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

Abstract

Transforming growth factor-␤1 (TGF-␤1) is the most important cytokine involved in the promotion of myelofibrosis. Mechanisms leading to its local activation in the bone marrow environment remain unclear. As a recent study has highlighted the role of thrombospondin-1 (TSP-1) in platelet-derived TGF-␤1 activation, we investigated the role of TSP-1 in the TPO high murine model of myelofibrosis. Two groups of engrafted mice, WT TPO high and Tsp-1-null TPO high , were constituted. All mice developed a similar myeloproliferative syndrome and an increase in total TGF-␤1 levels in the plasma and in extracellular fluids of marrow and spleen. Surprisingly, we were able to detect the active form of TGF-␤1 in Tsp-1-null TPO high mice. Accordingly, these mice developed marrow and spleen fibrosis, with intriguingly a higher grade than in WT TPO high mice. Our results show that TSP-1 is not the major activator of TGF-␤1 in TPOinduced myelofibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment. IntroductionTransforming growth factor-␤1 (TGF-␤1) 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-␤1 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-␤1 2 occurs mainly from megakaryocytes and, to a lesser extent, from monocytes. 10 Because both TGF-␤1 and TSP-1 are synthesized and stored in megakaryocyte ␣-granules, 11 abnormal and concomitant release of both molecules may hypothetically lead to pathologic local TGF-␤1 activation. As a recent study has highlighted the role of TSP-1 in platelet-derived TGF-␤1 activation, 12 we investigated the role of TSP-1 in TPO-induced myelofibrosis. MethodsWe 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-null 13 male littermates were infected with a retrovirus encoding murine TPO 9 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 ϭ 22) and Tsp-1-null/Tsp-1-null (ie, Tsp-1-null TPO high mice, n ϭ 15). Lethally irradiated hosts were engrafted with 4 to 8 ϫ 10 6 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...

Thrombospondin-1 triggers macrophage IL-10 production and promotes resolution of experimental lung injury

“…Our finding is consistent with recent observations where the absence of TSP-1 does not confer protection in models of fibrosis in which TGF-β1 is thought to be causally involved. 15,16 In both models, increased fibrosis was observed in the absence of TSP-1. 15,16 …”

“…14 It has been suggested that the impaired lung homeostasis in thbs1 −/− mice is due to a failure to activate latent TGF-β1 10,14 and, therefore, predisposing the mice to impaired tissue repair and unresolved inflammation. However, recent studies have shown that TSP-1 is not required for the activation of TGF-βl in vivo and thbs1 −/− mice are not protected from developing either pulmonary fibrosis or myelofibrosis, 15,16 both disease processes in which TGF-β1 has been implicated as a central regulator. Here, we present an alternative mechanism by which thbs1 −/− mice show impaired lung homeostasis.…”

Stromal SPARC contributes to the detrimental fibrotic changes associated with myeloproliferation whereas its deficiency favors myeloid cell expansion

“…19 Some of these proteins, such as thrombospondin-1 and osteopontin, whose role in the pathogenesis of myelofibrotic myeloid neoplasms has been postulated, share with SPARC detrimental effects on the BM stroma homeostasis related to their up-or down-regulation in hematologic malignancies. [51][52][53][54] Merging together our results from in situ expression analyses on human myeloid neoplasms and in vivo mouse models, we could hypothesize that the BM stroma response to myeloproliferative stimuli is entwined with the status of stromal SPARC expression. Increased or defective expression of SPARC in the BM stroma may exert different, yet detrimental, effects by fostering the development of fibrotic changes or inducing a flawed stromal niche permissive for the deregulated myeloid expansion, respectively.…”

The cytokines of pulmonary fibrosis: Much learned, much more to learn

“…To become functionally active, TGF-b needs to be activated from its latent state into a biologically active cytokine; such activation may occur through a spectrum of molecular processes, among which activation by aV-containing integrins plays the most important role, making them an ideal target for innovative antifibrotic therapies [108,109]. Thrombospondin is also an important activator of latent TGF-b [110,111], but is not required in all cases of tissue fibrosis, including in lung [112,113].…”