Matriptase generates a tissue damage response via promoting Gq signalling, leading to RSK and DUOX activation

  1. MA Jiajia
  2. Claire A. Scott
  3. HO Ying Na
  4. Harsha Mahabaleshwar
  5. Katherine S. Marsay
  6. Changqing Zhang
  7. Christopher K. J. Teow
  8. NG Ser Sue
  9. Weibin Zhang
  10. Vinay Tergaonkar
  11. Lynda J. Partridge
  12. Sudipto Roy
  13. Enrique Amaya
  14. Tom J. Carney

  • Curated by eLife

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    Evaluation Summary:

    This manuscript unravels a detailed bipartite signaling mechanism, activation of which results in epithelial inflammation and cell motility. The paper is potentially of broad interest to cancer biologists and epithelial cell biologists. The data generated using the combination of genetic analyses, chemical inhibitors, and state-of-the-art confocal microscopy is of exceptionally high quality and supports the majority of the claims made in this paper.

    (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

Tissues respond to damage by increasing inflammation and epithelial cell motility. How damage detection and responses are orchestrated is unclear. Overexpression of the membrane bound protease, Matriptase, or mutation of its inhibitor, Hai1, results in inflamed epithelia, in which cells have increased motility and are prone to carcinoma. How Matriptase leads to these cellular outcomes is unknown. We demonstrate that zebrafish hai1a mutants show increased H 2 O 2 , NfκB signalling, and IP 3 R-mediated calcium flashes, and that these promote inflammation, but do not generate epithelial cell motility. In contrast, inhibition of the Gq subunit rescues both the hai1a inflammation and epithelial phenotypes, with the latter recapitulated by the DAG analogue, PMA. We demonstrate that hai1a has elevated pERK, inhibition of which rescues the epidermal defects. Finally, we identify RSK kinases as pERK targets disrupting adherens junctions in hai1a mutants. Our work maps novel signalling cascades mediating the potent effects of Matriptase on epithelia, with implications for tissue damage response and carcinoma progression.

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  1. eLife

    Author Response:

    Reviewer #1 (Public Review):

    The paper by Ma et al. uses a combination of proteomics, morpholinos and inhibitor studies to organize a pathway by which matriptase overactivation in Hai-1 mutants leads to epidermal clumps and defects, peroxidation, and inflammation. They find that Hai-1 mutants have upregulated H2O2, calcium signalling and pERK activation, which are mediated through Gq and RSK. Other studies have suggested how Hai-1 mutants over-activate matriptase to cause epidermal clumping and shedding, associated with increased inflammation. This study uses a series of morpholino and inhibitor studies to more mechanistically order this pathway. Understanding the downstream pathway of matriptase activation, upregulated in wound healing and cancer, could reveal a better understanding of its roles in these processes Overall, it is an interesting study, logically laid out, with convincing data.

    The model at the end and the discussion propose that the inflammation and the epidermal pathways are in parallel but from it seems more likely that inflammation results directly from epidermal defects, which should act like a wound. If this is true and if these embryos soak up more fluorescent dextran compared to wild type embryos, it would seem that this could be a linear rather than parallel pathway.

    We have tested this using fluorescein dextran and methylene blue permeability assays (Zhang, J et al., (2015) Exp Dermatol, 24: 605-610; Richardson, R., et al. (2013) The Journal of Investigative Dermatology, 133(6), 1655–1665). Whilst larval fin wounds show strong uptake of the dyes, we were unable to show robust staining in hai1a mutants. This data has been added as Figure 9 - figure supplement 2. The lack of overt dye penetration made it difficult to draw conclusions from this as it is still possible there is permeability problems, just that we cannot detect it through these assays. We think more pertinent was the fact that we can rescue the epithelial defects robustly (eg with MAPK or PKC inhibition) without completely rescuing the inflammation phenotype, which you would expect if inflammation was purely a consequence of epithelial defects. Finally, new data added in response to a request by reviewer #2 has shown that the increase in Ca++ and H2O2 occurs very early, prior to epidermal phenotypic presentation (New panels 1L and 3H). As these are well described pro-inflammatory drivers in zebrafish, we believe this adds to the case that the inflammation is, to some extent, independent of the epithelial defects. We thus think it unlikely that inflammation in hai1a is solely due to epithelial defects. HOWEVER, there is clear evidence from us and others that PKC and MAPK activation can also promote inflammation. We have added remarks on this to the discussion

  2. eLife

    Evaluation Summary:

    This manuscript unravels a detailed bipartite signaling mechanism, activation of which results in epithelial inflammation and cell motility. The paper is potentially of broad interest to cancer biologists and epithelial cell biologists. The data generated using the combination of genetic analyses, chemical inhibitors, and state-of-the-art confocal microscopy is of exceptionally high quality and supports the majority of the claims made in this paper.

    (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.)

