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International Journal of Molecular... Apr 2020The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily...
The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.
Topics: Aging; Animals; Cell Survival; Cells, Cultured; Gene Expression Regulation, Developmental; Hedgehog Proteins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoclasts; Osteogenesis; Osteoporosis; Pyridines; Pyrimidines; Receptor, Macrophage Colony-Stimulating Factor; Signal Transduction; Smoothened Receptor; Thiophenes; Up-Regulation; Veratrum Alkaloids; X-Ray Microtomography; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2
PubMed: 32326611
DOI: 10.3390/ijms21082745 -
International Journal of Molecular... Jul 2020Stromal‑epithelial interaction serves a pivotal role in normal prostate growth, as well as the onset of benign prostatic hyperplasia (BPH). The present study aimed to...
Cyclopamine functions as a suppressor of benign prostatic hyperplasia by inhibiting epithelial and stromal cell proliferation via suppression of the Hedgehog signaling pathway.
Stromal‑epithelial interaction serves a pivotal role in normal prostate growth, as well as the onset of benign prostatic hyperplasia (BPH). The present study aimed to explore the role of cyclopamine in the proliferation and apoptosis of epithelial and stromal cells in rats with BPH by blocking the Hedgehog signaling pathway. Cyclopamine (an inhibitor of the Hedgehog signaling pathway) was administered in a rat model of BPH, and the expression of Ki67 (proliferation factor) was determined by immunohistochemistry. In addition, epithelial and stromal cells were separated and cultured in order to investigate the role of cyclopamine in the progression of BPH. The expression of Hedgehog signaling pathway‑ and apoptosis‑related genes, including basic fibroblastic growth factor (b‑FGF) and transforming growth factor β (TGF‑β), was evaluated using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Cell proliferation, cell cycle and apoptosis were analyzed using an MTT assay and flow cytometry. We identified upregulated Ki67 expression and activated Hedgehog signaling pathway in rats with BPH. Cyclopamine inhibited the activation of the Hedgehog signaling pathway. In response to cyclopamine treatment, epithelial and stromal cell proliferation was inhibited; this was concomitant with decreased Ki67, TGF‑β, and b‑FGF expression. On the other hand, epithelial cell apoptosis was enhanced, which was associated with increased Bax and reduced Bcl‑2 expression. Based on these findings, we proposed that cyclopamine may serve as a potential therapeutic agent in the treatment of BPH. Cyclopamine could inhibit epithelial and stromal cell proliferation, and induce epithelial cell apoptosis by suppressing the Hedgehog signaling pathway.
Topics: Animals; Apoptosis; Cell Cycle; Cell Proliferation; Epithelial Cells; Flow Cytometry; Hedgehog Proteins; Immunohistochemistry; Male; Prostatic Hyperplasia; Rats; Rats, Sprague-Dawley; Signal Transduction; Stromal Cells; Veratrum Alkaloids
PubMed: 32319534
DOI: 10.3892/ijmm.2020.4569 -
Bioscience Reports May 2020Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was...
Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3'-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.
Topics: Adult Stem Cells; Animals; Apoptosis; Cartilage, Articular; Cell Proliferation; Cells, Cultured; Chondrocytes; Hedgehog Proteins; MicroRNAs; Multigene Family; Parathyroid Hormone-Related Protein; Patched-1 Receptor; Primary Cell Culture; Rabbits; Veratrum Alkaloids
PubMed: 32319512
DOI: 10.1042/BSR20191654 -
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 -
International Journal of Molecular... Jun 2020Sonic hedgehog (Shh) is pivotally important in embryonic and adult blood vessel development and homeostasis. However, whether Shh is involved in atherosclerosis and...
Sonic hedgehog (Shh) is pivotally important in embryonic and adult blood vessel development and homeostasis. However, whether Shh is involved in atherosclerosis and plays a role in endothelial apoptosis induced by oxidized low‑density lipoprotein (ox‑LDL) has not been reported. The present study used recombinant Shh‑N protein (rShh‑N) and a plasmid encoding the human Shh gene (phShh) to investigate the role of Shh in ox‑LDL‑mediated human umbilical vein endothelial cell (HUVEC) apoptosis. The present study found that ox‑LDL was able to induce apoptosis in HUVECs and that Shh protein expression was downregulated. Furthermore, pretreatment with rShh‑N or transfection with phShh increased anti‑apoptosis protein Bcl‑2 expression and decreased cell apoptosis. These protective effects of rShh‑N could be abolished by cyclopamine, which is a hedgehog signaling inhibitor. Furthermore, a co‑immunoprecipitation assay was performed to demonstrate that Shh interacted with NF‑κB p65 in HUVECs. Additionally, ox‑LDL upregulated the phosphorylation of NF‑κB p65 and inhibitor of NF‑κB‑α (IκBα), and these effects decreased notably following rShh‑N and phShh treatment. Together, the present findings suggested that Shh serves an important protective role in alleviating ox‑LDL‑mediated endothelial apoptosis by inhibiting the NF‑κB signaling pathway phosphorylation and Bcl‑2 mediated mitochondrial signaling.
Topics: Apoptosis; Cells, Cultured; Down-Regulation; Hedgehog Proteins; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins, LDL; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Transcription Factor RelA; Up-Regulation; Veratrum Alkaloids
PubMed: 32186749
DOI: 10.3892/ijmm.2020.4542 -
Molecules (Basel, Switzerland) Mar 2020Peimine (also known as verticine) is the major bioactive and characterized compound of , a traditional Chinese medicine that is most frequently used to relieve a cough....
