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PDGFRb regulation in murine myelofibrosis
expression during the development of myelofibrosis in Gata-1low mice. Ptpn11, encoding the phosphatase SHP-2, has been described as positive regulator by mediating binding of Grb2 and thus promotes ERK signaling.33,34 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.35,36 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 defi- cient 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 PDGFRb 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-1low mice, implicating the importance of TC-PTP also with regard to PDGFRb regulation. TC-PTP is ubiquitously expressed but shows strong expression in cells of the different hematopoietic lineages. We observed TC-PTP expression in hematopoi- etic cells, megakaryocytes as well as in stromal cells with- in the bone marrow. In addition to PDGFRb, TC-PTP dephosphorylates EGFR and JAK-STAT signaling compo- nents.37,38 Importantly, there are several lines of evidence showing TC-PTP is involved in a number of bone marrow alterations.23,24,39 Ultimately, a pharmacological approach with a highly efficient modulator of TC-PTP activity in vivo will be needed to provide evidence for TC-PTP contri- bution to the pathogenesis of myelofibrosis, which is a limitation of our study. A direct pharmacological interven- tion of TC-PTP in vivo, however, is not currently available. Consistent with other studies,21 we were able to show a counter-regulation of PDGFRb by TC-PTP in fibroblasts in vitro. Ptpn2 KD resulted in enhanced PDGFRb tyrosine phosphorylation at Y751, which serves as a binding site for PI3K.40 Conclusively, we detected an increase in down- stream AKT activation as a central mediator of cell prolif- eration. PDGFRb 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 PDGFRb tyrosine phosphory- lation at Y1021, resulting in enhanced downstream PLCγ1 activation, suggesting a possible role of downstream pro- tein kinase C and Ca2+ signaling.
We observed that Ptpn2 KD fibroblasts cultured in com-
plete growth medium containing 10% FBS did not have an apparent superiority in proliferation. However, we detect- ed 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.42,43 Furthermore, emphasizing the role of TC-PTP in hematopoietic cells, TC-PTP controls T-cell proliferation.44 Our data, based on a moderate KD, indi- cate 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 progres- sion. The data acquired by PLA are generally in good agreement with our data acquired by multiplex staining, as another antibody-based method. While some discrep- ancies remained, further optimization regarding this tis- sue-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 analyt- ical methods should carefully consider pre-analytic pro- cessing 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 repre- sent previously unrecognized contributors that control PDGF signaling in myelofibrosis. As this study focused on PDGFRb–TC-PTP interaction within the bone marrow in situ microenvironment, future examination of PDGFRb regulation by TC-PTP in primary stromal cells from mouse models and from PMF patients will help to eluci- date the precise role of TC-PTP in the development of bone marrow disease.
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.
References
1. O'Sullivan JM, Harrison CN. Myelofibrosis: clinicopathologic features, prognosis, and management. Clin Adv Hematol Oncol. 2018;16(2):121-131.
2. Tefferi A. Pathogenesis of myelofibrosis with myeloid metaplasia. J Clin Oncol. 2005;23(33):8520-8530.
3. Kleppe M, Kwak M, Koppikar P, et al. JAK-
STAT pathway activation in malignant and nonmalignant cells contributes to MPN pathogenesis and therapeutic response. Cancer Discov. 2015;5(3):316-331.
4. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disor- ders. Lancet. 2005;365(9464):1054-1061.
5. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid
metaplasia. PLoS Med. 2006;3(7):e270.
6. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J
Med. 2013;369(25):2379-2390.
7. Caenazzo A, Pietrogrande F, Polato G, et al.
Changes in the mitogenic activity of platelet-derived growth factor(s) in patients with myeloproliferative disease. Acta Haematol. 1989;81(3):131-135.
8. Reilly JT. Pathogenesis of idiopathic
haematologica | 2020; 105(8)
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