AbstractThere is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor β (PDGFRβ) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRβ signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRβ signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRβ and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRβ–PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRβ tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRβ by protein tyrosine phosphatases as endogenous PDGFRβ antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRβ-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRβ–T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRβ phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.
Primary myelofibrosis (PMF) is a malignant hematologic disorder characterized by the clonal proliferation of hematopoietic stem cells (HSC) in the bone marrow. Patients display symptoms of ineffective hematopoiesis such as anemia, thrombocytopenia and related extramedullary hematopoiesis resulting in splenomegaly. The bone marrow of PMF patients shows dysplastic megakaryocytes, neoangiogenesis and, as a central pathological feature, progressive fibrosis.1 The development of myelofibrosis is mainly ascribed to the overproduction of pro-fibrotic cytokines and growth factors by malignant immature cells of the megakaryocytic lineage. As a consequence, fibroblasts proliferate and produce extensive amounts of extracellular matrix (ECM) components, leading to impaired hematopoietic function of the bone marrow.2
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 aberrations. 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.
Platelet-derived growth factors (PDGF) have been implicated in the progression of bone marrow fibrosis.87 PDGF-A and -B, as well as PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ) expression is increased in the bone marrow of PMF patients, regardless of driver mutations.109 The PDGF system comprises five dimeric ligands: PDGF-AA, -AB, -BB, -CC and -DD.11 The two cognate transmembrane receptors, PDGFRα and PDGFRβ, dimerize upon ligand binding and cross-phosphorylate intracellular tyrosine residues. These phosphorylated residues serve as binding sites for downstream signaling components and activate, among others, phospholipase C γ (PLCγ), phosphatidyl inositol 3-kinase (PI3K), and JAK-STAT signaling.12 A large proportion of PDGF derive from megakaryocytes, from where the ligands act on their receptors in a paracrine and autocrine manner.13 Whereas PDGFRα binds PDGF-A, -B and -C, PDGFRβ can only be activated by PDGF-B and -D. Therefore, depending on the ligand dimers, PDGFRα and PDGFRβ can form homo- and, if co-expressed, heterodimers.14 Distinct functions of PDGFRα and PDGFRβ have been ascribed to the discrete, cell-type specific expression of the receptors. The receptors are predominantly expressed by cells of mesenchymal origin; within the bone marrow, PDGFRα expression is highest in megakaryocytes, whereas PDGFRβ is almost exclusively expressed in fibroblasts.109 Therefore, PDGFRβ has been attributed a major role in the proliferation of bone marrow 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 tyrosine phosphatases (PTP) dephosphorylate intracellular tyrosine residues of PDGF receptors and negatively regulate PDGF signaling.
PDGFRβ expression in activated fibroblasts correlates with the grade of myelofibrosis in the bone marrow of PMF patients.10 However, the mechanisms of transformation from malign clonal proliferation of HSC to myelofibrosis and the involvement of the PDGF system are not fully understood. In particular, the expression dynamics of PDGFRβ, 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-1 mouse model for PMF, this study concentrates on PDGFRβ and its relevance in stromal cell proliferation. These mice show reduced Gata-1 expression in megakaryocytes, which have a high proliferation rate while remaining immature and releasing reduced platelet numbers. Therefore, Gata-1 mice develop fibrosis in the bone marrow that resembles the development of myelofibrosis in PMF patients.16 For a detailed characterization, we performed whole transcriptome analyses, protein expression and localization analyses in pre-fibrotic, early fibrotic and overt fibrotic bone marrow. Furthermore, we applied a proximity ligation assay (PLA) as a novel, sensitive technique for the quantification of protein expression, interaction of PDGFRβ with its ligand PDGF-B, as well as PDGFRβ tyrosine phosphorylation and the interaction of PDGFRβ with T-cell protein tyrosine phosphatase (TC-PTP) for a detailed evaluation of PDGFRβ activation status in bone marrow fibrosis in situ. Finally, we provide evidence for the regulation of PDGFRβ by TC-PTP in fibroblasts in vitro.
Gata-1 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.
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-1 and the age-matched WT control group were determined using unpaired Student t-test. For comparison of multiple groups, analysis 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 statistically significant.
Development of myelofibrosis in the bone marrow of Gata-1low mice
We determined different stages of bone marrow fibrosis in a cohort of Gata-1 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-1 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-1 mice, we stained murine femoral bone marrow for reticulum fibers (Figure 1I). We did not observe an apparent accumulation of reticulum fibers in the bone marrow of Gata-1 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-1 mice. To monitor the induction of collagen production in the bone marrow of Gata-1 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 fibrotic bone marrow and remained increased in overt fibrotic bone marrow of 15-month-old Gata-1 mice.
