRE1 deletion promotes hepatic steatosis progression (PMID: 21407177 and PMID: 29764990), contradicting the authors' findings. Besides, the authors did not deplete IRE1 specifically in the liver. Therefore, they made a misleading conclusion without solid evidence. The authors basically depleted IRE1 in the whole body of these experimental mice upon poly I:C injection. The authors must not conclude that hepatic Ern1 deficiency suppresses the progression of liver steatosis without considering the contributions of other organs in the phenotypes that you observed. Also, the HFD-treated mice would develop liver fibrosis 50 weeks post-feeding (PMID: 37270060). It is unclear what other treatments the authors used to accelerate the fibrotic liver phenotype, as shown in Fig. 5D. The authors should show body weight and liver weight over body weight in the result section. Besides, hepatic cholesterol, serum triglyceride, and cholesterol need to be measured in these mice to clarify how deleting IRE1 in the whole body can suppress hepatic steatosis, but liver-specific deletion of IRE1 promotes fatty liver. Without clarifying this issue, it is unclear how hepatic IRE1 deficiency can reduce steatosis and liver fibrosis.
    7. The authors suggested that ablation of IRE1 expression increased the levels of intracellular GFP-collagen as compared with control cells (Fig. 6C). How did the authors quantify the results? It is not clear if KO really increased the intracellular collagen levels. As the authors showed in Fig. 6C, WT-NT, and WT-GFP-collagen-untreated have no overlap of green fluorescence. However, KO-NT and KO-GFP-collagen-untreated still have an overlap of green fluorescence, indicating that some cells are not GFP-positive. In this case, how could authors conclude that IRE1-KO cells have a more than 2-fold increase of green fluorescence change compared to WT? Besides, Fig. 6F suggested that secreted collagens increased in KO cells, contradicting the authors' previous data in Fig. 2, 4, and 5. Why did you use U2OS, Hepa1-6, and Huh7 in these studies? Should the collagen be secreted by hepatic stellate cells?
    8. In Fig. 7, the authors suggested that IRE1 KO promotes the levels of collagen inside cells using the whole cell lysate. Interestingly, they indicated that IRE1 deficiency suppressed TGFb1-induced collagen production using whole cell lysates (Fig. 4D). It is really confusing if IRE1 KO promotes or suppresses collagen production or secretion. Also, Fig. 7C did not support that IRE1-KO reduced collagen secretion. Besides, what cells did the authors use for these studies? Are they hepatic stellate cells?
    9. It is interesting to see the positive correlation between XBP1 and P4HB mRNA expression. However, it is still unclear if IRE1 deficiency could downregulate P4HB mRNA expression, given its RNase function. Thus, it would be essential to determine how IRE1 regulates P4HB expression before analyzing the correlation using human datasets. Besides, Fig. 8D did not suggest that XBP1 expression levels are really correlated with chronic liver disease progression, given that its correlation scores with AST and ALT are 0 and -0.01, respectively.

    Significance

    The current study aimed to investigate the possible contribution of the unfolded protein response (UPR), the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum (ER), to collagen biogenesis and liver disease. The authors targeted the ER sensor, inositol requiring transmembrane kinase/endoribonuclease 1 alpha (IRE1), using the IRE1Lox/Lox, Mx1Cre/- mouse strain where Cre is induced with 3 poly I:C injections. After confirmation of depleting IRE1 in the liver, they challenged mice with a high dose of carbon tetrachloride (CCl4). Phenotype analysis revealed the deleterious role of IRE1 in the liver in acute liver damage. Then, the authors determined the biological consequence of IRE1 deletion on the progression of experimental liver fibrosis. Consistently, IRE1 plays a promotive role in the CCl4-induced liver fibrosis mouse model since the results suggest that IRE1 knockout mice have reduced collagen deposition and AST/ALT activity (two major liver damage enzymes). To determine the potential mechanisms underlying the protective effects of IRE1 deficiency, the authors did mass spectrometry-based quantitative proteomics of liver samples from the experimental mice. Through bioinformatic analysis, they determined that protein and mRNA expression levels of P4HB decreased in IRE1 deficiency mice. Besides, in vitro and ex vivo studies suggested that IRE1 deficiency downregulated collagen. They further investigated the impact of IRE1 deficiency in liver steatosis in a mouse model fed a high-fat diet (HFD). Similarly, IRE1 deficiency suppressed liver steatosis and fibrosis in this mouse model. To clarify how IRE1 modulates collagen expression, the authors generated IRE1 knockout cell (KO) lines. They found that IRE1 KO cells may cause misfolding and intracellular accumulation of collagen inside the cells, reducing its secretion. Finally, they suggested the correlation of IRE1 associated gene (XBP1) and PH4B but not PH4A1 in human patients with non-alcoholic steatohepatitis (NASH). Overall, the current study may provide a potential molecular linkage between IRE1 and collagen in chronic liver disease progression. However, the inconsistent data presentation, poor data quality, and lack of suitable animal models make the current manuscript's impact on the field of chronic liver disease low.

