ALBERT

Description: Langmuir DOI: 10.1021/la400768s

Description: Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo . A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS ALL . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS ALL data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo . This article is protected by copyright. All rights reserved

Description: Biochemistry DOI: 10.1021/acs.biochem.5b01341

Description: Highly variable expression of mesoderm specific transcript ( Mest ) in adipose tissue among genetically homogeneous mice fed an obesogenic diet, and its positive association with fat mass expansion, suggests that Mest is an epigenetic determinant for the development of obesity. Although the mechanisms by which MEST augments fat accumulation in adipocytes have not been elucidated, it has sequence homology and catalytic peptide motifs which suggests that it functions as an epoxide hydrolase or as a glycerol- or acylglycerol-3-phosphate acyltransferase. To better understand MEST function, detailed studies were performed to precisely define the intracellular organelle localization of MEST using immunofluorescence confocal microscopy. Lentiviral-mediated expression of a C-terminus Myc-DDK-tagged MEST fusion protein expressed in 3T3-L1 preadipocytes/adipocytes, and ear-derived mesenchymal stem cells (EMSC) from mice was observed in the endoplasmic reticulum (ER) membranes and is consistent with previous studies showing endogenous MEST in the membrane fraction of adipose tissue. MEST was not associated with the Golgi apparatus or mitochondria; however, frequent contacts were observed between MEST-positive ER and mitochondria. MEST-positive domains were also shown on the plasma membrane (PM) of non-permeabilized cells but they did not co-localize with ER-PM bridges. Post-adipogenic differentiated 3T3-L1adipocytes and EMSC showed significant co-localization of MEST with the lipid droplet surface marker perilipin at contact points between the ER and lipid droplet. Identification of MEST as an ER-specific protein that co-localizes with lipid droplets in cells undergoing adipogenic differentiation supports a function for MEST in the facilitation of lipid accumulation and storage in adipocytes. This article is protected by copyright. All rights reserved

Description: FGF applied as a single growth factor to quiescent mouse fibroblasts induces a round of DNA replication, however continuous stimulation results in arrest in the G1 phase of the next cell cycle. We hypothesized that FGF stimulation induces the establishment of cell memory, which prevents the proliferative response to repeated or continuous FGF application. When a 2-5 day quiescence period was introduced between primary and repeated FGF treatments, fibroblasts failed to efficiently replicate in response to secondary FGF application. The establishment of “FGF memory” during the first FGF stimulation did not require DNA synthesis, but was dependent on the activity of FGF receptors, MEK, p38 MAPK and NFκB signaling, and protein synthesis. While secondary stimulation resulted in strongly decreased replication rate, we did not observe any attenuation of morphological changes, Erk1/2 phosphorylation and cyclin D1 induction. However, secondary FGF stimulation failed to induce the expression of cyclin A, which is critical for the progression from G1 to S phase. Treatment of cells with a broad range histone deacetylase inhibitor during the primary FGF stimulation rescued the proliferative response to the secondary FGF treatment suggesting that the establishment of “FGF memory” may be based on epigenetic changes. We suggest that “FGF memory” can prevent the hyperplastic response to cell damage and inflammation, which are associated with an enhanced FGF production and secretion. “FGF memory” may present a natural obstacle to the efficient application of recombinant FGFs for the treament of ulcers, ischemias and wounds. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: Transient FGF stimulation of various cell types results in FGF memory – a sustained blockage of efficient proliferative response to FGF and other growth factors. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates proinflammatory NFκB signaling, which is also critical for FGF memory formation. The search for FGF-induced mediators of FGF memory revealed that FGF stimulates HDAC-dependent expression of the inflammatory cytokine IL1α. Similarly to FGF, transient cell treatment with recombinant IL1α inhibits the proliferative response to further FGF and EGF stimulation, but does not prevent FGF receptor-mediated signaling. Interestingly, like cells pretreated with FGF1, cells pretreated with IL1α exhibit enhanced restructuring of actin cytoskeleton and increased migration in response to FGF stimulation. IRAP, a specific inhibitor of IL1 receptor, and a neutralizing anti-IL1α antibody prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. A similar effect results following treatment with the anti-inflammatory agents aspirin and dexamethasone. Thus, FGF memory is mediated by proinflammatory IL1 signaling. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound-induced hyperplasia. This article is protected by copyright. All rights reserved