Differential expression of receptor tyrosine kinases and their ligands in myelofibrosis
Since receptor tyrosine kinases (RTK) and RTK-activating ligands have been attributed an important role in myelofibrosis, we investigated transcriptomic changes by RNA sequencing (RNAseq) analyses with a focus on RTK (Figure 2A) and their cognate ligands (Figure 2B). Interestingly, a high number of RTK showed a significant increase in gene expression; among which Ptk7, Tie1, Flt1, Fgfr1 and the PDGF receptors Pdgfra and Pdgfrb. Although many ligands were not significantly regulated in early fibrotic bone marrow of Gata-1 mice, we observed an induction of Ptn, Efnb1, Pgf, Angpt2, Angpt4, Igf2 and Efna2. To classify transcriptionally up-regulated genes to their biological function, we further performed gene ontology enrichment analyses (Figure 2C). Interestingly, genes implicated in PDGF binding (Pdgfa, Pdgfb, Pdap1, Pdgfra, Pdgfrb, Col1a1, Col1a2, Col2a1, Col3a1, Col4a1, Col5a1 and Col6a1) were most over-represented within the up-regulated genes, followed by ECM structural constituents and genes referring to collagen binding.
Expression dynamics of platelet-derived growth factor signaling components during the development of myelofibrosis
Platelet-derived growth factors and their receptors are linked to bone marrow fibrosis, as was inferred from their increased expression in PMF patients.109 However, data addressing the expression dynamics during the develop ment of myelofibrosis are sparse. In order to characterize the expression pattern of PDGF signaling components at different stages of bone marrow fibrosis, we analyzed gene expression of the ligand and receptor genes in the bone marrow of Gata-1 mice by quantitative PCR (qPCR). Here, we observed that gene expression of both receptor genes Pdgfra and Pdgfrb was highly induced in early fibrotic bone marrow from 10-month-old Gata-1 mice and remained increased in overt fibrotic bone marrow of 15-month-old Gata-1 mice (Figure 3A and B). Interestingly, Pdgfrb gene expression was significantly decreased in pre-fibrotic bone marrow of 5-month-old Gata-1 mice, as also detected for Col1a1 gene expression. qPCR analyses of the ligand genes Pdgfa and Pdgfb revealed a major increase in ligand gene expression at the early fibrotic stage (Figure 3C and D). We again observed a decrease in Pdgfa gene expression in pre-fibrotic bone marrow, whereas Pdgfa gene expression was increased in early and overt fibrotic bone marrow. Pdgfb expression was significantly up-regulated only in early fibrotic bone marrow and remained at nearly baseline level in pre-fibrotic and overt fibrotic bone marrow of Gata-1 mice.
To validate the gene expression data, we further analyzed protein expression using an in situ proximity ligation assay (PLA) in a single recognition approach (Figure 3E). By this method, two oligonucleotide-coupled secondary antibodies (PLA probes) detect a single primary antibody. Through ligation, oligonucleotides are joined to a circle when in close proximity and serve as template for polymerization. A polymerase replicates the DNA circles and a concatemeric product is generated. Fluorescently-labeled nucleotides enable the detection of a rolling circle product (RCP), which can be visualized and quantified as a distinct fluorescent dot.1817 Corresponding negative controls, positive controls, and results from the quantitative analyses by proximity ligation assay (PLA) in comparison to quantified protein expression data acquired from multiplex staining are shown in Online Supplementary Figures S1-S3. Although we observed a heterogenic protein expression in Gata-1 mice, there was a steady increase in PDGF receptor protein expression during the development of myelofibrosis (Figure 3F and G; original PLA images are shown in Online Supplementary Figures S4). When analyzing PDGF-A and PDGF-B protein expression by single recognition PLA, we again observed high heterogeneity among age-matched Gata-1 mice. We did not detect a significant increase in PDGF-A expression, while PDGF-B protein expression was significantly increased in overt fibrotic bone marrow of 15-month-old Gata-1 mice (Figure 3H and I, original PLA images are shown in Online Supplementary Figures S4).
