Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells |
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Authors: | Christina Patlaka Heike Becker Maria Norgård Staffan Paulie Annica Nordvall-Bodell Pernilla Lång Göran Andersson |
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Institution: | 1. Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden;2. Division of Pathology, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden;3. Mabtech AB, Box 1233, SE 131 28 Nacka Strand, Sweden |
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Abstract: | Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors. |
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Keywords: | FGF Fibroblast growth factor GAG Glycosaminoglycans GPI Glycosylphosphatidylinositol HS Heparan sulfate HSPG Heparan sulfate proteoglycans IBMX Isobutylmethylxanthine LAMP-1 Lysosomal-associated membrane protein 1 MSCs Mesenchymal stem cells MDC Monodansylcadaverine MVBs Multi-vesicular bodies PG Proteoglycans TRAP Tartrate resistant acid phosphatase |
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