Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.

Energy expenditure and bone formation share a common sensitivity to AP-1 transcription in the hypothalamus.

The regulation of bone and fat homeostasis and its relationship to energy expenditure has recently been the focus of increased attention because of its potential relevance to osteoporosis, obesity, and diabetes. Although central effectors within the hypothalamus have been shown to contribute to the regulation of both energy balance and bone homeostasis, little is known of the underlying mechanisms, including the possible involvement of transcriptional factors within the hypothalamus. Transgenic mice overexpressing ΔFosB, a splice variant of the AP-1 transcription factor FosB with mixed agonist-antagonistic properties, have increased energy expenditure and bone mass. Because these mice express ΔFosB in bone, fat, and hypothalamus, we sought to determine 1) whether overexpression of ΔFosB within the hypothalamus was sufficient to regulate energy expenditure and whether it would also regulate bone mass, and 2) whether these effects were the result of antagonism to AP-1. Our results show that stereotactic injection of an adeno-associated virus vector to restrict overexpression of ΔFosB to the ventral hypothalamus of wild-type mice induced a profound increase in both energy expenditure and bone formation and bone mass. This effect was phenocopied, at an even stronger level, by overexpression of a dominant-negative DNJunD, a pure AP-1 antagonist. Taken together, these results suggest that downregulation of AP-1 activity in the hypothalamus profoundly increases energy expenditure and bone formation, leading to both a decrease in adipose mass and an increase in bone mass. These findings may have physiological implications because ΔFosB is expressed and regulated in the hypothalamus.

Human hepatocellular carcinoma in a mouse model: assessment of tumor response to percutaneous ablation by using glyceraldehyde-3-phosphate dehydrogenase antagonists.

PURPOSE: To characterize tumor response to percutaneous injection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antagonists in a mouse model of human hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee. Luciferase (luc) gene-expressing Hep3B tumor-bearing athymic nude mice were randomly divided into four groups of six mice each. Tumor-specific GAPDH inhibition was achieved by using percutaneous injection of GAPDH antagonists-3-bromopyruvate (3-BrPA) or GAPDH-specific short hairpin RNA (shRNA). Tumor response to treatment was assessed by using bioluminescence imaging and analysis of GAPDH function and apoptotic markers (caspase-3, caspase-9, and positive staining for terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling). HCC samples from 34 patients were obtained from the Johns Hopkins tumor bank, as approved by the Institutional Review Board, for GAPDH expression analysis. Statistical analysis was performed by using a two-sample t test or Spearman rank correlation coefficient. RESULTS: In vitro, 3-BrPA affected Hep3B cell viability (half maximal inhibitory concentration = 0.15 mmol/L), and GAPDH shRNA suppressed (45.5%) colony formation. In vivo, percutaneous injection of GAPDH antagonists into luc-Hep3B tumors decreased bioluminescence imaging signal and viability (3-BrPA, P < .0001; GAPDH shRNA, P = .03). The 3-BrPA treatment primarily inhibited GAPDH activity (74.5%) compared with its expression (34.3%), whereas GAPDH shRNA inhibited both activity (60.6%) and expression (44.4%). Targeted inhibition of GAPDH by using 3-BrPA or shRNA induced apoptosis. HCC samples from patients demonstrated a strong correlation between GAPDH upregulation and the proto-oncogene c-jun expression (r = 0.543, P = .003). CONCLUSION: Percutaneous injection of GAPDH antagonists induces apoptosis and blocks Hep3B tumor progression, which demonstrates the therapeutic potential of targeting GAPDH in human HCC.

Establishment and assessment of a simple and easily reproducible incision model of spinal cord neuron cells in vitro.

A growing number of in vitro models have been introduced to study the mechanisms of spinal cord injury. A potential drawback of these models is that they are difficult to reproduce. In this study, an in vitro incision model was established using primary cultured neuronal cells from fetal rat spinal cords. The neurons were subjected to incision in a simple and reproducible way. To assess whether this model could simulate the responses of spinal cord neuron cells in vivo after a spinal cord transection, apoptosis, and the expression of immediate early genes were detected in the neurons at various time points after injury. The results indicated that: (1) significantly more terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were observed at 1, 3, and 7 d following injury and (2) the expression of both c-Jun and c-Fos was induced 10 min after incision and had markedly higher levels 2 h post-injury. These results suggested that our model can partially imitate the responses of in vivo neuronal cells after a spinal cord transection and such models may facilitate further understanding of biochemical and cellular events associated with spinal cord injury.

Assessment of the role of activator protein-1 on transcription of the mouse steroidogenic acute regulatory protein gene.

cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5'-CRE half-sites within the cAMP-responsive region (-151/-1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-beta. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.