To visualize the cell type-specific expression of the PDGF signaling components, we performed multiplexed staining of PDGF signaling components in the bone marrow (Figure 4). We observed PDGFRα expression predominantly in megakaryocytes, whereas PDGFRβ marked spindle-shaped stromal cells in early and overt fibrotic bone marrow of Gata-1 mice. Staining of the ligand PDGF-A showed expression in a wide variety of different hematopoietic cells, whereas PDGF-B mainly derived from megakaryocytes, suggesting a paracrine effect of PDGF-B on PDGFRβ in bone marrow fibrosis.
Whereas megakaryocyte dysplasia and proliferation is a defining feature of PMF, fibroblast proliferation leading to a progressive fibrosis is the key pathological aspect of PMF. Given the distinct expression of PDGFRβ in stromal cells of early fibrotic and fibrotic bone marrow, we further concentrated on the dynamics of PDGFRβ and its ligand PDGF-B.
Ligand-activation and regulation of PDGFRβ during the development of myelofibrosis
The increased protein expression of PDGFRβ and its ligand PDGF-B in overt myelofibrosis prompted us to investigate the interaction of both signaling components in situ. To analyze PDGFRβ–PDGF-B binding, we performed a PLA using combined primary antibodies detecting PDGFRβ and PDGF-B (Figure 5A). The assay allows for the detection and quantification of signals and showed an increased interaction of receptor and ligand in overt fibrotic bone marrow of 15-month-old Gata-1 mice (Figure 5B, original PLA images are shown in Online Supplementary Figures S5). This is in accordance with enhanced protein expression of both PDGFRβ and PDGF-B in the bone marrow of 15-month-old Gata-1 mice and suggests an increased activation of intracellular signaling in the overt fibrotic stage. To further analyze the activation status of the receptor, we applied a PLA combining PDGFRβ and phosphotyrosine-targeting primary antibodies to analyze PDGFRβ phosphorylation in situ (Figure 5C). Surprisingly, we did not observe increased PDGFRβ tyrosine phosphorylation at any stage of myelofibrosis in Gata-1 mice (Figure 5D, original PLA images are shown in Online Supplementary Figure 5). To verify these results, we performed both an enzyme-linked immunosorbent assay (ELISA) with isolated protein from fresh frozen material and in situ staining for PDGFRβ phosphorylation at Y. In line with the PLA data, no differences in tyrosine phosphorylation were detectable between the WT animals and Gata-1 mice at all ages (data not shown). These results suggest the presence of further counter-regulatory mechanisms, such as PTP. Therefore, to investigate the contribution of the different PTP regarding the PDGFRβ phosphorylation status, we screened RNAseq data from 10-month-old Gata-1 mice and WT controls for differential expression of all classical PTP and the dual-specific phosphatase Pten. These analyses included the six PTP which are known to target PDGFRβ.2219 However, Ptpn1 (encoding PTP1B), Ptpn2 (encoding TC-PTP), Ptpn6 (encoding SHP-1), Ptpn11 (encoding SHP-2), Ptpn12 (encoding PTP-PEST), and Ptprj (encoding DEP-1) were not differentially expressed in the bone marrow of early fibrotic, 10-month-old mice (data not shown). To complement these data with gene expression analyses for Gata-1 mice of all ages, and to overcome the small sample size (n=3 mice per group) in the RNAseq analyses, we performed qPCR analyses for Ptpn1, Ptpn2, Ptpn6, Ptpn11, Ptpn12, and Ptprj. Indeed, in the gene expression analyses we observed distinct expression dynamics among gene expression of these PTP (Figure 5E-J). Overall, the data generated by qPCR from early fibrotic bone marrow of 10-month-old Gata-1 mice displayed pronounced biological variances, possibly contributing to the lack of significance within the RNAseq data. Interestingly, Ptpn1 and Ptprj gene expression, analyzed by qPCR, showed decreased expression in pre-fibrotic bone marrow. There was an increased expression of Ptpn11 and Ptpn12 in early fibrotic bone marrow. In overt fibrotic bone marrow, Ptpn2 and Ptpn11 gene expression was enhanced. Since TC-PTP (Ptpn2) has previously been ascribed an essential role in normal hematopoietic function,2423 we further focused on PDGFRβ regulation by TC-PTP. Given the conclusive increase in Ptpn2 gene expression in overt fibrotic bone marrow, we next analyzed TC-PTP protein expression in the different fibrotic stages in Gata-1 mice. Quantitative analysis of TC-PTP protein expression by PLA confirmed an increased expression in early and overt myelofibrosis (Figure 6A, original PLA images are shown in Online Supplementary Figure S5). In addition, in situ imaging showed ubiquitous expression of TC-PTP in the bone marrow of Gata-1 mice (Figure 6B). TC-PTP staining was positive in a wide variety of hematopoietic cells, megakaryocytes and in spindle-shaped cell structures, raising the question if TC-PTP directly regulates PDGFRβ in bone marrow stromal cells. To determine the interaction of PDGFRβ and TC-PTP, we again applied the sensitive proximity ligation technique (Figure 6C) and detected increased interaction of PDGFRβ and TC-PTP in early and overt fibrotic bone marrow of Gata-1 mice (Figure 6D, original PLA images are shown in Online Supplementary Figure S5).
