Mer Expression Research Articles

Mer activation ameliorates nerve injury-induced neuropathic pain by regulating microglial polarization and neuroinflammation via SOCS3 in male rats.

Accumulating evidence has demonstrated that M1 microglial polarization and neuroinflammation worsen the development of neuropathic pain. However, the mechanisms underlying microglial activation during neuropathic pain remain incompletely understood. Myeloid-epithelial-reproductive tyrosine kinase (Mer), which is a member of the Tyro-Axl-Mer (TAM) family of receptor tyrosine kinases, plays a crucial role in the regulation of microglial polarization. However, the effect of Mer on microglial polarization during neuropathic pain has not been determined. In this study, western blotting, immunofluorescence analysis, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA) were used to examine the role of Mer in pain hypersensitivity and microglial polarization in rats with chronic constriction injury (CCI) of the sciatic nerve. The results indicated that Mer expression in microglia was prominently increased in the spinal cords of rats subjected to CCI. Furthermore, treatment with recombinant protein S (PS, an activator of Mer) alleviated mechanical allodynia and thermal hyperalgesia, promoted the switch in microglia from the M1 phenotype to the M2 phenotype, and ameliorated neuroinflammation in rats subjected to CCI. However, the use of suppressor of cytokine signalling 3 (SOCS3) siRNA abolished these changes. These results indicated that Mer regulated M1/M2 microglial polarization and neuroinflammation and may be a potential target for treating neuropathic pain.

Phosphorylation of Ack1 by the Receptor Tyrosine Kinase Mer.

Ack1 is a nonreceptor tyrosine kinase that is associated with cellular proliferation and survival. The receptor tyrosine kinase Mer, a member of the TAM family of receptors, has previously been reported to be an upstream activator of Ack1 kinase. The mechanism linking the two kinases, however, has not been investigated. We confirmed that Ack1 and Mer interact by co-immunoprecipitation experiments and found that Mer expression led to increased Ack1 activity. The effect on Ack1 was dependent on the kinase activity of Mer, whereas mutation of the Mer C-terminal tyrosines Y867 and Y924 did not significantly decrease the ability of Mer to activate Ack1. Ack1 possesses a Mig6 Homology Region (MHR) that contains adjacent regulatory tyrosines (Y859 and Y860). Using synthetic peptides, we showed that Mer preferentially binds and phosphorylates the MHR sequence containing phosphorylated pY860, as compared to the pY859 sequence. This suggested the possibility of sequential phosphorylation within the MHR of Ack1, as has been observed previously for other kinases. In cells co-expressing Mer and Ack1 MHR mutants, the Y859F mutant had higher activity than the Y860F mutant, consistent with this model. The interaction between Mer and Ack1 could play a role in immune cell signaling in normal physiology and could also contribute to the hyperactivation of Ack1 in prostate cancer and other tumors.

Вариабельность ответа костно-минерального обмена у женщин с хирургической менопаузой на менопаузальную гормональную терапию

Patients with surgical menopause rapidly develop signs of osteopenia and osteoporosis. Hormone replacement therapy (HRT) for menopause increases bone mineral density (BMD). However, the positive metabolic effects of HRT vary widely. The variability of response to HRT may depend on the sensitivity of these cells to steroid hormones, i.e., the level of steroid receptors in them. An indirect indicator of the sensitivity of osteoclasts to HRT components can be the level of transcription of estradiol and progesterone receptors in peripheral blood mononuclear cells (PBMCs). Objective. To determine the transcriptome of PBMCs in patients with surgical menopause against the background of HRT using a combination of 1 mg estradiol and 5 mg dydrogesterone in cyclic regimen and to study its effect on BMD. Patients and methods. A total of 16 women aged 40–55 years with a duration of surgical menopause ranging from 12 months to 6 years were examined. An inclusion criterion for this study was the absence of contraindications for HRT. The subject of the study was determination of BMD by dual-energy X-ray absorptiometry and expression levels of estradiol and progesterone receptor genes by polymerase chain reaction (PCR). Results. Comparative analysis of estrogen and progesterone gene expression in PBMCs revealed a difference in gene expression: a 2-fold decrease in membrane estradiol receptor (mER) expression (p = 0.0420), a 2-fold decrease in nuclear estradiol receptor (ERβ) expression (p = 0.0295), and a 3-fold decrease in nuclear progesterone receptor (PR-B) expression (p = 0.0480) in PBMCs of patients in the group with no HRT effect on BMD. Conclusion. Estradiol (mER and ERβ) and progesterone (PR-B) receptor mRNA levels in PBMCs of women with surgical menopause may serve as a prognostic marker of treatment efficacy. Key words: menopause, hormone replacement therapy, bone mineral density, peripheral blood mononuclear cells, dydrogesterone, estradiol

