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Nature Cell Biology Jan 2015The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One...
The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK-STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.
Topics: Adipocytes, Brown; Adipocytes, White; Animals; Bone Morphogenetic Protein 7; Cell Differentiation; Cells, Cultured; Gene Expression Profiling; Hedgehog Proteins; Humans; Interferon-gamma; Ion Channels; Janus Kinase 1; Janus Kinase 3; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Proteins; Obesity; Oxazines; Phosphorylation; Piperidines; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Pyrroles; STAT1 Transcription Factor; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Uncoupling Protein 1; Veratrum Alkaloids
PubMed: 25487280
DOI: 10.1038/ncb3075 -
Cellular Physiology and Biochemistry :... 2017Hypoxic microenvironment, a common feature of hepatocellular carcinoma (HCC), can induce HIF-1α expression and promote the epithelial-mesenchymal transition (EMT) and...
BACKGROUND/AIMS
Hypoxic microenvironment, a common feature of hepatocellular carcinoma (HCC), can induce HIF-1α expression and promote the epithelial-mesenchymal transition (EMT) and invasion of cancer cells. However, the underlying molecular mechanisms have not fully elucidated.
METHODS
HCC cells were cultured under controlled hypoxia conditions or normoxic conditions. Transwell assays were used to examine the migration and invasion capacity. HIF-1α siRNA, cyclopamine (a SMO antagonist) and GLI1 siRNA were used to inhibit HIF-1α transcription or Hh signaling activation.
RESULTS
In present study, we first observed a strongly positive correlation between HIF-1α and GLI1 expression in HCC tissues. Then, we showed that hypoxia significantly promoted EMT process and invasion of HCC cells, associated with activating the non-canonical Hh pathway without affecting SHH and PTCH1 expression. HIF-1α knockdown mitigated hypoxia-induced SMO and GLI1 expression, EMT invasion of HCC cells. Moreover, the SMO inhibitor or GLI1 siRNA also reversed the hypoxia-driven EMT and invasion of HCC cells under hypoxia condition. Here, we show that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in HCC cells. In addition, we found that hypoxia increased ROS production and that ROS inhibitors (NAC) blocked GLI1-dependent EMT process and invasion under hypoxic conditions. To determine a major route of ROS production, we tested whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is involved in hypoxia-induced ROS production. NOX4 expression was found to be increased at both mRNA and protein levels in hypoxic HCC cells. Furthermore, siRNA-mediated knockdown of NOX4 expression abolished hypoxia induced ROS generation and GLI1-dependent activation and invasion of HCC cells.
CONCLUSION
Our findings indicate that hypoxia triggers ROS-mediated GLI1-dependent EMT progress and invasion of HCC cells through induction of NOX4 expression. Thus, hypoxia-driven ROS mediated non-canonical Hh signaling may play an important role in the initiation of EMT and provides a potential marker for cancer prevention and treatment.
Topics: Cadherins; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver; Liver Neoplasms; Microscopy, Fluorescence; NADPH Oxidase 4; Oxidative Stress; Patched-1 Receptor; RNA Interference; RNA, Small Interfering; Reactive Oxygen Species; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids; Vimentin
PubMed: 29237157
DOI: 10.1159/000485821 -
Proceedings of the National Academy of... Aug 2021In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult...
In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of , a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.
Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain; Gene Expression Regulation; Gliosis; Hedgehog Proteins; Mice; Mice, Transgenic; Selective Estrogen Receptor Modulators; Smoothened Receptor; Spinal Cord; Tamoxifen; Veratrum Alkaloids
PubMed: 34417306
DOI: 10.1073/pnas.2017779118 -
Journal of Bone and Mineral Research :... Nov 2007Specific oxysterols have been shown to be pro-osteogenic and anti-adipogenic. However, the molecular mechanism(s) by which oxysterols inhibit adipogenic differentiation...
UNLABELLED
Specific oxysterols have been shown to be pro-osteogenic and anti-adipogenic. However, the molecular mechanism(s) by which oxysterols inhibit adipogenic differentiation is unknown. We show that the anti-adipogenic effects of osteogenic oxysterol, 20(S)-hydroxycholesterol, are mediated through a hedgehog-dependent mechanism(s) and are associated with inhibition of PPARgamma expression.
INTRODUCTION
Multipotent bone marrow stromal cells (MSCs) are common progenitors of osteoblasts and adipocytes. A reciprocal relationship between osteogenic and adipogenic differentiation may explain the increased adipocyte and decreased osteoblast formation in aging and osteoporosis. We have previously reported that specific oxysterols stimulate osteogenic differentiation of MSCs while inhibiting their adipogenic differentiation.
