Phosphoproteomics of cellular mechanosensing reveals NFATC4 as a regulator of myofibroblast activity

Laura F. Mattner, Zhen Zeng, Christoph H. Mayr, Meshal Ansari, Xin Wei, Sara Asgharpour, Anita A. Wasik, Nikolaus Kneidinger, Mircea-Gabriel Stoleriu, Jürgen Behr, Julien Polleux, Ali Önder Yildirim, Gerald Burgstaller, Matthias Mann, Herbert B. Schiller

Posted on: 21 February 2023

Preprint posted on 13 February 2023

Alumni

Phosphoproteomic analysis of human lung fibroblasts identifies a stiffness threshold above which fibroblasts undergo fibrosis-associated changes and finds NFATC4 as a potential therapeutic target.

Selected by Rob Hynds

Categories: bioinformatics, cell biology, molecular biology

Background

Pulmonary fibrosis is a chronic and progressive lung disease that is characterized by the formation of excessive scar tissue in the lungs. For patients, this leads to breathing difficulties, a reduced supply of oxygen to the body and ultimately death due to respiratory failure. While the causes of pulmonary fibrosis are not fully defined, it is believed that repeated injury to epithelial cells in the alveolus triggers an abnormal wound healing response that leads to the accumulation of scar tissue.

Lung fibroblasts are the cell type primarily responsible for producing the extracellular matrix that forms fibrotic lesions. Fibroblasts undergo characteristic phenotypic changes including proliferation, increased extracellular matrix (e.g. collagens) production and conversion to myofibroblasts. In tissue homeostasis and repair, physical forces can induce cellular responses to contribute to tissue regeneration. However, mechanical forces can also contribute to pathological changes and fibrosis. Fibroblasts reside in and contribute to the maintenance of the extracellular matrix environment and therefore experience and contribute to changes in the physical properties of tissue. Although fibroblasts are known to be mechanosensitive, the specific signalling pathways that drive changes in fibroblast phenotype in response to increased tissue stiffness are not well understood. Characterising the signalling events induced by stiffness changes is an opportunity to develop more effective anti-fibrotic therapies.

Key Findings

Proteome-wide characterisation of phosphorylation events on substrates with varied stiffness

The authors seeded normal human lung fibroblasts (CCL-151 cells) on fibronectin-coated PDMS substrates of five defined stiffnesses and subjected cells to proteome-wide assessment of phosphorylation 120 minutes after seeding. As substrate stiffness increased, more phosphorylation events were found, including those known to be involved infibroblast mechanosensing, such as at sites on myosin regulatory light chain and paxillin. In total, 1631 events on 731 proteins were significantly altered by substrate stiffness with a 1% false discovery rate, with a large majority of these representing phosphorylation at serine residues. Pathway analyses showed that 8 kPa represents a threshold above which kinase activity across multiple signalling pathways increases substantially.

The experiment in CCL-151 cells was repeated using a freshly isolated primary fibroblast culture from an adult patient. Analysis of the combined dataset allowed a focus upon high confidence events but interesting variability was also observed between the two lines, paving the way for future studies looking at phosphoproteomic hetereogeneity within and between disease states.

Time-resolved analysis of fibroblast cell spreading

Next, phosphorylation events across the course of cell spreading on stiff substrates were investigated, adding temporal resolution to the analysis of mechanoresponsive phosphosites. Multiple clusters of sites were identified that occurred in stereotypic temporal order during cell spreading. Integration of this dataset with the previous analysis of stiffness responsiveness identified 227 sites that were significantly altered in both datasets, suggesting their central roles in lung fibroblast mechanosensing.

Identification of NFATC4 as transcriptional modulator of the fibrotic phenotype

Finally, the authors zoomed in on NFATC4, a transcription factor identified in their datasets that hadn’t previously been associated with fibrotic responses. Phosphorylation of NFATC4 S213/S217 increased with increasing stiffness in lung fibroblasts, and had been shown in prior studies to activate transcriptional regulation by NFATC4; thus making NFATC4 a likely mediator of fibrotic phenotypes. Consistent with this, knockdown experiments showed reductions in aSMA expression and collagen I production in response to both substrate stiffness and TGFb stimulation, as well as reduced invasion into and contraction of collagen gels.

Why I think this preprint is important

This preprint describes proteome-wide lung fibroblast phosphorylation events in response to increasing substrate stiffness and during cell spreading. NFATC4 is identified as a novel regulator of lung fibroblast mechanosensing and further elucidation of the pathway in which it acts might open new avenues for anti-fibrotic therapy. This study will therefore be an important reference resource for the lung fibrosis field and lays the groundwork for further functional studies that determine the significance of both the signalling pathways and specific phosphorylation events described.

