Chromatin access regulates the formation of Müller glia-derived progenitor cells in the retina.

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein, we investigate the expression and functions of S-adenosylhomocysteine hydrolase (SAHH; AHCY) and histone methyltransferases (HMTs) during the formation of Müller glia (MG)-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1 and AHCYL2, and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to convey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the differentiation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

Keratinocyte-derived microvesicle particles mediate ultraviolet B radiation-induced systemic immunosuppression.

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.

Thermal Burn Injury Generates Bioactive Microvesicles: Evidence for a Novel Transport Mechanism for the Lipid Mediator Platelet-Activating Factor (PAF) That Involves Subcellular Particles and the PAF Receptor.

Thermal burn injuries are an important environmental stressor that can result in considerable morbidity and mortality. The exact mechanism by which an environmental stimulus to skin results in local and systemic effects is an area of active research. One potential mechanism to allow skin keratinocytes to disperse bioactive substances is via microvesicle particles, which are subcellular bodies released directly from cellular membranes. Our previous studies have indicated that thermal burn injury of the skin keratinocyte in vitro results in the production of the lipid mediator platelet-activating factor (PAF). The present studies demonstrate that thermal burn injury to keratinocytes in vitro and human skin explants ex vivo, and mice in vivo generate microvesicle particles. Use of pharmacologic and genetic tools indicates that the optimal release of microvesicles is dependent upon the PAF receptor. Of note, burn injury-stimulated microvesicle particles do not carry appreciable protein cytokines yet contain high levels of PAF. These studies describe a novel mechanism involving microvesicle particles by which a metabolically labile bioactive lipid can travel from cells in response to environmental stimuli.

Gemcitabine Induces Microvesicle Particle Release in a Platelet-Activating Factor-Receptor-Dependent Manner via Modulation of the MAPK Pathway in Pancreatic Cancer Cells.

Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.