PDK1 plays a vital role on hematopoietic stem cell function

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

Cell cycle analysis. Freshly isolated BM cells were stained using antibodies against Sca-1, c-kit, CD34, Flt3 and lineage markers to identify HSCs and MPPs. Antibody-labeled cells were subsequently incubated with DAPI and Ki67 to determine the cell cycle profile. The Ki67 antibody allows for the separation of cells in G0 and G1 stages, and co-staining with DAPI allows for the separation of S/G2/M cell populations. Cells were analyzed using a LSR II flow cytometer (BD Biosciences).
Apoptosis assay. BM cells from groups were incubated with antibodies against Sca-1, c-kit, CD34, Flt3 and linage markers to identify HSCs and MPPs. Antibody-labeled cells were washed and incubated with Annexin V and DAPI at room temperature followed by flow cytometry analysis using an LSR II flow cytometer.
Real-time RT-PCR. mRNA expression levels were quantified using real-time RT-PCR with SYBR Green PCR Master Mix. Changes in relative gene expression between groups were calculated using the 2 −∆∆CT method normalized to GAPDH expression. Statistical analyses. Significant differences in parameters were assessed between groups using unpaired Student's test. Significance is denoted with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), and P > 0.05 was considered non-significant (NS).

Results
PDK1 deficiency in mice results in increased phenotypic HSCs and decreased progenitor cells. We generated PDK1 conditional knockout mice Vav-Cre;PDK1 fl/fl (PDK1 Δ/Δ ) to explore the roles of PDK1 in murine HSCs. PDK1 fl/fl (WT) mice were used as a control. Real-time PCR confirmed the efficient excision of the PDK1 gene in PDK1 Δ/Δ mice (Fig. 1A). The BM cellularity, splenocytes and thymocytes were decreased after PDK1 deletion ( Fig. 1B-D). WBC, lymphocyte and platelet number were also decreased in PDK1 deficient mice (Fig. 1E-H). PDK1 Δ/Δ mice were smaller than wild-type controls and exhibited a larger spleen and smaller thymus (Fig. 1G-I). H&E staining revealed evidence of extramedullar hematopoiesis in PDK1-deficient spleens (Fig. 1K).
FACS analysis revealed that the percentage of LSK (Lin − c-kit + Sca-1 + ) cells and LK (Lin − c-kit + Sca-1 − ) cells in PDK1 Δ/Δ mice were comparable to those of the control mice ( Fig. 2A,B). Further examination of the frequency of HSCs and HPCs in BM using flow cytometry revealed significant increases in phenotypic LT-HSCs and ST-HSCs but substantial decreases in MPPs (Fig. 2C,D, Figure S1A,B) and CMPs after PDK1 deletion (Fig. 2E,F). These results indicated that the loss of PDK1 significantly perturbed steady-state hematopoiesis.