TC-PTP in PDGFRβ signaling and proliferation in fibroblasts in vitro
Our findings of increased PDGFRβ and TC-PTP expression and interaction led us to further analyze the regulation of PDGFRβ by TC-PTP in fibroblasts in vitro. We knocked down Ptpn2 in NIH-3T3 fibroblasts and stimulated cells with PDGF-BB (Figure 7A). Transfection with Ptpn2-targeting siRNA resulted in a moderate but significant knockdown (KD) compared to cells transfected with non-targeting (NT) siRNA (Figure 7B). We observed a consecutive increase in PDGFRβ phosphorylation at Y and downstream AKT signaling in Ptpn2 KD cells (Figure 7C and F). However, there was no substantial effect on downstream ERK signaling (Figure 7G). PDGFRβ phosphorylation at Y, as well as downstream PLCγ activation, was increased in Ptpn2 KD cells (Figure 7D and E). We further monitored proliferation of Ptpn2 KD cells (Figure 7H) and did not find any evident differences in proliferation in cells cultured in complete growth medium containing 10% fetal bovine serum (FBS). However, when cells were exposed to serum-reduced medium (1% FBS), Ptpn2 KD cells showed increased growth rates compared to NT control cells.
In this study, we provide detailed analyses of the expression patterns of PDGFRβ signaling in the bone marrow of Gata-1 mice at different fibrotic disease stages using RNAseq, qPCR, in situ protein expression analyses, multiplex staining and PLA. Early and overt fibrotic bone marrow was characterized by increased expression of PDGF signaling components and overt fibrosis by an increase in PDGFRβ–PDGF-B interaction. Since PDGFRβ tyrosine phosphorylation levels were not increased, the regulation of PDGFRβ by PTP was investigated. Ptpn2 gene as well as TC-PTP protein expression was increased in fibrotic bone marrow of Gata-1 mice. Furthermore, enhanced PDGFRβ–TC-PTP interaction was observed in early and overt myelofibrosis, potentially counteracting PDGFRβ phosphorylation. Likewise, Ptpn2 KD increased PDGFRβ tyrosine phosphorylation at Y and Y and resulted in enhanced downstream AKT and PLCγ1 signaling in fibroblasts. Furthermore, Ptpn2 KD cells showed a growth condition-dependent increase in expansion rate. Thus, while PDGF signaling is differentially regulated during PMF, PTP seem novel and so far unrecognized components in disease development. Previously not applied in bone marrow, PLA might add to diagnostics as a novel technique.
Intense efforts have been made to understand the mechanisms leading to PMF, focusing on genetic analysis. However, the PMF-associated driver mutations leading to aberrant activation of JAK-STAT signaling are not unique to PMF but also occur in other MPN, namely essential thrombocythemia and polycythemia vera. Although one of the driver mutations is sufficient to induce PMF in patients, they are often accompanied by other mutations and epigenetic changes.2625 However, there is still no distinct molecular marker available for the respective MPN, emphasizing that the underlying mechanisms directing the different MPN are not yet understood. The discovery of JAK-STAT-associated mutations led to the development of JAK inhibitors and since its US Food and Drug Administration approval in 2011, the JAK1/2 inhibitor ruxolitinib has become part of combined standard therapy for PMF patients. Long-term treatment with ruxolitinib reduces spleen size and prolongs the overall survival of PMF patients.27 However, there is no improvement or reversal of bone marrow fibrosis. Furthermore, efficacy of ruxolitinib is limited by drug resistance,28 and JAK inhibition does not abrogate clonal proliferation.29 Recently, PDGFRα signaling was shown to remain active despite JAK2 inhibition in vivo and is a cell-intrinsic bypass for maintaining downstream ERK signaling upon ruxolitinib treatment.30 To date, allogeneic stem cell transplantation is the only curative treatment for PMF; however, transplantation is only suitable for a subset of high-risk patients and limited by comorbidities and donor availability.3231
Gata-1 mice were characterized by a marked thrombocytopenia, splenomegaly and progressive anemia starting before 5 months of age. Enrichment analyses of RNAseq data from early fibrotic bone marrow of Gata-1 mice revealed that genes implicated in PDGF binding are most over-represented within the up-regulated genes. We further observed enhanced PDGFRβ and PDGF-B protein expression at 15 months of age, along with an increase in PDGFRβ–PDGF-B interaction, analyzed by PLA.
