ABSTRACT
The Hedgehog (Hh) signaling pathway plays important roles in various physiological functions. Several malignancies, such as basal cell carcinoma (BCC) and medulloblastoma (MB), have been linked to the aberrant activation of Hh signaling. Although therapeutic drugs have been developed to inhibit Hh pathway-dependent cancer growth, drug resistance remains a major obstacle in cancer treatment. Here, we show that the newly identified, 2-{3-[1-(benzylsulfonyl)-1,2,3,6-tetrahydropyridin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone analog (LKD1214) exhibits comparable potency to vismodegib in suppressing the Hh pathway activation. LKD1214 represses Smoothened (SMO) activity by blocking its ciliary translocation. Interestingly, we also identified that it has a distinctive binding interface with SMO compared with other SMO-regulating chemicals. Notably, it maintains an inhibitory activity against the SmoD477H mutant, as observed in a patient with vismodegib-resistant BCC. Furthermore, LKD1214 inhibits tumor growth in the mouse model of MB. Collectively, these findings suggest that LKD1214 has the therapeutic potential to overcome drug-resistance in Hh-dependent cancers.
ABSTRACT
Obesity is a chronic metabolic disease that is prevalent worldwide, causing complications that affect the quality of life and longevity of humans. Currently, the low bioavailability upon subcutaneous injection of an appetite suppressant, liraglutide, and health problems in the locally injected region remain to be overcome. In this study, we developed a novel hyaluronic acid-based liraglutide-encapsulated triple-layer microneedle (TLM) as a painless and patient-friendly long-term drug delivery system. In contrast to previous anti-obesity microneedle approaches, this TLM is composed of three layers for complete skin insertion, protecting the encapsulated liraglutide from environmental stresses. Daily topical application of the liraglutide-loaded TLM significantly reduced body weight and improved body composition in a mouse model of high-fat diet-induced obesity. Additionally, it ameliorated diet-induced hepatic steatosis in obese mice. This novel TLM could promote a glucagon-like peptide-1 drug release system for long-term daily administration with relatively higher patient compliance compared to subcutaneous injection.
Subject(s)
Hyaluronic Acid , Liraglutide , Mice , Animals , Humans , Liraglutide/therapeutic use , Liraglutide/pharmacology , Hyaluronic Acid/therapeutic use , Quality of Life , Obesity/drug therapy , Diet, High-FatABSTRACT
Liraglutide, a human glucagon-like peptide-1 (GLP-1) analog, is promising for safely treating type 2 diabetes mellitus (T2DM), compared to insulin, by significantly reducing the risk of glucose-dependent hypoglycemia. Concerns related to injection prevent T2DM patients from taking liraglutide regularly, even though once-a-day subcutaneous (SC) injections. Dissolving microneedles (DMNs) are promising substitutes for SC injection and for improving patient convenience. However, there are two fundamental limitations: the low drug delivery due to incomplete insertion and loss of drug activity during DMN fabrication. Here, it is shown that an egg microneedle (EMN) designed with three functional layered structures can maintain the maximum activity of the loaded compound during DMN fabrication and deliver it completely into the skin, with the base layer allowing the complete delivery of liraglutide, and the shell layer maintaining the drug activity by mimicking the role of albumin in eggs. In a diabetic mouse model, liraglutide administration via EMN exhibited similar effect when compared to that of injection. Therefore, EMN-mediated liraglutide administration is a good potential option for replacing liraglutide injections in T2DM treatment.
