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. 2018 Nov 19;9(1):4862.
doi: 10.1038/s41467-018-07268-w.

"V体育平台登录" Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway

Etienne Boulter  1 , Soline Estrach  2 , Floriane S Tissot  2 , Marco L Hennrich  3   4 , Lionel Tosello  2 , Laurence Cailleteau  2 , Laura R de la Ballina  2 , Sabrina Pisano  2 , Anne-Claude Gavin  3   4 , Chloé C Féral  5
Affiliations

Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway

Etienne Boulter et al. Nat Commun. .

Abstract

Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2 VSports手机版. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis. .

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
CD98hc depletion impairs RhoA activation and cell response to mechanical force application on integrins. a, b Loss of CD98hc impairs cell stiffening triggered by successive pulses of mechanical force applied to integrins. Red brackets, maximal bead displacement shown for a control experiment. (Mean + s.e.m., n = 2 with at least 25 beads each, ***P < 0.001 ****P < 0.0001 in a two-way ANOVA). c Genetic deletion of CD98hc in dermal fibroblasts impairs RhoA activation triggered by mechanical force application on integrins using fibronectin (FN)-coated paramagnetic beads, one experiment representative of n = 2. d Re-expression of WT CD98hc in CD98hc-null fibroblasts rescues mechanically coupled RhoA activation, one experiment representative of n = 3. e Genetic deletion of CD98hc in dermal fibroblasts impairs RhoA activation triggered by mechanical force application on integrins using either collagen I (ColI)-coated or FN-coated paramagnetic beads, one experiment representative of n = 3. f Loss of CD98hc in fibroblasts does not affect FCS-induced RhoA activation, one experiment representative of n = 2. g Loss of CD98hc in fibroblasts does not affect Rac1 regulation by mechanical forces. h, i Direct mechanical stimulation of CD98hc with RL388-coated magnetic beads does not trigger RhoA activation nor does it stimulate cell stiffening. i Means are plotted with s.e.m. as error bars from n = 12 beads, statistical analysis was performed in a two-way ANOVA. j mTORC1 inhibition with rapamycin 100 µM for 1 h does not impair mechanically coupled RhoA activation using FN-coated magnetic beads, one experiment representative of n = 2
Fig. 2
Fig. 2
CD98hc depletion impairs rigidity sensing. a Representative morphology of control or CD98hc-null dermal fibroblasts grown on FN-coated hydrogels of specified elastic modulus. Scale bar is 50 µm. Cell area is plotted as means with s.e.m. as error bars (n = 40–54 cells, **P < 0.01, ****P < 0.0001 in a one-way ANOVA). b Loss of CD98hc in fibroblasts impairs RhoA activation upon substrate stiffening. c, d Loss of CD98hc impairs cell contractility (c) and rigidity sensing (d). Traction force microscopy was performed on subconfluent cells. Black and black hatched bar, control cells, gray and gray hatched bar, CD98hc-null cells (mean + s.e.m., n = 3, **P < 0.01 in a one-way ANOVA). Scale bar is 50 µm. e Loss of CD98hc impairs YAP/Taz activation by mechanical cues as measured by ANKRD1 and CTGF transcript quantification by RT-qPCR. Black bars, control cells and gray bars, CD98hc-null cells (mean + s.e.m., n = 3, ****P < 0.0001 in a Student’s t test). f Forcing RhoA activation using CNF1 in CD98hc-null cells rescues YAP/Taz activation by mechanical cues. Means are plotted with s.e.m. as error bars. n = 2 **P < 0.01 in a Student’s t test
Fig. 3
Fig. 3