However, we did not detect an increase in PDGFRβ tyrosine phosphorylation in the bone marrow of Gata-1 mice. This observation raised the question as to whether other mechanisms are involved in the regulation of the receptor in fibrotic bone marrow. Since a number of PTP have been identified, which site-selectively dephosphorylate PDGFRβ, namely PTP1B, TC-PTP, SHP-1, SHP-2, PTP-PEST and DEP-1,2219 an increased expression of these PTP might be responsible for the absence of an increased PDGFRβ phosphorylation. Indeed, our data showed distinct dynamics in gene expression of these PTP.
We observed decreased Ptpn1 and Ptprj gene expression in pre-fibrotic bone marrow in Gata-1 mice. The relevance of these PTP as potential diagnostic markers could be validated in a translational clinical approach. In contrast, Ptpn2, Ptpn11 and Ptpn12 showed an increased gene expression during the development of myelofibrosis in Gata-1 mice. Ptpn11, encoding the phosphatase SHP-2, has been described as positive regulator by mediating binding of Grb2 and thus promotes ERK signaling.3433 Therefore, interpretation of enhanced Ptpn11 expression is complex and context dependent. Ptpn12, coding for PTP-PEST, has previously been implicated in dendritic cell migration and macrophage fusion.3635 However, data on Ptpn12 involvement in bone marrow malignancies are scarce. Ptpn2 and its gene product TC-PTP, however, play a pivotal role in normal hematopoietic and stromal cell function, as emphasized by studies using TC-PTP deficient mice. Homozygous mice die 3-5 weeks after birth with severe defects in hematopoiesis.24 Transplantation experiments further suggest that TC-PTP knockout leads to changes in the bone marrow microenvironment, which impede normal HSC function.24 This supports the findings in our study, which indicate that TC-PTP expression in bone marrow stromal cells and the interaction with PDGFRβ might be important mediators of changes in the bone marrow microenvironment during the development of myelofibrosis. Ptpn2 gene expression was increased in overt fibrotic bone marrow of Gata-1 mice, implicating the importance of TC-PTP also with regard to PDGFRβ regulation. TC-PTP is ubiquitously expressed but shows strong expression in cells of the different hematopoietic lineages. We observed TC-PTP expression in hematopoietic cells, megakaryocytes as well as in stromal cells within the bone marrow. In addition to PDGFRβ, TC-PTP dephosphorylates EGFR and JAK-STAT signaling components.3837 Importantly, there are several lines of evidence showing TC-PTP is involved in a number of bone marrow alterations.392423 Ultimately, a pharmacological approach with a highly efficient modulator of TC-PTP activity in vivo will be needed to provide evidence for TC-PTP contribution to the pathogenesis of myelofibrosis, which is a limitation of our study. A direct pharmacological intervention of TC-PTP in vivo, however, is not currently available. Consistent with other studies,21 we were able to show a counter-regulation of PDGFRβ by TC-PTP in fibroblasts in vitro. Ptpn2 KD resulted in enhanced PDGFRβ tyrosine phosphorylation at Y, which serves as a binding site for PI3K.40 Conclusively, we detected an increase in downstream AKT activation as a central mediator of cell proliferation. PDGFRβ phosphorylation also activates Ras and downstream ERK signaling;41 however, we did not observe increased ERK signaling in Ptpn2 KD cells. Ptpn2 KD further led to increased PDGFRβ tyrosine phosphorylation at Y, resulting in enhanced downstream PLCγ1 activation, suggesting a possible role of downstream protein kinase C and Ca signaling.
We observed that Ptpn2 KD fibroblasts cultured in complete growth medium containing 10% FBS did not have an apparent superiority in proliferation. However, we detected increased growth rates in Ptpn2 KD cells exposed to reduced-serum media containing 1% FBS. This suggests that under conditions of high abundance of growth factor differences in proliferation in Ptpn2 KD cells are abolished, while these are apparent during serum-deprivation. Other studies using murine skin cancer models showed that TC-PTP controls proliferation and survival via AKT and STAT3 activation.4342 Furthermore, emphasizing the role of TC-PTP in hematopoietic cells, TC-PTP controls T-cell proliferation.44 Our data, based on a moderate KD, indicate that more discrete changes in TC-PTP expression controls cell growth mainly when the availability of growth components is limited.
