Nonsense mutations are present in 10% of patients with CF, produce a premature termination codon in CFTR mRNA causing early termination of translation, and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study, we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels, demonstrates absence of CFTR function, and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly, CFTR restoration is observed in G542X intestinal organoids treated with G418, an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations.
Tissue fibrosis in many organs results from altered and excessive extracellular matrix (ECM) protein deposition 1. Concomitant with ECM expansion, resident lipid-filled cells including mature adipocytes are lost in human and mouse fibrosis2-5, yet the mechanisms that drive mature adipocyte lipid loss and their contribution to tissue fibrosis are unknown. Here, we identify an early, fibro-protective role of mature adipocyte lipolysis driven by Wnt signaling during fibrosis onset. Using chemical and genetic mouse models of skin fibrosis, we show that fibrotic stimuli induce and maintain lipolysis in mature dermal adipocytes. Loss of the lipolytic rate-limiting enzyme adipocyte triglyceride lipase (ATGL)6,7 in murine dermal adipocytes exacerbates bleomycin-induced fibrosis development. Adipocyte lipolysis is stimulated in the early stages of Wnt signaling-induced skin fibrosis and by Wnt agonists in vitro. Furthermore, deletion or inhibition of the Wnt target gene, CD26/Dipeptidyl peptidase 4 (DPP4) prevented Wnt-induced lipolysis and skin fibrosis in mice. Notably, DPP4 expression correlates with skin fibrosis severity in human patients. Thus, we propose that adipocyte-derived fatty acids and the Wnt-DPP4 axis act as essential regulators of ECM homeostasis within tissues and provide a therapeutic avenue to manipulate fibrosis.
Fibrotic diseases contribute to approximately 45% of deaths in Europe and North America, and are characterized by an accumulation of extracellular matrix and loss of lipid-filled cells. Dermal fibrosis is a debilitating disease which serves as a model for studying universal trends in fibrosis such as lipid depletion. Intradermal adipocytes comprising the dermal white adipose tissue (DWAT) have diverse functions impacted by their lipid content. The mechanisms governing DWAT lipid depletion are not known nor are the effects of lipid depletion in causing fibrosis. Wnt signaling is dysregulated among fibrotic tissues and sustained Wnt signaling in mouse dermis is sufficient to cause dermal fibrosis including DWAT lipid depletion. Here we tested the following hypothesis: Wnt signaling and Wnt-responsive factors cause DWAT lipid depletion and subsequent dermal thickening. We developed a genetic mouse model of Wnt activation in the dermis resulting in inducible and reversible dermal thickening preceded by DWAT lipid depletion, enabling us to identify mediators of Wntinduced lipid depletion. Wnt activation in intradermal adipocytes in vitro leads to lipid loss by induction of lipolysis, as indicated by a three-fold increase in free glycerol, suggesting Wnt signaling causes cell autonomous lipolytic effects. Additionally, conditioned media from Wnt-activated fibroblasts stimulate lipid loss in intradermal adipocytes in vitro, implicating intercellular communication in fibrotic adipocyte lipid loss. Finally, transient inhibition of a candidate Wnt-responsive factor, CD26/DPPIV, in genetic and chemical models of fibrosis leads to preservation or faster recovery of lipid in DWAT and reduced dermal thickness in vivo, demonstrating that Wnt-DPPIV modulates lipid homeostasis in adipocytes impacting neighboring dermal fibroblasts. Thus, we propose to treat dermal fibrosis in a pre-clinical model with FDA-approved drugs by targeting a new cellular player in fibrosis.
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