Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis

Abstract

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the "bad seed in bad soil" hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

Figure 1.

CD9 expression is deregulated during PMF dysmegakaryopoiesis. (A) Expression of CD9 transcripts normalized to RPL38 by QRT-PCR in CD34⁺ cells from healthy donors (HD) and primary myelofibrosis (PMF) patients. (B) CD9 membrane expression (MFI) by flow cytometry on CD34⁺ cells purified from the peripheral blood (PB) of HD and PMF patients and CD9 membrane expression ranking (gray bars: PMF patients; white bars: HD). (C) Correlation between the CD9 membrane expression level on PMF CD34⁺ cells and the patient’s platelet count. (D) CD9 expression level on HD and PMF circulating platelets determined by flow cytometry (MFI). (E) Co-expression of CD9 and CD41 on PMF megakaryocytes derived from HD and PMF circulating CD34⁺ cells (D10 of culture). (F) Membrane expression analysis of CD9 on megakaryocytes (Mks) derived from CD34⁺ cells purified from the bone marrow (BM) or PB of HD and PMF patients (D10 of culture) by flow cytometry. (G) Western blot analysis and quantification of CD9 expression in Mks derived from circulating CD34⁺ cells from HD and PMF patients (D10 of culture).

Figure 2.

CD9 is involved in PMF dysmegakaryopoiesis. Effect of monoclonal antibody (mAb) ligation of surface CD9 (clone-SYB; 10 μg/mL) on (A) PMF megakaryocyte (Mk) colonies obtained from PMF circulating CD34⁺ cells in semi-solid collagen culture and (B) in liquid cultures (D8–10 of culture) on cell maturation (Giemsa) as well as CD41 and CD62p membrane expression (MFI) determined by flow cytometry, (C) on percentage of apoptotic annexin V⁺ cells and Bcl-XL expression level (MFI) determined by flow cytometry, (cells were gated on megakaryocytic cells derived from PMF CD34⁺ cell culture in the presence of a cocktail of cytokines including IL3, IL6, IL11 and TPO; a red quadrant corresponding to the addition of annexin V⁺ and pre-aptototic propidiumiodide⁻ (IP) cells and of annexin V⁺ and post-apoptotic IP⁺ cells was drawn to define the total proportion of apoptotic cells), (D) on GATA-1 expression as shown by QRT-PCR and western blot analyses and by immunofluorescence microscopy and (E) on modulation of c-myb expression by QRT-PCR analysis.

Figure 3.

CD9 controls Akt/MAPK balance during PMF dysmegakaryopoiesis. Analysis by cytometry, microscopy and western blot of (A) phospho-Akt (Thr308 and Ser473), - GSK3β (Ser9) levels (MFI), or (B) phospho-p38 (Thr180 and Tyr182) and phospho-JNK levels (MFI) in megakaryocytes (Mk) derived from PMF circulating CD34⁺ cells (D6 of culture in the presence of Tpo) at 2, 24, and 48 h after anti-CD9 monoclonal antibody (mAb) (clone-SYB; 10 μg/mL) treatment. (A and B) Data were normalized either to IgG control values or to total protein level. (B) QRT-PCR analysis of AP1 transcript expression level in Mk derived from PMF circulating CD34⁺ cells, treated or not with anti-CD9 mAb (clone-SYB; 10 μg/mL) and normalized to actin level.

Figure 4.

CD9 mediates PMF megakaryocyte-mesenchymal stromal cell interactions. (A) Analysis of CD9 transcript (QRT-PCR) and membrane expression (flow cytometry) levels in MSC isolated from the bone marrow of PMF patients (BM-MSC). (B) Effect of 2 days of co-culture between PMF megakaryocytes (Mk) derived from CD34⁺ culture (at D6 of Mk culture) and BM-MSC on the number of PMF Mk in suspension. (C) Effect of surface CD9 ligation by anti-CD9 monoclonal antibody (mAb) (clone-SYB; 10 μg/mL) on the proportion of PMF Mk derived from CD34⁺ culture in apoptosis (annexin V⁺ cells) and in the cell cycle phases (S+G2M cells) as well as on the cell count of non-adherent PMF Mk in the co-culture with BM MSC. (D) Effect of surface CD9 ligation by anti-CD9 mAb on the proportion of adherent PMF CD45⁺ hematopoietic cells obtained after co-culture on BM MSC isolated from HD or PMF patients and on adherent PMF Mk in co-culture with MSC isolated from the BM of either HD or PMF patients as shown by Giemsa staining and immunostaining (CD41⁺ Mk, in pink and CD105⁺ MSC, in orange).

Figure 5.

CD9 mediates PMF megakaryocyte migration in response to CXCL12. (A) Expression of CXCR4 on CD41⁺ PMF megakaryocytes (Mk) derived from PMF circulating CD34⁺ cells (D8–10 of culture) by flow cytometry and microscopy as compared to Mk derived from healthy donors (unmobilized peripheral blood or bone marrow). (B) CD9 and CXCR4 coexpression on CD41^low and CD41^high PMF cells. (C) Effect of surface CD9 ligation by anti-CD9 monoclonal antiboby (mAb) (clone-SYB; 10 μg/mL) on CXCR4 and CXCL12 transcript expression normalized to RPL38 by QRT-PCR in Mk derived from PMF circulating CD34⁺ cells (D8–10 of culture). (D) Effect of silencing CD9 using siRNA CD9 (1 μg/10⁶ cells) in Mk derived from PMF circulating CD34⁺ cells (D8–10 of culture) on the CD9 transcript and protein expression levels analyzed by QRT-PCR (at 48 h) and by flow cytometry (at 72 h), respectively, and on the percentage of migrating PMF Mk in a Boyden chamber in response to CXCL12 (100 ng/mL) as compared to passive diffusion and on the phospho-FAK (Tyr 576/577) expression level (MFI).