Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine

Abstract

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine-(PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol-(GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain. † This work was supported by USPHS Grant DE13576. * Address correspondence to this author. . battagli@biochem.dental.upenn.edu. HHS Public Access Author manuscriptBiochemistry. Author manuscript; available in PMC 2016 January 29. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptVertebrate skeletal growth occurs largely through a highly regulated process known as endochondral ossification. During this process mesenchymal cells condense and differentiate into chondrocytes to form a cartilage template. Chondrocytes proliferate and then undergo a complex maturation process associated with an increase in cell size (hypertrophy). This hypertrophic stage is characterized by upregulation of the expression of genes encoding ALPase, 1 type X collagen, and matrix metalloproteinase-13, followed by mineralization of the extracellular matrix. The zones of proliferating, prehypertro...

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

“…These results suggest that, under physiological conditions, TMEM16F plays an important role in microparticle shedding. TMEM16F dependency has been similarly observed during bone tissue mineralization (39), during which PtdSer-rich matrix vesicles containing concentrated phosphate and Ca 2+ ions bud off from the plasma membranes (40). Our EM images revealed that the rod-like extensions that formed on the surface of the activated platelets were released in a TMEM16F-dependent manner, consistent with the possibility that microparticle release from the activated platelets is associated with morphological changes.…”

Inactivation of anoctamin-6/Tmem16f, a regulator of phosphatidylserine scrambling in osteoblasts, leads to decreased mineral deposition in skeletal tissues

“…At least for vesicle-producing hypertrophic chondrocytes, it has been demonstrated that they express phospholipid scramblase 1 and expose PS to the cell surface. (47) Similarly, mineralization-competent matrix vesicles released from vascular smooth muscle cells in an arteriosclerosis culture model contain increased levels of externalized PS, (67) supporting the importance of PS exposure in tissue mineralization. How Ano6 regulates osteoblast mineralization is difficult to predict, but one could speculate that Ano6-dependent PS translocation facilitates matrix vesicle release from the cell membrane, a prerequisite for initiating the mineralization core.…”

“…(46)(47)(48)(49)(50) Furthermore, at least in hypertrophic chondrocytes, the release of mineralization-competent matrix vesicles coincides with the translocation of PS from the inner to the outer leaflet of the plasma membrane. (47) This process, denoted as phospholipid scrambling, is regulated by members of the phospholipid scramblase protein family. Interestingly, for the pro B cell line Ba/F3, it has recently been shown that Ano6 regulates inside-out scrambling of PS in response to an increase in intracellular calcium levels.…”

“…(49,50,63,64) In addition, according to the current view, mineralization of chondrocytes and osteoblasts is initiated in extracellular, lipid-bilayer enclosed matrix vesicles that are rich in PS (65) and are released through budding from the plasma membrane. (47,66) In these matrix vesicles, calcium and phosphate ions are actively concentrated to generate an environment for the regulated nucleation of hydroxyapatite crystals. At least for vesicle-producing hypertrophic chondrocytes, it has been demonstrated that they express phospholipid scramblase 1 and expose PS to the cell surface.…”

Proteoliposomes in nanobiotechnology

“…In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts" (Simons and Ikonen 1997;Simons and Toomre 2000;Giocondi et al 2007;Lingwood and Simons 2010), ordered microdomains enriched in sphingolipids, glycosphingolipids, and cholesterol (Simons and Toomre 2000;Damek-Poprawa et al 2006). In order to better understand the role of the lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM), and ganglioside (GM1) (Bolean et al 2010(Bolean et al , 2011.…”