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
The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.
Subject(s)
Protein Kinase Inhibitors , Protein Serine-Threonine Kinases , YAP-Signaling Proteins , Animals , Humans , Mice , Cell Proliferation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Stem Cells/metabolism , Transcription Factors/metabolism , YAP-Signaling Proteins/agonists , YAP-Signaling Proteins/drug effects , YAP-Signaling Proteins/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacologyABSTRACT
Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17alpha-ethinyl estradiol (EE, 10 microg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.
Subject(s)
Endocrine Disruptors/toxicity , Estrogens, Non-Steroidal/toxicity , Phenols/toxicity , Surface-Active Agents/toxicity , Administration, Oral , Animals , Body Weight/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Endocrine Disruptors/metabolism , Estradiol/blood , Estrogens, Non-Steroidal/metabolism , Estrous Cycle/drug effects , Estrous Cycle/physiology , Female , Gene Expression/drug effects , Humans , Oligonucleotide Array Sequence Analysis , Organ Size/drug effects , Phenols/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/drug effects , Receptors, Estrogen/metabolism , Surface-Active Agents/metabolism , Toxicity Tests , Uterus/drug effects , Uterus/pathologyABSTRACT
A chemically induced model of ovarian failure has been developed in rodents, and was used to test whether or not anti-Müllerian hormone (AMH) can be used as a non-invasive measure of primordial follicle numbers. Repeated exposures of mice to 4-vinylcyclohexene diepoxide (VCD) induce loss of primordial and earliest growing ovarian follicles. An accelerated exposure regimen was used to eliminate small ovarian follicles in C57BL6/J mice (240mg VCD/kg/day, 5 days, i.p.). Follicle populations were determined and correlated with circulating AMH levels. Exposures decreased only primordial and small primary follicles by 96% on day 16 after initiating exposures, followed by almost complete follicle elimination on days 37-100. AMH levels in VCD-exposed mice were similar to vehicle-treated mice on day 16, but became significantly lower or undetectable at later time points. Thus, AMH correlated well with growing follicle numbers. AMH only correlated with primordial follicles at time points after ovarian insult at which their loss led to decreased growing follicle numbers.
Subject(s)
Anti-Mullerian Hormone/blood , Cyclohexenes/toxicity , Ovarian Follicle/drug effects , Vinyl Compounds/toxicity , Animals , Anti-Mullerian Hormone/metabolism , Biomarkers/blood , Biomarkers/metabolism , Body Weight/drug effects , Female , Immunohistochemistry , Mice , Mice, Inbred C57BL , Ovarian Follicle/metabolism , Ovarian Follicle/pathology , Time FactorsABSTRACT
BACKGROUND: During organogenesis the embryo is highly sensitive to oxidative stress. We hypothesize that oxidative stress and activation of a redox-sensitive transcription factor, activator protein-1 (AP-1), are early indicators of embryonic stress in response to a teratogenic insult. 5-Bromo-2'-deoxyuridine (BrdU) was chosen as a model teratogen to test this hypothesis; BrdU is a thymidine analog that is incorporated into replicating DNA. METHODS: Timed pregnant CD1 mice were given vehicle or BrdU (400, 600, 800, or 1000 mg of BrdU/kg of body weight) on gestation day 9 (GD 9). Oxidative stress, assessed as the ratio of glutathione disulfide (GSSG) to reduced glutathione (GSH), and AP-1 DNA binding activity (c-Fos- and c-Jun-dependent DNA binding) were measured in the maternal livers and embryos 0.5, 3, and 6 hr after treatment. External and skeletal malformations were assessed on GD 18. N-acetylcysteine, a glutathione precursor, was coadministered with BrdU to further explore the relationship between teratogenicity and redox homeostasis. RESULTS: BrdU exposure produced a dose-dependent increase in skeletal malformations, which included polydactyly, and delayed ossification of the sternebrae and vertebrae. Exposure to teratogenic doses of BrdU depleted GSH concentrations and increased oxidative stress, as assessed by the GSSG:GSH ratio, in both maternal livers and embryos. While c-Jun DNA binding activity in embryos was not affected, c-Fos DNA binding activity was elevated significantly 3 hr after BrdU exposure. Coadministration of N-acetylcysteine decreased the skeletal malformations and AP-1 DNA binding activity induced by BrdU. CONCLUSIONS: BrdU exposure induced an embryonic stress response manifested as an increase in oxidative stress and AP-1 DNA binding activity; these data support the hypothesis that disturbances in redox homeostasis mediate the response of the conceptus to a teratogenic insult.