PDK1-deficient HSCs fail to reconstitute the hematopoietic system upon transplantation.
Colony-Forming Cell (CFC) assays were performed to determine the colony-forming abilities of PDK1-deficient progenitor cells in vitro to investigate whether the loss of PDK1 affected their function. PDK1 Δ/Δ BM cells gave rise to fewer CFU-GM and CFU-GEMM colonies when compared with control BM cells in MethoCult GF M3434 medium (Fig. 3A), demonstrating that the loss of PDK1 impairs the colony-forming ability of PDK1-deficient cells in vitro.
BM cells from PDK1 ∆/∆ and WT mice (CD45.2 + ) were transplanted into lethally irradiated recipients (CD45.1 + ) to evaluate the effect of PDK1 on HSC reconstitution ability ( Figure S2A). BM cells from PDK1-deficient mice failed to reconstitute the hematopoietic system in recipient mice, while the WT BM cells fully rescued the lethally irradiated mice (Fig. 3B). We didn't found any significant different in homing assay ( Figure S2B), suggesting that the impaired reconstitute ability in recipients by PDK1 knockout BM cells might not be due to their homing defect. with wild-type competitive cells (CD45.1 + ) ( Figure S2C). The recipient mice displayed extremely reduced percentages of PDK1 ∆/∆ -derived total donor cells, CD3 + , B220 + and myeloid cells in the peripheral blood (PB) at various time points after transplantation (Fig. 3C-G). The chimerism of BM cells was examined using flow cytometric analyses 6 months after transplantation. PDK1 ∆/∆ -derived BM cells were almost absent in recipient BM ( Figure S2D-F), but the control and competitive cells generated normal proportions of hematopoietic cells. These results suggest that PDK1-deficient HSCs fail to reconstitute hematopoiesis in vivo upon transplantation.
To determine the HSC function after PDK1 deletion, we transplanted 300 LT-HSCs from PDK1 ∆/∆ or WT mice and competitor cells into lethally irradiated recipient mice. We found that PDK1 deficient HSCs loss the ability to reconstitution in recipients where control group showed the normal self-renew ability (Fig. 3H,I). These results indicate that PDK1 is vital for HSC reconstitution. PDK1 deficiency is dominant over mTORC2 deficiency. PDK1 phosphorylates Akt at its T308 residue. Therefore, we examined the related protein phosphorylation levels in LSKs and HSCs using flow cytometry. Phosphorylation at the T308 residue of Akt was lower in PDK1-deficient HSCs ( Figure S3A), but the phosphorylation level of S473 was comparable to control ( Figure S3B). Notably, a downstream effector of Akt, S6 protein, exhibited decreased phosphorylation levels, which indicates an impairment of Akt signaling transduction after PDK1 gene loss ( Figure S3C). Phosphorylation of P44/P42 and Stat3 was altered after the loss of PDK1, which suggests a potential role of PDK1 in the p38-MAPK and Jak-Stat signaling pathways ( Figure S3D,E).
To explore how mTORC2 and/or PDK1 influence Akt function in HSCs, we generated Rictor Δ/Δ PDK1 Δ/Δ (DKO) mice in conjunction with Rictor Δ/Δ and PDK1 Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. In addition to the defective colony-forming ability of Rictor Δ/Δ PDK1 Δ/Δ progenitors (Fig. 4A), lethally irradiated recipient mice transplanted with whole bone marrow cells from PDK1 Δ/Δ or Rictor Δ/Δ PDK1 Δ/Δ mice failed to survive compared with WT or Rictor Δ/Δ BM cell transplantations (Fig. 4B). Competitive transplantation experiments revealed an impaired reconstitution ability of Rictor Δ/Δ PDK1 Δ/Δ HSCs after transplantation (Fig. 4C-F), which indicates a long-term hematopoiesis defect after Rictor/PDK1 deletion, consistent with PDK1 Δ/Δ HSCs. Therefore, our data suggest that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function compared with Rictor/mTORC2. PDK1 deficiency results in less quiescent HSC. We examined the cell cycle status of HSCs using Ki67 to categorize HSCs in resting or active cell cycle stages during cellular proliferation to explore the mechanism of PDK1 regulation of HSCs. The percentage of cells in G0 stage was decreased significantly in PDK1 ∆/∆ HSCs compared with control HSCs, and this result was characterized by a reduction in the Ki67 − G0 fraction (Fig. 5A-C). PDK1 ∆/∆ HSCs were also enriched in G1 and S/G2/M phases, which suggest an increase in HSC exit from their quiescent state (Fig. 5C). We further confirmed this in an in vitro Brdu incorporation assay and found that PDK1 deficiency decreased the G0 fraction of LSKs (Fig. 5D). Next, we examined the proportion of HSC undergoing apoptosis. We found a comparable percentage of Annexin V + DAPI − PDK1 ∆/∆ and WT HSCs (Fig. 5E-G, Figure S4A,B). These findings indicate that the loss of PDK1 altered HSC cell cycle status to be less quiescent.  8,20,21 . Therefore, we assessed ROS levels in PDK1-deficient HSCs by measuring intracellular ROS levels using 2′-7′-dichlorofluorescein diacetate (DCF-DA) staining 22,23 . Notably, we found that PDK1-deficient LSKs and HSCs exhibit significantly reduced ROS levels compared to that of control HSCs (Fig. 6A,B).
We treated PDK1-deficient BM cells with various concentrations of BSO in vitro to increase cellular ROS levels and examined the colony-forming ability of HSCs after treatment to probe whether reduced cellular ROS levels were responsible for the impaired function of PDK1 Δ/Δ hematopoietic stem and progenitor cells. The colony counts of PDK1-deficient BM cells treated with 0.01 μM and 0.02 μM BSO increased significantly, which indicates recovery of the colony-forming ability with increasing ROS levels (Fig. 6C). Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 μM. Next we raised ROS level by BSO treatment with sorted HSCs in vitro. We found the increased colony size (Fig. 6D) and number (Fig. 6E) of PDK1-deleted HSCs upon BSO treatment, indicating the impaired colony-forming ability of PDK1-deficient HSCs was partially rescued by increased ROS level.