Subject(s)
Diabetes Mellitus, Type 2 , Liraglutide , Mice , Animals , Humans , Liraglutide/pharmacology , Liraglutide/therapeutic use , Diabetes Mellitus, Type 2/drug therapy , Administration, Cutaneous , Skin , Glucagon-Like Peptide 1ABSTRACT
Primary cilia have essential roles as signaling centers during development and adult homeostasis. Disruption of ciliary structure or function causes congenital human disorders called ciliopathies. Centriolar distal appendage (DAP) proteins are important for anchoring cilia to the membrane. However, the exact functions of DAP during in vivo ciliogenesis and animal development remain poorly understood. Here, we showed that the DAP component sodium channel and clathrin linker 1 (Sclt1) mutant mice had abnormal craniofacial and limb development with postnatal lethality. In mutant embryos, most of the affected tissues had defects in DAP recruitment to the basal body and docking to the membrane that resulted in reduced ciliogenesis and disrupted hedgehog (Hh) signaling in limb bud mesenchymal cells. However, limb digit formation and ciliogenesis in Sclt1 mutant mice were differentially affected between the fore- and hindlimb buds. The forelimbs developed normally in Sclt1 mutants, but the hindlimbs had preaxial polydactyly. Heterozygous loss of Cep83, another core DAP component, in Sclt1 mutant mice, caused forelimb and hindlimb polydactyly. These findings revealed the tissue-specific differential requirement of DAPs. Taken together, these results indicated that during limb development the ciliary base components, DAPs, play an essential role in ciliogenesis and Hh signaling in vivo in a position-dependent manner.
ABSTRACT
BACKGROUND: Ciliogenesis-associated kinase 1 (CILK1) is a ciliary gene that localizes in primary cilia and regulates ciliary transport. Mutations in CILK1 cause various ciliopathies. However, the pathogenesis of CILK1-deficient kidney disease is unknown. METHODS: To examine whether CILK1 deficiency causes PKD accompanied by abnormal cilia, we generated mice with deletion of Cilk1 in cells of the renal collecting duct. A yeast two-hybrid system and coimmunoprecipitation (co-IP) were used to identify a novel regulator, kinesin light chain-3 (KLC3), of ciliary trafficking and cyst progression in the Cilk1-deficient model. Immunocytochemistry and co-IP were used to examine the effect of KLC3 on ciliary trafficking of the IFT-B complex and EGFR. We evaluated the effects of these genes on ciliary trafficking and cyst progression by modulating CILK1 and KLC3 expression levels. RESULTS: CILK1 deficiency leads to PKD accompanied by abnormal ciliary trafficking. KLC3 interacts with CILK1 at cilia bases and is increased in cyst-lining cells of CILK1-deficient mice. KLC3 overexpression promotes ciliary recruitment of IFT-B and EGFR in the CILK1 deficiency condition, which contributes to the ciliary defect in cystogenesis. Reduction in KLC3 rescued the ciliary defects and inhibited cyst progression caused by CILK1 deficiency. CONCLUSIONS: Our findings suggest that CILK1 deficiency in renal collecting ducts leads to PKD and promotes ciliary trafficking via increased KLC3.
Subject(s)
Polycystic Kidney Diseases , Mice , Animals , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/metabolism , Kidney/metabolism , Cilia/metabolism , Mutation , ErbB Receptors/metabolismABSTRACT
Seborrheic keratosis, which is a benign tumor composed of epidermal keratinocytes, develops common in the elderly. Uric acid generated by upregulated guanine deaminase (GDA) has been identified to cause UV-induced keratinocyte senescence in seborrheic keratosis. Seborrheic keratosis is also frequently pigmented. Growing evidences indicate that hyperuricemia is a risk factor of acanthosis nigricans, an acquired skin hyperpigmentation. The objective of this study was to investigate role of GDA and its metabolic end product, uric acid, in hyperpigmentation of patients with seborrheic keratosis using their lesional and non-lesional skin specimen sets and cultured primary human epidermal keratinocytes with or without GDA overexpression or uric acid treatment. GDA-overexpressing keratinocytes or their conditioned media containing uric acid increased expression levels of MITF and tyrosinase in melanocytes. Uric acid released from keratinocytes was facilitated by ABCG2 transporter with the help of PDZK1 interaction. Released uric acid was taken by URAT1 transporter in melanocytes, stimulating melanogenesis through p38 MAPK activation. Overall, GDA upregulation in seborrheic keratosis plays a role in melanogenesis via its metabolic end product uric acid, suggesting that seborrheic keratosis as an example of hyperpigmentation associated with photoaging.
