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Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP

Nature Cell Biology volume 21, pages 338–347 (2019)Cite this article

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Abstract

Extracellular matrix (ECM) mechanical cues have powerful effects on cell proliferation, differentiation and death. Here, starting from an unbiased metabolomics approach, we identify synthesis of neutral lipids as a general response to mechanical signals delivered by cell–matrix adhesions. Extracellular physical cues reverberate on the mechanical properties of the Golgi apparatus and regulate the Lipin-1 phosphatidate phosphatase. Conditions of reduced actomyosin contractility lead to inhibition of Lipin-1, accumulation of SCAP/SREBP to the Golgi apparatus and activation of SREBP transcription factors, in turn driving lipid synthesis and accumulation. This occurs independently of YAP/TAZ, mTOR and AMPK, and in parallel to feedback control by sterols. Regulation of SREBP can be observed in a stiffened diseased tissue, and contributes to the pro-survival activity of ROCK inhibitors in pluripotent stem cells. We thus identify a general mechanism centered on Lipin-1 and SREBP that links the physical cell microenvironment to a key metabolic pathway.

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Fig. 1: Actomyosin contractility and ECM mechanical cues regulate lipid synthesis.
Fig. 2: ECM mechanical cues regulate SREBP1/2 target genes.
Fig. 3: ECM mechanical cues regulate lipid synthesis by controlling SREBP1/2 activation.
Fig. 4: ECM mechanical cues regulate SCAP accumulation to the Golgi apparatus.
Fig. 5: ECM mechanical cues regulate Lipin-1/ARF1 activity.
Fig. 6: Actomyosin contractility regulates SREBP through Lipin-1.
Fig. 7: The Golgi apparatus responds to extracellular physical cues and intracellular force application.

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Code availability

No custom codes were used in this study. All codes are indicated in the appropriate Methods sections and references.

Data availability

Microarray data generated here have been deposited in GEO under accession code GSE107275. Metabolomics and targeted lipidomics data have been deposited in the Figshare database (https://doi.org/10.6084/m9.figshare.7338764). The previously generated gene expression data analysed for Fig. 3i can be found in GEO under accession code GSE90051 (secondary data set). Source data for Figs. 1–7 and Supplementary Figs. 1–7 are provided in Supplementary Table 3. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank Y. Chen, K. Mori, G. DelSal, B. Viollet, H. Louvel, E. Greotti, A. DeMatteis, R. Venditti, A. Hausser, R. Rizzuto, D. Vecellio Reane, L. Scorrano, L. Pernas, C. Cheng, E. Melloni, M. Pende, R. Talha, R. Ogawa, J. Goldstein P. Espenshade and W. Shao for protocols and materials, S. Giulitti for generous help with hydrogels, M. Pellegrini and I. Zorzan for help with hPSC cultures and G. Martello and M. Montagner for thoughtful discussions. This work was supported by AIRC IG-15307 and IG-21392, WCR 15-1192, CARIPARO Eccellenza 2017 and University of Padua BIRD grants to S.D., an AIRC Hard ROCK Café Fellowship to G.S., the UPMC ‘Interface pour le Vivant’ doctoral programme to S.M. and an AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to S.B.

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Authors and Affiliations

  1. Department of Molecular Medicine (DMM), University of Padua, Padua, Italy

    Patrizia Romani, Irene Brian, Giulia Santinon, Arianna Pocaterra & Sirio Dupont

  2. Department of Pharmacological and Biomolecular Sciences (DiSFeB), University of Milan, Milan, Italy

    Matteo Audano, Silvia Pedretti & Nico Mitro

  3. Institut Curie, PSL Research University, CNRS, UMR 144, Paris, France

    Samuel Mathieu & Jean-Baptiste Manneville

  4. Sorbonne Université, UPMC University Paris 06, CNRS, UMR 144, Paris, France

    Samuel Mathieu & Jean-Baptiste Manneville

  5. Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy

    Mattia Forcato & Silvio Bicciato

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Contributions

P.R. and I.B. performed experiments and analysed data with help from G.S. and A.P. M.A., S.P. and N.M. performed metabolic measurements and advised on the interpretation of metabolic data. S.M. and J.B.M. performed Golgi micromanipulations. M.F. and S.B. performed bioinformatics analyses. S.D. and P.R. planned experiments. S.D. coordinated and supervised the project, and wrote the paper.

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Correspondence to Sirio Dupont.

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Supplementary Figure 1 Lipid accumulation upon ROCK/MLCK inhibition is a general response.

a, Immunofluorescence for F-actin bundles with Phalloidin in MCF10ATk1 cells treated for 6 hours with DMSO or with 20 μM Y27632 and 20 μM ML7 (YM). At least 50 cells per condition. Scale bar: 10 μm. b, qPCR of the YAP/TAZ target CTGF in MCF10ATk1 cells treated as in a for 24 hours. Data are relative to GAPDH levels. Mean expression in controls was set to 1, and all other samples are relative to this. n = 4 per condition, pooled across 2 independent experiments. c, TUNEL assay on MCF10ATk1 cells treated for 24 hours with YM. n = 4 independent biological samples per condition, pooled across 2 independent experiments. d, EdU incorporation assay on MCF10ATk1 cells treated 24 hours with YM. n = 4 independent biological samples per condition. pooled across 2 independent experiments. e, Heatmap of lipid molecules displaying significant accumulation by global metabolomics (mean fold>2.5 P<0.05) in MCF10ATk1 cells treated 6 or 24 hours with YM (n=6 biologically independent samples), as compared to an equivalent dose of DMSO (D, n = 4 biologically independent samples). Each column represents an independent biological replicate; each line represents a single metabolite. AcC acylcarnitines; GP phospholipids; LysoGP lyso-phospholipids; MG monoacylglycerols; DG diacylglycerols; SL sphingolipid metabolism; ST sterols. Triglycerides were not part of this analysis. f-h, lyso-phosphatidylcholine (lyso-PC, f) and ceramide (Cer, g and h) levels in MCF10ATk1 cells treated 24 hours with YM or DMSO, measured by targeted lipidomics. Only the five most abundant and lyso-PC are shown. n=5 biologically independent samples per condition. i, Cholesterol (Filipin) and neutral lipids (ORO) accumulation in MCF10ATk1 mammary epithelial treated with YM at lower concentrations for 24 hours. j, Close-up of a representative YM-treated MCF10ATk1 cell transfected with the lipid-droplet marker Perilipin3-RFP and stained with Bodipy493/503 neutral lipid stain. k, Treatment of cells with YM together with the pan-caspase inhibitor Z-VAD-FMK (ZVAD, 20 μM) excludes activation of SREBP by caspases1. l, Time-course of lipid accumulation upon YM treatment. m, Cholesterol accumulation in multiple cell lines treated with YM for 24 hours: RPE1 human immortalized retinal pigment epithelium; MDA231 human metastatic breast cancer; 3T3L1 mouse primary fibroblasts; MCF10A human immortalized mammary epithelium; HEK293 human immortalized embryonic kidney; WI38 human adult primary fibroblasts. n, Neutral lipid accumulation in multiple cells treated as in m. Scale bars 5 μm. The images in panels a, i-n are representative of at least two independent experiments with similar results. Data are mean and SD or mean and single points; unpaired two-tailed Student’s t-tests. Source data and quantification of images are providedin Supplementary Table 3.

Supplementary Figure 2 YAP/TAZ activity is inconsequential for lipid synthesis and SREBP activation in response to inhibition of ROCK/MLCK.