MATERIALS AND METHODS
The M2-10B4 (M2) murine pluripotent bone MSC line was used to assess the inhibitory effects of 20(S)-hydroxycholesterol (20S) and sonic hedgehog (Shh) on peroxisome proliferator-activated receptor gamma (PPARgamma) and adipogenic differentiation. All results were analyzed for statistical significance using ANOVA.
RESULTS AND CONCLUSIONS
Treatment of M2 cells with the osteogenic oxysterol 20S completely inhibited adipocyte formation induced by troglitazone after 10 days. PPARgamma mRNA expression assessed by RT-qPCR was significantly induced by Tro after 48 (5-fold) and 96 h (130-fold), and this induction was completely inhibited by 20S. In contrast, 20S did not inhibit PPARgamma transcriptional activity in M2 cells overexpressing PPARgamma and retinoid X receptor (RXR). To elucidate the molecular mechanism(s) by which 20S inhibits PPARgamma expression and adipogenic differentiation, we focused on the hedgehog signaling pathway, which we previously showed to be the mediator of osteogenic responses to oxysterols. The hedgehog signaling inhibitor, cyclopamine, reversed the inhibitory effects of 20S and Shh on troglitazone-induced adipocyte formation in 10-day cultures of M2 cells by 70% and 100%, respectively, and the inhibitory effect of 20S and Shh on troglitazone-induced PPARgamma expression was fully reversed at 48 h by cyclopamine. Furthermore, 20S and Shh greatly inhibited PPARgamma2 promoter activity induced by CCAAT/enhancer-binding protein alpha overexpression. These studies show that, similar to the induction of osteogenesis, the inhibition of adipogenesis in murine MSCs by the osteogenic oxysterol, 20S, is mediated through a hedgehog-dependent mechanism(s).
Topics: Adipogenesis; Animals; Bone Marrow Cells; CCAAT-Enhancer-Binding Protein-alpha; Cell Line; Hedgehog Proteins; Hydroxycholesterols; Mice; PPAR gamma; Retinoid X Receptors; Stromal Cells; Veratrum Alkaloids
PubMed: 17638575
DOI: 10.1359/jbmr.070710 -
Science Translational Medicine Jun 2015FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and...
FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML.
Topics: Animals; Cell Compartmentation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Progression; Drug Synergism; Gene Duplication; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Leukemia, Myeloid, Acute; Mice; Mutant Proteins; Myeloproliferative Disorders; Niacinamide; Nuclear Proteins; Phenylurea Compounds; Receptors, G-Protein-Coupled; STAT5 Transcription Factor; Signal Transduction; Smoothened Receptor; Sorafenib; Stem Cells; Veratrum Alkaloids; Zinc Finger Protein Gli2; fms-Like Tyrosine Kinase 3
PubMed: 26062848
DOI: 10.1126/scitranslmed.aaa5731 -
Oncogene Nov 2017The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup...
The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.
Topics: Anilides; Animals; Cell Line, Tumor; Cerebellar Neoplasms; Disease Models, Animal; HEK293 Cells; Hedgehog Proteins; Humans; Male; Medulloblastoma; Mice; Mice, Transgenic; Pyridines; Random Allocation; SOXB1 Transcription Factors; Small Molecule Libraries; TRPC Cation Channels; Transfection; Tumor Suppressor Protein p53; Veratrum Alkaloids; Wnt Proteins; Wnt Signaling Pathway
PubMed: 28714964
DOI: 10.1038/onc.2017.232 -
The Journal of General Physiology Mar 1988The state dependence of Na channel modification by the alkaloid neurotoxin veratridine was investigated with single-channel and whole-cell voltage-clamp recording in...
The state dependence of Na channel modification by the alkaloid neurotoxin veratridine was investigated with single-channel and whole-cell voltage-clamp recording in neuroblastoma cells. Several tests of whole-cell Na current behavior in the presence of veratridine supported the hypothesis that Na channels must be open in order to undergo modification by the neurotoxin. Modification was use dependent and required depolarizing pulses, the voltage dependence of production of modified channels was similar to that of normal current activation, and prepulses that caused inactivation of normal current had a parallel effect on the generation of modified current. This hypothesis was then examined directly at the single-channel level. Modified channel openings were easily distinguished from normal openings by their smaller current amplitude and longer burst times. The modification event was often seen as a sudden, dramatic reduction of current through an open Na channel and produced a somewhat flickery channel event having a mean lifetime of 1.6 s at an estimated absolute membrane potential of -45 mV (23 degrees C). The modified channel had a slope conductance of 4 pS, which was 20-25% the size of the slope conductance of normal channels with the 300 mM NaCl pipette solution used. Most modified channel openings were initiated by depolarizing pulses, began within the first 10 ms of the depolarizing step, and were closely associated with the prior opening of single normal Na channels, which supports the hypothesis that modification occurs from the normal open state.
