ABSTRACT
Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
Subject(s)
Cell Cycle Proteins/genetics , Epidermis/drug effects , Re-Epithelialization/drug effects , Skin Ulcer/drug therapy , Small Molecule Libraries/pharmacology , Transcription Factors/genetics , Wounds, Nonpenetrating/drug therapy , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Disease Models, Animal , Epidermis/metabolism , Epidermis/pathology , Fluorescence Resonance Energy Transfer , Gene Expression Regulation , High-Throughput Screening Assays , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/pathology , Male , Mice , Mice, Inbred C57BL , Primary Cell Culture , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Precursors/antagonists & inhibitors , Protein Precursors/genetics , Protein Precursors/metabolism , Re-Epithelialization/genetics , Skin Ulcer/genetics , Skin Ulcer/metabolism , Skin Ulcer/pathology , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcription, Genetic , Wounds, Nonpenetrating/genetics , Wounds, Nonpenetrating/metabolism , Wounds, Nonpenetrating/pathologyABSTRACT
R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.
