The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Iterated composite relative importance of all Connections Map proteins with altered expression. Proteins previously shown in Figures 11 and 12 to best discriminate control from SN50-treated explants were subjected to further PNN analysis to determine which proteins are most discriminating in defining the SN50-treated E15 + 2 phenotype. Refer to Table 2 for the direction and magnitude of change for each protein.
- The iterated composite relative importance of all Connections Map proteins with altered expression as a consequence of NF-κB inhibition was then determined (Fig. 13).
- Regarding the ERK1/2 pathway, we found a greater than 2-fold increase of activated ERK2 in SN50-treated glands (Fig. 13) associated with a 1.5-fold increase in total ERK2 protein (Table 2).
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Connections Map. This signaling map reflects the pathways investigated in SMGs. Known and putative connections are based on references [6], [11], [23], [36], [76]-[108].
- These SMG cellular and extracellular components may be visualized as a Connections Map which details the functional relationships within and between pathways (Fig. 1).
- Complex networks of biological signaling pathways (Fig. 1) emerge from the interconnections of simple pathways under local control [15-17].
- With the present experiments, we sought a glimpse of the extraordinarily complex behaviors of a focused signaling network (Fig. 1).
- Of these, we focused our attention on those signal transduction, cell cycle, and apoptosis transcripts related to the Connections Map (Fig. 1).
- Cyclin D2, Cdc25a, and PCNA promote cell division; p57 inhibits cell division (Fig. 1).
- BMPs inhibit cell proliferation via downstream Smad1/5/8 proteins whereas Smad7 inhibits TGF-β and activin signaling (Fig. 1).
- Further, we utilized PNN analysis to determine the iterated composite relative importance among Connections Map (Fig. 1) transcripts which have altered expression as a consequence of inhibition of NF-κB translocation into the nucleus (Fig. 10).
- The declining PCNA and GR reflect the sharp decline in cell proliferation and branching; the increasing BMP1 and BMP3b similarly reflects inhibition of cell proliferation (Fig. 1).
- As shown in Table 2, we find 18 proteins which have both a 1.5-fold or greater change with NF-κB inhibition and are specifically related to the Connections Map (Fig. 1).
- Raf plays a key role in the Ras signaling pathway (Fig. 1).
- Further, both the SHP-2/Ras and JAK/STAT3 pathways are activated by IL-6R/gp130 signaling (Fig. 1).
- Considering the outcome of this study relative to the Connections Map (Fig. 1), it is apparent that NF-κB nuclear translocation is functionally integral to a genetic network with broadly related, rather than independent, components.
- Specifically, we assigned those genes related to the Connections Map (Fig. 1) that have a 1.5 or greater fold-change to functional groups (i.e., cell cycle, apoptosis, signal transduction, etc.) which have biological significance.
- We used PNN analyses to determine which Connection Map (Fig. 1) transcripts or proteins with altered expression best discriminate CONT from SN50-treated explants with 100% sensitivity and specificity [69].
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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NF-κB immunolocalization during embryonic SMG development. A. Pseudoglandular Stage. B. Canalicular and Terminal Bud Stages. During embryonic mouse SMG development, the SMG primordium branches by repeated furcation at the distal ends of successive buds to produce a bush-like structure comprised of a network of elongated epithelial branches and terminal epithelial buds surrounded by loosely packed mesenchyme in the Pseudoglandular Stage. We evaluated the spatial distribution of NF-κB (p65) protein in the Pseudoglandular Stage (A) and demonstrated that NF-κB is diffusely distributed throughout the branching epithelia, and to a lesser degree, in the mesenchyme. As development continues, the SMG epithelia branches and buds hollow out by epithelial cell apoptosis during the Canalicular and Terminal Bud Stages to form the ductal system and presumptive acini. Because the embryonic SMG develops by repeated epithelial end bud branching, the morphogenetic state of terminal bud clusters differs between SMG regions, dependent on the time of branch formation. Thus both the Canalicular (double arrows) and the Terminal Bud (double arrowheads) Stages can be seen in B. In the Canalicular Stage (B, double arrows), NF-κB p65 is primarily immunodetected in the central region of the terminal buds. By contrast, NF-κB p65 is diffusely distributed in Early Terminal Bud Stage (B, double arrowheads) epithelia in terminal buds which exhibit lumina. Similar localization patterns were immunodetected for NF-κB p50 protein (not shown). Bar: 50 μm.