Description: ABSTRACT FGF1 is a nonclassically released growth factor that regulates carcinogenesis, angiogenesis and inflammation. In vitro and in vivo, FGF1 export is stimulated by cell stress. Upon stress, FGF1 is transported to the plasma membrane where it localizes prior to transmembrane translocation. To determine which proteins participate in the submembrane localization of FGF1 and its export, we used immunoprecipitation mass spectrometry to identify novel proteins that associate with FGF1 during heat shock. The heat shock-dependent association of FGF1 with the large protein AHNAK2 was observed. Heat shock induced the translocation of FGF1 and AHNAK2 to the cytoskeletal fraction. In heat-shocked cells, FGF1 and the C-terminal fragment of AHNAK2 colocalized with F-actin in the vicinity of the cell membrane. Depletion of AHNAK2 resulted in a drastic decrease of stress-induced FGF1 export but did not affect spontaneous FGF2 export and FGF1 release induced by the inhibition of Notch signaling. Thus, AHNAK2 is an important element of the FGF1 nonclassical export pathway. This article is protected by copyright. All rights reserved

Description: Background: Exosomes are microvesicles that play important roles in intercellular communications in both normal and tumor cells via their cargoes, which include microRNA (miRNA) and proteins. miRNAs play critical regulatory roles in hematopoiesis, and their abnormal expression is correlated with several hematological malignancies, including acute myeloid leukemia (AML). Exosome-derived miRNAs and proteins are increasingly recognized for their prognostic biomarker potential. Exosomes are being evaluated for their potential as novel drug-delivery vehicles due to their endogenous nature and ability to carry small molecule drugs. Aims: We aim to characterize HSC-derived exosomes, investigate the biomarker potential of exosomes derived from AML patient samples (by examining their proteomic and miRNA profiles) and utilize exosomes as innovative drug delivery vehicles with the ability to eliminate leukemic stem cells in a targeted manner. Methods: Secreted exosomes from murine bone marrow HSCs were isolated from conditioned medium and visualized using confocal microscopy. We isolated exosomes and performed miRNA profiling, using qPCR, and LC-MS/MS proteomic analysis to characterize the constituents. We also isolated exosomes from the CD34+ cells of three AML patient samples and profiled 372 of the most abundantly expressed miRs in these cells compared to normal CD34+ cells. We analyzed the proteome of these exosomes as well. In order to assess the utility of exosomes as drug carriers, exosomes from bone marrow-derived OP9 stromal cells were transfected with Daunorubicin (1ug/ul). Normal CD34+ cells and patient-derived AML samples (from n=2 patients) were treated with varying doses of the drug-loaded exosomes. Drug-loaded exosomes uptake was tracked with a Texas Red siRNA. After 24 hours, cells were screened for apoptosis. To test the feasibility of targeted exosomes, OP9 cells were exposed to AML patient samples for 48 hours. The patient cells were then removed and, 24 hours later, "trained" stromal cell-derived exosomes were isolated from the media and transfected with Daunorubicin. These patient-specific, exosomes were plated with both the corresponding patient's CD34+ cells as well as normal CD34+ primary cells. After 24 hours, apoptosis was measured. Results: miRNA profiling of murine bone marrow showed miR-21a, miR-92a and miR-25 were most abundant in exosomes. Proteomic LC-MS/MS analysis revealed presence of exosome-associated novel proteins such as Syntenin-1. Syntenin-1, which is known to bind IL-5R and promote myelopoiesis, was present in significantly higher levels in HSCs compared to myeloid progenitors, implying a functional role for exosome derived Syntenin-1. miRNA profiling in AML samples revealed distinct signature profiles. Importantly, exosome derived miRs such as -1290, -375, -205 and -21-that are known prognostic markers in cancers such as prostate, ovarian and hepatocellular carcinoma-were significantly upregulated in all the three exosome-derived AML samples. The drug-loaded exosomes were successful in inducing significant apoptosis in two patient samples tested. These drug-loaded exosomes also induced cell death in CD34+ normal cells when compared to control exosomes. However, the patient-trained exosomes specifically eliminated 92% of CD34+ AML patient cells, while causing significantly less cell death (44%) of normal CD34+ primary cells exposed to drug-loaded, patient-trained exosomes. Summary/Conclusion: Taken together, our data predict important functional roles for exosome-derived Syntenin-1 in regulating lineage specific hematopoietic differentiations. Furthermore, for the first time, we have identified highly upregulated select exosome-derived miRs from AML patient samples whose prognostic value has been recently reported for other cancers, making these miRs promising candidates for AML biomarkers as well. Finally, stromal cell-derived, drug-loaded exosomes are not only able to induce apoptosis in AML patient samples, but they can effectively be trained by leukemic cells to favor uptake resulting in targeted elimination of leukemic over normal CD34+ cells. Disclosures No relevant conflicts of interest to declare.