Topics: Animals; Electric Conductivity; Ion Channels; Membrane Potentials; Neuroblastoma; Sodium; Tumor Cells, Cultured; Veratridine; Veratrine
PubMed: 2454286
DOI: 10.1085/jgp.91.3.421 -
Organic Letters May 2020Progress toward a convergent approach for the enantioselective synthesis of the alkaloid jervine is presented. The two requisite fragments were stereoselectively and...
Progress toward a convergent approach for the enantioselective synthesis of the alkaloid jervine is presented. The two requisite fragments were stereoselectively and efficiently fashioned from economical and readily available reagents. Key reactions include (a) a highly diastereoselective Ireland-Claisen rearrangement to establish the necessary relationship between the amine and methyl group on the tetrahydrofuran E-ring; (b) a diastereoselective selenoetherification reaction that enabled the assembly of the D/E oxaspiro[4.5]decene in the needed configuration; and (c) an enzymatic desymmetrization of an abundant achiral diol en route to a key four-carbon building block as a practical alternative to a protected Roche ester reduction.
Topics: Chemistry Techniques, Synthetic; Cyclization; Stereoisomerism; Veratrum Alkaloids
PubMed: 32286835
DOI: 10.1021/acs.orglett.0c00972 -
The American Journal of Pathology Oct 2018Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog...
Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog signaling, a major pathway regulated by primary cilia, in biliary cystogenesis in Caroli disease remains unknown. Using the polycystic kidney (PCK) rat as an animal model of Caroli disease, this study investigated the involvement of Hedgehog signaling in its pathogenesis. In vitro experiments revealed that PCK cholangiocytes overexpressed Smoothened, Gli1, and Gli1's target molecule cyclin D1. The nuclear expression of Gli1, Gli2, and Gli3 was observed in PCK cholangiocytes by immunocytochemistry. An immunohistochemical analysis using liver sections confirmed the overexpression of Smoothened and cyclin D1, and the nuclear expression of the Gli proteins in the biliary epithelium of PCK rats as well as human Caroli disease. The treatment of PCK cholangiocytes with cyclopamine inhibited cell proliferative activity that was associated with the inhibition of nuclear translocation of Gli1 and Gli2, and reduced cyclin D1 expression. The in vivo administration of cyclopamine to PCK rats decreased abnormally elevated serum liver enzymes, and significantly attenuated bile duct dilation as well as kidney cyst formation. These results suggest that cholangiocyte hyperproliferation is causally associated with the aberrant activation of Hedgehog signaling, and the inhibition of the signaling has potential as a therapeutic strategy for biliary cystogenesis in Caroli disease.
Topics: Animals; Bile Ducts; Caroli Disease; Cell Proliferation; Cells, Cultured; Cyclin D1; Enzyme Inhibitors; Hedgehog Proteins; Male; Polycystic Kidney, Autosomal Recessive; Rats; Signal Transduction; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3
PubMed: 30036521
DOI: 10.1016/j.ajpath.2018.06.014 -
PloS One 2015Primary Cilia (PC) are a very likely place for signal integration where multiple signaling pathways converge. Two major signaling pathways clearly shown to signal...
Primary Cilia (PC) are a very likely place for signal integration where multiple signaling pathways converge. Two major signaling pathways clearly shown to signal through the PC, Sonic Hedgehog (Shh) and PDGF-Rα, are particularly important for the proliferation and differentiation of oligodendrocytes, suggesting that their interaction occurs in or around this organelle. We identified PC in rat oligodendrocyte precursor cells (OPCs) and found that, while easily detectable in early OPCs, PC are lost as these cells progress to terminal differentiation. We confirmed the interaction between these pathways, as cyclopamine inhibition of Hedgehog function impairs both PDGF-mediated OPC proliferation and Shh-dependent cell branching. However, we failed to detect PDGF-Rα localization into the PC. Remarkably, ciliobrevin-mediated disruption of PC and reduction of OPC process extension was counteracted by recombinant Shh treatment, while PDGF had no effect. Therefore, while PDGF-Rα-dependent OPC proliferation and survival most probably does not initiate at the PC, still the integrity of this organelle and cilium-centered pathway is necessary for OPC survival and differentiation.
Topics: Animals; Cell Differentiation; Cells, Cultured; Cilia; Hedgehog Proteins; Oligodendroglia; Platelet-Derived Growth Factor; Quinazolinones; Rats, Sprague-Dawley; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction; Veratrum Alkaloids
PubMed: 26218245
DOI: 10.1371/journal.pone.0133567