a, Staining for cholesterol in MCF10ATk1 depleted of YAP/TAZ. Scale bar: 10 μm. Quantifications and n in Supplementary Table 2. Scale bar 10 μm. b, Staining for cholesterol in MCF10ATk1 stably infected with pBABE TAZ 4SA or empty pBABE control, and treated with YM for 24 hours. Slightly enhanced lipids in TAZ 4SA cells is in line with Ref. 2. Quantifications and n in Supplementary Table 2. Scale bar: 10 μm. c, 8XGTIIC-luciferase reporter assay for YAP/TAZ activity in MDA231 cells transfected with control siRNA (siCO.), YAP/TAZ siRNA (siYT), cotransfected with the YAP/TAZ inhibitor NF2/Merlin expression plasmid, or treated with YM. Mean expression in the control was set to 1, and all other samples are relative to this; n = 4 independent biological samples pooled across 2 independent experiments for each bar; unpaired Mann-Whitney tests. d,e,f, qPCR in MCF10ATk1 (d) MDA231 (e) and 3T3L1 (f) cells treated for the indicated times with YM, FM or DMSO. Data are relative to GAPDH levels; mean expression in control cells was set to 1, and all other samples are expressed relative to this; n ≥ 4 independent biological samples pooled across 2 independent experiments for each bar; multiple unpaired two-tailed Student’s t-tests. g,h, LDLR-luciferase reporter assay for SREBP activity in MDA231 cells transfected in g with control siRNA (siCO.), YAP/TAZ siRNA (siYT) or SREBP1/2 siRNAs (siSREBP A and B), in h with a NF2/Merlin expression plasmid. Mean expression in the control was set to 1, and all other samples are relative to this; n≥4 independent biological samples pooled across at least 2 independent experiments for each bar; unpaired Mann-Whitney tests. i, Uptake of the fluorescent Bodipy-C11 fatty acid was measured by flow cytometry in MCF10ATK1 cells treated with DMSO (D) or YM for 24 hours. n=8 independent biological samples pooled across 3 independent experiments; unpaired two-tailed Student’s t-test. Increased fatty acid uptake is also evident from accumulation of lipid species containing linoleic acid, an essential fatty acid provided by the serum (see Ref. {Romani:2018hf}). j, MCF10ATK1 cells were treated with DMSO or YM for 24 hours with 13C2-acetate in the culture medium and subjected to mass-spectrometry analysis of total fatty acids. M+8 and M+10 indicate fatty acids isotopologs containing 8 or 10 13carbon atoms respectively, which derive from acetate through acetyl-CoA and fatty acid synthesis. Mean isotopolog levels in DMSO-treated cells were set to 1, and levels in YM-treated cells are expressed relative to this; n = 5 independent biological samples per condition in one single experiment; unpaired two-tailed Student’s t-tests. The images in panels a and b are representative of at least two independent experiments with similar results. Data are mean and single points. Source data in Supplementary Table 3.

Supplementary Figure 3 ECM mechanical cues regulate lipid synthesis by controlling SREBP1/2 activation.

a, A simplified scheme illustrating how SREBP is regulated by sterols (see text). Left: sterols retain SCAP/SREBP at the ER by binding SCAP and Insig1 proteins; a minor portion of SCAP/SREBP complexes continuously shuttles through the Golgi apparatus and is transported back to the ER3,4. Right: absence of sterols induces SCAP/SREBP accumulation at the Golgi apparatus where SREBP are cleaved by Site-1 Protease (S1P), releasing the transcriptionally-active nuclear form of SREBP. b, LDLR-luciferase in MDA231 cells transfected with the indicated siRNAs in combination with expression plasmids encoding for mouse full-length SREBP1 or SREBP2, whose cDNA is insensitive to siSREBP mixes A and B. Mean expression in the control was set to 1, and all other samples are relative to this; data are mean and single points; n≥4 independent biological samples per condition; unpaired Mann-Whitney tests. c, Accumulation of cholesterol (Filipin) and neutral lipids (ORO) in MCF10ATk1 cells treated with YM for 24 hours is inhibited by knockdown of SREBP1/2. Quantifications and n in Supplementary Table 2. Scale bars 5 μm. d, Co-localization of endogenous SREBP2 with an ER marker (KDEL-mCherry, false colors) in control cells (DMSO), or with a Golgi marker (GM130) upon 2 hours of treatment with YM in MCF10ATk1 cells. Scale bars 10 μm. At least 30 cells per condition. e, Quantification of the ER, Golgi and nuclear localization of endogenous SREBP2 during the time-course YM treatment shown in Fig. 3d. Data are mean with range; n = 5 independent slides per condition; unpaired Mann-Whitney tests for Golgi localization. f, Representative immunofluorescence for endogenous SREBP2 in 3T3L1 cells treated with YM, blebbistatin or plated on soft ECM hydrogels for 6 hours. At least 50 cells per condition. Scale bars 10 μm. g, Representative immunofluorescence for endogenous SREBP2 in RPE1 cells treated with YM or plated on soft ECM hydrogels for 6 hours. At least 50 cells per condition. Scale bars 10 μm. h, Western blotting for endogenous mature SREBP2 in RPE1 cells treated with YM or FM. i, Quantification of SREBP2 nuclear localization in the presence of vehicle (EtOH), cycloheximide (CHX) and CHX + YM shown in Fig. 3f. n = 5 independent slides per condition; data are mean and single points; unpaired Mann-Whitney test. j, Luciferase assay with a highly unstable luciferase mRNA/protein reporter (CMV-luc2P_ARE) in MCF10ATk1 cells. Cells were treated for 3 hours with vehicle (EtOH) or 100 μg/ml cycloheximide (CHX) before harvesting. Mean expression in EtOH was set to 1; n = 6 independent biological samples per condition; data are mean and single points; unpaired Mann-Whitney test. The images in panels b, d, f, g and h are representative of at least two independent experiments with similar results. All n values are pooled across independent experiments. Source data in Supplementary Table 3.