In this study, we applied a PLA as a novel technique to analyze in situ alterations in bone marrow disease progression. The data acquired by PLA are generally in good agreement with our data acquired by multiplex staining, as another antibody-based method. While some discrepancies remained, further optimization regarding this tissue-specific approach are desired. This refers in particular to fluorescent signals, which are distinct from the clearly recognizable RCP. Those are most likely not ascribed to primary antibody binding but are caused by binding of oligonucleotides conjugated to secondary antibodies (PLA probes). Indeed, such signals have also been observed using DNA probes in in situ hybridization (FISH) approaches on bone marrow tissue and are associated with eosinophils.45 Future studies in patient material are warranted to evaluate the applicability of the PLA as a diagnostic tool in early disease stages, to monitor disease progression and response to JAK inhibition. These analytical methods should carefully consider pre-analytic processing such as specific decalcification protocols and archiving conditions for long-term storage of specimens.
In summary, PDGF signaling components display major alterations in bone marrow fibrosis. While PDGF and their cognate receptors are dynamically regulated, PTP represent previously unrecognized contributors that control PDGF signaling in myelofibrosis. As this study focused on PDGFRβ–TC-PTP interaction within the bone marrow in situ microenvironment, future examination of PDGFRβ regulation by TC-PTP in primary stromal cells from mouse models and from PMF patients will help to elucidate the precise role of TC-PTP in the development of bone marrow disease.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/8/2083
- Funding The authors would like to thank the Stiftung für Pathobiochemie und Molekulare Diagnostik for funding to KK and the Sonnenfeld Stiftung (Berlin) for a doctoral scholarship to FK.
- Received May 14, 2019.
- Accepted October 29, 2019.
- O’sullivan JM, Harrison CN. Myelofibrosis: clinicopathologic features, prognosis, and management. Clin Adv Hematol Oncol. 2018; 16(2):121-131. Google Scholar
- Tefferi A. Pathogenesis of myelofibrosis with myeloid metaplasia. J Clin Oncol. 2005; 23(33):8520-8530. PubMedhttps://doi.org/10.1200/JCO.2004.00.9316Google Scholar
- Kleppe M, Kwak M, Koppikar P. JAK- STAT pathway activation in malignant and nonmalignant cells contributes to MPN pathogenesis and therapeutic response. Cancer Discov. 2015; 5(3):316-331. PubMedhttps://doi.org/10.1158/2159-8290.CD-14-0736Google Scholar
- Baxter EJ, Scott LM, Campbell PJ. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005; 365(9464):1054-1061. PubMedhttps://doi.org/10.1016/S0140-6736(05)71142-9Google Scholar
- Pikman Y, Lee BH, Mercher T. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006; 3(7):e270. PubMedhttps://doi.org/10.1371/journal.pmed.0030270Google Scholar
- Klampfl T, Gisslinger H, Harutyunyan AS. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013; 369(25):2379-2390. PubMedhttps://doi.org/10.1056/NEJMoa1311347Google Scholar
- Caenazzo A, Pietrogrande F, Polato G. Changes in the mitogenic activity of platelet-derived growth factor(s) in patients with myeloproliferative disease. Acta Haematol. 1989; 81(3):131-135. PubMedGoogle Scholar
- Reilly JT. Pathogenesis of idiopathic myelofibrosis: role of growth factors. J Clin Pathol. 1992; 45(6):461-464. PubMedhttps://doi.org/10.1136/jcp.45.6.461Google Scholar
- Bock O, Loch G, Busche G, von Wasielewski R, Schlue J, Kreipe H. Aberrant expression of platelet-derived growth factor (PDGF) and PDGF receptor-alpha is associated with advanced bone marrow fibrosis in idiopathic myelofibrosis. Haematologica. 2005; 90(1):133-134. PubMedGoogle Scholar
