Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K

SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K+ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a down-regulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from WT but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, noncanonical Hh signaling mediated by Gi proteins regulates K+ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias. © 2018 Cheng et al

PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function

Enhancement of cutaneous wound healing by Dsg2 augmentation of uPAR secretion

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. While low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in non-healing venous ulcers, overexpression has been observed in both melanomas and non-melanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor (uPAR). Dsg2 induced uPAR expression in the skin of transgenic compared to wild-type mice. Wound healing further enhanced uPAR in both epidermis and dermis with concomitant increase in the pro-healing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through uPAR-related signaling cascades

Induction of Gli1 by SmoA1 and Gα₁₃QL.

<p>C3H10T1/2 cells were transduced with recombinant adenoviruses encoding LacZ, SmoA1, or Gα₁₃QL (MOI = 10) in the presence or absence of DEAE-Dextran (10 μg/ml) as noted. At 2 days following transduction, RNA was extracted for PCR with primers specific for Gli1 and, as a control for loading, GAPDH. Arrows denote anticipated sizes of the products. Shown is one experiment, which is representative of three total.</p

Stress fiber formation in response to SmoA1 and Gα₁₃QL.

<p>C3H10T1/2 cells grown on coverslips were serum-starved for 12 h then incubated with recombinant adenoviruses encoding LacZ, SmoA1, or Gα₁₃QL (MOI = 10) in the presence of DEAE-Dextran (10 μg/ml). They were serum-starved an additional 18 h and fixed with paraformaldehyde. F-actin was stained with FITC-Phalloidin, and nuclei were counterstained with DAPI. ‘Merge’ is pseudocolored. Shown is one experiment representative of three total. Few or no stress fibers were identified in the absence of transduction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197442#pone.0197442.s002" target="_blank">S2 Fig</a>).</p

Changes in transcripts for selected genes in C3H10T1/2 cells following introduction of SmoA1 and Gα₁₃QL, or exposure to purmorphamine.

<p>Changes in transcripts for selected genes in C3H10T1/2 cells following introduction of SmoA1 and Gα₁₃QL, or exposure to purmorphamine.</p

Enhancement of adenoviral transduction by polycations.

<p>C3H10T1/2 cells were transduced with recombinant adenoviruses expressing LacZ (AdV·LacZ) or GFP (AdV·GFP) in the presence or absence of polycations. (A) Cells were transduced with AdV·LacZ at the indicated MOIs in the absence of polycation. The cells were lysed 2 days later, and LacZ activity was measured. Activity is expressed as units per mg cell lysate. The data are averages of three different experiments, each carried out in triplicate, ± SEM; <i>p</i> <0 .05 for MOIs ≥ 20 relative to no virus. (B) Cells were transduced with AdV·LacZ at an MOI of 10 at the indicated concentrations of polybrene and DEAE-dextran. The cells were lysed 2 days later, and LacZ activity was measured. The data are from an individual experiment that represents three experiments total, each carried out in triplicate but using slightly different sets of concentrations of polycations, ± SEM; <i>p</i> < 0.01 at all concentrations of polycation relative to no polycation. (C) Cells were transduced with AdV·LacZ at the indicated MOIs in the presence of 10 μg/ml DEAE-Dextran. The cells were lysed 2 days later, and LacZ activity was measured. The data are averages of four different experiments, each carried out in triplicate, ± SEM; <i>p</i> < 0.05 for MOIs ≥ 2.5 relative to no virus. (D) Cells were transduced with AdV·GFP at the indicated MOIs without or with polybrene (5 μg/ml) or DEAE-dextran (10 μg/ml). The cells were fixed 2 days later, counterstained with DAPI, and evaluated as the number of GFP- relative to DAPI-positive cells by fluorescence microscopy. The data are from an individual experiment that represents three experiments total, each carried out using a minimum of 10 random fields for each data point, but using slightly different sets of concentrations of polycations, ± SEM; <i>p</i> < 0.01 for MOIs ≥ 5 in the presence of polycation relative to absence of polycation.</p

PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1&minus;/&minus; mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function

Partial Truncation of the C-Terminal Domain of PTCH1 in Cancer Enhances Autophagy and Metabolic Adaptability

The Hedgehog receptor, Patched1 (PTCH1), is a well-known tumour suppressor. While the tumour suppressor’s activity is mostly ascribed to its function as a repressor of the canonical Smoothened/Gli pathway, its C-terminal domain (CTD) was reported to have additional non-canonical functions. One of them is the reduction of autophagic flux through direct interaction with the Unc-51, like the autophagy activating kinase (ULK) complex subunit autophagy-related protein-101 (ATG101). With the aim of investigating whether this function of PTCH1 is important in cancer cell fitness, we first identified frameshift mutations in the CTD of PTCH1 in cancer databases. We demonstrated that those mutations disrupt PTCH1 interaction with ATG101 and increase autophagic flux. Using deletion mutants of the PTCH1 CTD in co-immunoprecipitation studies, we established that the 1309–1447 region is necessary and sufficient for interaction with ATG101. We next showed that the three most common PTCH1 CTD mutations in endometrial, stomach and colon adenocarcinomas that cause frameshifts at S1203, R1308 and Y1316 lack the ability to interact with ATG101 and limit autophagic flux, determined by bafilomycin A1-sensitive accumulation of the autophagy markers LC3BII and p62. We next engineered PTCH1 indel mutations at S1223 by CRISPR/Cas9 in SW620 colon cancer cells. Comparison of two independent clones harbouring PTCH1 S1223fs mutations to their isogenic parental cell lines expressing wild-type PTCH1 showed a significant increase in basal and rapamycin-stimulated autophagic flux, as predicted by loss of ATG101 interaction. Furthermore, the PTCH1 CTD mutant cells displayed increased proliferation in the presence of rapamycin and reduced sensitivity to glycolysis inhibitors. Our findings suggest that loss of the PTCH1-ATG101 interaction by mutations in the CTD of PTCH1 in cancer might confer a selective advantage by stimulating autophagy and facilitating adaptation to nutrient deprivation conditions