HOXC4 up-regulates NF-κB signaling and promotes the cell proliferation to drive development of human hematopoiesis, especially CD43+ cells

Abstract

The hematopoietic function of HOXC4 has not been extensively investigated. Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenitor cells. CD34−CD43+ cells could be clearly classified into CD34−CD43 low and CD34−CD43 high sub-populations at D14. The former cells had greater myelogenic potential, and their production was not significantly influenced by induction of HOXC4 . By contrast, the latter cells had greater potential to differentiate into megakaryocytes and erythroid cells, and thus had properties of erythroid–megakaryocyte common progenitors, which abundance was increased by ∼2-fold when HOXC4 was induced from D10. For CD34−CD43 low , CD34+CD43+, and CD34−CD43 high sub-populations, CD43 level served as a natural index for the tendency to undergo hematopoiesis. Induction of HOXC4 from D10 caused more CD43+ cells sustain in S-phase with up-regulation of NF-κB signaling, which could be counteracted by inhibition of NF-κB signaling. These observations suggested that promotion of hematopoiesis by HOXC4 is closely related to NF-κB signaling and a change in cell-cycle status, which containing potential of clinical applications.

The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-β and 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 .…”

“…After cell counting. 5 × 10 4 cells in suspension were mixed well on methylcellulose (H4320, STEM CELL) containing 1% Antibiotic–Antimycotic (Gibco) and cytokines, as previously described 23 – 25 , and then 1.1 ml of the mixture was divided into each 35-mm Petri dishes at a final concentration of 5 × 10 4 cells per dish. The cells were incubated for 12–14 days at 37 °C in a 5% CO 2 /95% humidity incubator.…”

“…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.…”