TBK1 regulates YAP/TAZ and fibrogenic fibroblast activation.

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-ß-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.

Selective YAP/TAZ inhibition in fibroblasts via dopamine receptor D1 agonism reverses fibrosis.

Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gαs-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice.

CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis.

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFß and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFß- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying.

BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.

Human airway organoid engineering as a step toward lung regeneration and disease modeling.

Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-ß1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues.

Platelet-Derived Growth Factor Receptor-α Regulates Proliferation of Gastrointestinal Stromal Tumor Cells With Mutations in KIT by Stabilizing ETV1.

BACKGROUND & AIMS: In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. METHODS: We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. RESULTS: KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA. CONCLUSIONS: In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.

Loss of Kitlow progenitors, reduced stem cell factor and high oxidative stress underlie gastric dysfunction in progeric mice.

Gastrointestinal functions decline with ageing leading to impaired quality of life, and increased morbidity and mortality. Neurodegeneration is believed to underlie ageing-associated dysmotilities but the mechanisms have not been fully elucidated. We used progeric mice deficient in the anti-ageing peptide Klotho to investigate the contribution of key cell types of the gastric musculature to ageing-associated changes in stomach function and the underlying mechanisms. Klotho expression, enteric neurons, interstitial cells of Cajal (ICC), smooth muscle cells and electrical activity were assessed by immunofluorescence, confocal microscopy, 3-dimensional reconstruction, flow cytometry, quantitative RT-PCR, Western immunoblotting and intracellular recordings. Gastric emptying of solids was analysed by the [13C]octanoic acid breath test. Circulating and tissue trophic factors were measured by enzyme immunoassays and quantitative RT-PCR. The role of oxidative stress was investigated in organotypic cultures. Klotho expression was detected in gastric glands, myenteric neurons and smooth muscle cells. Progeric Klotho-deficient mice had profound loss of ICC and ICC stem cells without a significant decrease in neuron counts, expression of neuronal nitric oxide synthase or smooth muscle myosin. Slow wave amplitude and nitrergic inhibitory junction potentials were reduced while solid emptying was unchanged. Klotho-deficient mice were marantic and had low insulin, insulin-like growth factor-I and membrane-bound stem cell factor. Klotho deficiency accentuated oxidative stress and ICC loss. We conclude that Klotho-deficient, progeric mice display a gastric phenotype resembling human ageing and involving profound ICC loss. Klotho protects ICC by preserving their precursors, limiting oxidative stress, and maintaining nutritional status and normal levels of trophic factors important for ICC differentiation.

CD206-positive M2 macrophages that express heme oxygenase-1 protect against diabetic gastroparesis in mice.

BACKGROUND & AIMS: Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. METHODS: Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. RESULTS: Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages. CONCLUSIONS: CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.

Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation.

Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in beta-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and beta-casein synthesis but not the synergistic stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of beta-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3'-untranslated region of beta-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity.