Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function

Abstract

Background: The function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it’s expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated. Methods: ADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR). Results: Inhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles. Conclusions: Ceramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays. [MediaObject not available: see fulltext.]