Peimine (also known as verticine) is the major bioactive and characterized compound of , a traditional Chinese medicine that is most frequently used to relieve a cough. Nevertheless, its molecular targets and mechanisms of action for cough are still not clear. In the present study, potential targets of peimine for cough were identified using computational target fishing combined with manual database mining. In addition, protein-protein interaction (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using, GeneMANIA and Database for Annotation, Visualization and Integrated Discovery (DAVID) databases respectively. Finally, an interaction network of drug-targets-pathways was constructed using Cytoscape. The results identified 23 potential targets of peimine associated with cough, and suggested that MAPK1, AKT1 and PPKCB may be important targets of pemine for the treatment of cough. The functional annotations of protein targets were related to the regulation of immunological and neurological function through specific biological processes and related pathways. A visual representation of the multiple targets and pathways that form a network underlying the systematic actions of peimine was generated. In summary, peimine is predicted to exert its systemic pharmacological effects on cough by targeting a network composed of multiple proteins and pathways.
Topics: Cevanes; Computational Biology; Cough; Drugs, Chinese Herbal; Gene Expression Profiling; Humans; Medicine, Chinese Traditional; Models, Biological; Protein Interaction Maps; Signal Transduction
PubMed: 32131410
DOI: 10.3390/molecules25051105 -
Plants (Basel, Switzerland) Feb 2020L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to...
L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of mediated with is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of .
PubMed: 32033134
DOI: 10.3390/plants9020191 -
Journal of Pharmaceutical Analysis Oct 2019Jervine, a novel steroidal alkaloid from L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors...
Jervine, a novel steroidal alkaloid from L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors and enterohepatic circulation of jervine in rats. A rapid and simple ultra-high performance liquid chromatography-tandem mass spectrometric method was developed and validated for quantification of jervine and alpinetin (internal standard) in rat plasma. After extraction from rat plasma by a simple protein-precipitation method, the analyte was separated on a C column (2.1 mm × 50 mm, 1.7 μm) using water with 0.1% formic acid and acetonitrile as the mobile phase delivered at a flow rate of 0.4 mL/min. Jervine and alpinetin were determined in the positive mode with multiple reaction monitoring (MRM) of the ion transitions at 426.3 → 108.8 and 271.0 → 166.9, respectively. Molecular docking method was used to investigate the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The method was well validated within acceptance limits including specificity, matrix effect, recovery, precision, accuracy, and stability, and was successfully applied to the pharmacokinetic study of jervine after oral and intravenous administration to rats. Jervine presented a small volume of distribution, fast absorption, high oral bioavailability, and enterohepatic circulation. The enterohepatic circulation was first observed in veratrum alkaloids, and was further investigated by molecular docking studies, which was related to the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The pharmacokinetic properties and enterohepatic circulation of jervine in rats provided a significant basis for the drug-drug interaction and toxicity study in the future.
PubMed: 31929946
DOI: 10.1016/j.jpha.2019.04.004 -
Circulation. Genomic and Precision... Feb 2020Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can...
BACKGROUND
Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants.
METHODS
We have developed and validated a method to perform a deep mutational scan of variants in , which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells.
RESULTS
In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores.
CONCLUSIONS
These experiments demonstrate the accuracy of a high-throughput in vitro scan of variant function, which can be used to identify deleterious variants in and other ion channel genes.
Topics: Cardiotonic Agents; DNA Mutational Analysis; HEK293 Cells; Humans; Marine Toxins; Mutation; NAV1.5 Voltage-Gated Sodium Channel; Ouabain; Oxocins; Pharmacogenomic Testing; Veratridine
PubMed: 31928070
DOI: 10.1161/CIRCGEN.119.002786 -
Leukemia Apr 2020Accumulation of DNA damage and myeloid-skewed differentiation characterize aging of the hematopoietic system, yet underlying mechanisms remain incompletely understood....
Accumulation of DNA damage and myeloid-skewed differentiation characterize aging of the hematopoietic system, yet underlying mechanisms remain incompletely understood. Here, we show that aging hematopoietic progenitor cells particularly of the myeloid branch exhibit enhanced resistance to bulky DNA lesions-a relevant type of DNA damage induced by toxins such as cancer drugs or endogenous aldehydes. We identified aging-associated activation of the Hedgehog (Hh) pathway to be connected to this phenotype. Inhibition of Hh signaling reverts DNA damage tolerance and DNA damage-resistant proliferation in aged hematopoietic progenitors. Vice versa, elevating Hh activity in young hematopoietic progenitors is sufficient to impair DNA damage responses. Altogether, these findings provide experimental evidence for aging-associated increases in Hh activity driving DNA damage tolerance in myeloid progenitors and myeloid-skewed differentiation. Modulation of Hh activity could thus be explored as a therapeutic strategy to prevent DNA damage tolerance, myeloid skewing, and disease development in the aging hematopoietic system.
Topics: Aging; Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Cells, Cultured; DNA Damage; Female; Hedgehog Proteins; Hematopoiesis; Hematopoietic Stem Cells; Humans; Mice; Mice, Inbred C57BL; Veratrum Alkaloids
PubMed: 31728056
DOI: 10.1038/s41375-019-0641-3