MER Signaling Axis: A Novel Therapeutic Target in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues with a highly variable disease course. B-cell receptor (BCR)-signal inhibitors, specifically Ibrutinib which targets Bruton's tyrosine kinase (BTK) and Idelalisib which targets PI3Kδ, are effective in relapsed/refractory CLL patients. Despite BCR-signal-inhibitors are changing the management of CLL, these agents are not curative and resistance can develop, often leading to the transformation of CLL into more aggressive B-cell lymphomas limiting their therapeutics options. Thus, improved understanding of the nature and extent of other active receptor tyrosine kinase (RTK) signals in CLL cells is critical for development of additional therapies to treat these high-risk patients. Our recent discovery of MER, a RTK of the TAM (Tyro3, AXL, MER) family, in CLL cells may offer such an option. In order to analyze MER expression status in CLL cells from previously untreated CLL patients, we employed western blot, flow cytometry and qRT-PCR analyses. We detected low to high level expression of MER in CLL cells from ~50% of CLL patients as compared to normal, purified B cells. We also found that MER in CLL cells remained constitutively phosphorylated (active). Furthermore, our preliminary analysis suggested that MER expression in CLL cells may be positively associated with high-risk, unmutated IGHV CLL patients. In vitro ligation of Gas6 (growth arrest-specific gene 6), a specific ligand for all the TAM receptors, robustly activated MER as compared to AXL or Tyro3. Interestingly, activation of MER preferentially activated Erk1/2, NF-κB, and BTK of the BCR pathway in CLL cells. Partial depletion of MER in CLL cells using a specific-siRNA resulted in inhibition of P-Erk1/2, P-NF-κB and P-BTK, but not P-AKT, suggesting that MER may crosstalk with the BCR signaling pathway in CLL cells likely via interaction with CD79a, LYN or BTK. To address this hypothesis, we immunoprecipitated MER from CLL cell lysates and found a direct complex formation between MER and LYN. We also analyzed leukemic B cells from the Eµ-TCL1 transgenic mice which recapitulate highly aggressive human CLL for the expression of MER by western blot/flow cytometry. Results showed robust expression of MER in TCL1 leukemic B cells purified from mice with advanced CLL. Finally, treatment of CLL mice with a high-affinity MER inhibitor (UNC-2025) via oral gavage significantly reduced leukemic tumor burden. In total, our findings suggest that expression/activation of MER may potentiate CLL cell survival via a crosstalk with the BCR signal thus, could be an attractive therapeutic target for the progressive CLL patients.

Inhibition of Mer exacerbates early brain injury by regulating microglia/macrophage phenotype after subarachnoid hemorrhage in mice

BackgroundPolarization of microglia/macrophages toward the pro-inflammatory phenotype is a crucial contributor to neuroinflammation after subarachnoid hemorrhage (SAH). Mer belongs to the TAM receptor tyrosine kinases family, which is known to play a significant role in the resolution of inflammation. However, the effect and mechanism of Mer after SAH remain unclear. In this study, we explored the effect of Mer on modulating the microglia/macrophage phenotype and neuroinflammation and possible potential mechanism after SAH. Method: Endovascular perforation model of SAH was performed. There are 3 parts in this study. Firstly, the time course of Mer expression was determined within 72 hours after SAH. Secondly, the effect of Mer downregulation on brain water content, neurological function, and microglial polarization was evaluated at 24 h after SAH. Thirdly, the neuroprotective effects of pharmacological Mer agonist were assessed. Result: The expression of Mer increased after SAH, and was prominently localized in microglia/macrophages. Treatment with Mer siRNA increased pro-inflammatory phenotype and decreased anti-inflammatory phenotype of microglia/macrophage, thus resulted in exacerbation of neurological deficits and brain edema after SAH. Mechanistically, the downregulation of Mer inhibited the downstream anti-inflammatory signals, SOCS1/SOCS3, by decreasing phosphorylated STATs. Conclusion: Mer is involved in the microglia/macrophage polarization and inflammation resolution after SAH, and that mechanism, at least in part, may contribute to the involvement of the STATs/SOCSs pathway.

