Airway Hydration, Apical K+ Secretion, and the Large-Conductance, Ca2+-activated and Voltage-dependent Potassium (BK) Channel

Large-conductance, calcium-activated, and voltage-gated K + (BK) channels are expressed in many tissues of the human body, where they play important roles in signaling not only in excitable but also in nonexcitable cells. Because BK channel properties are rendered in part by their association with four β and four γ subunits, their channel function can differ drastically, depending on in which cellular system they are expressed. Recent studies verify the importance of apically expressed BK channels for airway surface liquid homeostasis and therefore of their significant role in mucociliary clearance. Here, we review evidence that inflammatory cytokines, which contribute to airway diseases, can lead to reduced BK activity via a functional down-regulation of the γ regulatory subunit LRRC26. Therefore, manipulation of LRRC26 and pharmacological opening of BK channels represent two novel concepts of targeting epithelial dysfunction in inflammatory airway diseases

CRISPR-Cas9 screen identifies mechanisms of BET bromodomain inhibitor sensitivity including manganese

BET Bromodomain Inhibitors hold promise as therapeutic agents in inflammation and cancer but clinical studies show adverse side-effects at high, sustained dose. Clinical success requires further mechanistic understanding of inhibition of BET bromodomains and biomarkers to optimize efficacious dosing. To uncover the mechanisms of sensitivity and resistance to BETi, we employed a whole-genome CRISPR-Cas9 proliferation screen using colorectal cancer cells. We identify the mTOR signaling pathway as a key determinant of BETi sensitivity and that two Ca/Mn transporters mediate resistance. This later finding led to the discovery that extracellular manganese regulates sensitivity to BETi and that exposure of cells to BETi dose dependently increases intracellular manganese concentration. Our results describe new molecular pathways mediating BETi action and suggest several potential avenues for biomarker discovery

Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating, often dose limiting side effect of radiation treatment. A mouse model of mucositis, induced by gamma irradiation, leads to weight loss and tissue damage, similar to that observed in patients. This model reflects the human ailment as it responds to keratinocyte growth factor (KGF), the standard of care treatment. Culturing of intestinal crypt organoids derived from primary cells allowed the development of a 3D assay to monitor the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay closely resembles the mouse model as KGF and Roof Plate-Specific Spondin-1 (RSPO1) enhanced the recovery of crypt organoids following radiation. Screening identified tool compounds that increased the survival of organoids post radiation. Repeated testing of these compounds revealed that the organoids changed their response over time. To investigate this adaptive behavior, intestinal organoid cultures were studied over time. Samples of organoids at various time points were used to prepare mRNA for unbiased transcriptome analyses. This expression profiling revealed a number of genes and pathways that were modulated over time, providing a rationale for the altered sensitivity of the intestinal crypt organoid cultures. This report describes the development of an in vitro assay that reflects the response of disease to therapeutic treatment. The assay was miniaturized and used to identify bioactive tool compounds, which served as probes to interrogate the patho-physiology of organoids over prolonged culture conditions. In vitro disease models based on primary 3D cell cultures represent valuable tools to identify potential drug targets and bioactive hits

IRF2 is a master regulator of human keratinocyte stem cell fate

SUMMARY Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and provision of differentiated cells. Human epidermal keratinocytes retain stem cell potential in vitro but this is highly variable and lost over time suggesting extrinsic and intrinsic regulation. As transcription factor regulatory circuits have been shown to govern cell identity and are sufficient to induce pluripotency or transdifferentiate cell types, we sought to define changes in transcriptional circuitry between two populations of keratinocytes with intrinsic high and low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate the transcription factor IRF2 as antagonistic to stemness and show that its knockdown in keratinocytes with low stem cell potential is sufficient to increase self-renewal, migration and ability to generate 3D human skin constructs. These data suggest that transcription factor regulatory circuits, in addition to maintaining cell identity, control cellular plasticity and could offer potential for therapeutic modulation of cell functio

R-Spondin Potentiates Wnt/b-Catenin Signaling Through Orphan Receptors LGR4 and LGR5

The Wnt/b-catenin signaling pathway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/b-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced b-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced b-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated b-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications

R-Spondin Potentiates Wnt/β-Catenin Signaling through Orphan Receptors LGR4 and LGR5

<div><p>The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4−/− crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.</p> </div

BET bromodomain inhibitors regulate keratinocyte plasticity

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. [Figure not available: see fulltext.

RSPO1 interacts with the extracellular domain of LGR4 and LGR5.

<p>(a) RSPO1 binds to cells overexpressing LGR4 or LGR5. HEK293T cells transiently overexpressing HA-tagged LGR4 or LGR5 were incubated with RSPO1-GFP-conditioned medium for 1 h at 37°C and subjected to immunofluorescence analysis. RSPO1-GFP only binds to cells overexpressing LGR4 or LGR5. Also note the co-localization of RSPO1-GFP and LGR4 or LGR5 in some intracellular vesicular structures. (b) Co-immunoprecipitation of LGR4-ECD and RSPO1. HEK293T cells were transiently transfected with plasmids expressing either C-terminally V5-6xHis-tagged LGR4-ECD or Fc-tagged RSPO1. Supernatants of these cultures and of control non-transfected HEK293T cells were mixed in combinations as indicated and subjected to Fc-pulldown experiments. Eluates (E), flow-through fractions (F) and input lysates (I) were analyzed by Western Blot analyses. Immunoreagents against IgG or V5 were applied to detect RSPO1 (a-Fc) and LGR4-ECD (α-V5), respectively. The position of the 62 kDa marker band is indicated on the right.</p