ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis

Abstract

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling.

Fig. 1. Differentiation of physical properties and comprehensive lipidomes of hMSC PMs.

(A) Sigmoidal fits (solid lines) to phase separation data (50 to 100 vesicles per point) for quantification of phase separation temperature (T_misc). (B) Quantification of T_misc after 14 days of MSC differentiation; colors denote seven individual human donors. (C) Normalized C-laurdan emission spectra of GPMVs obtained from differentiated MSCs. a.u., arbitrary units. (D) Quantification of membrane order by C-laurdan GP; colors denote four individual human donors. (E) Differentiation-induced changes [log₁₀(Diff/Undiff)], with red and blue values indicating an increase and decrease in individual lipid species, respectively. (F) Comparison of lipid classes in PMs after 14 days of MSC differentiation; inset shows expanded y axis for clarification. (G) PM GPL length (combined number of carbons in the two acyl chains) after MSC differentiation; inset shows mole percent (mol %) of lipids ≥36 carbons in each cell type. (H) PM GPL unsaturation after MSC differentiation; inset shows mol % of lipids with three or more unsaturations. (I) PCA of lipid structural classes and features in the three cell lineages with the loading plot (summary of variables) superimposed on the score plot for the samples. Filled circles are individual human donors, and “x” are individual variables contributing to the principal components. Table S2 shows the variable loadings defining PC1 and PC2. (J) Mol % of membrane lipids containing ω-3 PUFA [DHA or eicosapentaenoic acid (EPA)] as one of the acyl chains. All data in (F) to (H) and (J) are means ± SD for three to four human donors. ***P < 0.001, **P < 0.01, *P < 0.05 determined by Student’s t test compared to Undiff in (F) to (J).

Fig. 2. ω-3 DHA induces biophysical and biochemical remodeling of MSC membranes.

(A) Lipids containing DHA (22:6) or EPA (20:5) are significantly increased in membranes from MSCs supplemented with 20 μM DHA. (B) Distribution of the GPL classes of ω-3–containing lipids. (C) DHA-induced changes [log₁₀(DHA-treated/untreated)], with red and blue values indicating an increase and decrease of each individual lipid species, respectively. (D) DHA-induced changes to GPL unsaturation as a mol % of all non–DHA/EPA-containing GPLs. Inset shows mol % of saturated acyl chains in non–DHA/EPA-containing lipids. (E) Effect of DHA supplementation on lipid classes in MSC membranes. (F) Unbiased PCA of the lipid structural class and features in the three cell lineages (untreated and DHA-supplemented). Circles represent individual human donors. Table S3 shows the variable loadings defining PC1 and PC2. The dashed lines represent the effects of DHA. (G) Temperature-dependent phase separation in GPMVs isolated from MSCs as a function of supplementation with DHA or OA. (H) Comparison between T_misc for fatty acid–supplemented undifferentiated MSCs and those induced to differentiate into osteoblasts. ns, not significant. (I) C-laurdan GP images of isolated GPMVs from untreated and DHA-supplemented MSCs. (J) Quantification of the GP of the disordered microdomains. Data in (A) to (F) are means ± SD for three human donors. Data in (G) to (H) are means ± SD for at least five human donors. Data plotted in (J) are means ± SD of individual vesicles from one representative experiment of three independent trials. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Supplementation with DHA potentiates osteogenic differentiation.

(A and B) The extent of osteogenesis was followed during differentiation by automated image processing of ossified matrix deposition. (A) Bright-field images (left) were automatically thresholded (middle) to calculate the area covered by deposited osteogenic matrix (red overlay). (B) Effect of fatty acid supplementation on osteogenic deposition by osteoblast-differentiated MSCs as a function of differentiation time. Significances shown are unpaired t tests for individual time points (+DHA compared to untreated). (C) Representative images of MSCs stained with Alizarin Red S to visualize insoluble calcium deposits after 14 days of osteogenic differentiation with or without fatty acid supplementation. (D) Automated quantification of Alizarin Red staining as a function of fatty acid supplementation. (E) Protein expression of the osteogenic master regulator transcription factor RUNX2 is enhanced by DHA supplementation; Western blotting of protein expression 3 days after induction of differentiation with or without concomitant DHA supplementation. (F and G) Transcription of osteoblast markers (F) BSP2 and (G) ALP is enhanced by DHA, as measured by qPCR at 14 days after induction of differentiation. (B and D) Data are means ± SD of n > 4 independent experiments for at least three human donors. (E) Data are means ± SD of n = 3 experiments on two human donors. (F and G) Data are means ± 95% confidence interval of n = 5 independent experiments on three human donors. ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 4. Supplementation with DHA induces osteogenic gene and protein expression profile via alterations of PM signaling, notably via Akt.

