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. 2015 Mar;29(3):615-24.
doi: 10.1038/leu.2014.254. Epub 2014 Sep 2.

FHL2 regulates hematopoietic stem cell functions under stress conditions

Y Hou  1 , X Wang  2 , L Li  3 , R Fan  2 , J Chen  4 , T Zhu  5 , W Li  1 , Y Jiang  6 , N Mittal  1 , W Wu  1 , D Peace  1 , Z Qian  1
Affiliations

FHL2 regulates hematopoietic stem cell functions under stress conditions

Y Hou et al. Leukemia. 2015 Mar.

Abstract

FHL2, a member of the four and one half LIM domain protein family, is a critical transcriptional modulator. Here, we identify FHL2 as a critical regulator of hematopoietic stem cells (HSCs) that is essential for maintaining HSC self-renewal under regenerative stress. We find that Fhl2 loss has limited effects on hematopoiesis under homeostatic conditions. In contrast, Fhl2-null chimeric mice reconstituted with Fhl2-null bone marrow cells developed abnormal hematopoiesis with significantly reduced numbers of HSCs, hematopoietic progenitor cells (HPCs), red blood cells and platelets as well as hemoglobin levels. In addition, HSCs displayed a significantly reduced self-renewal capacity and were skewed toward myeloid lineage differentiation. We find that Fhl2 loss reduces both HSC quiescence and survival in response to regenerative stress, probably as a consequence of Fhl2-loss-mediated downregulation of cyclin-dependent kinase-inhibitors, including p21(Cip) and p27(Kip1). Interestingly, FHL2 is regulated under the control of a tissue-specific promoter in hematopoietic cells and it is downregulated by DNA hypermethylation in the leukemia cell line and primary leukemia cells. Furthermore, we find that downregulation of FHL2 frequently occurs in myelodysplastic syndrome and acute myeloid leukemia patients, raising a possibility that FHL2 downregulation has a role in the pathogenesis of myeloid malignancies.

