Epithelial Membrane Protein 3 (EMP3), a 4‐transmembrane glycoprotein, first gained attention as a putative tumor suppressor. Accumulating evidence, however, points to a more tumor promotive function of EMP3. The biological function of EMP3 remains largely unclear. To elucidate more of EMP3’s interaction network, we performed a Yeast‐Two‐Hybrid (Y2H) screening, followed by validation of candidate interactors by Biomolecular Fluorescence Complementation (BiFC) and Proximity Ligation Assay (PLA). Furthermore, we generated stable EMP3 knockdown cell lines and measured cell proliferation, migration and sensitivity to apoptosis induction as well as the expression and activation levels of important signal pathway components. The Y2H screening yielded 10 novel interactions of EMP3, eight of which could also be detected by BiFC and PLA interaction assays. All newly discovered interaction partners are involved in signaling or trafficking regulation. Most notably, FLOT1 and HTATIP2 have well described roles in the regulation of EGFR signaling. In addition, knockdown of EMP3 resulted in reduced levels of p‐AKT, p‐ERK and p‐EGFR, attenuated cell proliferation and migration and sensitized cells to apoptosis induction by TRAIL and Staurosporine. Based on these observations we hypothesize that EMP3 might be involved in the regulation of receptor‐tyrosine‐kinase mediated mitogenic signaling.
Three-dimensional bioprinting of an endocrine pancreas is a promising future curative treatment for patients with insulin secretion deficiency. In this study, we present an end-to-end concept from the molecular to the macroscopic level. Building-blocks for a hybrid scaffold device of hydrogel and functionalized polycaprolactone were manufactured by 3D-(bio)printing. Pseudoislet formation from INS-1 cells after bioprinting resulted in a viable and proliferative experimental model. Transcriptomics showed an upregulation of proliferative and ß-cell-specific signaling cascades, downregulation of apoptotic pathways, overexpression of extracellular matrix proteins, and VEGF induced by pseudoislet formation and 3D-culture. Co-culture with endothelial cells created a natural cellular niche with enhanced insulin secretion after glucose stimulation. Survival and function of pseudoislets after explantation and extensive scaffold vascularization of both hydrogel and heparinized polycaprolactone were demonstrated in vivo. Computer simulations of oxygen, glucose and insulin flows were used to evaluate scaffold architectures and Langerhans islets at a future perivascular transplantation site.
BackgroundCocaine use disorder (CUD) is characterized by a loss of control over cocaine intake and is associated with structural, functional, and molecular alterations in the human brain. At the molecular level, epigenetic alterations are hypothesized to contribute to the higher-level functional and structural brain changes observed in CUD. Most evidence of cocaine-associated epigenetic changes comes from animal studies while only a few studies have been performed using human tissue.MethodsWe investigated epigenome-wide DNA methylation (DNAm) signatures of CUD in human post-mortem brain tissue of Brodmann area 9 (BA9). A total of N = 42 BA9 brain samples were obtained from N = 21 individuals with CUD and N = 21 individuals without a CUD diagnosis. We performed an epigenome-wide association study (EWAS) and analyzed CUD-associated differentially methylated regions (DMRs). To assess the functional role of CUD-associated differential methylation, we performed Gene Ontology (GO) enrichment analyses and characterized co-methylation networks using a weighted correlation network analysis. We further investigated epigenetic age in CUD using epigenetic clocks for the assessment of biological age.ResultsWhile no cytosine-phosphate-guanine (CpG) site was associated with CUD at epigenome-wide significance in BA9, we detected a total of 20 CUD-associated DMRs. After annotation of DMRs to genes, we identified Neuropeptide FF Receptor 2 (NPFFR2) and Kalirin RhoGEF Kinase (KALRN) for which a previous role in the behavioral response to cocaine in rodents is known. Three of the four identified CUD-associated co-methylation modules were functionally related to neurotransmission and neuroplasticity. Protein-protein interaction (PPI) networks derived from module hub genes revealed several addiction-related genes as highly connected nodes such as Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1), and Jun Proto-Oncogene, AP-1 Transcription Factor Subunit (JUN). In BA9, we observed a trend toward epigenetic age acceleration (EAA) in individuals with CUD remaining stable even after adjustment for covariates.ConclusionResults from our study highlight that CUD is associated with epigenome-wide differences in DNAm levels in BA9 particularly related to synaptic signaling and neuroplasticity. This supports findings from previous studies that report on the strong impact of cocaine on neurocircuits in the human prefrontal cortex (PFC). Further studies are needed to follow up on the role of epigenetic alterations in CUD focusing on the integration of epigenetic signatures with transcriptomic and proteomic data.
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