Ulinastatin promotes macrophage efferocytosis and ameliorates lung inflammation via the ERK5/Mer signaling pathway.

Acute lung injury (ALI) is a pneumonic response characterized by neutrophil infiltration. Macrophage efferocytosis is the process whereby macrophages remove apoptotic cells, and is required for ALI inflammation to subside. The glycoprotein ulinastatin (UTI) has an anti‐inflammatory effect during the acute stages of ALI, but its effect on efferocytosis and the subinflammatory stage of ALI is unclear. Extracellular signal‐regulated kinase 5 (ERK5) is a key protein in efferocytosis, and we thus hypothesized that it may be activated by UTI to regulate efferocytosis and the resolution of pneumonia. To test this hypothesis, here we monitored phagocytosis of macrophages through in vivo and in vitro experiments. Pulmonary edema, neutrophil infiltration, protein exudation, and inflammatory factor regression were observed on days 1, 3, 5, and 7 in vivo. RAW264.7 cells were pretreated with different concentrations of UTI and ERK5 inhibitors, and the expression of tyrosine‐protein kinase Mer (Mer) protein on macrophage membrane was detected. UTI increased the phagocytosis of apoptotic neutrophils by macrophages in vitro and in vivo, and promoted the resolution of pneumonia. The protein expression of ERK5 and Mer increased with UTI concentration, while the expression of Mer was down‐regulated by ERK5 inhibitors. Therefore, our results suggest that UTI enhances efferocytosis and reduces lung inflammation and injury through the ERK5/Mer signaling pathway, which may be one of the targets of UTI in the treatment of lung injury.

Abstract 3642: The aged lung promotes reactivation from metastatic melanoma dormancy

Abstract Our previous data have indicated that while tumors grow more slowly in aged mouse skin, they form more lung metastases. We queried whether the lung microenvironment undergoes age-related changes that provide a permissive niche for metastatic outgrowth. Our data showed that secreted factors by lung fibroblasts changed during aging that promoted proliferation of melanoma cells via expression of the tyrosine kinase receptor MER. Further investigation of this MER-high phenotype revealed that these melanoma cells induce dramatic changes to the lung microenvironment via secretion of its primary ligand PROS1, which creates a more permissive niche for metastasis. It first acts in a paracrine manner to induce proliferation of previously dormant melanoma cells in the lung. MER induced secretion of PROS1 by melanoma cells also regulates the production of extracellular matrix (ECM) from lung fibroblasts. Specifically, healthy human lung fibroblasts produce thick, unified cellular derived matrices (CDMs) in vitro that are growth-restrictive to melanoma cells. Treatment of lung fibroblasts with conditioned media from MER-High melanoma cells or with recombinant-PROS1 alters CDM density and orientation, which resulted in a CDM that dramatically increased melanoma proliferation. Finally, we found that MER induced metastatic outgrowth of melanoma cells within the lungs of mice decreased CD4 and CD8 T-cells whilst increasing infiltration of and immunosuppressive T-regulatory cells (Tregs) and Myeloid Derived Suppressor Cells (MDSCs). Overall, we find that age-induced activation of melanoma cells towards a MER-high state within the lung can induce a cancer phenotype which dramatically alters fibroblast ECM production and immune cell infiltration to promote aggressive and efficient metastatic outgrowth and a growth permissive microenvironment for dormant melanoma cells. Citation Format: Mitchell Fane, Ashani Weeraratna. The aged lung promotes reactivation from metastatic melanoma dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3642.

