Abstract
Efforts to understand the molecular mechanisms underlying the differentiation of hematopoietic progenitor cells into mature blood cells have focused mainly on late events that largely reflect the differentiated state of the cells. In order to evaluate early changes of the gene expression profile of HSPC subjected to differentiation stimuli, transcriptional profiles of immuno-magnetically sorted bone marrow CD34+ HSPC were generated before and after 12 and 40 hours of culture with supplemented media favoring myeloid or erythroid commitment. Four independent samples were pooled and submitted to each treatment, and cells aliquots were subjected to RNA extraction and to methylcellulose cultures. CD34+ HSPC without previous stimulation generated about equal percentages (50% each) of erythroid (BFU-E) and myeloid (CFU-GM) colonies, whereas upon erythroid stimulation the median percentages of BFU-E, CFU-GM and mixed colonies (CFU-Mix) were, respectively, 61%, 38% and 1% for the12 hour treatment and 83%, 17% and 0% for 40 hours treatment. Conversely, upon myeloid stimulation, respectively, 37%, 61% and 2% colonies were observed after 12 hours and 23%, 61% and 14% for 40 hours treatment. These results indicate that after 12 and 40 hours of treatment, the genetic program of those cells were shifted towards the desired phenotype. Serial analysis of gene expression (SAGE) was employed to generate four independent libraries, each with more than 60,000 tags sequenced, representing more than 12,000 annotated transcripts. By analyzing the differentially regulated transcripts between the control CD34+ HSPC and the stimulated cells, we observed a set of genes that were initially up-regulated at 12 hours but were then down-regulated at 40 hours, exclusively after myeloid stimuli. Among those we found transcripts for IL1B, LTB, TNFRSF4 and NFKB2. Additionally, the receptor for LTB and the inhibitor of NF-κB signaling NFKBIA (IKBA) were respectively, up and down modulated at both time points. All those transcripts code for signaling proteins of the nuclear factor kappa B (NF-κB) pathway. More specifically, NFKB2 is a subunit of the NF-κB transcription factor (TF) that together with RELB mediates the non-canonical NF-κB pathway. The up-regulation followed by a down regulation was confirmed for NFKB2 and also demonstrated for RELB and NFKB1, indicating that the NF-κB pathway could be involved in the early commitment of CD34+ HSPC towards the myeloid lineage. To test this hypothesis, interference RNA (RNAi) against NFKB2 and control RNAi were transfected into BM CD34+ HSPC. Cells submitted to transfection with RNAi were stimulated towards the myeloid lineage and subjected to evaluation on methylcellulose cultures. Transcript levels of NFKB2 and RELB (a transcription target of NFKB2) were shown to be down-modulated, confirming the successful inhibition of NFKB2. After inhibition of NFKB2, the percentage of CFU-GM and BFU-E colonies shifted from 53% and 47% on control cells, respectively, to 22% and 78%. Altogether, our results indicate that NFKB2 has a role in the early commitment of CD34+ HSPC towards the myeloid lineage, directly inducing the differentiation program or, alternatively, protecting early myeloid progenitors from apoptosis.
NAD Modulates DNA Methylation and Cell Differentiation