Supplementary Figure 4 Localization and function of core SREBP regulators upon inhibition of ROCK/MLCK.

a, Immunofluorescence localization of transfected HA-tagged S1P in MCF10ATk1 cells treated with DMSO or with YM (Y27632+ML7) for 6 hours. Calreticulin stains the endoplasmic reticulum (ER). At least 50 cells per condition. Scale bar 10 μm. b, Immunofluorescent localization of endogenous Insig1 in RPE1 cells treated with DMSO or with YM for 6 hours. Similar results were obtained in MCF10ATk1 cells. At least 50 cells per condition. Scale bar 10 μm. c, Immunofluorescent localization of transfected MYC-tagged SCAP in MCF10ATk1 cells treated with DMSO or with YM for 6 hours. At least 50 cells per condition. Scale bar 10 μm. d, qPCR analysis of SCAP and of established SREBP target genes in MCF10ATk1 48 hours after transfection with two independent siRNAs targeting SCAP (A and B). Data are relative to GAPDH levels; mean expression levels in controls was set to 1, and all other samples are relative to this; n=2 independent biological samples; data are mean and single points. e, Filipin staining of hPSC plated as single-cells or cultured as colonies and treated for 24 hours with 10 μM Y27632. Quantifications and n in Supplementary Table 2. Scale bar 20 μm. The images in panels a, b, c, and e are representative of at least two independent experiments with similar results. n values are pooled across independent experiments. Source data in Supplementary Table 3.

Supplementary Figure 5 ROCK/MLCK inhibition induces phenotypes different from NPC1 inhibition and similar to Lipin-1 inhibition.

a, Filipin staining in MCF10ATk1 cells treated with DMSO, YM or with the U18666A NPC1 inhibitor (3 μM) for the indicated times. NPC1 inhibition blocks the transport of extracellular LDL-cholesterol to the ER, causing lysosomal cholesterol accumulation. At least 30 cells per condition. Scale bar 10 μm. g, MCF10ATk1 cells were treated as in f. Arrowheads indicate cholesterol accumulation (blue) in structures encircled by the lysosomal membrane marker LAMP2 (red). At least 30 cells per condition. Scale bar 10 μm. h, immunofluorescence for OSBP-mCherry transfected in MCF10ATk1 cells. OSBP is a cytosolic protein that accumulates to the Golgi apparatus when the levels of cholesterol in the ER are low; U18666A and serum deprivation (NO FCS) serve as positive controls for decreased ER cholesterol levels. On the right: cells were scored based on differential localization of OSBP At least 24 cells were scored per condition. Scale bar 10 μm. d, A simplified scheme illustrating how SREBP is regulated by Lipin-1 and ARF1 (see text). e, qPCR validation of the effects of COPI siRNAs. Data are relative to GAPDH levels; mean expression in controls was set to 1, and all other samples are relative to this; data are mean and single points; n = 4 independent biological samples per condition; unpaired Mann-Whitney tests. f, Phenotypic appearance of the Golgi apparatus in MCF10ATk1 cells treated for 24 hours with YM, as assayed with the Golgin97 or Giantin markers. DAPI (blue) serves as nuclear counterstain. At least 50 cells per condition. Scale bar 10 μm. g, Phenotypic appearance of Golgi membranes (GFP-Rab6) in RPE1 cells treated for 24 hours with YM or with 100 μM Propranolol. At least 30 cells per condition. Scale bar 10 μm. h, Localization of transfected KDEL-mCherry in GFP-Rab6 RPE1 cells treated for 6 hours with YM or with Propranolol. At least 30 cells per condition. The images in panels a, b, c, f, g, and h are representative of at least two independent experiments with similar results. n values are pooled across independent experiments. Source data in Supplementary Table 3.

