Page 108 - 2020_08-Haematologica-web

F. Kramer et al.
Abberantly activated janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling has been identified as a driver of clonal cells in PMF patients.3 Somatic mutations in JAK2,4 the thrombopoietin receptor MPL5 and CALR6 are the most prevalent genetic aberra- tions. These, however, are also frequently found in other myeloproliferative neoplasms (MPN), making a definite diagnosis of early PMF problematic. Thus, efforts are directed towards novel and valid diagnostic markers.
assay (PLA) as a novel, sensitive technique for the quan- tification of protein expression, interaction of PDGFRb with its ligand PDGF-B, as well as PDGFRb tyrosine phosphorylation and the interaction of PDGFRb with T- cell protein tyrosine phosphatase (TC-PTP) for a detailed evaluation of PDGFRb activation status in bone marrow fibrosis in situ. Finally, we provide evidence for the regu- lation of PDGFRb by TC-PTP in fibroblasts in vitro.
Methods
Mouse model
Gata-1low mice were purchased from Jackson Laboratory (Bar Harbour, ME, USA) and bred according to standards of the animal facility at the Center for Cardiovascular Research of Charité – Universitätsmedizin Berlin (Berlin, Germany). All littermates were genotyped using polymerase chain reaction (PCR) according to the standard protocol provided by Jackson. Hemizygous males and age-matched wild-type (WT) littermates were euthanized at 5, 10 and 15 months of age.
Further material and methods used in this study are described in the Online Supplementary Appendix.
Statistical analysis
Results presented as boxplots show the median, with whiskers representing minima and maxima; bar graphs show mean and standard deviation. Statistical differences between a Gata-1low and the age-matched WT control group were determined using unpaired Student t-test. For comparison of multiple groups, analy- sis of variance with post hoc Tukey correction was used. Statistical analyses were performed using GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA). P<0.05 was considered sta- tisticallysignificant.
Results
Development of myelofibrosis in the bone marrow of Gata-1low mice
We determined different stages of bone marrow fibrosis in a cohort of Gata-1low mice, which served as time points for all subsequent analyses: 5 months, 10 months and 15 months of age. Mice of all ages were normal in body weight (Figure 1A). As early as 5 months of age, Gata-1low mice showed a pronounced splenomegaly (Figure 1B), whereas liver weight remained normal at all ages (Figure 1C). Mice displayed time-dependent, progressive anemia (Figure 1D) and a slight increase in leukocytes starting at month 10 (Figure 1E). Platelets were markedly reduced at all ages (Figure 1F). To determine fibrotic stages in Gata- 1low mice, we stained murine femoral bone marrow for reticulum fibers (Figure 1I). We did not observe an appar- ent accumulation of reticulum fibers in the bone marrow of Gata-1low mice at month 5, whereas there was a time- dependent increased deposition of fibers at month 10 and 15. Hence, month 5 was defined as a pre-fibrotic, month 10 as an early fibrotic, and month 15 as an overt fibrotic stage in Gata-1low mice. To monitor the induction of colla- gen production in the bone marrow of Gata-1low mice, we analyzed type I collagen Col1a1 and type III collagen Col3a1 gene expression by qPCR (Figure 1G and H). We observed a significant decrease in Col1a1 gene expression in the pre-fibrotic stage. However, a marked increase in gene expression of the two collagens was detected in early
Platelet-derived growth factors (PDGF) have been implicated in the progression of bone marrow fibrosis.7,8 PDGF-A and -B, as well as PDGF receptor a (PDGFRa) and PDGF receptor b (PDGFRb) expression is increased in the bone marrow of PMF patients, regardless of driver mutations.9,10 The PDGF system comprises five dimeric ligands: PDGF-AA, -AB, -BB, -CC and -DD.11 The two cognate transmembrane receptors, PDGFRa and PDGFRb, dimerize upon ligand binding and cross-phos- phorylate intracellular tyrosine residues. These phospho- rylated residues serve as binding sites for downstream signaling components and activate, among others, phos- pholipase C γ (PLCγ), phosphatidyl inositol 3-kinase (PI3K), and JAK-STAT signaling.12 A large proportion of PDGF derive from megakaryocytes, from where the lig- ands act on their receptors in a paracrine and autocrine manner.13 Whereas PDGFRa binds PDGF-A, -B and -C, PDGFRb can only be activated by PDGF-B and -D. Therefore, depending on the ligand dimers, PDGFRa and PDGFRb can form homo- and, if co-expressed, het- erodimers.14 Distinct functions of PDGFRa and PDGFRb have been ascribed to the discrete, cell-type specific expression of the receptors. The receptors are predomi- nantly expressed by cells of mesenchymal origin; within the bone marrow, PDGFRa expression is highest in megakaryocytes, whereas PDGFRb is almost exclusively expressed in fibroblasts.9,10 Therefore, PDGFRb has been attributed a major role in the proliferation of bone mar- row stromal cells in myelofibrosis. Different mechanisms are involved in the regulation of PDGF signaling, e.g. injury and pro-inflammatory cytokines affect expression of the ligands and receptors.15 Furthermore, protein tyro- sine phosphatases (PTP) dephosphorylate intracellular tyrosine residues of PDGF receptors and negatively regu- late PDGF signaling.
PDGFRb expression in activated fibroblasts correlates with the grade of myelofibrosis in the bone marrow of PMF patients.10 However, the mechanisms of transforma- tion from malign clonal proliferation of HSC to myelofi- brosis and the involvement of the PDGF system are not fully understood. In particular, the expression dynamics of PDGFRb, the interaction with the ligand PDGF-B, and a possible regulation by PTP during the development of bone marrow fibrosis have not been thoroughly addressed. Using the Gata-1low mouse model for PMF, this study concentrates on PDGFRb and its relevance in stro- mal cell proliferation. These mice show reduced Gata-1 expression in megakaryocytes, which have a high prolif- eration rate while remaining immature and releasing reduced platelet numbers. Therefore, Gata-1low mice develop fibrosis in the bone marrow that resembles the development of myelofibrosis in PMF patients.16 For a detailed characterization, we performed whole transcrip- tome analyses, protein expression and localization analy- ses in pre-fibrotic, early fibrotic and overt fibrotic bone marrow. Furthermore, we applied a proximity ligation
2084
haematologica | 2020; 105(8)