Subject(s)
Guanine Deaminase/genetics , Hyperpigmentation/genetics , Keratosis, Seborrheic/genetics , Uric Acid/metabolism , Aged , Cells, Cultured , Epidermal Cells/metabolism , Female , Gene Expression Regulation, Enzymologic/genetics , Humans , Hyperpigmentation/complications , Hyperpigmentation/pathology , Keratinocytes/metabolism , Keratosis, Seborrheic/complications , Keratosis, Seborrheic/pathology , Male , Melanocytes/metabolism , Middle Aged , Skin/metabolismABSTRACT
Defective primary cilia cause a range of diseases known as ciliopathies, including hearing loss. The etiology of hearing loss in ciliopathies, however, remains unclear. We analyzed cochleae from three ciliopathy mouse models exhibiting different ciliogenesis defects: Intraflagellar transport 88 (Ift88), Tbc1d32 (a.k.a. bromi), and Cilk1 (a.k.a. Ick) mutants. These mutants showed multiple developmental defects including shortened cochlear duct and abnormal apical patterning of the organ of Corti. Although ciliogenic defects in cochlear hair cells such as misalignment of the kinocilium are often associated with the planar cell polarity pathway, our results showed that inner ear defects in these mutants are primarily due to loss of sonic hedgehog signaling. Furthermore, an inner ear-specific deletion of Cilk1 elicits low-frequency hearing loss attributable to cellular changes in apical cochlear identity that is dedicated to low-frequency sound detection. This type of hearing loss may account for hearing deficits in some patients with ciliopathies.
Subject(s)
Cell Polarity/physiology , Ciliopathies/physiopathology , Gene Expression Regulation, Developmental/genetics , Hearing Loss/physiopathology , Hedgehog Proteins/metabolism , Animals , Cilia/metabolism , Disease Models, Animal , Embryo, Mammalian/metabolism , Hair Cells, Auditory/metabolism , Mice , Wnt Signaling Pathway/physiologyABSTRACT
BACKGROUND: Polycystic kidney disease (PKD) involves renal cysts arising from proliferating tubular cells. Autophagy has been recently suggested as a potential therapeutic target in PKD, and mammalian target of rapamycin (mTOR) is a key negative regulator of autophagy. However, the effect of autophagy regulation on cystogenesis has not been elucidated in PKD mice. METHODS: Clinical validation was performed using GEO datasets and autosomal dominant polycystic kidney disease (ADPKD) patient samples. Newly established PKD and LC3 transgenic mice were used for in vivo verifications, and additional tests were performed in vitro and in vivo using multiple autophagy drugs. FINDINGS: Neither autophagy stimulation nor LC3 overexpression alleviated PKD. Furthermore, we observed the inhibitory effect of an autophagy inhibitor on cysts, indicating its possible therapeutic use in a specific group of patients with ADPKD. INTERPRETATION: Our findings provide a novel insight into the pathogenesis related to autophagy in PKD, suggesting that drugs related to autophagy regulation should be considered with caution for treating PKD. FUNDING SOURCES: This work was supported by grants from the Bio & Medical Technology Development Program; the Collaborative Genome Program for Fostering New Post-Genome Industry of the NRF; the Basic Science Program.
