Neutral sphingomyelinase 1 regulates cellular fitness at the level of ER stress and cell cycle

  1. Dolma Choezom
  2. Julia Christina Gross

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Abstract

Neutral sphingomyelinase 1 (nSMase1) belongs to the sphingomyelinase enzyme family that hydrolyzes sphingomyelin to produce signaling active lipid ceramide and phosphorylcholine. The molecular characterization and biological function of nSMase1 remain poorly studied. Here, we report that nSMase1 (gene name: SMPD2) knockdown reduces LAMP1 at the mRNA levels and is required for initiating a full-potential unfolded protein response under ER stress. Additionally, SMPD2 KD dramatically reduces the global protein translation rate. We further show that SMPD2 KD cells are arrested in the G1 phase of the cell cycle and that two important cell cycle regulating processes - PI3K/Akt pathway and Wnt signaling pathway are altered. Taken together, we propose a role for nSMase1 in buffering ER stress and modulating cellular fitness via cell cycle regulation.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors show that nSMase1 (gene name: SMPD2) knockdown reduces LAMP1 at the mRNA levels, causes inefficient activation of the UPR upon ER stress, arrests cells in the G1 phase, reduces the level of phosphorylated Akt, downregulates the Wnt signaling pathway, and reduces the overall protein translation in both HeLa cells and HCT116 cells. Although these findings are potential interesting, these findings do not define the biological role for nSMase1. Moreover, it is unclear how nSMase1 knockdown causes these changes.

    Specific comments:

    1. The authors do not provide any evidence showing that "nSMase1 knockdown" actually occurs in HeLa cells or HCT116 cells. Does siRNA reduce the levels of nSMase1 mRNA and protein?
    2. Many western blots lack quantification, such as Figures 1A, 1G, 3B, and 4K.
    3. Figure 3A shows that the effects of nSMase1 knockdown on cell apoptosis are very modest.
    4. Is there any explanation how nSMase1 knockdown dramatically reduces protein translation?
    5. It could be better to assess the UPR by performing western bolt for PERK, ATF6 and IRE1.

    Significance

    The authors show that nSMase1 (gene name: SMPD2) knockdown reduces LAMP1 at the mRNA levels, causes inefficient activation of the UPR upon ER stress, arrests cells in the G1 phase, reduces the level of phosphorylated Akt, downregulates the Wnt signaling pathway, and reduces the overall protein translation in both HeLa cells and HCT116 cells. Although these findings are potential interesting, these findings do not define the biological role for nSMase1. Moreover, it is unclear how nSMase1 knockdown causes these changes.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this paper, the authors sought to investigate the biological role of neutral sphingomelinase 1 (nSMase1; SMPD2) in two established cell lines, with a focus on viability and response to cell stress. The authors reduced the level of SMPD2 in HeLa and HCT116 cells, with the use of siRNA and validated the efficiency of this knockdown. They followed up with a characterization on autophagic activity, unfolded protein response (UPR) pathway and cell cycle progression in SMPD2-KD cells. The approach is rational and the statistical methods used are sound.

    The authors showed that SMPD2-KD in both cell lines resulted in a significant reduction of LAMP1, a lysosomal-associated protein. However, this reduction did not affect the lysosomal activity of the cells, with the turnover lysosomal-associated proteins, LC3B-II and P62, shown to be unchanged under both starved and normal conditions. Similarly, measurement of lysotracker puncta also revealed no changes in lysosomal acidification. Furthermore, the authors showed that the downregulation of LAMP1 in SMPD2-KD HeLa cells occurs at the transcriptional level, as the inhibition of proteasomal and lysosomal activity, using MG132 and Bafilomycin-A, respectively, did not impact LAMP1 protein levels.

    The authors went on to show that the induction of ER stress in HeLa cells using thapsigargin and tunicamycin resulted in the increase in SMPD2 protein. In SMPD2-KD cells, thapsigargin and tunicamycin treatment resulted in lower levels of ER stress markers, spliced version of XBP-1, ATF4 and EDEM mRNA, when compared to 'control' (taken to be SMPD2 intact) cells. Despite the reduced induction of ER stress in SMPD2-KD cells by thapsigargin and tunicamycin, the viability of these cells was found to be significantly lower than thapsigargin- and tunicamycin-treated control cells. These findings led the authors to conclude that an inability to mount a UPR response was detrimental to cell viability. A problematic inference.

