Severity of Megakaryocyte-Driven Osteosclerosis in Mpig6b-Deficient Mice Is Sex-Linked

Abstract

Patients with chronic myelofibrosis often suffer from osteosclerosis, which is associated with bone pain and may lead to bone marrow failure. The pathogenesis of myelofibrosis is linked to aberrant megakaryocyte development and function. Null and loss‐of‐function mutations in MPIG6B, which codes for the inhibitory heparan sulfate receptor G6b‐B, result in severe macrothrombocytopenia, large megakaryocyte clusters, and focal primary myelofibrosis in mice and humans. We investigated the development of osteosclerosis in Mpig6b null (Mpig6b−/−) mice. Although male and female Mpig6b−/− mice presented with elevated bone marrow megakaryocyte number and macrothrombocytopenia, female Mpig6b−/− mice developed progressive splenomegaly starting at 8 weeks of age. Micro–computed tomography (μCT) of femurs showed that female Mpig6b−/− mice had increased cortical thickness and reduced bone marrow area starting at 8 weeks of age and developed occlusion of the medullary cavity by trabeculae by 16 weeks of age. In contrast, male Mpig6b−/− mice developed only a small number of trabeculae in the medullary cavity at the proximal diaphysis and demonstrated a temporary decrease in bone volume fraction and trabecular thickness at 16 weeks. Ovariectomy of 10‐week‐old female Mpig6b−/− mice prevented the development of medullary cavity osteosclerosis, whereas orchiectomy of male Mpig6b−/− mice did not exacerbate their disease. Importantly, ovariectomized female Mpig6b−/− mice also demonstrated improvement in spleen weight compared to sham‐operated Mpig6b−/− mice, establishing estrogen as a contributing factor to the severity of the megakaryocyte‐driven osteosclerosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

G6b-B regulates an essential step in megakaryocyte maturation

“…3–7 Our results demonstrate a previously unrecognized key function of G6b-B in MK maturation by regulating cell size, DMS development, receptor levels and gene expression. These findings thus challenge the previous concept, which proposed a rather unaffected development of G6b-B deficient MKs 2, 4, 8, 20 , and provide an unexpected mechanistic explanation for the severe macrothrombocytopenia in Mpig6b mut mice, as a direct consequence of an overall MK maturation defect. We observed an increased number of immature MKs in the bone marrow, which might help to better understand the cause of myelofibrosis in G6b-B null mice and patients, as an accumulation of immature MKs has been described as the main driver of this complex disease.…”

“…We thus identified a spontaneous single nucleotide mutation within Mpig6b resulting in a phenotype, which faithfully recapitulates Mpig6b -/and Mpig6b diY/F mice. 2,8,9 Analyzing BM-derived MKs from Mpig6b mut mice in vitro, we found that the number of proplatelet-forming cells was significantly reduced compared to the wildtype (WT) (Figure S2ad). The cytoskeleton of proplatelet-forming Mpig6b mut MKs, however, appeared morphologically comparable.…”

“…Absence of G6b-B led to a reduction in surface expression levels of platelet membrane glycoproteins (GPs) ( Table S1 ), infinite tail bleeding times, myelofibrosis, splenomegaly and additional osteosclerosis in female Mpig6b mut mice ( Figure S1c-f ).We thus identified a spontaneous single nucleotide mutation within Mpig6b resulting in a phenotype, which faithfully recapitulates Mpig6b −/− and Mpig6b diY/F mice. 2, 8, 9…”

“…2 Deletion of the Mpig6b locus in mice results in macrothrombocytopenia and myelofibrosis, 2 which was recently recapitulated in humans with disease-causing null-variants within MPIG6B. [3][4][5][6][7] It was hypothesized that G6b-B affects a terminal step in platelet production, 2,8 however, mechanistic insights are yet lacking. By characterizing a spontaneous Mpig6b mutant together with a newly generated Mpig6b-null mouse line we provide unexpected experimental evidence that establishes G6b-B as a central regulator for MK maturation, which can explain the complex phenotype of these mice.…”

G6b-B regulates an essential step in megakaryocyte maturation

“…[3][4][5][6][7] Based on unaltered ploidy and TPO-induced Erk1/2 activation, reduced proplatelet formation and spreading of Mpig6b -/-MKs in vitro, it was hypothesized that G6b-B does not play a role in MK development, but in a terminal step in platelet production. 2,8 However, the underlying molecular mechanism how this receptor regulates thrombopoiesis remained unknown. By characterizing a spontaneous Mpig6b mutant together with a newly generated Mpig6b-null mouse line we provide unexpected experimental evidence that establishes G6b-B as a central regulator of transcription during MK maturation, which can explain the complex phenotype of these mice.…”