CD98hc is required for proper skin mechanical homeostasis in vivo. a Representative skin sagittal sections of CD98hcfl/fl or Fsp1Cre, CD98hcfl/fl mice stained with H&E. Scale bar is 50 µm. b Genetic deletion of CD98hc in dermal fibroblasts alters dermal mechanical properties. Young’s elastic modulus (E) of the dermis was measured by AFM with a spherical probe (mean with s.d. as error bars, n = 5 and 4 mice, respectively, *P < 0.05 in a Student’s t test). c E magnitude heatmaps of areas of dermis scanned by AFM using a pyramidal probe. d, e Loss of CD98hc in dermal fibroblasts reduces collagen assembly as observed by picrosirius red staining under transmitted or polarized light (d), fiber length (e) (mean with s.d. as error bars, at least 300 fibers were counted with n = 3 **P < 0.01 in a Welch’s t test (e)). f Relative mRNA levels of LOX was measured by RT-qPCR on total mRNA extracted from skin of CD98hcfl/fl or Fsp1Cre, CD98hcfl/fl mice (mean with s.d. as error bars, n = 6 ****P < 0.0001 in a Student’s t test). g, h Loss of CD98hc in dermal fibroblasts impairs elastin fibers assembly as observed on skin sagittal sections stained for elastin. Scale bar is 50 µm. Means are plotted with s.d. as error bars. At least 300 fibers were measured on sections from n = 3 mice. ****P < 0.0001 in a Student’s t test. i Relative mRNA levels of LOX was measured by RT-qPCR on total mRNA extracted from control or CD98hc-null dermal fibroblasts grown FN-coated hydrogels of indicated stiffness for 24 h. n = 2, means are plotted with s.e.m. as error bars, **P < 0.01, ****P < 0.0001 in a Student’s t test. j Total cell lysates from control or CD98hc-null dermal fibroblasts grown FN-coated hydrogels of indicated stiffness for 24 h were resolved by SDS–PAGE and analyzed by Western blotting. k Relative LOX activity was measured in cell lysates from control or CD98hc-null dermal fibroblasts grown FN-coated hydrogels of indicated stiffness for 24 h. n = 2, means are plotted with s.e.m. as error bars. **P < 0.01, ***P < 0.001 in a two-way ANOVA
Fig. 4
Fig. 4
CD98hc regulates sphingolipid biosynthesis and integrin mechanosensing. a Schematic strategy to identify CD98hc mutants regulating simultaneously integrin mechanosensing and cell metabolism. b Re-expression of C109S CD98hc mutant in CD98hc-null cells but not of C330S mutant rescues mechanically coupled RhoA activation, one experiment representative of n = 2. c Quantification of RhoA activation in control, C109S or C330S-expressing CD98hc-null cells. Means are plotted with s.e.m. as error bars from n = 2 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 in a two-way ANOVA. d Volcano plot depicting metabolites fold changes versus p value in C330S versus CD98hc re-expressing CD98hc-null cells. Pink, long chain based sphingoids, ceramides and phytoceramides; magenta, glycosphingolipids and sphingomyelins; green, amino acid metabolism. Numbered metabolites are analogous to the numbering in Fig. 5i. 1, 3-ketosphinganine; 2, sphinganine; 3, N-palmitoyl-sphinganine; 4, N-palmitoyl-sphingosine; 5, palmitoyl dihydrosphingomyelin; 6, sphingosine. P values were calculated using a Student’s t test, values are reported in Supplementary Data 1. e Inhibition of S1P synthesis with dimethyl-sphingosine (DMS) treatment at 10 µM for 1 h does not impair mechanically coupled RhoA activation triggered by FN-coated paramagnetic beads, one experiment representative of n = 2. f Sphingolipid and cholesterol depletion impairs mechanically coupled RhoA activation, one experiment representative of n = 2. g, h Exogenous supply of C12 sphingomyelin to C330S-expressing cells restores mechanically coupled RhoA activation. H is representative of n = 3 experiments. Means are plotted with s.e.m. as error bars in I from n = 3 experiments. *P < 0.05 in a Student’s t test
Fig. 5
Fig. 5
CD98hc determines the level of DES2 in fibroblasts. a–d Loss of CD98hc or expression of C330S mutant reduce the expression of DES2 and induces DES1. One experiment representative of at least n = 3 experiments. e–h Levels of DES1 and DES2 mRNA as measured by RT-qPCR on total mRNA from CD98hc-null cells or C330S reconstituted null cells. Means are plotted with s.e.m. as error bars from, respectively, n = 6 or n = 3 experiments. Differences were not statistically different in a Student’s t test. i Schematic representation of the de novo sphingolipid biosynthesis pathway presenting variations of metabolites. Numbers refer to metabolites depicted and numbered in the volcano plot of Fig. 4d. Metabolites and p values are reported in Supplementary Data 1. P values were calculated using a Student’s t test. DES1/2 variations were extracted from results presented in Fig. 5a–d and Supplementary Figure 5A–D. j, k Hsp90 inhibition in control cells reduces the level of DES2 expressed in cells. Right panel, means are plotted with s.e.m. as error bars from n = 3 experiments, **P < 0.01 in a Student’s t test. l Grp78 and Grp94 co-immunoprecipitate with DES2 upon loss of CD98hc or geldanamycin treatment. Control, CD98hc-null, or C330S-expressing cells were treated with geldanamycin for 16 h prior to cell lysis and DES2 immunoprecipitation. Precipitates were loaded on SDS–PAGE and analyzed by Western blot. One experiment representative of n = 2