    Major concern:

    1. The authors suggested that P450 levels are not changed in the liver samples of IRE1 KO mice compared to the wildtype (WT) ones. It has been demonstrated that IRE1 activation reduced P450 expression levels (PMID: 22291093). Can the authors explain the inconsistent findings?
    2. Why did the authors challenge mice with CCl4 for 12 weeks? The CCl4-induced liver fibrosis model would have a severe fibrotic liver phenotype post the 8-week CCl4 challenge. Did the author check if IRE1 deficient mice have less liver injury than wildtype (WT) mice at early time points? Also, could you check the liver fibrosis phenotype using another typical liver fibrosis mouse model? The HFD-feeding mouse model would induce liver fibrosis after challenging mice for 50 weeks (PMID: 37270060).
    3. What are the mouse numbers, age, and sex in the CCl4 studies? In Fig. 2, several figures have inconsistent mouse numbers for the data presentation.
    4. There are many downregulated and upregulated target genes. What is the rationale for focusing on downregulated P4HB in IRE1 deficient mice, given that IRE1 has an RNase domain functioning in posttranscriptional regulation? In this case, how does IRE1 depletion upregulate P4HB mRNA expression?
    5. The author suggested that TGFb1 would promote collagen generation, but targeting IRE1 would reduce P4HB and the TGFb1-mediated collagen generation. The Fig. 4D does not support the statement. However, the authors' data demonstrated that TGFb1 cannot promote IRE1 and P4HB expression (Fig. 4G). If TGFb1 cannot promote collagen generation through activating IRE1 and P4HB, how can targeting IRE1 reduce TGFb1-induced collagen (Fig. 4H)?
    6. The authors stated that hepatic Ern1 deficiency suppresses the progression of liver steatosis. In fact, it has been reproducibly demonstrated that hepatic IRE1 deletion promotes hepatic steatosis progression (PMID: 21407177 and PMID: 29764990), contradicting the authors' findings. Besides, the authors did not deplete IRE1 specifically in the liver. Therefore, they made a misleading conclusion without solid evidence. The authors basically depleted IRE1 in the whole body of these experimental mice upon poly I:C injection. The authors must not conclude that hepatic Ern1 deficiency suppresses the progression of liver steatosis without considering the contributions of other organs in the phenotypes that they observed. Also, the HFD-treated mice would develop liver fibrosis 50 weeks post-feeding (PMID: 37270060). It is unclear what other treatments the authors used to accelerate the fibrotic liver phenotype, as shown in Fig. 5D. The authors should show body weight and liver weight over body weight in the result section. Besides, hepatic cholesterol, serum triglyceride, and cholesterol need to be measured in these mice to clarify how deleting IRE1 in the whole body can suppress hepatic steatosis, but liver-specific deletion of IRE1 promotes fatty liver. Without clarifying this issue, it is unclear how hepatic IRE1 deficiency can reduce steatosis and liver fibrosis.
    7. The authors suggested that ablation of IRE1 expression increased the levels of intracellular GFP-collagen as compared with control cells (Fig. 6C). How did the authors quantify the results? It is not clear if KO really increased the intracellular collagen levels. As the authors showed in Fig. 6C, WT-NT and WT-GFP-collagen-untreated have no overlap of green fluorescence. However, KO-NT and KO-GFP-collagen-untreated still have an overlap of green fluorescence, indicating that some cells are not GFP-positive. In this case, how could authors conclude that IRE1-KO cells have a more than 2-fold increase of green fluorescence change compared to WT? Besides, Fig. 6F suggested that secreted collagens increased in KO cells, contradicting the authors' previous data in Fig. 2, 4, and 5. Why did you use U2OS, Hepa1-6, and Huh7 in these studies? Should the collagen be secreted by hepatic stellate cells?
    8. In Fig. 7, the authors suggested that IRE1 KO promotes the levels of collagen inside cells using the whole cell lysate. Interestingly, they indicated that IRE1 deficiency suppressed TGFb1-induced collagen production using whole cell lysates (Fig. 4D). It is really confusing if IRE1 KO promotes or suppresses collagen production or secretion. Also, Fig. 7C did not support that IRE1-KO reduced collagen secretion. Besides, what cells did the authors use for these studies? Are they hepatic stellate cells?
    9. It is interesting to see the positive correlation between XBP1 and P4HB mRNA expression. However, it is still unclear if IRE1 deficiency could downregulate P4HB mRNA expression, given its RNase function. Thus, it would be essential to determine how IRE1 regulates P4HB expression before analyzing the correlation using human datasets. Besides, Fig. 8D did not suggest that XBP1 expression levels are correlated with chronic liver disease progression, given that its correlation scores with AST and ALT are 0 and -0.01, respectively.
  4. Version published to 10.1101/2023.05.02.538835v1 on bioRxiv