Subject(s)
Autophagy , Cysts/pathology , Polycystic Kidney Diseases/etiology , Polycystic Kidney Diseases/pathology , Animals , Autophagy/genetics , Cell Line , Computational Biology , Disease Models, Animal , Disease Susceptibility , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Mice , Mice, Transgenic , Phosphorylation , Polycystic Kidney Diseases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , ZebrafishABSTRACT
Expression of immune checkpoint ligands (ICLs) is necessary to trigger the inhibitory signal via immune checkpoint receptors (ICRs) in exhausted T cells under tumor immune microenvironment. Nevertheless,to our knowledge, ICL expression profile in cancer patients has not been investigated. Using previously reported RNA-seq data sets, we found that expression of ICLs was patient specific but their coexpression can be patterned in non-small-cell lung cancers (NSCLCs). Since the expression of PD-L1 and poliovirus receptor (PVR) among various ICLs was independently regulated, we could stratify the patients who were treated with anti-PD-1 later into 4 groups according to the expression level of PD-L1 and PVR. Of interest, high PVR and low PVR expressions in PD-L1-expressing patients enriched nonresponders and responders to PD-1 blockade, respectively, helping in further selection of responders. Using a genetically engineered cancer model, we also found that PVR-deficient and PD-L1-sufficient tumor-bearing mice were highly sensitive to anti-PD-1 therapy, whereas PVR-sufficient and PD-L1-deficient tumor-bearing mice were resistant to anti-PD-1 therapy. Taken together, our study provides a concept that combinatorial expression patterns of PVR and PD-L1 are key determinants for PD-1 blockade and furthermore suggest a better therapeutic usage of immune checkpoint blockades (ICBs).
Subject(s)
B7-H1 Antigen/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Programmed Cell Death 1 Receptor/genetics , Receptors, Virus/genetics , Aged , Animals , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immune Checkpoint Inhibitors/pharmacology , Male , Mice , Middle Aged , Tumor Microenvironment/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Cell cycle-related kinase (CCRK) has a conserved role in ciliogenesis, and Ccrk defects in mice lead to developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, retinal degeneration, and polycystic kidney. Here, we found that Ccrk is highly expressed in mouse trachea and bronchioles. Ccrk mutants exhibited pulmonary hypoplasia and abnormal branching morphogenesis in respiratory organ development. Furthermore, we demonstrated that Ccrk mutant lungs exhibit not only impaired branching morphogenesis but also a significant sacculation deficiency in alveoli associated with reduced epithelial progenitor cell proliferation. In pseudoglandular stages, Ccrk mutant lungs showed a downregulation of Hedgehog (Hh) signaling and defects in cilia morphology and frequency during progenitor-cell proliferation. Interestingly, we observed that activation of the Hh signaling pathway by small-molecule smoothened agonist (SAG) partially rescued bud morphology during branch bifurcation in explants from Ccrk mutant lungs. Therefore, CCRK properly regulates respiratory airway architecture in part through Hh-signal transduction and ciliogenesis. [BMB Reports 2020; 53(7): 367-372].
Subject(s)
Cilia/metabolism , Cyclin-Dependent Kinases/metabolism , Hedgehog Proteins/metabolism , Animals , Cell Cycle/physiology , Cell Division/physiology , Cell Proliferation/physiology , Cilia/genetics , Embryo, Mammalian/metabolism , Embryonic Development/physiology , Hedgehog Proteins/genetics , Lung/embryology , Lung/metabolism , Mice/embryology , Mice, Inbred C3H , Morphogenesis , Neural Tube/embryology , Signal Transduction/physiology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome is a genetic disorder associated with congenital defects of the endocrine, cerebral, and skeletal systems in humans. ECO syndrome is caused by mutations of the intestinal cell kinase (ICK) gene, which encodes a mitogen-activated protein (MAP) kinase-related kinase that plays a critical role in controlling the length of primary cilia. Lack of ICK function disrupts transduction of sonic hedgehog (SHH) signaling, which is important for development and homeostasis in humans and mice. Craniofacial structure abnormalities, such as cleft palate, are one of the most common defects observed in ECO syndrome patients, but the role of ICK in palatal development has not been studied. METHODS: Using Ick-mutant mice, we investigated the mechanisms by which ICK function loss causes cleft palate and examined pharmacological rescue of the congenital defects. FINDINGS: SHH signaling was compromised with abnormally elongated primary cilia in the developing palate of Ick-mutant mice. Cell proliferation was significantly decreased, resulting in failure of palatal outgrowth, although palatal adhesion and fusion occurred normally. We thus attempted to rescue the congenital palatal defects of Ick mutants by pharmacological activation of SHH signaling. Treatment of Ick-mutant mice with an agonist for Smoothened (SAG) rescued several congenital defects, including cleft palate. INTERPRETATIONS: The recovery of congenital defects by pharmacological intervention in the mouse models for ECO syndrome highlights prenatal SHH signaling modulation as a potential therapeutic measure to overcome congenital defects of ciliopathies.
