Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K 553 and K 573 ). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes. (Blood. 2010;115: 1254-1263) IntroductionHematopoiesis is tightly regulated by several cytokines and growth factors to ensure that numbers of circulating blood cells remain constant under normal conditions. In many hematologic disorders, cytokine and growth factor signaling is dysfunctional, resulting in the overproduction or underproduction of 1 or more blood cell lineages. Thrombopoietin (Tpo) is a hematopoietic cytokine that, via its receptor c-Mpl, supports hematopoietic stem cell maintenance and proliferation and is the primary regulator of megakaryopoiesis. 1,2 Absence of Tpo signaling results in thrombocytopenia, reduced numbers of transplantable stem cells, and eventually aplastic anemia in humans. [3][4][5] Conversely, excessive Tpo signaling, usually due to mutations in c-Mpl or its secondary signaling proteins, results in hyperproliferation of numerous cell lineages, causing myeloproliferative syndromes. 6-8 Therefore, the control of Tpo-mediated signaling is critical in maintaining physiologic numbers of circulating blood cells.Protein phosphatases, suppressors of cytokine signaling (SOCS) proteins, and inhibitory intracellular mediators are all mechanisms that contribute to the negative regulation of cytokine signaling. [9][10][11][12] However, the process of receptor internalization and degradation is one of the quickest and most effective ways in which activated receptors are negatively regulated. We recently demonstrated a mechanism for Tpo-stimulated c-Mpl internalization, through the interaction of adaptor protein 2 with YRRL motifs located at Y 521 and Y 591 in the c-Mpl intracellular domain; elimination of these sites significantly reduced degrad...

Chronic lymphocytic leukaemia (CLL) is a clonal disorder of mature B cells. Most patients are characterised by an indolent disease course and an anergic phenotype of their leukaemia cells, which refers to a state of unresponsiveness to B cell receptor stimulation. Up to 10% of CLL patients transform from an indolent subtype to an aggressive form of B cell lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here we show that B cell-specific ablation of Nfat2 leads to the loss of the anergic phenotype culminating in a significantly compromised life expectancy and transformation to aggressive disease. We further define a gene expression signature of anergic CLL cells consisting of several NFAT2-dependent genes including Cbl-b, Grail, Egr2 and Lck. In summary, this study identifies NFAT2 as a crucial regulator of the anergic phenotype in CLL.

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation and differentiation through binding to and stimulation of the cell surface receptor c-MPL. Activating mutations in c-MPL constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-MPL that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study we focused on c-MPL tyrosine 591 (Y591). We found Y591 of wild-type c-MPL to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using an SH2/PTB domain binding microarray, we identified novel c-MPL binding partners for phosphorylated Y591, including Src homology phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through siRNA treatment of Ba/F3-MPL cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by SYK. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.

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BackgroundFew studies have been conducted to address the impact of systemic lupus erythematodes (SLE) or systemic sclerosis (SSc) on sexual function (1,2), and so far none in Germany.ObjectivesTo assess and compare sexual dysfunction (SDF) in female patients with SLE or SSc, to correlate sexual function with disease characteristics and depression, and to evaluate a short questionnaire (Qualisex) as a screening test.MethodsFemale patients with systemic lupus erythematodes or systemic sclerosis in two German tertiary university hospitals were evaluated with a self-designed questionnaire on various sexual and gynaecological aspects, a 19-item version of the Female Sexual Function Index (FSFI), the 9-item questionnaire Qualisex, and the Beck’s depression inventory in a prospective study.Results171 female patients were included into the study. Among them 83 suffered from SSc (mean age 48.50 years), and 88 from SLE (mean age 39.65 years). Organ involvement and immunosuppressive medication was frequent in both groups. 34.9% of SSc patients and 28.4% of SLE patients ever received cyclophosphamide. Disease duration was significantly longer in SLE patients (13.17 vs 9.85 years in SSc patients, p=0.021). No significant differences between SSc and SLE were found as to educational background, BDI depression categories, marital status, or number of children. Only 9.6% of SSc patients and 14.8% of SLE patients had ever discussed sexual problems with their physician, whereas 52% of all patients thought that these were relevant in relation to their disease. Significantly more SSc patients would wish to discuss sexuality with their physician more intensively (37.3% vs 28.4% in SLE patients, p=0.011). 79.5% of SSc patients and 79.4% of SLE patients were in a constant relationship; 62.6% (52 of 83) of SSc patients and 67.0% (59 of 88) of SLE patients were sexually active. Impeding factors like vaginal stenosis, sicca, impaired mobility of pelvis, or pain did not differ significantly between SSc and SLE. Among the 51 sexually active and evaluable SSc patients a mean FSFI of 25.53 (±5.06) was found, with a FSFI value below the cut off defining SDF (<26.55) in 49% of patients, which did not differ significantly compared to SLE patients (n=59, mean FSFI 26.92 (±5.17), SDF in 45.8%). More patients who suffered from SDF had at least mild depression defined by BDI than patients without SDF (71.2% vs. 46.4%, p=0.016). SSc patients showed a numerically better Qualisex than SLE patients (2.98 (±2.24) vs. 3.16 (±2.45) with 0 meaning no impairment and 10 severe impairment). The Qualisex correlated significantly with the FSFI (r=-0.367; p<0.001) with numerically better correlation in SSc patients (r=-0.407, p=0.001 vs r=-0.340, p=0.004).ConclusionSexual Dysfunction (SDF) is a frequent problem in female patients with SSc and SLE. Frequency and nature are comparable between the two entities. Addressing sexual issues during medical consultation is an unmet need. The Qualisex constitutes a short questionnaire, which is suitable for SDF screening.Refe...

Purpose of the Review Elevated levels of anti-phospholipid (aPL) antibodies are the most important criterion in the diagnosis of anti-phospholipid syndrome (APS) and are usually responsible for promoting the risk of thrombotic complications. Now, in the course of the global coronavirus disease 2019 (COVID-19) pandemic, measurable aPL antibodies have also been detected in a noticeable number of patients showing a variety ranging from studies with only isolated positive tests to cohorts with very high positivity. Thus, the question arises as to whether these two different clinical pictures may be linked. Recent Findings The ambivalent results showed a frequent occurrence of the investigated aPL antibodies in COVID-19 patients to an individually varying degree. While some question a substantial correlation according to their results, a number of studies raise questions about the significance of a correlation of aPL antibodies in COVID-19 patients. Within the scope of this review, these have now been described and compared with each other. Summary Ultimately, it is necessary to conduct further studies that specifically test aPL antibodies in a larger context in order to make subsequent important statements about the role of APS in COVID-19 and to further strengthen the significance of the described comparisons.

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