MMP9 inhibition increases erythropoiesis in RPS14-deficient del(5q) MDS models through suppression of TGF-β pathways

Abstract

The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number of colony forming unit-erythroid colonies and the CD71⁺ erythroid population from RPS14 knockdown human BMCD34⁺ cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71⁺ population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factor β (TGF-β) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-β inhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71⁺ population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-β signaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.

Graphical abstract

Figure 1.

MMP9 inhibitors increase the erythroid development in RPS14-deficient models. (A) Wild-type (WT) embryos stained with o-dianisidine showed strong brown signal on the yolk sac, indicating normal hemoglobin levels. rps14^−/− mutant embryos (MT) stained with o-dianisidine showed weak brown signal on the yolk sac, indicating reduced hemoglobin levels. MT treated with different concentrations of mmp9 inhibitors showed improved staining signal of hemoglobin compared with DMSO-treated rps14^−/− MT. All embryos are ventral views. (B-C) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 or Luc control. After 1 day of transduction, cells were treated by MMP9 inhibitors. 9-I-L, MMP9-I 5 nM; 9-I-H, MMP9-I 1 μM; 9-II-L, MMP9-II 10 nM; 9-II-H, MMP9-II 10 μM. (B) Cells were sorted for GFP+ at 4 days after treatment. A total of 1500 GFP+ cells were plated in methylcellulose media specific for CFU-E colonies and cultured for 1 week. Colonies were counted by an investigator blinded to the conditions. (C) Cells were analyzed for the CD71 and the CD11b expression by using flow cytometry at 6 days after treatment with MMP9 inhibitors. Data are representative of 3 independent transduction experiments. *P < .05, **P < .01, ***P < .001. ERY media, erythroid media; MY media, myeloid media.

Figure 2.

MMP9 is upregulated in RPS14-deficient cells resulting in decreased erythroid populations. Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 or Luc control. (A) After 5 days of transduction, cells were sorted for GFP+. RNA was collected and analyzed by using RT-qPCR. (B) Protein was collected from sorted cells and analyzed by using western blot analysis. β-Actin was used as a loading control. (C) RNA was collected from rps14-deficient and wild-type zebrafish embryos and analyzed by using RT-qPCR. (D) RNA was isolated from bone marrow CD34⁺ cells from patients with del(5q) MDS or healthy control subjects and analyzed by using RT-qPCR. (E) GFP+-sorted cells were cultured for an additional 2 days, and culture media were collected for an MMP9 ELISA. (F) After 7 days of transduction, cells were treated by using GolgiPlug and GolgiStop for 12 hours. Cells were stained with MMP9 antibody and analyzed according to flow cytometry. (G) Human BMCD34⁺ cells were cultured in liquid culture media. After 5 days of culture, cells were treated by rMMP9 for 2 days and analyzed for the CD71 expression by using flow cytometry. (H) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 and MMP9. After 7 days of transduction, cells were analyzed for the CD71 expression by using flow cytometry. Data are representative of 3 independent transduction experiments. *P < .05, **P < .01, ***P < .001.

Figure 3.

MCP1 regulates MMP9 expression in RPS14-deficient cells. Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 or Luc control. (A) After 5 days of transduction, cells were sorted for GFP+. RNA was collected and analyzed by using RT-qPCR. (B) After 5 days of transduction, cells were stained with MCP1 antibody and analyzed by using flow cytometry. (C) Cells sorted per GFP+ were cultured for an additional 2 days, and culture media were collected for an MCP1 ELISA. (D) Human BMCD34⁺ cells were cultured in liquid culture media. After 1 day of culture, cells were treated by rMCP1 for 4 days. RNA was collected and analyzed by using RT-qPCR. (E) Human BMCD34⁺ cells were cultured in liquid culture media. After 1 day of culture, cells were treated by rMCP1 for 4 days. Cells were stained with MMP9 antibody and analyzed by using flow cytometry. (F) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 and MCP1. After 7 days of transduction, cells were stained with MMP9 antibody and analyzed by using flow cytometry. Data are representative of 2 independent transduction experiments. *P < .05, **P < .01, ***P < .001.

Figure 4.

Inhibition of TGF-β signaling increases erythroid development in RPS14-deficient models. Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 or Luc control. (A) After 5 days of transduction, cells were sorted for GFP+ and were cultured for an additional 2 days. Culture media were collected for a TGF-β ELISA. (B) After 5 days of transduction, cells were sorted for GFP+. Protein was collected and analyzed by using western blot analysis. β-Actin was used as a loading control. (C) After 3 days of transduction, cells were treated by SB431542 at the indicated concentration for 4 days and analyzed for the CD71 expression by using flow cytometry. (D) After 1 day of transduction, cells were treated by 2 μM of SB431542 for 4 days and sorted for GFP+. A total of 4000 cells of GFP+ cells were plated in methylcellulose media specific for CFU-E colonies and cultured for 1 week. Colonies were counted by an investigator blinded to the conditions. (E) Wild-type (WT) embryos stained with o-dianisidine showed strong brown signal on the yolk sac, indicating normal hemoglobin levels. rps14^−/− mutant embryos (MT) stained with o-dianisidine showed weak brown signal on the yolk sac, indicating reduced hemoglobin levels. MT treated with 2 concentrations of SB431542 showed improved staining signal of hemoglobin compared with DMSO-treated rps14^−/− MT. All embryos are ventral views. (F) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14 and ALK5. After 7 days of transduction, cells were analyzed for the CD71 expression by using flow cytometry. Data are representative of 3 independent transduction experiments. *P < .05, **P < .01, ***P < .001.

Figure 5.

MMP9 inhibitors attenuate activated TGF-β signaling in human RPS14-deficient cells and in bone marrow cells from a patient with del(5q) MDS. (A) Human BMCD34⁺ cells were cultured in liquid culture media. After 5 days of culture, cells were treated by rMMP9 for 2 days, and protein was collected for western blot analysis. (B) Human BMCD34⁺ cells were cultured in liquid culture media. After 3 days of culture, cells were treated by rMMP9 for 2 days followed by SB431542 treatment for an additional 2 days. At 7 days after culture, cells were analyzed for the CD71 expression by using flow cytometry. (C) Human BMCD34⁺ cells were transduced with lentivirus carrying shRNAs against RPS14. After 5 days of transduction, cells were treated by MMP9 inhibitors for 2 days and were analyzed for pSMAD2/3 or SMAD2/3 expression by using flow cytometry. (D) RNA was isolated from bone marrow CD34⁺ cells from a patient with del(5q) MDS or healthy control subjects and analyzed by using RT-qPCR. (E) BMCD34⁺ cells from a patient with del(5q) MDS or a healthy control subject were cultured in liquid culture media. After 1 day of culture, cells were treated by MMP9 inhibitors for 4 days. Cells were cultured for an additional 4 days without MMP9 inhibitors and then analyzed for CD71 and CD235a expressions by using flow cytometry. (F) BMCD34⁺ cells from a patient with del(5q) MDS or a healthy control subject were cultured in liquid culture media. After 1 day of culture, cells were treated by MMP9 inhibitors for 4 days and then analyzed for pSMAD2/3 or SMAD2/3 expression by using flow cytometry. (G) Model of the defective erythroid development in RPS14 deficiency through increased MMP9 expression. Data are representative of 3 independent transduction experiments (A-C) or 1 independent experiment (D-F). *P < .05, **P < .01, ***P < .001.