Subject(s)
Central Nervous System Diseases/congenital , Central Nervous System Diseases/metabolism , Endocrine System Diseases/congenital , Endocrine System Diseases/metabolism , Hedgehog Proteins/metabolism , Signal Transduction , Smoothened Receptor/agonists , Animals , Cell Proliferation , Central Nervous System Diseases/genetics , Cilia/metabolism , Cleft Palate/pathology , Disease Models, Animal , Embryo, Mammalian/abnormalities , Embryo, Mammalian/pathology , Endocrine System Diseases/genetics , Gene Expression Regulation, Developmental , Mice, Knockout , Models, Biological , Mutation/genetics , Palate/abnormalities , Palate/embryology , Palate/ultrastructure , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/metabolismABSTRACT
Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.
Subject(s)
Cilia/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Animals , CRISPR-Cas Systems , Fibroblast Growth Factors/metabolism , HEK293 Cells , Hedgehog Proteins/metabolism , Humans , Mice , Mice, Knockout , Models, Animal , Molecular Docking Simulation , NIH 3T3 Cells , Phosphorylation , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/genetics , Proteomics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Receptors, Fibroblast Growth Factor/genetics , Signal TransductionABSTRACT
Precise modulation of polymer brush in its thickness and grafting density can cause unexpected cell behaviors and regulated bioactivities. Herein, a nanoscale poly(dimethylsiloxane) (PDMS) brush was employed to use as a controllable material for cell adhesion. Facile fabrication of ultrathin monolayer PDMS nanobrush on an underlying substrate facilitated regaining cell adhesion through long-range cell attractive forces such as the van der Waals forces. We showed that cell adhesion is diminished by increasing the number of nanobrush layers, causing a gradual decrease of the effectiveness of the long-range force. The result demonstrates that ultrathin PDMS nanobrush can either promote or inhibit cell adhesion, which is required for various biomedical fields such as tissue-engineering, anti-fouling coating, and implantable biomaterials and sensors.
Subject(s)
Dimethylpolysiloxanes/chemistry , Nanostructures/chemistry , Tissue Engineering/instrumentation , Animals , Biocompatible Materials , Biosensing Techniques , Cell Adhesion , Cell Culture Techniques , Cell Line, Tumor , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Materials Testing , Mice , Microscopy, Confocal , Microscopy, Fluorescence , NIH 3T3 Cells , Oxygen/chemistry , Polymers/chemistry , Surface Properties , Tissue Engineering/methodsABSTRACT
Whole-transcriptome analysis and western blotting of sauchinone-treated HepG2 cells demonstrated that sauchinone regulated genes relevant to cholesterol metabolism and synthesis. In particular, it was found that the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) was downregulated, and the expression of low density lipoprotein receptor (LDLR) was upregulated in sauchinone-treated HepG2 cells. Consequently, LDL-cholesterol (LDL-C) uptake was increased. As a transcriptional regulator of PCSK9 expression, sterol regulatory elements binding protein-2 (SREBP-2) was proposed by transcriptome analysis and western blotting. Oral administration of sauchinone increased hepatic LDLR through PCSK9 inhibition in obese mice and showed the reduced serum LDL-C levels and downstream targets of SREBP-2. Thus, it is evident that sauchinone reduces hepatic steatosis by downregulating the expression of hepatic PCSK9 via SREBP-2.
