Pharmacological Targeting of the RAGE-NFκB Signalling Axis Impedes Monocyte Activation under Diabetic Conditions through the Repression of SHP-2 Tyrosine Phosphatase Function.

Monocytes play a vital role in the development of cardiovascular diseases. Type 2 diabetes mellitus (T2DM) is a major CVD risk factor, and T2DM-induced aberrant activation and enhanced migration of monocytes is a vital pathomechanism that leads to atherogenesis. We recently reported the upregulation of SHP-2 phosphatase expression in mediating the VEGF resistance of T2DM patient-derived monocytes or methylglyoxal- (MG, a glucose metabolite and advanced glycation end product (AGE) precursor) treated monocytes. However, the exact mechanisms leading to SHP-2 upregulation in hyperglycemic monocytes are unknown. Since inflammation and accumulation of AGEs is a hallmark of T2DM, we hypothesise that inflammation and AGE-RAGE (Receptor-for-AGEs) signalling drive SHP-2 expression in monocytes and blockade of these pathways will repress SHP-2 function. Indeed, monocytes from T2DM patients revealed an elevated SHP-2 expression. Under normoglycemic conditions, the serum from T2DM patients strongly induced SHP-2 expression, indicating that the T2DM serum contains critical factors that directly regulate SHP-2 expression. Activation of pro-inflammatory TNFα signalling cascade drove SHP-2 expression in monocytes. In line with this, linear regression analysis revealed a significant positive correlation between TNFα expression and SHP-2 transcript levels in T2DM monocytes. Monocytes exposed to MG or AGE mimetic AGE-BSA, revealed an elevated SHP-2 expression and co-treatment with an NFκB inhibitor or genetic inhibition of p65 reversed it. The pharmacological inhibition of RAGE was sufficient to block MG- or AGE-BSA-induced SHP-2 expression and activity. Confirming the importance of RAGE-NFκB signalling in regulating SHP-2 expression, the elevated binding of NFκB to the SHP-2 promoter-induced by MG or AGE-BSA-was reversed by RAGE and NFκB inhibition. Besides, we detected elevated RAGE levels in human and murine T2DM monocytes and monocytes exposed to MG or AGE-BSA. Importantly, MG and AGE-BSA treatment of non-T2DM monocytes phenocopied the aberrant pro-migratory phenotype of T2DM monocytes, which was reversed entirely by either SHP-2- or RAGE inhibition. In conclusion, these findings suggest a new therapeutic approach to prevent accelerated atherosclerosis in T2DM patients since inhibiting the RAGE-NFκB-SHP-2 axis impeded the T2DM-driven, SHP-2-dependent monocyte activation.

The Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitor Empagliflozin Reverses Hyperglycemia-Induced Monocyte and Endothelial Dysfunction Primarily through Glucose Transport-Independent but Redox-Dependent Mechanisms.

PURPOSE: Hyperglycaemia-induced oxidative stress and inflammation contribute to vascular cell dysfunction and subsequent cardiovascular events in T2DM. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin significantly improves cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is known to be expressed on cells other than the kidney cells, we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate hyperglycaemia-induced dysfunction of these cells. METHODS: Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs), and fetoplacental endothelial cells (HPECs) were used as the EC model cells. Cells were exposed to hyperglycaemic conditions in vitro in 40 ng/mL or 100 ng/mL empagliflozin. The expression levels of the relevant molecules were analysed by RT-qPCR and confirmed by FACS. Glucose uptake assays were carried out with a fluorescent derivative of glucose, 2-NBDG. Reactive oxygen species (ROS) accumulation was measured using the H2DFFDA method. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. RESULTS: Both primary human monocytes and endothelial cells express SGLT-2. Hyperglycaemic conditions did not significantly alter the SGLT-2 levels in monocytes and ECs in vitro or in T2DM conditions. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly, but not significantly, suppressed glucose uptake by monocytes and endothelial cells. However, we detected the significant suppression of hyperglycaemia-induced ROS accumulation in monocytes and ECs when empagliflozin was used to inhibit SGLT-2 function. Hyperglycaemic monocytes and endothelial cells readily exhibited impaired chemotaxis behaviour. The co-treatment with empagliflozin reversed the PlGF-1 resistance phenotype of hyperglycaemic monocytes. Similarly, the blunted VEGF-A responses of hyperglycaemic ECs were also restored by empagliflozin, which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. The induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by hyperglycaemic monocytes and endothelial cells, and a general antioxidant N-acetyl-L-cysteine (NAC) was able to mimic the effects of empagliflozin. CONCLUSIONS: This study provides data indicating the beneficial role of empagliflozin in reversing hyperglycaemia-induced vascular cell dysfunction. Even though both monocytes and endothelial cells express functional SGLT-2, SGLT-2 is not the primary glucose transporter in these cells. Therefore, it seems likely that empagliflozin does not directly prevent hyperglycaemia-mediated enhanced glucotoxicity in these cells by inhibiting glucose uptake. We identified the reduction of oxidative stress by empagliflozin as a primary reason for the improved function of monocytes and endothelial cells in hyperglycaemic conditions. In conclusion, empagliflozin reverses vascular cell dysfunction independent of glucose transport but could partially contribute to its beneficial cardiovascular effects.

Altered Cholesterol and Lipid Synthesis Mediates Hyperinflammation in COVID-19.

Recent data have revealed that fructose-rich diet triggers inflammation and lipid synthesis. Furthermore, lipid metabolism, cholesterol synthesis and sterol regulatory element binding protein-2 (SREBP-2) activation correlates with coronavirus disease 2019 (COVID-19)-induced cytokine storm. High fructose consumption result in SREBPs activation, altered cholesterol and lipid synthesis and may establish an innate immune memory in the cells, leading to severe COVID-19 in patients with obesity.

Trained immunity as a novel approach against COVID-19 with a focus on Bacillus Calmette-Guérin vaccine: mechanisms, challenges and perspectives.

COVID-19 is a severe health problem in many countries and has altered day-to-day life in the whole world. This infection is caused by the SARS-CoV-2 virus, and depending on age, sex and health status of the patient, it can present with variety of clinical symptoms such as mild infection, a very severe form or even asymptomatic course of the disease. Similarly to other viruses, innate immune response plays a vital role in protection against COVID-19. However, dysregulation of innate immunity could have a significant influence on the severity of the disease. Despite various efforts, there is no effective vaccine against the disease so far. Recent data have demonstrated that the Bacillus Calmette-Guérin (BCG) vaccine could reduce disease severity and the burden of several infectious diseases in addition to targeting its primary focus tuberculosis. There is growing evidence for the concept of beneficial non-specific boosting of immune responses by BCG or other microbial compounds termed trained immunity, which may protect against COVID-19. In this manuscript, we review data on how the development of innate immune memory due to microbial compounds specifically BCG can result in protection against SARS-CoV-2 infection. We also discuss possible mechanisms, challenges and perspectives of using innate immunity as an approach to reduce COVID-19 severity.

LXR Activation Induces a Proinflammatory Trained Innate Immunity-Phenotype in Human Monocytes.

Objectives: The concept of trained innate immunity describes a long-term proinflammatory memory in innate immune cells. Trained innate immunity is regulated through reprogramming of cellular metabolic pathways including cholesterol and fatty acid synthesis. Here, we have analyzed the role of Liver X Receptor (LXR), a key regulator of cholesterol and fatty acid homeostasis, in trained innate immunity. Methods and Results: Human monocytes were isolated and incubated with different stimuli for 24 h, including LXR agonists, antagonists and Bacillus Calmette-Guerin (BCG) vaccine. After 5 days resting time, cells were restimulated with the TLR2-agonist Pam3cys. LXR activation did not only increase BCG trained immunity, but also induced a long-term inflammatory activation by itself. This inflammatory activation by LXR agonists was accompanied by characteristic features of trained innate immunity, such as activating histone marks on inflammatory gene promoters and metabolic reprogramming with increased lactate production and decreased oxygen consumption rate. Mechanistically, LXR priming increased cellular acetyl-CoA levels and was dependent on the activation of the mevalonate pathway and IL-1ß signaling. In contrast to mevalonate pathway inhibition, blocking fatty acid synthesis further increased proinflammatory priming by LXR. Conclusion: We demonstrate that LXR activation induces a proinflammatory trained immunity phenotype in human monocytes through epigenetic and metabolic reprogramming. Our data reveal important novel aspects of LXR signaling in innate immunity.

Hyperglycemia-induced endothelial dysfunction is alleviated by thioredoxin mimetic peptides through the restoration of VEGFR-2-induced responses and improved cell survival.

BACKGROUND: Diabetes mellitus is an important cardiovascular risk factor characterized by elevated plasma glucose levels. High glucose (HG) negatively influences endothelial cell (EC) function, which is characterized by the inability of ECs to respond to vascular endothelial growth factor (VEGF-A) stimulation. We aimed to identify potential strategies to improve EC function in diabetes. METHODS AND RESULTS: Human umbilical cord endothelial cells (HUVECs) were subjected to hyperglycemic milieu by exposing cells to HG together with glucose metabolite, methylglyoxal (MG) in vitro. Hyperglycemic cells showed reduced chemotactic responses towards VEGF-A as revealed by Boyden chamber migration assays, indicating the development of "VEGF resistance" phenotype. Furthermore, HG/MG-exposed cells were defective in their general migratory and proliferative responses and were in a pro-apoptotic state. Mechanistically, the exposure to HG/MG resulted in reactive oxygen species (ROS) accumulation which is secondary to the impairment of thioredoxin (Trx) activity in these cells. Pharmacological and genetic targeting of Trx recapitulated VEGF resistance. Functional supplementation of Trx using thioredoxin mimetic peptides (TMP) reversed the HG/MG-induced ROS generation, improved the migration, proliferation, survival and restored VEGF-A-induced chemotaxis and sprouting angiogenesis of hyperglycemic ECs. Importantly, TMP treatment reduced ROS accumulation and improved VEGF-A responses of placental arterial endothelial cells isolated from gestational diabetes mellitus patients. CONCLUSIONS: Our findings suggest a putative role for Trx in modulating EC function and its functional impairment in HG conditions contribute to EC dysfunction. Supplementation of TMP could be used as a novel strategy to improve endothelial cell function in diabetes.