Questions for the authors

Q1. The manuscript focuses on phosphorylation sites that increase with substrate stiffness but it seems that dephosphorylation is almost as common a result of increasing stiffness. Do the authors have any sense of whether those events regulate different pathways/processes to phosphorylation events?
Q2. Did knockdown of NFATC4 affect the viability and/or proliferation of cultured lung fibroblasts?
Q3. Prior to experiments, fibroblasts were cultured on extremely stiff tissue culture plastic. Do the authors think that this might change the threshold at which (i.e. sensitize or de-sensitize cells to stiffness)?
Q4. Do cells still spread on the very soft 0.5 kPa and 2 kPa substrates? Presumably the number and/or maturity of focal adhesions that form are different in those substrates, explaining a subset of signalling events?

doi: https://doi.org/10.1242/prelights.33812

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Author's response

Dr. Herbert Schiller shared

Thanks for selecting our preprint!

A1. In fact, increases in phosphorylation were more common than dephosphorylation. But indeed, we identified a cluster of phosphorylation sites (cluster 2 in Fig2A), which were high on soft substrates and gradually declined towards stiffer substrates. Interestingly, this cluster was enriched for RhoGDI signaling suggesting the regulation of Rho GTPases by substrate stiffness involves the dephosphorylation of RhoGDIs. We also found some exclusively enriched sites on specific kinases in this cluster such as MAPK1 (also known as ERK2), PAK1, PDK1, PKN2, and PRPF4B. For instance, MAPK1 T185 was undetectable on stiff substrates and thus likely dephosphorylated.

A2. Yes, knockdown of NFATC4 had the tendency to promote proliferation of cultured lung fibroblasts. In our hands, knockdown of NFATC4 resulted in a slight increase in cell proliferation in three different lung fibroblast cell lines, compared to the cells treated with negative control siRNA (siNC) (by WST-8 assay). Previous studies showed that NFATC4 can regulate cell proliferation in different cellular contexts (Cole et al.,2020; Moreno et al., 2015). Further investigation is needed to fully understand the underlying mechanisms involved.

Cole, A.J., Iyengar, M., Panesso-Gómez, S., O’Hayer, P., Chan, D., Delgoffe, G.M., Aird, K.M., Yoon, E., Bai, S. and Buckanovich, R.J., 2020. NFATC4 promotes quiescence and chemotherapy resistance in ovarian cancer. JCI Insight, 5(7).
Moreno, M., Fernández, V., Monllau, J.M., Borrell, V., Lerin, C. and De La Iglesia, N., 2015. Transcriptional profiling of hypoxic neural stem cells identifies calcineurin-NFATc4 signaling as a major regulator of neural stem cell biology. Stem Cell Reports, 5(2), pp.157-165.

A3. Great question. Indeed, studies on mesenchymal stem cells have demonstrated a potential mechanical memory of cells that could sensitize or desensitize cellular stiffness thresholds (Yang et al., 2014). Another previous study on fibroblasts evaluated the cell migration of fibroblasts that were pre-cultured on 2 kPa, 25 kPa gels or plastic dishes for 4 days. Fibroblasts precultured on stiff substrates showed altered migration behavior compared to soft, suggesting that the pre-culture conditions may affect the adaption of fibroblasts to new environments (Fig. 6C and D in Asano et al., 2017).

However, with the experimental design used in our phosphoproteomic analysis, we cannot answer whether previous culture of the lung fibroblasts in plastic dishes affected their signaling threshold.

Yang, C., Tibbitt, M.W., Basta, L., and Anseth, K.S., 2014. Mechanical memory and dosing influence stem cell fate. Nature Materials, 13, 645–652
Asano, S., Ito, S., Takahashi, K., Furuya, K., Kondo, M., Sokabe, M. and Hasegawa, Y., 2017. Matrix stiffness regulates migration of human lung fibroblasts. Physiological Reports, 5(9), p.e13281.

A4. As shown in Figure 1A and S3 the cells also spread and move around on soft 0.5kPa fibronectin coated PDMS. In fact, we think this represents the physiological stiffness range that lung fibroblasts encounter in vivo. You are correct that cells take longer to polarize and they also form smaller focal adhesions and contacts. As pointed out in the manuscript, we found a large number of adhesome proteins to be differentially phosphorylated which can indeed likely be linked to the differential state of the focal adhesions on soft and stiff.