Новая стратегия поиска гормональной терапии пролиферативных процессов эндометрия у пациенток в постменопаузе

The limited efficacy of hormone therapy for endometrial proliferative process (EPP) in postmenopausal patients and its side effects on the immune system functionalities have not been studied in detail. Here we assess the feasibility of hormone therapy for EPP in postmenopausal patients through evaluation of estradiol and progesterone receptor gene expression in endometrial tissue and peripheral blood mononuclear cells (PBMC). The study enrolled 92 postmenopausal patients with EPP, including 37 pts with glandular-fibrous polyps, 7 pts with non-atypical endometrial hyperplasia (EH), 8 pts with atypical endometrial hyperplasia (AEH), 31 pts with moderately differentiated adenocarcinoma and 9 pts with highly differentiated adenocarcinoma. The PBMC isolates and endometrial samples were tested for ER⍺, ERβ, mER, PRA, PRB, mPR and PGRmC1 expression by reverse real time polymerase chain reaction (RT–PCR). Differential changes in PBMC receptor profiles upon in vitro exposure to progesterone or mifepristone were determined for patients with endometrial polyps and healthy women. The results indicate elevated expression of ERα, ERβ, PRA, PRB, mPR and PGRmC1 by endometrial tissues in EH and elevated expression of mER, ER⍺ and PRA by PBMC in AEH, apparently reflecting suppressed functionalities of monocytes, macrophages, Т-cells and natural killer cells. Unaltered expression of the studied genes by PBMC in endometrial adenocarcinoma may reflect the incrementing tumor autonomy. In vitro, mifepristone inhibited ER⍺, ERβ, mPR, PGRmC1, PRA and PRB expression in PBMC isolated from patients with endometrial polyps. We suppose that such effects can mitigate the negative influence of sex steroid hormones on immunocompetent cells.

Phagocytosis promotes plaque

Neurodegeneration Many of the human genes associated with increased risk for Alzheimer's disease (AD) are exclusively expressed in the brain by microglia. Microglia express the tumor-associated macrophage (TAM) receptor tyrosine kinases Axl and Mer, which have been linked to AD. Huang et al. performed molecular, genetic, cell biological, and in vivo two-photon imaging analyses of AD mouse models crossed into Axl- and Mertk-knockout mouse mutants. Induced expression of Axl and Mer in plaque-associated microglia led to the recruitment of the TAM ligand Gas6 and to the formation of dense accumulations of phagocytosed amyloid within the microglia. An AD mouse model lacking Axl and Mer did not phagocytose amyloid as normal. Despite this, the mice developed fewer plaques than AD mice with normal microglia. Thus, TAM receptor signaling is required for microglial recognition of amyloid plaques, and, counterintuitively, TAM-driven microglial phagocytosis promotes plaque deposition and growth. Nat. Immunol. 22 , 586 (2021).

Microglia use TAM receptors to detect and engulf amyloid β plaques.

Two microglial TAM receptor tyrosine kinases - Axl and Mer - have been linked to Alzheimer’s disease, but their roles in disease have not been tested experimentally. We find that in Alzheimer’s disease and its mouse models, induced expression of Axl and Mer in amyloid plaque-associated microglia was coupled to induced plaque decoration by the TAM ligand Gas6 and its co-ligand phosphatidylserine. In the APP/PS1 mouse model of Alzheimer’s disease, genetic ablation of Axl and Mer resulted in microglia that were unable to normally detect, respond to, organize, or phagocytose amyloid beta plaques. These major deficits notwithstanding, TAM-deficient APP/PS1 mice developed fewer dense-core plaques than APP/PS1 mice with normal microglia. Our findings reveal that the TAM system is an essential mediator of microglial recognition and engulfment of amyloid plaques, and that TAM-driven microglial phagocytosis does not inhibit, but rather promotes, dense-core plaque development.

Efferocytosis Modulates Arginase-1 and Tyrosine Kinase Mer Expression in GM-CSF-Differentiated Human Macrophages.

We studied the expression of arginase-1 (Arg1) and tyrosine kinase Mer (MerTK) in GMCSF-differentiated human macrophage populations М0, М1(IFNγ), М2а(IL-4), and М2(low serum) generated under conditions of growth/serum factor deficiency. The maximum relative content of Arg1+ and MerTK+ cells was found in М2 macrophage populations: М2а(IL-4) and М2(low serum). As the uptake of apoptotic cells is the key mechanism of M2 polarization during M2(low serum) generation, we performed a special series of experiments and showed that incubation with allogeneic apoptotic neutrophils significantly increased the percentages of CD206+ macrophages co-expressing Arg1 and MerTK.

