Fak depletion in both hematopoietic and nonhematopoietic niche cells leads to hematopoietic stem cell expansion

Abstract

Hematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-Cre-inducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling Lin(-)Sca-1(+)c-kit(+) (LSK) cells is increased in Fak(-/-) mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from Fak(-/-) mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs (Lin(-)Sca-1(+)c-kit(+)CD34(-)Flk-2(-) cells) sorted from Fak(-/-) mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of Fak(fl/fl)Mx1-Cre(+) bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady-state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.

Figure 1. Efficient and stable deletion of floxed Fak allele in Fak^fl/flMx1-Cre⁺ BM compartments after 5 doses of pIpC injection

(A) Fak deletion was examined by PCR genotyping and western blotting of BM cells from pIpC treated mice. The genotyping PCR product consisted of floxed Fak (400bp band) and Cre-mediated excised fragment (326bp band). Western blot with ß-actin was included for loading controls. (B) Excision efficiency of floxed Fak gene sequence was monitored by qPCR. Genomic DNA was isolated from BM cells of pIpC injected WT (Fak^fl/fl) or KO (Fak^−/−Mx1-Cre⁺) mice. The DNA level of undeleted floxed Fak allele was normalized with internal control 18s ribosomal RNA. (C) Fak deletion in non-hematopoietic BM cells. Left, flow cytometric characterization of BM CD45⁻Ter119⁻ cells from a WT mouse. Right, PCR genotyping was performed with genomic DNA from CD45⁻Ter119⁻ and whole BM cells from a WT mouse (Lane 1 and 2), a KO mouse (Lane 3 and 4), or a KO mouse – 12 weeks post pIpC (Lane 5 and 6). (D) Left, flow cytometric analysis of EGFP expression in LSKs and Lin⁻c-Kit⁺ cells in BM of control (Fak^fl/flEGFP⁺) and knockout (Fak^−/−Mx1-Cre⁺EGFP⁺) mice. Right, PCR genotyping was performed with the genomic DNA isolated from LSKs, Lin⁻c-Kit⁺ and unsorted BM cells of a knockout mouse. All mice were 2 weeks post last pIpC injection unless otherwise indicated.

Figure 2. Fak deletion leads to increased LSK cells in BM

(A) BM cellularity was similar in Fak knockout (Fak^−/−Mx1-Cre+) and control (Fak^fl/fl) mice. BM cells were from two femurs and two tibias. (B) Representative flow cytometry profile of LSKs, CD34⁺LSKs and CD34⁻LSKs in BM of a control mouse. (C and D) Comparison of frequency and absolute number of LSKs, CD34⁺LSKs and CD34⁻LSKs in BM in control or knockout animals. n=7/genotype. (E-H) Comparison of total cells, number of CFU, frequency of LSKs and absolute number of LSKs in spleen of Fak knockout (Fak−/−Mx1-Cre+) and wild type (Fakfl/fl) mice. n≥7/genotype. (I-K) Comparison of number of CFU, frequency of LSKs and absolute number of LSKs in PB of Fak knockout (Fak−/−Mx1-Cre+) and wild type (Fakfl/fl) mice. n=5/genotype. All animals were 2 weeks after last pIpC treatment. *P<0.05. Data are mean±SD.

Figure 3. Fak^−/−Mx1-Cre+ mice have reduced quiescent LSK cells

(A) Left, representative cell cycle analysis (Hoechst and Ki-67 staining) on BM LSK cells of a wild type mouse. Right, % of WT or KO LSK cells in G0, G1 and S/G2/M phase. n=4/genotype. (B) Left, representative flow cytometry analysis of 7-AAD and Annexin V staining of WT BM LSK cells. Right, percentage of apoptotic cells in LSKs derived from freshly isolated BM cells. n≥3/genotype. All the animals were examined 2 weeks after last pIpC injection. *P<0.05. Data are mean±SD.

Figure 4. Fak-deleted BM Cells Exhibit Enhanced Engraftment of Long-term Duration

(A) Schematic diagram of competitive repopulation assay. (B) The chimerism of recipients was determined with PB cells at different time points. n≥5/genotype, *P<0.05. CD45.2⁺ cells were sorted from both BM and PB samples of recipients at 20 weeks after transplantation. Genomic DNA was extracted followed by PCR genotyping. Lane1 and 2, CD45.2⁺ cells from a KO recipient’s PB and BM; lane 3 and 4, CD45.2⁺ cells from a WT recipient’s PB and BM. (C) The chimerism was also measured in whole BM cells and PB granulocytes (B220⁻Gr-1⁺side scatter^high) of recipients at 20 weeks post transplantation. n≥5/genotype, *P<0.05. (D) Lineage reconstitution was comparable in WT and KO donor-derived (CD45.2⁺) cells at 20 weeks post transplantation. Data are mean±SD.

Figure 5. Fak Does Not Intrinsically Regulate Steady-State Hematopoiesis

(A) The chimeric mice were generated by transplanting 2×105 either floxed Fak (Fak^fl/flMx1-Cre+) or WT (Fak^fl/fl) BM cells along with same number of competitor BM cells into lethally irradiated recipient mice. Five weeks post transplantation, the mice were treated with pIpC to induce Fak deletion and PB chimerism was analyzed at the indicated time points. (B) The chimerisms of PB (Left), PB granulocytes (Upper Right) and LSKs in BM (Lower Right) were not statistically different between WT and KO recipients. P-values are 0.13, 0.30 and 0.92, respectively. CD45.2+ cells were sorted from PB samples of a WT (lane 1) and KO (lane 2) recipient at 20 weeks after transplantation. Genomic DNA was isolated followed by PCR genotyping. n=7/genotype. Data are mean±SEM.

Figure 6. Fak deletion in BM microenvironment niche cells alone does not affect HSC homeostasis

(A) Schematic diagram of reciprocal transplantation. (B) The chimerisms of PB (Left, P=0.63) and PB granulocytes (Right, P=0.55) were not statistically different between WT and KO recipients. (C) Both frequency and absolute number of LSKs in CD45.1+ donor-derived BM cells were similar between WT and KO recipients at 2 weeks post pIpC. Both p-values are 0.5. n≥3/genotype. Data are mean± SD.