Supplementary Figure 6 Effects of mTOR and AMPK inhibition on ROCK/MLCK-mediated regulation of SREBP.

a, LDLR-luciferase in MDA231 cells expressing Lipin-1 isoforms mutated in the known acetylation (Ac K/A, K476/646A) or SUMOylation (SUMO K/A, K616/646A) sites and treated with DMSO or FM. Mean expression in the control was set to 1, and all other samples are relative to this; data are mean and single points; n = 4 independent biological samples per condition, pooled between two independent experiments; unpaired Mann Whitney tests. b, qPCR for established SREBP target genes in MCF10ATk1 cells treated with DMSO, YM and Torin1 (500nM). Data are relative to GAPDH levels; mean expression in controls was set to 1, and all other samples are relative to this; n = 4 independent biological samples per condition, pooled between two independent experiments; multiple unpaired two-tailed Student’s t-tests. c, LDLR-luciferase in MDA231 cells treated with DMSO, YM and Torin1 (500nM). Mean expression in the control was set to 1, and all other samples are relative to this; data are mean and single points; n = 4 independent biological samples per condition, pooled between two independent experiments; unpaired Mann Whitney tests. d,e, qPCR for established SREBP target genes in MCF10ATk1 cells (d) transfected with control siRNA (siCo.) or with two independent mixes of siRNA targeting both AMPKa1 and AMPKa2 catalytic subunit genes (siAMPKa A and B), or in WT and AMPKa1/2-/- MEFs (e). Data are relative to GAPDH levels; mean expression controls was set to 1, and all other samples are relative to this; n = 4 independent biological samples per condition, pooled across two independent experiments; multiple unpaired two-tailed Student’s t-tests. Data are mean and single points. n.s. non significant (P > 0.05). Source data in Supplementary Table 3.

Supplementary Figure 7 Golgi rheology experimental set-up and controls.

a, Simplified diagram illustrating the technique to measure Golgi rheology. Left: an internalized bead (red) is immobilized in proximity to the Golgi membranes (green) by a laser optical trap (yellow cones). Right: when the stage and thus the whole cell is moved towards the bead (blue arrow), the Golgi apparatus displaces the bead away from the trap center (red arrow). Since the Golgi microenvironment has visco-elastic properties, the bead position partially relaxes in time attracted towards the trap center. b, Golgi rheology was measured in RPE1 cells freely spreading on fibronectin-coated glass (Large, n = 39 cells) or plated on micropatterned fibronectin islands restraining cell area and F-actin contractility (Small, 960 μm2 n = 28 cells; 490 μm2 n = 21 cells). GFP-Rab6-positive Golgi membranes were pushed towards a cytoplasmic bead, previously immobilized by an optical trap, by moving the microscope stage in a series of five 0.5µm steps in 1 min. The graph shows the averaged displacements of the bead as a function of time during the whole duration of the experiment. The graph corresponding to the first step (0<t<15) is shown at higher magnification in Fig. 7b. Gray shadows: s.e.m. error bars. c, Analysis of the relaxation curves obtained by moving the bead away from the Golgi (not shown), which serve as negative control. Large n = 28 cells; 960 μm2 n = 29 cells; 490 μm2 n = 26 cells. All n values are pooled across independent experiments. Data are mean and s.e.m.; two-tailed unpaired Student’s t-tests. Source data in Supplementary Table 3.

Supplementary Figure 8

Unprocessed Western Blots.

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Supplementary Information

Supplementary Figures 1–8, Supplementary Table title and legends, and Supplementary References.

Reporting Summary

Supplementary Table 1

Primer and siRNA sequences.

Supplementary Table 2

Antibodies

Supplementary Table 3

Statistics Source Data

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Romani, P., Brian, I., Santinon, G. et al. Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP. Nat Cell Biol 21, 338–347 (2019). https://doi.org/10.1038/s41556-018-0270-5

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