Rapamycin Alleviates the Symptoms of Multiple Sclerosis in Experimental Autoimmune Encephalomyelitis (EAE) Through Mediating the TAM-TLRs-SOCS Pathway.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Our research aimed to find an immunomodulatory therapy for MS. An experimental autoimmune encephalomyelitis (EAE) mouse model of MS was established induced with the syntheticmyelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55). Fifty C57BL/6 mice were randomly divided into the Normal group, EAE group, and Rapamycin group (EAE mice treated with three different doses of rapamycin). Hematoxylin and eosin staining and Weil myelin staining were performed on the brain tissues of mice after 21 days post-immunization. The protein expression of Gas6, Tyro3, Axl, Mer in paraventricular tissues were analyzed by immunohistochemistry. The mRNA and protein expression of Gas6, Tyro3, Axl, Mer, SOCS1, SOCS3, Toll-like receptor (TLR) 3, and TLR4 were detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. An enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory factors IFN-γ and IL-17. Rapamycin treatment could ameliorate the behavior impairment in EAE mice induced by MOG35-55. The expression of Gas6, Tyro3, Axl, Mer, SOCS1, and SOCS3 were decreased in EAE mice at 21 days post-immunization, while the expression of Gas6, Tyro3, Axl, and Mer in rapamycin group was higher than that in EAE group. It was accompanied by an increase in anti-inflammatory proteins SOCS1 and SOCS3, a decrease in the inflammatory proteins TLR-3, TLR-4 and in the amount of IFN-γ, and IL-17. Rapamycin injection relieved the nerve function of and the loss of myelin sheath in the EAE mice, mainly through mediating the TAM-TLRs-SOCS signaling pathway to regulate natural immunity.

Inhibition of adjuvant-induced TAM receptors potentiates cancer vaccine immunogenicity and therapeutic efficacy.

Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10+ DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8+ T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.

Crystal Structure of the Kinase Domain of MerTK in Complex with AZD7762 Provides Clues for Structure-Based Drug Development.

Aberrant tyrosine-protein kinase Mer (MerTK) expression triggers prosurvival signaling and contributes to cell survival, invasive motility, and chemoresistance in many kinds of cancers. In addition, recent reports suggested that MerTK could be a primary target for abnormal platelet aggregation. Consequently, MerTK inhibitors may promote cancer cell death, sensitize cells to chemotherapy, and act as new antiplatelet agents. We screened an inhouse chemical library to discover novel small-molecule MerTK inhibitors, and identified AZD7762, which is known as a checkpoint-kinase (Chk) inhibitor. The inhibition of MerTK by AZD7762 was validated using an in vitro homogeneous time-resolved fluorescence (HTRF) assay and through monitoring the decrease in phosphorylated MerTK in two lung cancer cell lines. We also determined the crystal structure of the MerTK:AZD7762 complex and revealed the binding mode of AZD7762 to MerTK. Structural information from the MerTK:AZD7762 complex and its comparison with other MerTK:inhibitor structures gave us new insights for optimizing the development of inhibitors targeting MerTK.

Gas6 Inhibits Toll-Like Receptor-Mediated Inflammatory Pathways in Mouse Microglia via Axl and Mer.