- In the Pseudoglandular Stage (~E14), NF-κB is primarily immunodetected in SMG branching epithelia, and, to a much lesser extent, in the mesenchyme (Fig. 2A).
- At the Canalicular Stage (~E15–16), NF-κB is primarily immunolocalized in the central regions of the terminal buds, and to a lesser extent, in the ductal cells facing the lumina (Fig. 2B, double arrows).
- By the Terminal Bud Stage (~E17–19), NF-κB is diffusely distributed throughout ductal and terminal bud epithelia (Fig. 2B, double arrowheads), with the intensity of immunostain being markedly diminished compared to the Canalicular Stage.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Comparison view of composite cDNA Expression Arrays. We analyzed differences in the relative abundance of transcript levels in control and 100 μg/ml SN50-treated E15 + 2 explants. This composite represents the changes in 3 independent experiments. It consists an array of boxes, each of which represents a specific gene. For a complete list of the genes and their position on this Expression Array, as well as the GenBank accession numbers, please see http://atlas.clontech.com. The color of each half-box reflects the calculated values for Gene Expression Ratio (top) and Gene Expression Differences (bottom): Red = upregulation; Blue = downregulation; Green = equal expression; Black = background level. Comparison is made between two composite arrays, each of which is a mean of 3 independent arrays. The signals of the composite SN50-treated SMG array is analyzed with respect to the composite control SMG array. In this comparison view, boxes which are black in the upper half indicate an "undefined" ratio because the signal for the SN50-treated SMG array is at the background level (i.e. signal intensity is less than the signal threshold, namely no evidence of gene expression).
- Of the 1176 transcripts assayed on these arrays (including transcription factors, cell cycle regulators, growth factors, etc.), 691 (~60%) demonstrated a 1.5-fold or greater increase or decrease in expression with SN50-induced NF-κB inhibition (Fig. 7).
- That is, we then subjected those transcripts with altered expression in each group (cell cycle, apoptosis, Ras/Raf, TGF-β family) previously shown in Figures 7 and 8 to be relatively important in defining the SN50 SMG phenotype to further PNN analysis.
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Relative importance of cell cycle, apoptosis, and signal transduction proteins with altered expression in defining control and SN50-treated phenotypes. These PNN analyses among cell cycle, apoptosis, or signal transduction proteins with altered expression identified which proteins best discriminate control from SN50-treated E15 + 2 explants. Refer to Table 2 for the direction and magnitude of change for each protein.
- PNN analysis shows that among cell cycle proteins with altered expression (Fig. 12A), the increased expression of Chk1, Chk2, and E2F1 best discriminates control from SN50-treated explants.
- Among apoptosis proteins with altered expression (Fig. 12B), PNN analysis demonstrates that increased expression of FAF and Caspase 6 best discriminates control from SN50-treated explants.
- Among signal transduction proteins with altered expression (Fig. 12C), PNN analysis shows that members of all three growth factor pathways (Ras/Raf; JAK/STAT; JNK) have high relative importance in discriminating control from SN50-treated explants.
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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SN50-treatment induces the ERK1/2 and Caspase 3 pathways. Quantitation of phospho-ERK1/2, phospho-cRaf, cleaved Caspase 3, and cleaved PARP protein expression in control (CONT) and 100 μg/ml SN50-treated E15 + 2 explants. Two independent experiments were conducted and the results are presented as a mean fold change relative to control protein. SN50 treatment induced a significant increase in activated ERK 1/2, Caspase 3, and PARP levels compared to control; no change was seen in activated c-Raf level.
- We found a significant increase (p < 0.05) in activated ERK1/2, Caspase 3, and PARP in SN50-treated explants (Fig. 14); no change was seen in activated c-Raf levels between control and SN50-treated explants.
- The greater than 2-fold increase in Caspase 3 activation is associated with a 1.8-fold increase in PARP cleavage (Fig. 14) and a 10-fold increase in apoptosis (Fig. 4B).
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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2-D Western blot multiprotein analyses of ~600 signal transduction and related proteins in E15+2 control and 100 μg/ml SN50-treated explants revealed significant changes in protein expression. Comparison of representative control (A, C) and SN50-treated (B, D) equivalent Western blots indicates marked qualitative and quantitative differences in the expression of specific signaling proteins with SN50 treatment compared to control.