Subject(s)
Benzopyrans/administration & dosage , Dioxoles/administration & dosage , Fatty Liver/drug therapy , Proprotein Convertase 9/genetics , Sterol Regulatory Element Binding Protein 2/genetics , Animals , Cholesterol/genetics , Cholesterol/metabolism , Cholesterol, LDL/genetics , Disease Models, Animal , Fatty Liver/blood , Fatty Liver/genetics , Fatty Liver/pathology , Gene Expression Regulation/drug effects , Gene Regulatory Networks/drug effects , Hep G2 Cells , Homeostasis/drug effects , Humans , Lipid Metabolism/drug effects , Liver/drug effects , Liver/metabolism , Liver/pathology , Mice , Receptors, LDL/genetics , Transcriptome/geneticsABSTRACT
Stress severely disturbs physiological and mental homeostasis which includes adult neurogenesis in hippocampus. Neurogenesis in hippocampus is a key feature to adapt to environmental changes and highly regulated by multiple cellular signaling pathways. The primary cilium is a cellular organelle, which acts as a signaling center during development and neurogenesis in adult mice. However, it is not clear how the primary cilia are involved in the process of restraint (RST) stress response. Using a mouse model, we examined the role of primary cilia in repeated and acute RST stress response. Interestingly, RST stress increased the number of ciliated cells in the adult hippocampal dentate gyrus (DG). In our RST model, cell proliferation in the DG also increased in a time-dependent manner. Moreover, the analysis of ciliated cells in the hippocampal DG with cell type markers indicated that cells that were ciliated in response to acute RST stress are neurons. Taken together, these findings suggest that RST stress response is closely associated with an increase in the number of ciliated neurons and leads to an increase in cell proliferation.
ABSTRACT
This study aimed to design an effective formulation for enhancing the tumor-targeted delivery of sorafenib. Three sorafenib-loaded liposomal formulations including uncoated liposome (SF-Lip), hyaluronic acid-coated liposome (HA-SF-Lip), and PEGylated hyaluronic acid-coated liposome (PEG-HA-SF-Lip) were developed with narrow size distribution and high encapsulation efficiency. The cellular uptake and cytotoxicity of HA-SF-Lip and PEG-HA-SF-Lip were greater than those of SF-Lip in MDA-MB-231 cells overexpressing CD44, whereas there were no significant differences in MCF-7 cells with low CD44 expression, indicating the CD44-mediated cellular uptake of coated liposomes. In comparison with sorafenib solution, PEG-HA-SF-Lip increased the systemic exposure and plasma half-life in rats by 3-fold and 2-fold, respectively. Consistently, PEG-HA-SF-Lip was the most effective for tumor growth inhibition through CD44 targeting in the MDA-MB-231 tumor xenograft mouse model. Taken together, the present study suggests that PEG-HA-SF-Lip might be effective for the tumor-targeted delivery of sorafenib with enhanced systemic exposure and longer blood circulation.
Subject(s)
Breast Neoplasms/drug therapy , Drug Carriers/chemistry , Drug Delivery Systems , Hyaluronic Acid/chemistry , Liposomes/chemistry , Polyethylene Glycols/chemistry , Sorafenib/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Survival , Female , Hemolysis/drug effects , Humans , Mice , Rats , Rats, Sprague-Dawley , Sorafenib/administration & dosage , Sorafenib/chemistry , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The Hedgehog (Hh) signaling pathway plays a key role in cell fate specification, proliferation, and survival during mammalian development. Cells require a small organelle, the primary cilium, to respond properly to Hh signals and the key regulators of Hh signal transduction exhibit dynamic localization to this organelle when the pathway is activated. Here, we investigate the role of Cell Cycle Related kinase (CCRK) in regulation of cilium-dependent Hh signaling in the mouse. Mice mutant for Ccrk exhibit a variety of developmental defects indicative of inappropriate regulation of this pathway. Cell biological, biochemical and genetic analyses indicate that CCRK is required to control the Hedgehog pathway at the level or downstream of Smoothened and upstream of the Gli transcription factors, Gli2 and Gli3. In vitro experiments indicate that Ccrk mutant cells show a greater deficit in response to signaling over long time periods than over short ones. Similar to Chlamydomonas mutants lacking the CCRK homolog, LF2, mouse Ccrk mutant cells show defective regulation of ciliary length and morphology. Ccrk mutant cells exhibit defects in intraflagellar transport (the transport mechanism used to assemble cilia), as well as slowed kinetics of ciliary enrichment of key Hh pathway regulators. Collectively, the data suggest that CCRK positively regulates the kinetics by which ciliary proteins such as Smoothened and Gli2 are imported into the cilium, and that the efficiency of ciliary recruitment allows for potent responses to Hedgehog signaling over long time periods.
