The receptor for interleukin-5 (IL-5R) is composed of a unique ␣ chain (IL-5R␣) expressed on eosinophils and basophils, associated with a ␤c subunit, which is shared by the receptors for IL-3 and granulocyte macrophagecolony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1␣ or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the ␣ and ␤c subunits of the IL-5R that the membrane-proximal regions of both subunits are required for STAT activation. Interestingly, a ␤c receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the ␤c receptor is not essential for STAT3 activation.Hematopoiesis is tightly regulated by a complex network of stromal interactions and by soluble polypeptide factors named cytokines. IL-3, 1 IL-5, and GM-CSF are cytokines that have diverse effects on the proliferation, differentiation, and activation of blood cells and their precursors (1-3). Whereas IL-3 and GM-CSF also have effects on other hematopoietic lineages (3, 4), the effect of IL-5 in humans is restricted to eosinophils and basophils. IL-5 is essential for eosinophil differentiation (5, 6) and plays an important role in the function of mature eosinophils (7-11). The specific effects of IL-5 on eosinophils and basophils are due to restricted expression of the low affinity IL-5R␣ receptor on these cell types (12, 13). The high affinity receptor for IL-5 (IL-5R) is composed of a unique ␣ subunit associated with a ␤c subunit that is shared with the receptors for IL-3 and GM-CSF (14). The ␤c subunit is essential for signal transduction (15, 16), but also, the ␣ chain transduces intracellular growth signals (17). Therefore, the common use of the ␤c subunit by IL-3, IL-5, and GM-CSF explains the partial observed functional redundancy of these cytokines (16, 18). However, postreceptor signal transduction pathways are not well defined and are likely to be composed of both mitogenic and differentiation signals. It is known that the ␤c subunit, like other cytokine receptors, does not contain intrinsic tyrosine kinase activity (19,20). However, one of the earliest events to occur after IL-3, IL-5, and GM-CSF stimulation is induction of protein tyrosine phosphorylation (19,21). This tyrosine phosphorylation is caused by the activation of ...

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.

Background Nivolumab is administered in a weight-based or fixed-flat dosing regimen. For patients with non-small cell lung cancer (NSCLC), a potential exposure-response relationship has recently been reported and may argue against the current dosing strategies. The primary objectives were to determine nivolumab pharmacokinetics (PK) and to assess the relationship between drug clearance and clinical outcome in NSCLC, melanoma, and renal cell cancer (RCC). Methods In this prospective observational cohort study, individual estimates of nivolumab clearance and the impact of baseline covariates were determined using a population-PK model. Clearance was related to best overall response (RECISTv1.1), and stratified by tumor type. Results Two-hundred-twenty-one patients with metastatic cancer receiving nivolumab-monotherapy were included of whom 1,715 plasma samples were analyzed. Three baseline parameters had a significant effect on drug clearance and were internally validated in the population-PK model: gender, BSA, and serum albumin. Women had 22% lower clearance compared to men, while the threshold of BSA and albumin that led to > 20% increase of clearance was > 2.2m 2 and < 37.5 g/L, respectively. For NSCLC, drug clearance was 42% higher in patients with progressive disease (mean: 0.24; 95% CI: 0.22–0.27 L/day) compared to patients with partial/complete response (mean: 0.17; 95% CI: 0.15–0.19 L/day). A similar trend was observed in RCC, however, no clearance-response relationship was observed in melanoma. Conclusions Based on the first real-world population-PK model of nivolumab, covariate analysis revealed a significant effect of gender, BSA, and albumin on nivolumab clearance. A clearance-response relationship was observed in NSCLC, with a non-significant trend in RCC, but not in melanoma. Individual pharmacology of nivolumab in NSCLC appears important and should be prospectively studied. Electronic supplementary material The online version of this article (10.1186/s40425-019-0669-y) contains supplementary material, which is available to authorized users.

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoi- IntroductionErythropoietin (EPO) is required for the survival, proliferation, and differentiation of erythroblasts. It acts with other growth factors and hormones to balance the expansion and terminal differentiation of the erythroid compartment. For instance, stem cell factor (SCF), a ligand of c-Kit, cooperates with EPO to delay differentiation and to enhance progenitor numbers. 1,2 The EPO receptor (EpoR) is a homodimer constitutively associated with Janus tyrosine kinase 2 (Jak2). Ligand binding induces a conformational change of the EpoR dimer, 3,4 resulting in the activation of Jak2, the phosphorylation of the EpoR on 8 tyrosine residues, and the recruitment of multiple signaling molecules. [5][6][7] Mice deficient in EPO, EpoR, or Jak2 lack definitive erythropoiesis. [8][9][10][11] Although this demonstrates a crucial role for EPO-induced Jak2 activation in erythropoiesis, the contribution of individual downstream signaling pathways to erythroblast proliferation, differentiation, and survival is less clear. Notably, mice expressing a truncated EpoR, lacking all tyrosine residues, are not anemic. 12 This may implicate that additional proteins, such as docking molecules, can form a complex with the EpoR to create a signaling platform with sufficient specificity to regulate the balance of erythroid expansion and differentiation.Candidates for this function are the Grb2-associated binder (Gab) family members. At present, 3 Gab family members have been identified-Gab1, Gab2, and Gab3. 13,14 Gab1 is ubiquitously expressed, whereas Gab2 and Gab3 expression are tissue specific. In the hematopoietic system, Gab3 is expressed in B cells and T cells and in the myeloid compartment, whereas Gab2 is expressed in all hematopoietic cells except T cells. Gab1 deficiency is embryonically lethal because of placental defects; Gab1Ϫ/Ϫ embryos are also characterized by reduced liver size and migration defects of muscle precursor cells. 15,16 In contrast, Gab2 deficiency results only in mild defects in mast cell and macrophage responses, 17,18 and Gab3-deficient mice show normal hematopoiesis. 19 Gab2 and Gab3 lack the c-Met binding sequence (MBS) of Gab1 and, therefore, may not complement Gab1 in c-Met signaling. Furthermore, the high homology between family members suggests that complementation by the ubiquitously expressed Gab1 may occur in Gab2/3 knockouts. On tyrosine phosphorylation, Gab proteins recruit signaling intermediates, among them Shc (Src homology-containing protein), Shp2 (Src-homology domain containing phosphatase), p85 subunit of phosphatidylinositol 3-kinase (PI3K), and phosp...

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