Notch intracellular domains form transcriptionally active heterodimeric complexes on sequence-paired sites.

Notch signaling is universally conserved in metazoans where it is important for a wide variety of both normal and abnormal physiology. All four mammalian Notch receptors are activated by a conserved mechanism that releases Notch intracellular domains (NICDs) from the plasma membrane to translocate to the nucleus. Once there, NICDs interact through highly conserved ankyrin domains to form head-to-head homodimers on Notch sensitive promoters and stimulate transcription. Due to the highly conserved nature of these Notch ankyrin domains in all four mammalian Notch proteins, we hypothesized that NICDs may also engage in heterodimerization. Our results reveal the presence of two NICD dimerization states that can both engage in homo and heterodimerization. Using a Co-IP approach, we show that all NICD's can form non-transcriptionally active dimers and that the N4ICD appears to perform this function better than the other NICDs. Using a combination of ChIP analysis and transcriptional reporter assays, we also demonstrate the formation of transcriptionally active heterodimers that form on DNA. In particular, we demonstrate heterodimerization between the N2ICD and N4ICD and show that this heterodimer pair appears to exhibit differential activity on various Notch sensitive promoters. These results illustrate a new diversification of Notch signaling mechanisms which will help us better understand basic Notch function.

Notch Intracellular Domains form Transcriptionally Active Heterodimeric Complexes on Sequence-paired Sites.

Notch signaling is universally conserved in metazoans where it is important for a wide variety of both normal and abnormal physiology. All four mammalian Notch receptors are activated by a conserved mechanism that releases Notch intracellular domains (NICDs) from the plasma membrane to translocate to the nucleus. Once there, NICDs interact through highly conserved ankyrin domains to form head-to-head homodimers on Notch sensitive promoters and stimulate transcription. Due to the highly conserved nature of these Notch ankyrin domains in all four mammalian Notch proteins, we hypothesized that NICDs may also engage in heterodimerization. Our results reveal the presence of two NICD dimerization states that can both engage in homo and heterodimerization. Using a Co-IP approach, we show that all NICD's can form non-transcriptionally active dimers and that the N4ICD appears to perform this function better than the other NICDs. Using a combination of ChIP analysis and transcriptional reporter assays, we also demonstrate the formation of transcriptionally active heterodimers that form on DNA. In particular, we demonstrate heterodimerization between the N2ICD and N4ICD and show that this heterodimer pair appears to exhibit differential activity on various Notch sensitive promoters. These results illustrate a new diversification of Notch signaling mechanisms which will help us better understand basic Notch function.

Use of integrated metabolomics, transcriptomics, and signal protein profile to characterize the effector function and associated metabotype of polarized macrophage phenotypes.

MΦs display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 MΦs and a broad set of M2 MΦs, comprehensive characterization of functional phenotype and associated metabotype driving this diverse MΦ activation remains. Herein, an ex vivo model was utilized to produce 6 MΦ functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 MΦs, and then polarized into M1 (IFN-γ/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) MΦs. The MΦs were profiled using a bioanalyte matrix of 4 cell surface markers, ∼50 secreted proteins, ∼800 expressed myeloid genes, and ∼450 identified metabolites relative to M0 MΦs. Signal protein and expressed gene profiles grouped the MΦs into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b MΦs shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO, and OXPHOS. Additionally, M2a, M2c, and M2d MΦs all profiled as tissue repair MΦs; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a MΦs, and angiogenesis and ECM assembly capabilities for M2b, M2c, and M2d MΦs. By integrating metabolomics into a systems analysis of MΦ phenotypes, we provide the most comprehensive map of MΦ diversity to date, along with the global metabolic shifts that correlate to MΦ functional plasticity in these phenotypes.

Metabolic immunomodulation of macrophage functional plasticity in nonhealing wounds.

PURPOSE OF REVIEW: Despite modern advances in medicine, nonhealing wounds are the number one cause of nontraumatic, lower-limb amputation. Nonhealing wounds are characterized by a healing process stalled between inflammation and tissue remodel/repair, a stage characterized by a shift in macrophage functional phenotype. Characterization of diversity in macrophage functional phenotype in wounds and metabolic contributions to macrophage polarization are discussed. RECENT FINDINGS: Macrophage functional diversity in phenotype has recently evolved from duality (classically activated, pro-inflammatory M1 and alternatively activated, anti-inflammatory M2) to include an additional four alternately activated subphenotypes (M2a, M2b, M2c and M2d). Metabolic pathway utilization shifts characterize macrophage polarization with resulting metabolic and immune outcomes impacting host-pathogen interactions during wound healing. SUMMARY: Recognition of the key role macrophage diversity plays in wound healing, along with better characterization of diverse macrophage phenotypes, will inform our understanding of pathogenicity in wound healing. Comprehensive profiling of the metabolism regulating macrophage polarization and host-pathogen interaction creates opportunity of discovery for innovative new diagnostics and therapeutics for treating nonhealing wounds.