Subject(s)
Cilia/genetics , Cyclin-Dependent Kinases/genetics , Kruppel-Like Transcription Factors/genetics , Morphogenesis/genetics , Smoothened Receptor/genetics , Animals , Cell Cycle/genetics , Cell Differentiation/genetics , Chlamydomonas/genetics , Embryonic Development/genetics , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Mice , Mutation , Nerve Tissue Proteins/genetics , Signal Transduction , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3 , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
In this study, the anti-inflammatory effects of mangosteen extract (MGE) on dextran sulfate sodium (DSS)-induced colitis in mice and nuclear factor (NF)-κB pathway modulation were investigated. Acute colitis was induced by administering 3% DSS in drinking water for 7 days, and three groups of Institute of Cancer Research mice were treated with 30 and 120 mg/kg MGE or 5-aminosalicylic acid for 7 days; an additional two groups of mice served as healthy and disease controls. The results indicated that MGE significantly prevented weight loss, reduced disease activity index scores, and preserved colon length compared with the findings in the untreated colitis group. MGE downregulated the NF-κB pathway by inhibiting the phosphorylation of IκB and IKK in a dose-dependent manner. These findings suggest that MGE alleviates ulcerative colitis by modulating the NF-κB pathway.
Subject(s)
Colitis, Ulcerative/drug therapy , Dextran Sulfate/chemistry , Garcinia mangostana/chemistry , NF-kappa B/immunology , Plant Extracts/administration & dosage , Animals , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/genetics , Colitis, Ulcerative/immunology , Dextran Sulfate/adverse effects , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred ICR , NF-kappa B/genetics , Signal TransductionABSTRACT
Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) of the colon as a target site. Previous reports regarding the efficacy of α-mangostin (αMG) to inhibit nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) as well as relatively high distribution to the colon suggested the therapeutic potential of this compound in UC model. In dextran sodium sulfate (DSS)-induced colitis mice (DSS mice), the disease activity index scores involving diarrhea, bloody stool, body weight reduction, and myeloperoxidase (MPO) activities of the esophagus and colon increased with the reduced colon length. Also histologic disturbances and changes of NF-κB and MAPK pathways including phosphorylation of IκB kinase, ERK1/2, SAPK/JNK and p38 were observed in the colon of the DSS mice. However, all of these impaired conditions in the DSS mice were restored by αMG treatment, and the intestinal metabolism of αMG decreased, increasing its distribution to the colons in the DSS mice compared with the control mice. All of these results suggest that high distribution of αMG in the colon might attenuate DSS-induced colitis by inhibiting NF-κB and MAPK pathways in the colon.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Colitis, Ulcerative/drug therapy , Colitis/drug therapy , Colon/metabolism , Xanthones/therapeutic use , Animals , Colitis/chemically induced , Colon/pathology , Dextran Sulfate , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , MAP Kinase Kinase 4/metabolism , Male , Mice , Mice, Inbred Strains , NF-kappa B/metabolism , Signal TransductionABSTRACT
In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in the subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells.