Thrombospondin-1 is not the major activator of TGF-β1 in thrombopoietin-induced myelofibrosis

Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine involved in normal tissue repair, and its sustained production induces fibrosis in numerous organs,¹  including myelofibrosis.²  Mechanisms leading to in vivo TGF-β1 activation (from inactive proforms) remain complex,³  involving interactions with integrins,^4-6  thrombospondin-1 (TSP-1),⁷  or shear forces.⁸  In a murine model of myelofibrosis obtained by thrombopoietin (TPO) overexpression,⁹  enhanced release of TGF-β1²  occurs mainly from megakaryocytes and, to a lesser extent, from monocytes.¹⁰  Because both TGF-β1 and TSP-1 are synthesized and stored in megakaryocyte α-granules,¹¹  abnormal and concomitant release of both molecules may hypothetically lead to pathologic local TGF-β1 activation. As a recent study has highlighted the role of TSP-1 in platelet-derived TGF-β1 activation,¹²  we investigated the role of TSP-1 in TPO-induced myelofibrosis.

We used the TPO^high murine model. All procedures were approved by the Institut Gustave Roussy ethics committee. Bone marrow (BM) cells from C57BL/6 wild-type (WT) or Tsp-1–null¹³  male littermates were infected with a retrovirus encoding murine TPO⁹  and engrafted into lethally irradiated WT or Tsp-1–null female hosts, respectively, leading to the following 2 engraftment combinations: WT/WT (ie, WT TPO^high mice, n = 22) and Tsp-1–null/Tsp-1–null (ie, Tsp-1–null TPO^high mice, n = 15). Lethally irradiated hosts were engrafted with 4 to 8 × 10⁶ cells in 2 independent experiments. Peripheral blood was analyzed every 4 weeks during 3 months using an automated blood counter calibrated for mouse blood (MS9, Melet Schloessing). Control mice included unmanipulated WT and Tsp-1–null mice. At weeks 8 and 12 after engraftment, mice were killed for histologic analysis. Reticulin fibers revealed by silver staining were quantified using ImageJ Version 1.42q software (National Institutes of Health). Microvascular density (MVD) was determined by CD34 staining.¹⁴  TPO and TGF-β1 levels in plasma or extracellular fluids were determined with the appropriate Quantikine kits from R&D Systems.

To assess the role of TSP-1 in the TGF-β1-induced BM fibrosis observed in TPO^high mice, 2 groups of engrafted mice with BM TPO-transduced cells, WT TPO^high and Tsp-1–null TPO^high, were constituted. The transduction efficiency of the progenitor cells, evaluated with a colony-forming assay, was similar in the 2 mouse genotypes (77% ± 5%). Four weeks after engraftment, the TPO concentrations in plasma of WT and Tsp-1–null TPO^high mice ranged from 1000- to 10 000-fold higher than in WT and Tsp-1–null control mice and remained elevated during the 3 months of follow-up (Figure 1A). The magnitude of this increase was comparable regardless of the TPO^high mice groups. Chimerism, analyzed by fluorescent in situ hybridization on the Y chromosome (BM donors were male, recipients were female) on whole nucleated BM cells at weeks 8 and 12, when mice were killed for histologic analysis, showed levels more than 90% and similar in both TPO^high mice groups (data not shown).

As expected from previous reports,^2,9,10,15  all TPO^high mice developed a similar myeloproliferative syndrome with thrombocytosis (Figure 1B), leukocytosis (Figure 1C), and splenomegaly associated with an increased number of blood and spleen progenitor cells (data not shown). However, Tsp-1–null TPO^high mice showed increased thrombocytosis at week 4, which then turned out to be comparable with WT TPO^high mice. It has been demonstrated that mice deficient in both TSP-1 and TSP-2 displayed a more rapid regeneration of thrombopoiesis after myelosuppression,¹⁶  extending previous in vitro results that indicated that TSPs negatively regulate megakaryopoiesis.¹⁷  Here, we cannot exclude that TSP-1 deficiency shortens platelet recovery under TPO stimulation. Nevertheless, we did not observe a further increase of megakaryocyte hyperplasia or thrombocytosis in Tsp-1–null TPO^high mice compared with WT TPO^high mice during disease progression.

Because development of fibrosis has been reported to be a direct consequence of high TGF-β1 levels in TPO^high mice²  and TSP-1 has been demonstrated to activate TGF-β1,⁷  we measured total and active forms of TGF-β1 in plasma and extracellular fluids from WT and Tsp-1–null TPO^high mice BM and spleen. Control Tsp-1–null mice exhibited a statistically significant increase in total TGF-β1 (Figure 1D) as formerly reported on the C57BL/6 background.¹⁸  As expected, the level of total TGF-β1 in the plasma of TPO^high mice increased as early as 4 weeks after engraftment and reached a level 2- to 4-fold higher than in control mice by week 12. No active form was detected in the plasma from control or TPO^high mice. Moreover, we observed an increased level in both total and active TGF-β1 in extracellular fluids from BM (Figure 1E-F) and spleen (Figure 1G-H) in TPO^high mice compared with control mice. Surprisingly, we were able to detect the active form of TGF-β1 in Tsp-1–null TPO^high mice, suggesting that alternative mechanisms are responsible for local TGF-β1 activation in this model of myelofibrosis.

As predicted by the detection of the active form of TGF-β1, Tsp-1–null TPO^high mice developed BM and spleen fibrosis (Figure 2A), with, intriguingly, a greater grade than in WT TPO^high mice (Figure 2B). Our results show that local activation of TGF-β1 in the hematopoietic environment leading to BM and spleen fibrosis occurs in the absence of TSP-1, according to a previous study reporting that TSP-1 was not a major activator of TGF-β1 in platelets.¹⁸  However, Ahamed et al recently demonstrated that TSP-1 contributes to TGF-β1 activation in both serum and platelet releasates under conditions of stirring and/or shear.¹²  Such mechanisms are probably not involved within the BM environment. TSP-1 has been clearly demonstrated to be the major activator of TGF-β1 in lung and renal fibrosis.^19,20  However, one can speculate that TGF-β1 could be regulated in a tissue-specific way, particularly in the megakaryocyte/platelet compartment. Numerous studies have shed light on the major role of integrins in TGF-β1 activation.^4-6  We hypothesize that β3 integrins expressed by platelets and megakaryocytes²¹  may compensate for the absence of TSP-1 in this model.

Unexpectedly, the fibrosis observed in Tsp-1–null TPO^high mice was more enhanced than the one observed in WT TPO^high mice (Figure 2B). To explain this surprising finding, we first hypothesized that this increased fibrosis was related to an augmentation of neoangiogenesis¹⁴  mediated by the antiangiogenic function of TSP-1.¹⁶  We therefore analyzed the MVD in BM. MVD in control Tsp-1–null was higher than in control WT (10 ± 4.7 vs 3.4 ± 1.6; P < .001), as expected. However, MVD observed in Tsp-1–null TPO^high mice (8.3 ± 4.4) did not rise above the one observed in control Tsp-1–null mice and was similar to MVD observed in WT TPO^high mice (5.7 ± 2.9). Thus, the increase of myelofibrosis in Tsp-1–null TPO^high mice cannot be explained by an augmentation of neoangiogenesis. We then hypothesized that the increase of BM fibrosis in Tsp-1–null TPO^high mice could be related to an enhanced TGF-β1-mediated response by Tsp-1–null BM stromal cells. Supporting this hypothesis, it has been demonstrated that β3^−/− mice exhibit a sustained TGF-β1 activation via an increase of TGF-β1 receptors I and II expression, leading to enhanced wound healing with reepithelialization.²²  To this purpose, we compared the expression of several TGF-β1-signaling pathway members in Tsp-1–null and WT BM stromal cells. Unfortunately, we did not clearly demonstrate a sustained TGF-β1 activation nor an increase in TGF-β1 receptors or collagen expression in Tsp-1–null stromal cells (data not shown). Thus, mechanisms leading to the increased myelofibrosis in Tsp-1–null mice remain unclear. However, similar greater amounts of fibrosis have been recently reported in lung of Tsp-1–null mice after bleomycin treatment.²³ 

Together, these results show that TSP-1 is not the major activator of TGF-β1 in TPO-induced BM fibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment.

The authors thank the staff of the animal facilities directed by Patrick Gonin, in particular Benoît Petit.

This work was supported by Inserm and La Ligue Nationale contre le Cancer (équipe labellisée 2004 and 2007). S.E. was supported by Association pour la Recherche sur le Cancer (fellowship).

Contribution: S.E. and O.W.-B. performed animal studies, enzyme-linked immunosorbent assay, fluorescence in situ hybridization, and Western blotting; O.B. performed mRNA analysis; M.T. performed histologic analysis; P.R. performed reticulin fiber quantification; P.G. performed statistical analysis; E.Z. and J.P. performed angiogenesis analysis; A.B. provided TSP-1null mice; J.-L.V. provided MPZenTPO virus-producing GP + E86 cells; W.V. and S.G. designed studies and analyzed data; and O.W.-B. analyzed data, generated figures, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Orianne Wagner-Ballon, Inserm U1009, Institut Gustave Roussy, PR1, 114 rue Edouard Vaillant, 94805 Villejuif, France; e-mail: orianne.wagnerballon@hmn.aphp.fr.