    The impact of SMPD2 KD in HeLa cells was also validated using annexin V/PI FACS and immunoblotting of cleaved caspase 3 and cleaved caspase 7. FACS analysis showed a significantly lower percentage of viable cells in SMPD2-KD cells but caspase activation did not occur. The authors also showed that this decreased viability could potentially result from cell cycle dysregulation, as the percentage of cells in the G1 phase was found to be significantly higher in SMPD2-KD cells when compared to the control cells. Furthermore, p21 and p27, which are G1 cell cycle arrest protein, were found to be upregulated in SMPD2. Further elucidation revealed a higher level of phosphorylated CHK2 and lower level of phosphorylated AKT, suggesting that cell cycle dysregulation in SMPD2-KD cells could be partially affected by the P13K/Akt pathway. The authors also showed that the impaired cell cycle progression could partially result from the canonical beta-catenin pathway, with SMPD2-KD cells showing lower level of Wnt activity.

    Overall, the author concluded that the knockdown of SMPD2 could affect cell viability through dysregulation of cell cycle progression while autophagic activity were unaffected by the loss of SMPD2. Noteworthily, the authors also showed a lower level of UPR response in thapsigargin- and tunicamycin-treated SMPD2-KD cells and concluded that this reduced response is detrimental to the cells and would lead to reduced cell viability.

    Major Comments:

    1. While the KD of SMPD2 did result in a lowering of nSMase1, the effect of the SMPD2 KD on other SMases remains unclear. Was the compensation from other SMases because of SMPD2 KD?
    2. Related to the first, most results are primarily based on siRNA mediated knockdown of SMase1, but there were no rescue experiments conducted to rule out Off-Target effects of the siRNA. This is a major concern as the conclusions on SMase1 role(s) are entirely based on the KD of SMase1. The control for each of the KDs were a generic siRNA pool (siCtrl) purchased from Dharmacon, rather than a scrambled sequence for each specific gene-targeted siRNA. This raises a slight concern.
    3. The authors showed lower levels of UPR markers in SMPD2 KD cells exposed to thapsigargin and tunicamycin and concluded that this 'failure' to mount an ER response is responsible for the observed decrease in cell viability. However, there is no conclusive evidence linking the two observations. It becomes more confusing when the inhibition of IRE1 activity with 4µ8C was observed to INCREASE viability in both SMPD2-KD and control cells. Does this not suggest that lowered level of UPR response in SMPD2 cells is beneficial?
    4. The Annexin V assay also revealed that there are lower percentage of viable cells in SMPD2-KD cells, even in the absence of thapsigargin or tunicamycin treatment. This suggest that the impact of SMPD2 knockdown on cell viability could be independent of ER stress.
    5. The use of established lines that grow extremely rapidly limits the conclusion of the paper. Furthermore, why was the cell cycle analysis done in non-synchronised cells? It would have been cleaner to pre-treat cells with nocodozole for a brief period, before continuing with culture (and treatment). The impact of SMPD2 on cell cycle arrest could be more convincing.
    6. The authors also showed that there was a significant decrease in ceramides in SMPD2-KD cells which on its own can induce ER stress (1). The involvement of ceramides in the lowered ER stress response in SMPD2-KD cells is confounding and needs further clarification.
    7. The authors showed a higher percentage of early apoptotic cells in SMPD2-KD cells using Annexin V assay but western immunoblotting of cleaved caspases 3 and 7 were inconclusive of apoptosis (or pre-apoptosis) in these cells. A further validation is required, eg. caspase 3/7 activity assay to confirm the immunoblot data.
    8. The authors pointed out the endogenous SMPD2 resides in the nuclear matrix, while the shift in subcellular localization to the ER membrane occur when SMPD2 is overexpressed. This premise led to the authors' speculate that upregulation of SMPD2 during ER stress is a crucial event in the maintenance of ER homeostasis. The authors need to validate this (not speculate) by showing SMPD2 localization in the presence, and absence, of thapsigargin and separately tunicamycin.
    9. The authors found that p21 and p27 were upregulated in SMPD2-KD cells which then contributed to the cell cycle arrest. But a validation of this conclusion is missing. Are levels of p21 and p27 normalised upon rescue of SMPD2 in KD cells?

    Minor Comments

    1. There are couple of quantification which could benefit from an increase in N number as the individual points were inconsistent e.g., Fig 2 D-F.
    2. The presentation of the results is confusing as work with the two different cell lines were placed in the same figure e.g. Fig 4E-G.
    3. The procedure for lysotracker is missing in the materials and methods.