Fig. 6
Fig. 6
Depletion of DES2 recapitulates most of defects observed upon depletion of CD98hc. a Depletion of DES2 by siRNA impairs RhoA activation by mechanical force application on integrins. Right panel, quantification of RhoA activation, means are plotted with s.e.m. as error bars from n = 2 experiments, *P < 0.05 in a two-way ANOVA. b Depletion of DES2 by shRNA impairs SFK phosphorylation at Y416. One experiment representative of n = 2. c Depletion of DES2 impairs mechanically induced GEF-H1 activation. One experiment representative of n = 2. d Depletion of DES2 triggers GEF-H1 sequestration in cell membrane. s supernatant, p membrane pellet. One experiment representative of n = 2. e DES2 depletion partially inhibits mechanically coupled YAP/Taz activation as measured by RT-qPCR of ANKRD1 transcript. Means are plotted with s.e.m. as error bars from n = 6 with *P < 0.05 and ****P < 0.0001 in a two-way ANOVA. f Depletion of DES2 impairs generation of traction forces. Traction force microscopy was performed on subconfluent cells. Scale bar is 50 µm
Fig. 7
Fig. 7
Loss of CD98hc impairs GEF-H1and Src family kinase activation, and integrin dynamics. a Loss of CD98hc impairs LARG and GEF-H1 activation by mechanical force application on integrins. One experiment representative of n = 2. b Loss of CD98hc impairs Src activity but does not affect ERK1/2 phosphorylation by mechanical force application on integrins. Src activity was monitored by tracking substrate FAK Y576 phosphorylation and SFK phosphorylation at Y416. c Loss of CD98hc induces constitutive recruitment of GEF-H1 to the membrane. s supernatant, p membrane pellet. One experiment representative of n = 3. d Control or CD98hc-null dermal fibroblasts were stimulated with FN-coated magnetic beads. Cells were lysed and lysates were fractionated into cytosolic (s) and membrane (p) fractions by centrifugation. Active RhoGEFs was pulled-down from each fraction with GST-RhoA17A beads. One experiment representative of n = 2. e, f Diffusion rate (t-half) of integrin α5 and β3 as calculated from the curve fit generated from the FRAP measurements performed on n = 27, 41, and 67; and 73, 36, and 57 adhesions, respectively, for control, CD98hc-null and C330S cells on integrin α5 and β3. Error bars are 95% CI calculated from that fit. g, h Integrin α5 and β3 mobile fraction as calculated from the curve fit generated from the FRAP measurements. Error bars are 95 CI calculated from that fit. i Trafficking of integrin β1 in control or CD98hc-null cells. Integrin β1 was labeled with Alexa 488 coupled antibody then integrin trafficking was chased for indicated time. Extracellular staining was quenched and only intracellular labeled integrin is observed. Scale bar is 50 µm
Fig. 8
Fig. 8
Loss of CD98hc affects GEF-H1 recruitment in membrane microdomains. a, b Loss of CD98hc impairs recruitment of src kinases (a) and GEF-H1 (b) in CEMM. One experiment representative of n = 2. c GEF-H1 and cholesterol colocalize in control cells (arrows). Endogenous GEF-H1 and cholesterol were stained using an anti-GEF-H1 antibody or filipin III respectively. Right panel is a magnified crop of the white squared box. Scale bar is 50 µm. d Cholesterol depletion induces accumulation of GEF-H1 in membranes. e C12SM exogenous supply induces a decrease in the amount of GEF-H1 constitutively recruited to membrane fractions in CD98hc-null cells to levels similar to that of control cells
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