- Bedekovics J, Kiss A, Beke L, Karolyi K, Mehes G. Platelet derived growth factor receptor-beta (PDGFRbeta) expression is limited to activated stromal cells in the bone marrow and shows a strong correlation with the grade of myelofibrosis. Virchows Arch. 2013; 463(1):57-65. Google Scholar
- Fredriksson L, Li H, Eriksson U. The PDGF family: four gene products form five dimeric isoforms. Cytokine Growth Factor Rev. 2004; 15(4):197-204. PubMedhttps://doi.org/10.1016/j.cytogfr.2004.03.007Google Scholar
- Heldin CH, Ostman A, Ronnstrand L. Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta. 1998; 1378(1):F79-113. PubMedhttps://doi.org/10.1016/s0304-419x(98)00015-8Google Scholar
- Wickenhauser C, Hillienhof A, Jungheim K. Detection and quantification of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) release by normal human megakaryocytes. Leukemia. 1995; 9(2):310-315. PubMedGoogle Scholar
- Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999; 79(4):1283-1316. PubMedhttps://doi.org/10.1152/physrev.19188.8.131.523Google Scholar
- Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev. 2004; 15(4):255-273. PubMedhttps://doi.org/10.1016/j.cytogfr.2004.03.006Google Scholar
- Vannucchi AM, Bianchi L, Cellai C. Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1(low) mice). Blood. 2002; 100(4):1123-1132. PubMedhttps://doi.org/10.1182/blood-2002-06-1913Google Scholar
- Soderberg O, Leuchowius KJ, Gullberg M. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods. 2008; 45(3):227-232. PubMedhttps://doi.org/10.1016/j.ymeth.2008.06.014Google Scholar
- Fredriksson S, Gullberg M, Jarvius J. Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol. 2002; 20(5):473-477. PubMedhttps://doi.org/10.1038/nbt0502-473Google Scholar
- Klinghoffer RA, Kazlauskas A. Identification of a putative Syp substrate, the PDGF beta receptor. J Biol Chem. 1995; 270(38):22208-22217. PubMedhttps://doi.org/10.1074/jbc.270.38.22208Google Scholar
- Markova B, Herrlich P, Ronnstrand L, Bohmer FD. Identification of protein tyrosine phosphatases associating with the PDGF receptor. Biochemistry. 2003; 42(9):2691-2699. PubMedhttps://doi.org/10.1021/bi0265574Google Scholar
- Persson C, Savenhed C, Bourdeau A. Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase. Mol Cell Biol. 2004; 24(5):2190-2201. PubMedhttps://doi.org/10.1128/MCB.24.5.2190-2201.2004Google Scholar
- Kappert K, Paulsson J, Sparwel J. Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation. FASEB J. 2007; 21(2):523-534. PubMedhttps://doi.org/10.1096/fj.06-6219comGoogle Scholar
- Wiede F, Chew SH, van Vliet C. Strain-dependent differences in bone development, myeloid hyperplasia, morbidity and mortality in ptpn2-deficient mice. PLoS One. 2012; 7(5):e36703. PubMedhttps://doi.org/10.1371/journal.pone.0036703Google Scholar
- You-Ten KE, Muise ES, Itie A. Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice. J Exp Med. 1997; 186(5):683-693. PubMedhttps://doi.org/10.1084/jem.186.5.683Google Scholar
- Vannucchi AM, Lasho TL, Guglielmelli P. Mutations and prognosis in primary myelofibrosis. Leukemia. 2013; 27(9):1861-1869. PubMedhttps://doi.org/10.1038/leu.2013.119Google Scholar
- Martinez-Calle N, Pascual M, Ordonez R. Epigenomic profiling of myelofibrosis reveals widespread DNA methylation changes in enhancer elements and ZFP36L1 as a potential tumor suppressor gene epigenetically regulated. Haematologica. 2019; 104(8):1572-1579. PubMedhttps://doi.org/10.3324/haematol.2018.204917Google Scholar
- Verstovsek S, Mesa RA, Gotlib J. Long-term treatment with ruxolitinib for patients with myelofibrosis: 5-year update from the randomized, double-blind, placebo-controlled, phase 3 COMFORT-I trial. J Hematol Oncol. 2017; 10(1):55. https://doi.org/10.1186/s13045-017-0417-zGoogle Scholar
- Harrison CN, Vannucchi AM, Kiladjian JJ. Long-term findings from COMFORT-II, a phase 3 study of ruxolitinib vs best available therapy for myelofibrosis. Leukemia. 2016; 30(8):1701-1707. https://doi.org/10.1038/leu.2016.148Google Scholar