    Significance

    This paper delves into the role of nSMase1 in the regulation of cell cycle and ER stress response in two cancer cell lines. Previous work had identified nSMase1 as an important initiator of apoptosis when exposed to environmental stressors. Activation of nSMase1 increased ceramide levels, which in turn led to increased apoptosis via the caspase pathway (2). The authors now provide an observation that the knockdown of nSMase1 would also reduce cell viability through dysregulation of cell cycle progression, even in the absence of environmental stressors. However, these findings remain inconclusive in proving that the failure to mount an ER stress response in SMDP2-KD cells leads to G1 phase arrest. The role of nSMase1 in lowering ceramides and using ceramides to link ER stress and cell cycle arrest remains interesting. Ceramide dysregulation in diseases such as diabetes and cardiovascular diseases is perhaps more relevant rather than cancer (use of appropriate cells). Overall, the significance of finding SMDP2-KD cells causing cell cycle arrest is limited because of the cells used. More work needs to be done before realising whether nSMase1 could potentially be a therapeutic target for lowering of ER stress, promoting cell proliferation (and the importance of cellular ceramide in this pathway).

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, the authors report on the role of neutral sphingomyelinase 1 (nSMase1) in regulating cell cycle through ER stress.

    Overall, the authors report interesting observations, but the manuscript falls shot to provide a coherent and comprehensive mechanism. Many of the experiments reported have large reproducibility variation and lack further supportive experiments to support the authors' claims. I recommend the authors to perform additional experiments to strengthen their claims and/or to tone down some of their conclusions. The manuscript will require a major revision before it is ready for publication to a specialized and a broad readership.

    Major points to address

    [Unfortunately, the authors didn't include page numbers nor line numbers so I will refer to the page numbers of the PDF file]

