Author: Suzi Birnbaum
Access the described article here: https://www.ncbi.nlm.nih.gov/pubmed/29134198
Plasma membrane (PM) composition is necessary for regulating cell function and cell signaling. Lipids (specifically phospholipids) are the main component of PMs, as taught even at the level of high school biology. Yet characterizations of lipid content, regulation, and overall modulation of PMs are widely unexplored for specific cell/tissue types. Included in this lack of information are the exact mechanistic relationships between environmental (aka dietary) lipids and lipid roles of PMs. A recent study reported in Science Advances by Levental et al. titled, “ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis” details a preliminary model for the regulation of cell-specific PM phenotypes as the result of environmental lipid regulation.
The authors of this paper have reported their results in a step-wise chronological fashion originating at the broad hypothesis that plasma membrane (PM) phenotypes reflect a cell’s environment and functionality. To correct for confounding variables such as cell origin, cell isolation, and culture conditions the authors utilized mesenchymal stem cells (MSC) in an in vitro environment. They identify two distinct cell lineages labeled adipocytes and osteoblasts, which share enough similarity, to the equivalently in vivo cell types, for PM characterization.
The three cell types (MSC, adipocyte, and osteoblast) were found to have distinct biophysical properties regarding their PMs which supported the basis for the initial hypothesis. Giant plasma membrane vesicles (GPMV) is a methodology that separates lipid components of membranes based on physical properties (such as microdomain content or PM fluidity) which can then be used for characterization of PMs (2). The researchers establish a more specific hypothesis that not only do PMs have characteristic microdomains (as identified from GPMV), but also, cells have specific biophysical and biochemical phenotypes. They obtained quantitative differences between the differentiated adipocyte, osteoblast, and the MSC lines by performing electrospray ionization (tandem) mass spectrometry (ESI-MS/MS) shotgun analysis (1). This methodology produces a global data set of chemical components, in this case called lipidomics. The authors extracted two main observations on PM development which were, lack of triacylglyceride (TAG) inclusion in PM makeup and a statistically significant difference in lipid development between osteoblasts, and MSCs or adipocytes. Osteoblasts show a cell-specific PM phenotype primarily characterized by longer carbon chains of glycerophosopholipids (GPL), and increased amount of polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA) an omega 3 PUFA. These results were affirmed by additional Western blotting experimentation and statistical analysis by principal component analysis (PCA). This primary osteoblast PM phenotype is distinct from the MSC and adipocyte phenotypes, which are more similar to each other.
Due to the unexpected significance of the omega 3 PUFAs in osteoblast PM phenotype the authors then pose that the newly identified osteoblastic PM phenotype was the result of PUFA incorporation. They repeated the ESI-MS/MS lipidomic experiments on the same three cell cultures, but this time the cell cultures had all been exposed to physiological levels of DHA in the media. What came about was data that validated the hypothesis that MSCs treated with the omega 3 PUFA developed an osteoblastic PM phenotype. DHA supplementation lead to lipidome remodeling, most significant of which were that monounsaturated and saturated lipid contents as well as increased cholesterol levels and organization (related to membrane stability). We can now see the beginning of a model wherein an osteoblast PM phenotype (from MSC differentiation) is regulated by environmental DHA.
To test this budding model, the researchers quantified osteogenic differentiation by detecting calcified nodules via bright-field microscopy, histological assays, and Western blotting. The findings were that DHA had the specific effects of enhancing matrix deposition and differentiation in osteoblasts in contrast to its lack of significant effect on adipocyte development. Now that we have established DHA-induced PM remodeling promotes osteogenesis, by what mechanism does the presence of environmental omega 3 PUFAs do so? To answer this question the authors report a top-down methodology by carrying out transcriptomic and proteomic investigations via microarray analysis. What may seem logical to the reader, but completely by surprise to the research team was an unexpected significantly positive relationship between transcriptional signatures of DHA-supplementation and osteogenesis. In other words, the global ‘omic’ data indicated that DHA-supplementation regulated gene expression in such a way that those genes were potentiating the osteogenesis-promoting genes (instead of directly signaling them).
So now that we have an established outline of a signaling model, what lies between the genes enhanced by environmental DHA presence and the osteogenesis-promoting genes? Gene set enrichment analysis (GSEA) and reverse phase protein array (RPPA) uncovered altered regulation genes involved in cell growth and metabolism (unsurprisingly enough), with the most statistically significant change in regulation was found to lie in the Akt pathway. This pathway is involved in critical cell signaling pathways to promote cell survival and proliferation (3), which was doubly confirmed by Western blotting.
In order to identify where Akt activation fits into the DHA-regulated genes and the osteogenesis-involved genes, the authors pose a hypothesis whereby the DHA-induced PM phenotype up-regulates Akt activation which in turn enhances osteogenesis in undifferentiated MSCs. The researchers report use of transmission electron microscopy (TEM) to obtain quantitative results on Akt levels and organization in cell PMs of baby hamster kidney cell samples. The forthcoming data indicated that the stabilized microdomains mediated by DHA presence not only induced an overall increase of Akt levels in the PM, but also increased the amount of nanoclustering within microdomains. Akt placement in the proposed model was affirmed by a host of Akt-pathway inhibition studies.
The culminated result of this multi-step, multi-hypothesis investigation is a preliminary model of DHA-mediated PM modelling which is posed to enhance Akt activation via stabilization of microdomains, which in turn enhances osteogenesis from MSCs leading to a differentiated cell-specific osteogenic PM phenotype.
Potential applications of this model include drug treatments in the event of osteoblast membrane irregularities or issues. Additionally, it opens the floor for being able to better understand environmental influence of fatty acids on membrane development as a study of diet. As we have learned in the course, Advanced Biochemistry 441, DHA is central to neuron membrane function and plays a role in the inflammation pathway. If those roles weren’t enough for dietary pressures of including more omega 3’s, but now we can begin to consider the importance of the roles in osteocyte and in turn bone development.
- Isaac, G. Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)-Based Shotgun Lipidomics. Methods Mol. Biol. Clifton NJ 2011, 708, 259–275.
- Levental, K. R.; Levental, I. Giant Plasma Membrane Vesicles: Models for Understanding Membrane Organization. Curr. Top. Membr. 2015, 75, 25–57.
- Song, G.; Ouyang, G.; Bao, S. The Activation of Akt/PKB Signaling Pathway and Cell Survival. J. Cell. Mol. Med. 2005, 9 (1), 59–71.
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