  3. eLife

    Reviewer #1 (Public Review):

    The paper by Ma et al. uses a combination of proteomics, morpholinos and inhibitor studies to organize a pathway by which matriptase overactivation in Hai-1 mutants leads to epidermal clumps and defects, peroxidation, and inflammation. They find that Hai-1 mutants have upregulated H2O2, calcium signalling and pERK activation, which are mediated through Gq and RSK. Other studies have suggested how Hai-1 mutants over-activate matriptase to cause epidermal clumping and shedding, associated with increased inflammation. This study uses a series of morpholino and inhibitor studies to more mechanistically order this pathway. Understanding the downstream pathway of matriptase activation, upregulated in wound healing and cancer, could reveal a better understanding of its roles in these processes
    Overall, it is an interesting study, logically laid out, with convincing data.

    The model at the end and the discussion propose that the inflammation and the epidermal pathways are in parallel but from it seems more likely that inflammation results directly from epidermal defects, which should act like a wound. If this is true and if these embryos soak up more fluorescent dextran compared to wild type embryos, it would seem that this could be a linear rather than parallel pathway.

  4. eLife

    Reviewer #2 (Public Review):

    The major consequences of Matriptase activation and some of the downstream signaling events have been documented previously. However, it has remained unclear how the epithelial architecture phenotype and inflammation manifest upon activation of Matriptase. This manuscript by Ma et al teases apart this aspect using elegant genetics in zebrafish, chemical inhibitors of signaling molecules, and microscopy to unravel the detailed signaling mechanism downstream of Matriptrase and its inhibitor Hai1. Importantly, it identifies two (interacting) arms of the pathway downstream of Par2 and Gαq/PLC, one operating via RSK and E-cadherin that controls epithelial motility and the other operating via IP3R/Duox/H2O2/NfkB pathway, which regulates inflammation. The authors further propose that the Hai1-Matriptase system functions as a sensor of tissue damage and regulates both inflammation and cell motility, the two hallmarks of wound damage and repair. These are important findings having implications in both cancer and skin inflammatory diseases such as psoriasis.

    Strengths:

    This is a systematic attempt to build the signaling cascade downstream to Matriptase/Hai1, by using proteomic analysis followed by systematic phenotypic analysis of various genetic conditions combined with inhibitors. The imaging is of exceptionally high quality, phenotypes or the phenotypic rescues are quantified and supported by statistical analysis. Barring the following exceptions (below), most of the claims are supported by solid experimental evidence.

    Weakness:

    The basic premise of this paper is that the two arms of the pathway downstream to Gαq/PLC regulate two phenotypic aspects viz. inflammation and cell motility. While the evidence is strong for the inflammation arm, the cell motility arm is based mostly on the similarity of the PMA phenotype with the hai1 loss of function phenotype. There is little evidence to suggest that increased DAG levels downstream of hai1 cause the cell motility phenotype. As it stands currently, the PMA phenotype could be completely independent of the hai1 phenotype. Additionally, as per the proposed signaling cascade (Fig. 11), an increase in H2O2 levels, Ca++ flashes, the activation of NfkB signaling, nuclear localization of phosphoRSK should precede the hai1 phenotypic manifestation. However, most of these parameters are evaluated after the phenotype is visible, making it impossible to parse out the cause and consequences. The inhibitors rescue either both or one of the phenotypic aspects but it's not clear whether they block the phenotypic manifestation. This is important to find out because the authors claim that the phenotype arises because of the activation of these two pathways. However, it's quite likely that these two arms of the cascade get activated after the phenotype arises and are essential to maintain the phenotypes.

  5. eLife

    Reviewer #3 (Public Review):

    This study by Ma and colleagues combines the power of zebrafish genetics with live imaging to dissect out the signalling pathways operating downstream of matriptase activation in the fish. Overexpression of this protease or mutation of its inhibitor Hai1a lead to an increase in both neutrophil and epidermal motility. The authors go on to show that these effects are due to distinct signalling pathways operating downstream of matriptase. The work offers an important insight into how Matriptase dysregulation could lead to human carcinomas and offers a mechanistic explanation for its ability to promote malignancy when overexpressed in mice.
    The authors successfully identify the key individual molecular players of two separate cascades, ultimately depicting a neat scenario. The experimental approaches are appropriate, thorough and rigorous. The manuscript contains a huge amount of data and the authors have done an excellent job on dissecting out the signalling pathways that regulate the different effects of matriptase activation, although presenting the study in a more concise way could make it more accessible to readers.

    One major concern is that the relevance of these findings to a tissue damage response - which is stated in the manuscript's title - are not clear or at least not clearly supported by the data. The authors don't show any real evidence that this is a true tissue damage response or show any data on how matriptase is activated (or Hai1a might be inactivated) downstream of damage/wounding. Overexpression of matriptase (or inactivation of Hai1a) leads to many of the same biological outcomes as wounding but claiming this is a tissue damage response without directly linking it to damage is not justified.

  6. Version published to 10.1101/2021.01.21.427549v1 on bioRxiv