Background: Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders. These cells survey the CNS and, under inflammatory conditions, become “activated” through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. In the present study, we examined the roles of the related TAM receptors, Mer and Axl, and of their ligand, Gas6, in the regulation of microglial pro-inflammatory TNF-α production and microglial morphology.Methods: Primary cultures of murine microglia of wild-type (WT), Mer−/− and Axl−/− backgrounds were stimulated by the TLR4 agonist, lipopolysaccharide (LPS) with or without pre-treatment with Gas6. Gene expression of TNF-α, Mer, and Axl was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) was used to measure TNF-α release from microglia. Immunofluorescence staining of β-actin and the microglial marker Iba1 was performed to reveal microglial morphological changes, with cellular characteristics (area, perimeter, Feret’s diameter, minimum Feret, roundness, and aspect ratio) being quantified using ImageJ software.Results: Under basal conditions, TNF-α gene expression was significantly lower in Axl−/− microglia compared to WT cells. However, all microglial cultures robustly responded to LPS stimulation with the upregulation of TNF-α expression to similar degrees. Furthermore, Mer receptor expression was less responsive to LPS stimulation when in Axl knockout cells. The presence of Gas6 consistently inhibited the LPS-induced upregulation of TNF-α in WT, Mer−/− and Axl−/− microglia. Moreover, Gas6 also inhibited LPS-induced changes in the microglial area, perimeter length, and cell roundness in wild-type cells.Conclusion: Gas6 can negatively regulate the microglial pro-inflammatory response to LPS as well as via stimulation of other TLRs, acting through either of the TAM receptors, Axl and Mer. This finding indicates an interaction between TLR and TAM receptor signaling pathways and reveals an anti-inflammatory role for the TAM ligand, Gas6, which could have therapeutic potential.

Immunohistochemical Evaluation and Clinicopathological Correlation of Mer and Axl Tyrosine Kinase TAM Receptors in Cutaneous Melanoma.

Malignant melanoma (MM) is potentially the most dangerous form of skin tumor. In the last few years, the so-called TAM receptors, a unique family of tyrosine kinase (TK) receptors, have become increasingly important. To evaluate Mer and Axl TAM receptor expression to find clinicopathological features that could explain the biological behavior of MM. Clinicopathological data were obtained from an MM electronic database at our Institute. We reviewed 24 cutaneous MM specimens. TAM receptor expression was assayed using immunohistochemistry. Combinative semiquantitative scoring was used for the evaluation of TAM receptor expression (MerTK and AxlTK). Appropriate statistical methods were used to evaluate a possible correlation between TAM receptor expression and the clinicopathological variables of the MM samples (univariate analysis and multivariate analysis). MerTK and AxlTK were expressed differently in the MM samples, with a major expression of the first receptor. The cells of the tumor microenvironment contributed to the majority of the total score. A significant association was found between AxlScore and the site of the tumor and between AxlScore and the variable ulceration; another correlation was found between MerScore and the following characteristics: pathological stage of the tumor (pT), sex, ulceration, and tumor-infiltrating lymphocytes. All correlations between the expression of MerTK and AxlTK with the clinical and histological variables of MM should be validated in a large group of people in order to increase the validity and the impact of our observations, with subsequently therapeutic implications in the era of the "targeted therapy."

Identification of pAKT as a pharmacodynamic marker for MER kinase in human melanoma G361 cells

BackgroundThe MER signaling pathway represents an attractive therapeutic target for human cancers. Growth arrest–specific protein 6 (GAS6)–induced MER phosphorylation is often unstable and difficult to detect without pervanadate pretreatment in human cancer cells, posing a challenge for the development of selective MER kinase inhibitors. Here, we identified phosphorylated AKT (pAKT) as a specific pharmacodynamic marker for MER kinase inhibitors in human melanoma G361 cells.MethodsThe expression of MER, TYRO3, and AXL were profiled among multiple human cancer cells. To determine whether they play a role in the activation of pAKT, MER and TYRO3 were selectively depleted by small, interfering RNA knockdown. In addition, using AKT phosphorylation as a readout, a high-throughput cell-based assay was established in G361 cells for evaluation of the potency of potential inhibitors of MER pathway activation.ResultsWe demonstrated that high levels of MER and TYRO3, but not AXL, were expressed in G361 cells. In these cells, pAKT was induced by GAS6 treatment, which could be reversed by AXL/MER inhibitors. We showed that GAS6-induced pAKT is only dependent on MER kinase, but not TYRO3, in G361 cells. Furthermore, we observed a correlation in potency between inhibition of pAKT in G361 cells and pMER in MER-overexpressing Ba/F3 cells by these inhibitors.ConclusionsIn summary, we have demonstrated that GAS6-induced pAKT is a possible pharmacodynamic marker for the inhibition of MER kinase, and we have successfully developed a cell-based functional assay for screening small-molecule inhibitors of MER kinase for potential therapeutic utility in treating GAS6/MER-deregulated human cancers.