(A) Scatterplot of genome-wide changes in gene expression induced by DHA supplementation compared to osteogenic differentiation. The strong transcriptional correlation between these two unrelated stimuli suggests that DHA induces osteogenic signaling in MSCs. (B) Genes varying by more than 30% due to either DHA supplementation or induction of osteoblastic differentiation in MSCs show significant overlap. Ontological categorization of these reveals not only enrichments in differentiation and ossification but also signaling and organization of the PM. (C) GSEA visualization of gene sets significantly up-regulated (red) or down-regulated (blue) by DHA supplementation in early osteogenic differentiation (day 3). Nodes represent gene sets, and lines represent overlap between gene sets. The labels summarize the pathways or cellular functions that comprise each of the hubs within the network. TGFβ, transforming growth factor–β; GTPases, guanosine triphosphatases; MAPK, mitogen-activated protein kinase; IFN, interferon; GFR, growth factor receptor; GAG, glycosaminoglycan. (D) RPPA was performed on lysates from MSCs early in osteogenesis (day 3 of induction) or treated with DHA for 3 days. Fold changes over untreated (log₂) are shown for all antibodies showing significantly (P < 0.05) different responses with osteogenic differentiation. Osteogenic changes parallel those induced by DHA across three human donors, illustrating the robustness of the result. (E) Covariance (see Materials and Methods for calculation details) between DHA- and osteogenesis-induced posttranslational modifications to signaling proteins (modified/total protein). Red and blue dots indicate signaling modifications that are up- or down-regulated, respectively, by both DHA and osteogenesis. Gray dots indicate those not changing with either treatment. Black dots represent those varying in opposing directions (for example, up-regulated in osteogenic and down-regulated in DHA). (F and G) Quantification of Western blots for phosphorylated Akt [Ser⁴⁷³ (F) and Thr³⁰⁸ (G); normalized to total Akt] 3 days after DHA supplementation or osteogenic induction. Data are means ± SD of at least four independent experiments on three human donors.

Fig. 5. DHA enhances Akt activation at the PM.

(A to C) PM sheets of BHK cells expressing GFP-PH-Akt were labeled with anti-GFP gold nanoparticles and imaged by TEM. (A) The abundance of Akt on the PM is shown by box-and-whisker plots of gold particles per square micrometer. (B and C) Spatial mapping of GFP-PH-Akt distribution on the same PM sheets. (B) Representative weighted mean K-function L(r) − r, where values above the 99% confidence interval (C.I.) (dashed line) indicate nonrandom clustering of the lipid probe. (C) Peak L(r) − r values, L_max, derived from K-function curves [as in (B)] confirm enhanced nanoclustering of PH-Akt induced by DHA. (D) BHK cells expressing Lyn-AktAR (Raft) and Kras-AktAR (Nonraft) treated with 20 μM DHA for 3 days and imaged by confocal microscopy with an excitation of 405 nm and emission in the range of 465 to 500 nm [cyan fluorescent protein (CFP)] and 515 to 550 nm [yellow fluorescent protein (YFP)]. YFP/CFP emission ratio (reflective of extent of FRET and thus Akt activation) for at least 25 cells per condition is shown. Data are means ± SD of four independent experiments. (E) Model for DHA-mediated promotion of osteogenesis. DHA induces membrane remodeling and stabilized raft microdomains, which leads to increased Akt abundance and clustering at the PM, thus enhancing Akt activation to potentiate osteogenic differentiation. (F) Relative osteogenic matrix deposition after 7 days of MSC differentiation in the presence of DHA and/or MK2206 (a specific Akt inhibitor), as in Fig. 3 (A and B). Data are means ± SD of three independent experiments on two human donors. (G) Relative osteogenic matrix deposition after 7 days of MSC differentiation in the presence of DHA and/or myriocin + Zaragozic acid (MZ) (low, 10 μM myriocin/5 μM Zaragozic acid; high, 25 μM myriocin/5 μM Zaragozic acid). Data are means ± SD of four independent experiments on two human donors. Significances in (A) and (C) are unpaired t tests, in (D) are paired t tests compared to untreated cells, and in (E) and (F) are paired t tests compared to DHA-treated cells. ***P < 0.001, **P < 0.01, *P < 0.05.