C‐Cbl regulates c‐MPL receptor trafficking and its internalization

Abstract Thrombocyte formation from megakaryocyte and their progenitor cells is tightly regulated by thrombopoietin (TPO) and its receptor c‐MPL, thereby maintaining physiological functionality and numbers of circulating platelets. In patients, dysfunction of this regulation could cause thrombocytopenia or myeloproliferative syndromes. Since regulation of this pathway is still not completely understood, we investigated the role of the ubiquitin ligase c‐Cbl which was previously shown to negatively regulated c‐MPL signalling. We developed a new conditional mouse model using c‐Cblfl/flPf4Cre mice and demonstrated that platelet‐specific knockout of c‐Cbl led to severe microthrombocytosis and impaired uptake of TPO and c‐MPL receptor internalization. Furthermore, we characterized a constitutive STAT5 activation c‐Cbl KO platelets. This study identified c‐Cbl as a potential player in causing megakaryocytic and thrombocytic disorders.


| INTRODUC TI ON
During hematopoiesis, the development of bone marrow progenitor cells into mature blood cells and maintenance of physiological numbers of circulating blood cells are tightly regulated by several cytokines and growth factors. In this context, thrombopoietin (TPO) and its corresponding receptor c-MPL play a central role during thrombopoiesis, the developmental process of thrombocyte formation from megakaryocyte (MK) and their progenitor cells. 1,2 Disruption of TPO signalling results in thrombocytopenia, reduction of hematopoietic stem cells and can even lead to a state of complete bone marrow failure, called aplastic anaemia. [3][4][5] In contrast, uncontrolled TPO signalling due to, for example, mutations in c-MPL or its downstream signalling proteins results in a hyperproliferative phenotype, which causes myeloproliferative syndromes. [6][7][8] Therefore, a tight control of TPO-mediated signalling is necessary to maintain physiological hematopoiesis.
The proto-oncogene c-CBL (casitas B cell lymphoma) is involved in ubiquitination of the TPO receptor c-MPL and thereby negatively regulating TPO signalling. 9 C-Cbl is expressed in hematopoietic cells and MKs and is part of the Cbl protein family, which carry evolutionary conserved RING finger domains and show E3 ubiquitin ligase activity. Ubiquitination of target proteins by c-Cbl is induced by its phosphorylation in response to TPO stimulation. [10][11][12][13][14] In case of losing the negative regulation of the TPO signalling, c-Cbl null mice show a clear expansion in hematopoietic stem/ progenitor cell numbers as well as being hyperproliferative and sensitive to TPO stimulation which induces STAT5 signalling. 15 In patients with myeloproliferative disorders such as essential thrombocythemia (ET), studies have shown an impaired platelet-dependent TPO clearance, suggesting that thrombocytosis in ET may be attributed to an alteration in TPO signalling via its receptor c-MPL. 16 Here, we report on a new genetic model to specifically delete c-Cbl in the MK lineage by using a conditional knockout strain with a platelet and MK specific Pf4 cre . C-Cbl fl/fl Pf4 Cre mice present with a microthrombocytosis and an accelerated platelet turnover along with faster platelet recovery after platelet depletion. The number of MKs was elevated along with the number of their progenitor cells in the bone morrow, without signs of myelofibrosis. Interestingly, there was no difference in spleen size compared to wild-type (WT) mice.
TPO receptor c-MPL surface expression was reduced on platelets of c-Cbl fl/fl Pf4 Cre mice while showing impaired internalization and markedly reduced ability to take up TPO, resulting in elevated TPO serum levels in these mice.

| Mice
Mice bearing a conditional floxed allele of c-Cbl (Cbl fl/fl ) C57BL/6-Cbl tm1Dejs (Australian Phenomics Facility) were crossed with Pf4 Cre mice were obtained from Jackson Laboratory (stock #008535) to obtain c-Cbl fl/fl Pf4 Cre . Homozygous c-Cbl fl/fl Pf4 Cre (KO) mice with megakaryocytic deletion of c-Cbl were used as the experimental cohort, while c-Cbl fl/fl (WT) mice without c-Cbl deletion served as controls. All mice were age-and sex-matched and were killed at the indicated time-points by overdosing inhalation anaesthesia.
Mice were maintained under specific pathogen-free conditions.
All animal experiments were performed with the authorization of the Institutional Animal Care and Use Committee of the University of Tübingen according to German federal and state regulations.

| Western blot
Protein lysates were isolated from purified B cells, platelets or in vitro generated MKs with NP-40 buffer (Thermo Scientific) containing 1 mmol/L PMSF (Sigma-Aldrich) and PI cocktail (Thermo Scientific) for 30 minutes on ice followed by 10 minutes centrifugation at 15 000 g. The supernatant was used for SDS-PAGE followed by Western blot analysis. Antibodies for c-CBL (#2747, 1:1000), Actin (8H10D10, 1:5000) (all from Cell Signaling) and c-MPL (#06-944; Merck) and horseradish peroxidase-coupled goat or swine anti-rabbit secondary antibody (Southern Biotech) were used.

| qRT-PCR
RNA was isolated from in vitro generated MKs and liver pellets using RNeasy plus mini kit (Qiagen) followed by reverse transcription (SuperScript II; Invitrogen). Quantitative PCR was performed

| Platelet analysis
Retroorbital blood was collected, and whole blood analysis was Blood count (cells/µL) Abbreviations: HGB, haemoglobin; MPV, mean platelet volume; PLT, platelets; RBC, red blood count; WBC, white blood count. a Statistical analysis with Student's t test.
b Statistical analysis with Mann-Whitney test.  Mann-Whitney test. An unpaired analysis of variance (ANOVA) was used to analyse the differences among group means. P-value of P < .05 (*) was used as cut-off for significance.

| c-Cbl knockout leads to microthrombocytosis and lymphocytosis
To determine the role of c-Cbl in thrombopoiesis, we generated c-Cbl fl/fl Pf4 Cre mice specifically lacking c-Cbl in the MK lineage ( Figure 1A). While isolated B cells exhibited normal c-CBL expression, knockout of c-CBL protein in MKs and platelets (Plts) was con-  The MKs in the c-Cbl fl/fl Pf4 Cre animals were slightly larger and hyperlobated ( Figures 2D and S4).

| c-Cbl fl/fl Pf4 Cre mice exhibit increased thrombopoiesis
After assessment of megakaryopoiesis, we further characterized the   (Table 2). showed severe defects in receptor internalization ( Figure 4H).

| D ISCUSS I ON
In   Internalization of TPO after binding and stimulation to c-MPL is an important method for regulating plasma TPO levels. [33][34][35][36] Interestingly, the R102P c-MPL mutation is known to lead to a loss In contrast to the autoregulation model of sera TPO, these levels are lower than expected in patients with immune thrombocytopenia 35,41 and higher in patients with ET. 42 Our data support a model that such disorders may be, in part, [Correction added on 09 November 2011 , after first online publication: Projekt DEAL funding statement has been added.]

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.