Эффероцитоз усиливает экспрессию аргиназы-1 и тирозинкиназы Mer ГМ-КСФ-дифференцированными макрофагами человека

We studied the expression of arginase-1 (Arg1) and tyrosine kinase Mer (MerTK) in GMCSF-differentiated human macrophage populations М0, М1(IFNγ), М2а(IL-4), and М2(low serum) generated under conditions of growth/serum factor deficiency. The maximum relative content of Arg1+ and MerTK+ cells was found in М2 macrophage populations: М2а(IL-4) and М2(low serum). As the uptake of apoptotic cells is the key mechanism of M2 polarization during M2(low serum) generation, we performed a special series of experiments and showed that incubation with allogeneic apoptotic neutrophils significantly increased the percentages of CD206+ macrophages co-expressing Arg1 and MerTK.

A critical role of the Gas6-Mer axis in endothelial dysfunction contributing to TA-TMA associated with GVHD

AbstractEndothelial dysfunction in the early phases of hematopoietic stem cell transplantation (HSCT) contributes to a common pathology between transplant-associated thrombotic microangiopathy (TA-TMA) and graft-versus-host disease (GVHD), which are serious complications of HSCT. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Serum Gas6 levels were significantly increased in HSCT patients with grade II to IV acute GVHD (aGVHD), and Gas6 and Mer expression levels were upregulated in aGVHD lesions of the large intestine and skin. The increased serum Gas6 levels were also correlated with elevated lactate dehydrogenase, d-dimer, and plasmin inhibitor complex values in HSCT patients with aGVHD. In human umbilical vein endothelial cells (ECs), exogenous Gas6 or the exposure of sera isolated from patients with grade III aGVHD to ECs induced the downregulation of thrombomodulin and the upregulation of PAI-1, as well as the upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, which were inhibited by UNC2250, a selective Mer tyrosine kinase inhibitor. In mouse HSCT models, we observed hepatic GVHD with hepatocellular apoptosis, necrosis, and fibrosis, as well as TA-TMA, which is characterized pathologically by thrombosis formation in the microvasculature of the liver and kidney. Of note, intravenous administration of UNC2250 markedly suppressed GVHD and TA-TMA in these mouse HSCT models. Our findings suggest that the Gas6-Mer axis is a promising target for TA-TMA after GVHD.

Csf1r or Mer inhibition delays liver regeneration via suppression of Kupffer cells.

IntroductionMurine Kupffer cells (KCs) comprise CD11bhi and F4/80hi subsets. Tissue-resident macrophages are known to express the tyrosine kinase receptors colony-stimulating factor 1 receptor (Csf1r) and Mer. However, the expression of Csf1r and Mer on KC subsets and the importance of these tyrosine kinases during liver regeneration (LR) are unknown.MethodsKCs from wild-type and Csf1r-GFP mice were characterized by flow cytometry. Partial hepatectomy (PH) was performed in mice treated with clodronate liposomes, a Csf1r small molecule inhibitor or depleting antibody, or a small molecule Mer inhibitor. Sera and livers were analyzed. The function of sorted KC subsets was tested in vitro.ResultsMer was specifically expressed on tissue-resident F4/80hi KCs, 55% of which also expressed Csf1r. Mer+Csf1r+ and Mer+Csf1r- KCs had distinct expression of macrophage markers. Csf1r inhibition in mice reduced F4/80hi KCs by approximately 50%, but did not affect CD11bhi KCs. Clodronate liposomes depleted F4/80hi KCs, but also altered levels of other intrahepatic leukocytes. Csf1r inhibition delayed LR, as demonstrated by a 20% reduction in liver-to-body weight ratios 7 days after PH. At 36h after PH, Csf1r inhibition increased serum ALT and histological liver injury, and decreased liver cell proliferation. A small molecule inhibitor of Mer did not alter the percentage of KCs or their proliferation and just modestly delayed LR. In vitro, Csf1r or Mer inhibition did not decrease KC viability, but did attenuate their cytokine response to stimulation.ConclusionsF4/80hi KCs are Mer+ and can be subdivided based on Csf1r expression. Csf1r or Mer inhibition each reduces KC cytokine production and delays LR.