Overexpression of HOXA9 upregulates NF-κB signaling to promote human hematopoiesis and alter the hematopoietic differentiation potentials

Abstract

Background The HOX genes are master regulators of embryogenesis that are also involved in hematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles in hematopoiesis and leukemogenesis. Methods We established HOXA9-inducible human embryonic stem cells (HOXA9/hESCs) with normal pluripotency and potential for hematopoiesis, which could be used to analyze gene function with high accuracy. HOXA9/hESCs co-cultured with aorta–gonad–mesonephros-derived stromal cells (AGM-S3) were induced to overexpress HOXA9 with doxycycline (DOX) at various times after hematopoiesis started and then subjected to flow cytometry. Results Induction of HOXA9 from Day 4 (D4) or later notably promoted hematopoiesis and also increased the production of CD34+ cells and derived populations. The potential for myelogenesis was significantly elevated while the potential for erythrogenesis was significantly reduced. At D14, a significant promotion of S phase was observed in green fluorescent protein positive (GFP+) cells overexpressing HOXA9. NF-κB signaling was also up-regulated at D14 following induction of HOXA9 on D4. All of these effects could be counteracted by addition of an NF-κB inhibitor or siRNA against NFKB1 along with DOX. Conclusions Overexpression of HOXA9 starting at D4 or later during hematopoiesis significantly promoted hematopoiesis and the production of myeloid progenitors while reduced the production of erythroid progenitors, indicating that HOXA9 plays a key role in hematopoiesis and differentiation of hematopoietic lineages.

The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-β and NF-κB signaling

“…When NF-κB signaling was inhibited by its inhibitor (QNZ) or by siRNA against NFKB1 , except for further increased apoptosis, all other effects of P18 overexpression at the late stage were counteracted (Fig. 6 b,c), which was very similar to the phenomenon caused by HOXC4 and HOXA9 overexpression at the late stage 23 , 24 , and indicated that these genes probably share common mechanisms to promote hematopoiesis with the aid of NF-κB signaling.…”

“…At the early stage P18 overexpression can block the early hematopoietic differentiation, which is similar to the effects of RUNX1b/c that can be partially rescued by inhibition of TGF-β signaling 1 . At the late stage it can broadly promote the hematopoietic differentiation, which is similar to the effects of HOXA9 and HOXC4 23 , 24 that can be counteracted by inhibition of NF-κB signal pathway, and enhance the expression level of key genes of this pathway. Therefore, we speculated that P18 function ought to be closely related to TGF-β and NF-κB signaling at the early and late stage of hematopoiesis respectively according to our previous researches and performed further exploration based it.…”

“…The co-cultures were grown for up to 14 days with 5% CO 2 at 37 °C, with a medium replacement once per day. The detailed procedure was defined previously 1 , 23 , 24 .…”

“…D10-induced P18 /hESCs co-cultured with AGM-S3 cells were detected by qRT-PCR at D14 using primers for NFKB1 and NFKB2 . QNZ, an inhibitor of the NF-κB signaling pathway (Selleck Inc) was dissolved in DMSO; siRNAs against NFKB1 (Sangon Biotech Inc, Shanghai, China) were described previously 23 , 24 . D10-induced P18 /hESC co-cultures were treated from D10 with 20 nM QNZ (or an equal volume of DMSO) or 20 nM siRNA against NFKB1 (siRNA NFKB-1 vs NFKB-3 = 1:1 mixture or an equal concentration of control siRNA), and changed fresh media every day.…”

“…Subsequent studies revealed that RUNX1b overexpression also changes the status of the cell cycle and increases the expression of some cell cycle regulators, including P18 (unpublished data). Hence, we sought to elucidate the function of P18 on different stages of hematopoiesis using a mature inducible expression system based on piggy Bac transposon and the AGM-S3 co-culture system that we established previously 1 , 23 , 24 . Our findings provide the first evidence that P18 overexpression can promote hematopoiesis, providing insight into the molecular mechanisms underlying CKI activity during hematopoietic differentiation.…”