ABSTRACT
CONTEXT.: Delta-like protein 3 (DLL3) is a protein that is implicated in the Notch pathway. OBJECTIVE.: To present data on DLL3 prevalence in small cell lung cancer and staining characteristics of the VENTANA DLL3 (SP347) Assay. In addition, the assay's immunoreactivity with other neoplastic and nonneoplastic tissues is outlined. DESIGN.: Individual formalin-fixed, paraffin-embedded specimens of small cell lung cancer and tissue microarrays comprising neoplastic and nonneoplastic tissues were procured. Sections were cut and stained with DLL3 (SP347) assay. The slides were examined to determine prevalence, staining characteristics, and immunoreactivity. RESULTS.: Cytoplasmic and/or membranous staining was observed in 1040 of 1362 specimens of small cell lung cancer (76.4%). Homogenous and/or heterogeneous and partial and/or circumferential granular staining with varied intensities was noted. Immunoreactivity was also observed in other neoplastic and nonneoplastic tissues. CONCLUSIONS.: Our study findings provided the profile of DLL3 staining characteristics that can be used for determining the level of DLL3 expression in small cell lung cancer.
Subject(s)
Antibodies, Monoclonal/immunology , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/pathology , Membrane Proteins/metabolism , Small Cell Lung Carcinoma/pathology , Animals , Cohort Studies , Humans , Immunohistochemistry , Lung Neoplasms/diagnosis , Paraffin Embedding , Rabbits , Small Cell Lung Carcinoma/diagnosis , Tissue Array AnalysisABSTRACT
5'-AMP-activated protein kinase (AMPK) is a metabolic fuel sensor that monitors cellular energy charge, while the vasculature is important for maintaining cellular energy homeostasis. Mice with muscle-specific inactive AMPK (AMPK DN) were used to investigate if AMPK regulates skeletal muscle capillarization and the angiogenic responses to exercise. Two hours of the AMP analogue AICAR (1.0 g kg(-1)) or systemic hypoxia (6% O(2)) increased vascular endothelial growth factor (VEGF) mRNA in wild-type (WT), but not in AMPK DN mice. In contrast, the increase in VEGF mRNA with acute exercise (1 h at 20 m min(-1), 10% gradient) was greater in AMPK DN compared to WT mice. Nuclear run-on assay demonstrated that exercise increased VEGF transcription, while hypoxia decreased VEGF transcription. There was no difference in VEGF transcription between WT and AMPK DN. There was a strong correlation between VEGF transcription and VEGF mRNA at rest and with exercise. Resting capillarization was lower in AMPK DN compared to WT. Wheel running (28 days) increased capillarization and this response was AMPK independent. Significant correlations between VEGF protein and muscle capillarization are consistent with VEGF being an important determinant of skeletal muscle capillarization. These data are to our knowledge the first to demonstrate in skeletal muscle in vivo that: (1) AMPK is necessary for hypoxia-induced VEGF mRNA stabilization, (2) acute exercise increases VEGF transcription, (3) inhibition of AMPK augments the VEGF mRNA response to acute exercise, and (4) AMPK regulates basal VEGF expression and capillarization, but is not necessary for exercise-induced angiogenesis.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic/physiology , Physical Conditioning, Animal/physiology , Vascular Endothelial Growth Factor A/metabolism , AMP-Activated Protein Kinases/genetics , Aminoimidazole Carboxamide/administration & dosage , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Body Weight/physiology , Capillaries/anatomy & histology , Capillaries/physiology , Catalytic Domain/genetics , Female , Gene Expression/drug effects , Hypoxia/physiopathology , Immunoblotting , Injections, Intraperitoneal , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/anatomy & histology , Muscle, Skeletal/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleotides/administration & dosage , Ribonucleotides/pharmacology , Vascular Endothelial Growth Factor A/geneticsABSTRACT
As the primary glucose transporter in skeletal muscle, GLUT4 is an important factor in the regulation of blood glucose. We previously reported that stimulation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased GLUT4 expression in muscle. GLUT4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) have been shown to be important for normal GLUT4 expression because deletion or truncation of the consensus sequences on the promoter causes depressed GLUT4 mRNA expression. This led to the current study to investigate possible roles for GEF and MEF2 in mediating the activation of GLUT4 gene transcription in response to AMPK. Here we show that, although AMPK does not appear to phosphorylate MEF2A, AMPK directly phosphorylates the GEF protein in vitro. MEF2 and GEF are activated in response to AMPK as we observed translocation of both to the nucleus after AICAR treatment. Nuclear MEF2 protein content was increased after 2 h, and GEF protein was increased in the nucleus 1 and 2 h post-AICAR treatment. Last, GEF and MEF2 increase in binding to the GLUT4 promoter within 2 h after AICAR treatment. Thus we conclude that GEF and MEF2 mediate the AMPK-induced increase in transcription of skeletal muscle GLUT4. AMPK can phosphorylate GEF and in response to AICAR, GEF, and MEF2 translocate to the nucleus and have increased binding to the GLUT4 promoter.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation , Glucose Transporter Type 4/genetics , Multienzyme Complexes/metabolism , Myogenic Regulatory Factors/genetics , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/genetics , AMP-Activated Protein Kinases , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Biological Transport/drug effects , Cell Nucleus/chemistry , Cell Nucleus/metabolism , DNA/metabolism , DNA-Binding Proteins/analysis , DNA-Binding Proteins/physiology , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , MEF2 Transcription Factors , Muscle, Skeletal/chemistry , Muscle, Skeletal/ultrastructure , Myogenic Regulatory Factors/analysis , Myogenic Regulatory Factors/physiology , Phosphorylation , Promoter Regions, Genetic/genetics , RNA, Messenger/analysis , Rats , Recombinant Proteins/metabolism , Ribonucleotides/pharmacology , Transcription Factors/analysis , Transcription Factors/physiologyABSTRACT
An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK alpha-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.