    1. In general, the authors report the efficiency of their knockdown after several experiments. It should be reported first before it is used for any of the experiments. For instance, knocking down siSMPD2 should be shown 1st before the experiment and not in panel H of Fig. 1.
    2. Some of the reported immunoblots are of poor quality and should be replace by a better replicate from their biological replicates or repeated to meet expected standards. Here are some examples:
      • (a)The band of LAMP1 in Fig. S1B should be strong and obvious based on the commercial antibody they used. Here, we are not even sure which of the 4 bands is LAMP1.
      • (b) In Fig. 1D, I am a bit concerned to see that the loading control GAPDH is uneven through the samples. Do the authors load the same amount of total protein for each sample?
      • (c) The detection of LC3B-II should include LC3B-I. Both are the same protein where the LC3B-II is covalently bound to PE.
    3. The authors concluded based on the data presented in Fig. 1D-F that "downsregulated LAMP1 does not affect lysosome function. The lysosomal function is not demonstrated from these experiments so the authors cannot make such conclusion.
    4. In Fig. S1G, H, the authors claims that SMPD2 KD affects cellular ceramide levels specifically at the ER. Microscopy is not the best approach to quantify ceramide levels. Ideally, the authors should use a biochemical approach to validate their finding such as TLC or LC/MS/MS.
    5. Four-hour treatment with tunicamycin (Tm) or thapsigargin (Tg) is rather small to provide enough time for cells to make sufficient proteins that will be visible by immunoblot. It is best to allow at least 12h of incubation for UPR-regulated proteins. However, 4h would be sufficient to monitor UPR-induced mRNA levels.
    6. The authors concluded that nSMase1 protein is increased upon ER stress. However, the increase is small. Was the nSMase1 mRNA level increased as well with Tm and Tg? What is the other protein that has been cut from the immunoblot of nSMase1 on top in DMSO and Tm lanes (Fig. 2A)? Also, nSMase1 predicted MW is 47 so I am bit puzzled why it runs below 40 KDa in Fig. 2A. Is the increase in nSMase1 upon Tm or Tg is specific to the UPR response such IRE1, PERK, or ATF6 branches? The variation between the replicates is enormous especially for PDI.
    7. I appreciate that the authors report the replicates for their experiments. However, the variation between biological is rather large for in vitro studies. Also, the authors shouldn't stress that they observe a small different when it is clearly not significant and that the variation is rather large. Many more replicates are needed to distinguish small unsignificant variations. Here are some examples:
      • (a) The authors reported that the upregulated of BiP by Tm or Tg is slightly impaired by SMPD2 KD (Fig. 2C, D). I don't see any significant difference. Large variation between replicates.
      • (b) The variation of spliced XBP1 is extremely high especially siCtrl with Tm (Fig. 2G). It should be very reproducible unless cell confluency was not consistent between replicates or that cells were overconfluent. It should be "XBP1" and not "XBP-1".
      • (c) Replicate variation for BiP, CHOP, SMPD2 is also problematic (Fig. 2).
      • (d) The authors stated "SMPD2 mRNA levels were slightly increased by tunicamycin and thapsigargin treatment (Fig. 2M)". Is the increase significant? It doesn't seem so and the variation between replicate is quite high.
      • (e) I don't see an increase in nSMases1 upon Tm treatment unlike the authors claim in Fig. 2A.
      • (f) The GAPDH band in fig. 4E contains a "bubble" so I am sure how the quantification can be meaningful.
    8. ATF4 mRNA level is not a good indicator of UPR activation. ATF4 mRNA is quite constant but the translation of ATF4 is induced upon PERK activation. Therefore, the authors should look at ATF4 protein levels.
    9. In Fig. 2, are the increase of all these UPR-upregulated genes significant for Tm and Tg compared to DMSO? Not indicated anywhere.
    10. In page 5, the authors stated "under ER stress conditions the LAMP1 mRNA remained significantly downregulated by SMPD2 KD". Is LAMP1 mRNA level significantly upregulated upon ER stress? It doesn't seem to be the case so I am wondering what this statement is implying.
    11. For the experiment reported in Fig. S1J, he inhibition should have been shown in the presence of Tm or Tg.
    12. What are the evidence that SMPD2 KD failed to activate the UPR upon ER stress? All the data in Fig. 2 demonstrate that the UPR is significantly induced with Tm and Tg in SMPD2 KD cells. Also the authors have to be cautious by using drugs that induce the ER stress as they have side effects. For instance, Tg dramatically increases the calcium levels in the cytosol which could affect SMPD2 KD cells independently of ER stress.
    13. I disagree with the authors interpretation of the data "a full-potential UPR signaling activation upon ER stress is not achieved in SMPD2 KD cells, and consequently their cellular fitness is impaired under ER stress conditions.". I am not sure what is their definition of "full-potential UPR" but I don't see any problems in the UPR activation with Tm or Tg in SMPD2 KD cells. Someone has to be very careful to interpret lower UPR activation but still significant activation of the UPR. Overall, the data related to ER stress and the UPR in SMPD2 KD is inconclusive and just a distraction.
    14. It should be clear in the text what is detected by flow cytometry for Fig. S2A.
    15. How many cells are included in the analysis of Fig. 3D? There should be at least 20 cells so at least 20 data points. It shouldn't be the average of cell diameter for each biological replicate. The difference is very small. Also, diameter is meaningless as most cells are uneven so it would be best to compare the area of each cell.
    16. In Fig. 3F, the experiment should include a control that induce cell cycle arrest such as nocodazole.
    17. The authors compared the levels of P21 and P27 at 72h and 96h. These 2 time point experiments were not done together so it is difficult to compare and to make any conclusion. Someone would have to analyse several time points to make any conclusion.
    18. Can we just say that they grow more slowly instead of claiming temporary cell cycle arrest in page 7? It just means that the cells are spending more time in G1 in KD SMPD2 compared to control.
    19. Some of the cell cycle experiments are not done correctly to make any conclusions. For instance, Chk2 is ATM substrate and is phosphorylated upon ATM signaling to enforce checkpoint arrest. Decrease in Chk2 phosphorylation typically means if in DNA damage context, ATR plays predominant role. Usually, ATM and ART are redundant kinases. There is no report of Chk2 phosphorylation from the referred publication.
    20. What is the rational of changing cell likes at Fig. 4H?
    21. The authors conclude that "SMPD2 KD seems to affect many cellular processes by downregulating their signaling components including Wnt signaling - which could explain the reduction in global protein translation and G1 cell cycle arrest". Global protein transcription and translation inhibition is typical of stressed cells. Therefore, their statement and findings are broad and failed to pinpoint to any major players or mechanism.

    Minor points to address

    There are some minor points that should be considered below before publication if they haven't been already addressed by the authors.

    [Unfortunately, the authors didn't include page numbers nor line numbers so I will refer to the page numbers of the PDF file]

    1. Page 3, should be "cancer cell line" and not "cancer line".
    2. Page 5, the authors should clarify what the inhibitor refers to in the sentence "We found BiP to be upregulated at the protein level by both inhibitors, which was slightly impaired by SMPD2 KD after 4 h of treatment".
    3. Fig. 3A, Y-axis labelling not clear.
    4. Fig. 3C, label of x-axis is missing.
    5. Page 7, it should be flow cytometry and not FACS.

    Significance

    The manuscript in its current form fails to provide an advance to the field as there is no coherent mechanism. Therefore, it is difficult to judge the target audience at this premature stage.

    My expertise includes endoplasmic reticulum stress, autophagy, lipid synthesis and regulation.

  5. Version published to 10.1101/2022.02.23.481585 on bioRxiv