- Deininger M, Radich J, Burn TC, Huber R, Paranagama D, Verstovsek S. The effect of long-term ruxolitinib treatment on JAK2p.V617F allele burden in patients with myelofibrosis. Blood. 2015; 126(13):1551-1554. PubMedhttps://doi.org/10.1182/blood-2015-03-635235Google Scholar
- Stivala S, Codilupi T, Brkic S. Targeting compensatory MEK/ERK activation increases JAK inhibitor efficacy in myeloproliferative neoplasms. J Clin Invest. 2019; 130:1596-1611. Google Scholar
- Kroger NM, Deeg JH, Olavarria E. Indication and management of allogeneic stem cell transplantation in primary myelofibrosis: a consensus process by an EBMT/ELN international working group. Leukemia. 2015; 29(11):2126-2133. PubMedhttps://doi.org/10.1038/leu.2015.233Google Scholar
- McLornan DP, Yakoub-Agha I, Robin M, Chalandon Y, Harrison CN, Kroger N. State-of-the-art review: allogeneic stem cell transplantation for myelofibrosis in 2019. Haematologica. 2019; 104(4):659-668. PubMedhttps://doi.org/10.3324/haematol.2018.206151Google Scholar
- Ronnstrand L, Arvidsson AK, Kallin A. SHP-2 binds to Tyr763 and Tyr1009 in the PDGF beta-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis. Oncogene. 1999; 18(25):3696-3702. PubMedhttps://doi.org/10.1038/sj.onc.1202705Google Scholar
- Dance M, Montagner A, Salles JP, Yart A, Raynal P. The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell Signal. 2008; 20(3):453-459. PubMedhttps://doi.org/10.1016/j.cellsig.2007.10.002Google Scholar
- Rhee I, Davidson D, Souza CM, Vacher J, Veillette A. Macrophage fusion is con-trolled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12. Mol Cell Biol. 2013; 33(12):2458-2469. PubMedhttps://doi.org/10.1128/MCB.00197-13Google Scholar
- Rhee I, Zhong MC, Reizis B, Cheong C, Veillette A. Control of dendritic cell migration, T cell-dependent immunity, and autoimmunity by protein tyrosine phosphatase PTPN12 expressed in dendritic cells. Mol Cell Biol. 2014; 34(5):888-899. PubMedhttps://doi.org/10.1128/MCB.01369-13Google Scholar
- Pike KA, Tremblay ML. TC-PTP and PTP1B: Regulating JAK-STAT signaling, controlling lymphoid malignancies. Cytokine. 2016; 82:52-57. PubMedhttps://doi.org/10.1016/j.cyto.2015.12.025Google Scholar
- Tiganis T, Bennett AM, Ravichandran KS, Tonks NK. Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase. Mol Cell Biol. 1998; 18(3):1622-1634. PubMedhttps://doi.org/10.1128/MCB.18.3.1622Google Scholar
- Bourdeau A, Trop S, Doody KM, Dumont DJ, Tremblay ML. Inhibition of T cell protein tyrosine phosphatase enhances inter-leukin-18-dependent hematopoietic stem cell expansion. Stem Cells. 2013; 31(2):293-304. PubMedhttps://doi.org/10.1002/stem.1276Google Scholar
- Panayotou G, Bax B, Gout I. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes. EMBO J. 1992; 11(12):4261-4272. PubMedGoogle Scholar
- Kashishian A, Kazlauskas A, Cooper JA. Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo. EMBO J. 1992; 11(4):1373-1382. PubMedGoogle Scholar
- Lee H, Morales LD, Slaga TJ, Kim DJ. Activation of T-cell protein-tyrosine phosphatase suppresses keratinocyte survival and proliferation following UVB irradiation. J Biol Chem. 2015; 290(1):13-24. PubMedhttps://doi.org/10.1074/jbc.M114.611681Google Scholar
- Lee H, Kim M, Baek M. Targeted disruption of TC-PTP in the proliferative compartment augments STAT3 and AKT signaling and skin tumor development. Sci Rep. 2017; 7:45077. Google Scholar
- Wiede F, La Gruta NL, Tiganis T. PTPN2 attenuates T-cell lymphopenia-induced proliferation. Nat Commun. 2014; 5:3073. PubMedhttps://doi.org/10.1038/ncomms4073Google Scholar
- Patterson S, Gross J, Webster AD. DNA probes bind non-specifically to eosinophils during in situ hybridization: carbol chromotrope blocks binding to eosinophils but does not inhibit hybridization to specific nucleotide sequences. J Virol Methods. 1989; 23(2):105-109. PubMedhttps://doi.org/10.1016/0166-0934(89)90124-9Google Scholar