A Regulatory Loop between β-Catenin and Extra-cellular Matrix Components during the Proliferative Phase of Cutaneous Wound Healing

β-Catenin is an important modulator of the cutaneous wound phenotype, where it is activated in dermal fibroblasts during the proliferative stage of repair. Despite its importance as a mediator of the healing process, the factors that regulate β-catenin during wound repair are not well understood, nor are the identities of its target genes. One possibility is the involvement of the extracellular matrix (ECM) as both a target and a regulator of β-Catenin activity. I investigated the existence of a regulatory loop between β-Catenin and the ECM in dermal fibroblasts during cutaneous wound repair. Fibroblasts cultured on various ECM components showed increased levels of β-Catenin activation, and this was found to occur through an integrin mediated, glycogen synthase kinase 3β dependent pathway. In vivo, wounds from mice that lack the EDA-fibronectin variant exhibited decreased β-Catenin signaling, fewer dermal fibroblasts, and decreased wound strength. These properties were rescued by genetic or pharmacologic activation of β-Catenin, using an EDA-fibronectin deficient mouse model that also expresses a conditionally stabilized form of β-Catenin, or the drug, Lithium Chloride. This suggests both, that ECM components regulate β-Catenin activity in dermal fibroblasts during healing, and that β-Catenin is an important modulator of cell behaviour and the resulting properties of the wound. Since fibronectin is a known transcriptional target of β-Catenin, it also suggests a regulatory loop between these two molecules. To investigate the existence of other ECM targets of β-Catenin during wound repair, we undertook a microarray analysis in wounds from conditional β-Catenin knockdown and stabilized mice. We found several ECM associated candidates that have not previously been identified as β-Catenin targets, and verified their direction of expression by real-time PCR. Together, this work suggests that β -catenin and ECM components interact in a regulatory loop to modulate fibroblast behaviour in the physiological context of wound repair.Ph

Enhancer RNA Expression in Response to Glucocorticoid Treatment in Murine Macrophages

Glucocorticoids are potent anti-inflammatory drugs; however, their molecular mode of action remains complex and elusive. They bind to the glucocorticoid receptor (GR), a nuclear receptor that controls gene expression in almost all tissues in a cell type-specific manner. While GR’s transcriptional targets mediate beneficial reactions in immune cells, they also harbor the potential of adverse metabolic effects in other cell types such as hepatocytes. Here, we have profiled nascent transcription upon glucocorticoid stimulation in LPS-activated primary murine macrophages using 4sU-seq. We compared our results to publicly available nascent transcriptomics data from murine liver and bioinformatically identified non-coding RNAs transcribed from intergenic GR binding sites in a tissue-specific fashion. These tissue-specific enhancer RNAs (eRNAs) correlate with target gene expression, reflecting cell type-specific glucocorticoid responses. We further associate GR-mediated eRNA expression with changes in H3K27 acetylation and BRD4 recruitment in inflammatory macrophages upon glucocorticoid treatment. In summary, we propose a common mechanism by which GR-bound enhancers regulate target gene expression by changes in histone acetylation, BRD4 recruitment and eRNA expression. We argue that local eRNAs are potential therapeutic targets downstream of GR signaling which may modulate glucocorticoid response in a cell type-specific way