- This technique allows for the densitometric analysis of about 600 signal transduction and other proteins simultaneously in each independent sample (Fig. 11).
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
Extra large 
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Relative importance of cell cycle and apoptosis transcripts with altered expression. The PNN analyses among cell cycle and apoptosis transcripts with altered expression identifies those transcripts which best discriminate control from SN50-treated E15 + 2 explants. Refer to Table 1 for the direction and magnitude of change for each transcript.
- Among the cell cycle transcripts with altered expression (Fig. 8A), PNN analysis shows that the increased expression of cyclin D2, p57, and Cdc25a, as well as decreased expression of PCNA, best discriminate control from SN50-treated explants.
- Among apoptosis transcripts with altered expression (Fig. 8B), PNN analysis demonstrates that downregulated Caspase 1 transcript, almost alone, best discriminates control from SN50-treated explants.
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Quantitation of cell proliferation and apoptosis in control and SN50-treated E15 + 2 SMG explants. The data presented here is the results of 3 independent samples. Mean ± SEM percent positive epithelium: each bar is the mean of 3 independent samples, each sample representing counts in 3 randomly selected regions of that sample; percents were arcsin transformed for analysis. A. SN50-treated SMG explants have an 81% decline in cell proliferation. B. SN50-treated SMG explants have a 10-fold increase in apoptosis.
- These 2-day SN50-treated explants demonstrate a highly significant 81% decline (t4 = 26.25; p < 0.001) in cell proliferation (Fig. 3A, B; Fig. 4A) and a significant 10-fold increase (t4 = 7.98; p < 0.001) in apoptosis (Fig. 3C, D; Fig. 4B).
- The significant decline in PCNA transcript (Table 1) is consistent with the highly significant (p < 0.001) decline in PCNA-defined cell proliferation (Fig. 4A).
- The significant decline in PCNA protein (Table 2) is consistent with the significant decline in PCNA transcript (Table 1) and PCNA-defined cell proliferation (Fig. 4A).
- It is reasonable, then, to assume that the high relative importance of diminished GR protein expression to the phenotype of SN50-treated SMGs is directly related to the significant (p < 0.001) decline in cell proliferation and branching (Fig. 3A, B; Fig. 4A).
- The greater than 2-fold increase in Caspase 3 activation is associated with a 1.8-fold increase in PARP cleavage (Fig. 14) and a 10-fold increase in apoptosis (Fig. 4B).
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Cell proliferation and apoptosis. E15 SMG primordia were cultured in the presence or absence of 100 μg/ml SN50 peptide for 2 days (E15 + 2) and cell proliferation and apoptosis was determined. A., B. Cell proliferation. There is a marked decrease in cell proliferation (PCNA positive/brown color) with SN50 treatment (B) compared to control (A). Note that these sections were counterstained in hematoxylin; thus the cytoplasm in non PCNA-positive cells appears blue. C., D. Apoptosis. SN50 treatment (D) induced a notable increase in apoptotic positive nuclei (dark brown color) in ductal and terminal bud epithelia compared to control (C). Note that since these sections were not counterstained; thus the cytoplasm appears light brown. Bar: 50 μm.
- After 2 days in culture, SN50-treated explants exhibit a substantial decrease in gland size and branching morphogenesis compared to controls (compare Fig. 3A to 3B).
- These 2-day SN50-treated explants demonstrate a highly significant 81% decline (t4 = 26.25; p < 0.001) in cell proliferation (Fig. 3A, B; Fig. 4A) and a significant 10-fold increase (t4 = 7.98; p < 0.001) in apoptosis (Fig. 3C, D; Fig. 4B).
- It is reasonable, then, to assume that the high relative importance of diminished GR protein expression to the phenotype of SN50-treated SMGs is directly related to the significant (p < 0.001) decline in cell proliferation and branching (Fig. 3A, B; Fig. 4A).
- Thus, if one focuses on the superimposition of the various layers of information, namely morphology, gene expression, protein expression, and protein activity (Figs. 3,4,5,6,7,8,9,10,11,12,13,14), one can visualize a coordinated, multidimensional response to inhibited NF-κB nuclear translocation.