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Figures

Figure 1
Figure 1. The effects of Fhl2 loss on the number and cell cycle status of HSCs and HPCs in BM
(A-B) The representative flow cytometric analysis of the frequency (A) and average absolute number (B) of HSCs, LSKs, HPCs, CMPs, GMPs and MEPs in BM cells from the control and Fhl2-/- mice (n=5, *, P<0.05). (C-E) The histograms show the distribution of HSCs (C), LSKs (D) and HPCs (E) in G1, S and G2/M phase in BM from control and Fhl2-/- mice (n=5, *, P<0.05). Cells were stained with DAPI. All mice were at age of 2 months. HPCs, LSKs, HSCs are defined as Lin-Sca-1-c-Kit+, Lin-Sca-1+c-Kit+ and LSK, CD150+CD48- respectively. CMPs, GMPs and MEPs are defined as Lin-c-kit+Sca-1- CD34+/loCD16/32int, Lin-c-kit+Sca-1- CD34+CD16/32+, and Lin-c-kit+Sca-1- CD34-CD16/32- respectively.
Figure 2
Figure 2. Loss of Fhl2 leads to intrinsic functional defects and impaired long-term engraftment of HSCs
(A) The diagram for the experimental design. (B) Histogram showing the relative ratio of donor (CD45.2+) versus competitor PB cells (CD45.2+ /CD45.2+) at 1 to 4 months after 1st, 2nd and 3rd transplantation(*, P<0.05, n=5). (C)Lineage differentiation in the recipient mice 4 month after 3rd transplantation. (D) The relative ratio of donor (CD45.2+) versus competitor (CD45.2+ /CD45.2+) in Lin-, HPC, LSK, HSC in BM at 4 months after 3rd transplantation (n=5; *, P<0.05; **, P<0.01).
Figure 3
Figure 3. Fhl2-deficient chimeric mice developed abnormal hematopoiesis
(A-B) The representative flow cytometric analysis of engraftment of CD45.2+ Fhl2-/- and control BM cells in total BM cells (A) and CD34-LSK cells (B) from CD45.1+ wildtype recipient mice (C) The number of white blood cells (WBC), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), basophils (BA), red blood cells (RBC), platelets (PLT), and hemoglobin (Hb) levels in PB from control and Fhl2-/- chimeric mice (n=7; *, P<0.05).
Figure 4
Figure 4. Decreased BM stem and progenitor cells in transplanted Fhl2-/- mice
(A-C) Representative FACS analyses (left) and average absolute number (right) of BM cells are shown for control Fhl2+/+ and mutant Fhl2-/- mice. Numbers in the FACS plots indicate percentages in the gated population cells. All BM cells were analyzed from the chimeric mice at 7-8 months post-transplantation (n=5; *, P<0.05).
Figure 5
Figure 5. Fhl2-deficient HSCs had a reduced number of quiescent cells and displayed an increased frequency of proliferation and apoptosis after transplantation
(A-C) The histograms show the distribution of HSCs (A), LSKs (B) and HPCs (C) in G1, S and G2/M phase in BM. Cells were stained with DAPI. (D) Representative flow cytometric analysis of LSK cells stained with Hoechst 33342 and Pyronin. (E) The histograms show the distribution of LSKs in G0, G1 and S-G2-M in BM. (F) The histograms depict the frequency of apoptosis in HPCs and LSKs in BM. All BM cells were isolated from the Fhl2+/+ and Fhl2-/- chimeric mice (n=4-7; *, P<0.05) at 6-7 months post-transplantation.
Figure 6
Figure 6. Down-regulation of CDK inhibitors in Fhl2-null LSKs
(A) qPCR analysis of the expression of CDKs and CDK inhibitors in Fhl2-null and wildtype control LSKs isolated from chimeric mice 6 months post-transplantation (n=3, p<0.05). LSKs are defined as Lin-Sca+c-Kit+ cells. (B) G0 status of Fhl2-null and wildtype control LSKs with forced expression of p21(Cip1), p27(Kip1) and p57(Kip2). The G0 status of infected cells was determined by flow cytometric analysis. **, P<0.005; *, P<0.05.
Figure 7
Figure 7. Downregulation of FHL2 in patients with AML and MDS
(A) Expression profile of FHL2 transcripts in PB mononuclear cells from 74 healthy individuals (left) and 542 AML patients (right) by microarray analysis (Average fold change is -2.288. P=6.28E-23). (B) FHL2 expression level in bone marrow cells from 46 patients with MDS and 10 healthy donors (Control, CTL) by qPCR. ACTB was used as the normalization gene. The square indicates the MDS patients who have a low FHL2 expression level.
Figure 8
Figure 8. Expression of FHL2 is associated with methylation status of the promoter region in myeloid leukemia cells
(A) B-FHL2 has a unique promoter region. The boxes represent different exons, and the black line represents intronic sequences. The common exons encoding the FHL2 protein, which are shared among these three transcripts, are shown in grey. The exons encoding the unique 5′ UTR are shown with boxes with different patterns for each transcript. The black boxes within the DRAL and FHL2 transcripts are identical. The numbers indicate the position within the GenBank nucleotide sequence [FHL2 (gi:42403584), DRAL (gi:11761688), and B-FHL2 (DQ307067)]. (B) Bisulfite sequencing of an amplicon from the promoter region of B-FHL2 containing 8 CpG dinucleotides in untreated KG-1 cells or cells treated with 5′-aza-dC. Each row presents a clone. Filled squares indicate methylated CpGs, whereas open squares refer to unmethylated CpGs. (C) Relative expression of B-FHL2 in untreated KG-1 cells, and cells treated with 5′-aza-dC was determined by qRT-PCR. The y-axis represents the ratio of fold-change in gene expression of FHL2 between treated and untreated KG-1 cells. (D) The expression of FHL2 in BM cells from AML patients and healthy individuals, as determined by qRT-PCR. (E) DNA methylation profile of the 8 CpG dinucleotides in the promoter region of B-FHL2 gene in multiple AML samples (AML1-AML11) and 2 control samples (Con1, Con2) from healthy individuals. The cycles represent the average methylation level of a specific CpG dinucleotide. Unmethylated, partially methylated, and fully methylated are indicated by open cycles, partially blacked cycles and blacked cycles respectively. (F) Relative expression of B-FHL2 in primary AML cells before and after treatment with 5′-aza-dC, as determined by qRT-PCR. The y-axis represents the ratio of FHL2 level between treated and untreated cells.
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