Profiling Changes in Gene Expression during Differentiation and Maturation of Monocyte-derived Dendritic Cells Using Both Oligonucleotide Microarrays and Proteomics

Abstract

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14؉ blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that ϳ40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca 2؉ binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.

Enhanced protein repair and recycling are not correlated with longevity in 15 vertebrate endotherm species

“…A further study examining several different heat shock protein mitochondrial RNAs (mRNAs) in Snell dwarf mice found no coordinated elevation of heat shock proteins (HSPs) in Snell dwarf tissues or fibroblasts (Swindell et al 2009). These latter mRNA data may not reflect protein levels, as changes in gene transcript levels often do not parallel changes in corresponding protein levels, even for transcriptionally regulated proteins (see for example Ideker et al 2001;Le Naour et al 2001). Investigation into HSP protein levels in this context is therefore probably still warranted.…”

Recent advances in blood‐related proteomics

“…Hierarchical cluster analyses of the data segregated the samples into two groups, one which contained only controls and the other only the patients. Combined oligonucleotide microarray and proteomic approaches have been used to study genes associated with dendritic cell differentiation [146,147]. Dendritic cells are antigen-presenting cells essential for the initiation of primary immune responses.…”

Kinetics of Gene Induction After FcεRI Ligation of Atopic Monocytes Identified by Suppression Subtractive Hybridization

“…Although this disadvantage was compensated by the analysis of a hundred clones, the method may yield limited information and some genes known to be overexpressed by Fc⑀RI triggering were not detected (31). Second, repressed genes as well as very weakly up-or downregulated genes might be missed; other methods like microarray techniques could be conducted to be combined with our strategy (32,33).…”