Discussion
We used conditional deletion of PDK1 gene in a hematopoietic system and found that the loss of PDK1 resulted in an impaired colony-forming ability in vitro and a defective short-term and long-term reconstitution ability after transplantation (Fig. 3). HSC quiescence is essential for their self-renewal ability 21,24,25 and the maintenance of HSC functions. Disturbed quiescence of HSCs impairs HSC functions 22,26 . Therefore, the disrupted cell cycle status, as detected in PDK1-deficient HSCs, may account for the impaired reconstitution ability of HSCs. We observed a reduced G0 phase of HSCs and an increased S/G2/M phase of PDK1 Δ/Δ HSCs, which was accompanied by an increase in HSC frequency. These results indicate that PDK1-deficient HSCs were less quiescent due to PDK1 loss. Fewer G0-phase HSCs in PDK1-deficient mice led to reduced HSC reconstitution ability, and the increased HSC proliferation likely occurs through feedback mechanisms because PDK1 deletion resulted in a significant loss of progenitor cells, mature B cells and T cells. This result is consistent with a previous study that HSCs in the G0 phase exhibited enhanced reconstitution ability than less quiescent HSCs 27 .
Lower HSC cellular ROS levels have been demonstrated to be essential for the maintenance of quiescent HSCs 8 . Notably, we found that PDK1-deficient HSCs exhibited lower ROS levels with an increased proportion of HSCs entering the cell cycle. This result likely occurred because the loss of PDK1 either interrupted the regulatory mechanism of adequate HSC ROS level maintenance or perturbed cell cycle regulation independently of ROS, which resulted in the loss of quiescence in HSCs. Moreover, we found that the colony count of PDK1-deficient cells in vitro increased when ROS levels were elevated by 0.01-0.02 μM BSO treatment, but the number of colonies decreased when BSO concentrations were above 0.03 μM (Fig. 6). This result suggests that ROS levels are precisely controlled in hematopoietic stem and progenitor cells, and that higher or lower ROS levels beyond the normal range are harmful to hematopoietic stem and progenitor cell functions and PDK1 plays an important role in this process. However, additional work is needed to completely elucidate the roles of ROS in HSCs.
Akt is a major downstream effector of PDK1. A previous study demonstrated that Akt1/Akt2 double-knockout HSCs exhibited only modest reduced reconstitution ability 9 . Here we showed that PDK1-deficient HSCs alone could not reconstitute the recipient mice, whereas Rictor/mTORC2-deficient HSCs successfully reconstituted hematopoiesis in lethally irradiated mice with minor defects in B cell and T cell differentiation 28,29 . We generated Rictor Δ/Δ PDK1 Δ/Δ to explore the possible differential downstream signaling roles of PDK1 and mTORC2 on HSCs that govern Akt activation. Notably, Rictor/PDK1 double-deficient HSCs exhibited very similar phenotypes as PDK1-deficient HSCs (Figs 3 and 4). This result indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSCs. The functional discrepancies in PDK1 and Akt-deficient HSCs may be attributed to other AGC kinases that are regulated by PDK1 5 . Other AGC kinases and PDK1 substrates, such as SGK and p70S6K might also contribute to the defective HSC function after PDK1 deletion 6 . Future studies about other potential downstream factors of PDK1 will improve the current understanding of the role of PDK1 on HSC function.