Hyperglycaemia-induced methylglyoxal accumulation potentiates VEGF resistance of diabetic monocytes through the aberrant activation of tyrosine phosphatase SHP-2/SRC kinase signalling axis.

Diabetes mellitus (DM) is a major cardiovascular risk factor contributing to cardiovascular complications by inducing vascular cell dysfunction. Monocyte dysfunction could contribute to impaired arteriogenesis response in DM patients. DM monocytes show blunted chemotactic responses to arteriogenic stimuli, a condition termed as vascular endothelial growth factor (VEGF) resistance. We hypothesize that methylglyoxal (MG), a glucose metabolite, induces monocyte dysfunction and aimed to elucidate the underlying molecular mechanisms. Human monocytes exposed to MG or monocytes from DM patients or mice (db/db) showed VEGF-resistance secondary to a pro-migratory phenotype. Mechanistically, DM conditions or MG exposure resulted in the upregulation of the expression of SHP-2 phosphatase. This led to the enhanced activity of SHP-2 and aided an interaction with SRC kinase. SHP-2 dephosphorylated the inhibitory phosphorylation site of SRC leading to its abnormal activation and phosphorylation of cytoskeletal protein, paxillin. We demonstrated that MG-induced molecular changes could be reversed by pharmacological inhibitors of SHP-2 and SRC and by genetic depletion of SHP-2. Finally, a SHP-2 inhibitor completely reversed the dysfunction of monocytes isolated from DM patients and db/db mice. In conclusion, we identified SHP-2 as a hitherto unknown target for improving monocyte function in diabetes. This opens novel perspectives for treating diabetic complications associated with impaired monocyte function.

Altered Cellular Metabolism Drives Trained Immunity.

Exposing innate immune cells to an initial insult induces a long-term proinflammatory response due to metabolic and epigenetic alterations which encompass an emerging new concept called trained immunity. Recent studies provide novel insights into mechanisms centered on metabolic reprogramming which induce innate immune memory in hematopoietic stem cells and monocytes.

Low density lipoprotein interferes with intracellular signaling of monocytes resulting in impaired chemotaxis and enhanced chemokinesis.

BACKGROUND: Hypercholesterolemia (HC) is an important cardiovascular risk factor characterized by elevated low density lipoprotein-cholesterol (LDL-C) plasma levels. HC negatively affects monocyte function by reducing their chemotactic response towards different growth factors. We aimed to elucidate the molecular mechanisms by which LDL induces monocyte dysfunction. METHODS AND RESULTS: Human monocytes exposed to either native (nLDL) or oxidized LDL (oxLDL) in vitro showed reduced chemotactic responses towards vascular endothelial growth factor A (VEGFA) and monocyte chemotactic protein-1 (MCP-1), but displayed enhanced random migration (chemokinesis). Mechanistically, the exposure to LDL resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and modulated MCP-1 and VEGFA-induced signaling in human monocytes. Furthermore, the aberrant p38 activation induced by oxLDL is due to the functional impairment of Dual Specificity Phosphatase-1 (DUSP-1). In the absence of LDL, the pharmacological inhibition of DUSP-1 alone was sufficient to recapitulate the accelerated chemokinetic and blunted chemotactic phenotype of monocytes. Finally, p38 MAPK inhibition in monocytes isolated from hyperlipidemic mice prevented the aberrant chemokinetic phenotype. CONCLUSIONS: Our data demonstrate that LDL induces monocyte chemokinesis of human monocytes by inducing mononuclear cell activation through the aberrant modulation of DUSP-1-p38/MAPK signaling axis. Moreover, our findings suggest that MCP-1/VEGFA-induced chemotaxis is reduced by LDL secondary to the impairment of ligand-induced signaling. These findings provide novel insight into hypercholesterolemia-associated vascular dysfunction and its potential involvement in the pathogenesis of atherosclerosis.

mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes.

Introduction: Cells of the innate immune system particularly monocytes and macrophages have been recognized as pivotal players both during the initial insult as well as the chronic phase of atherosclerosis. It has recently been shown that oxidized low-density lipoprotein (oxLDL) induces a long-term pro-inflammatory response in monocytes due to epigenetic and metabolic reprogramming, an emerging new concept called trained innate immunity. Changes in the cellular redox state are crucial events in the regulation of many physiologic functions in macrophages including transcription, differentiation and inflammatory response. Here we have analyzed the role of reactive oxygen species (ROS) in regulating this proinflammatory monocyte priming in response to oxLDL-treatment. Methods and Results: Human monocytes were isolated and incubated with oxLDL for 24 h. After 5 days of resting, oxLDL treated cells produced significantly more inflammatory cytokines upon restimulation with the TLR2-agonist Pam3cys. Furthermore, oxLDL incubation induced persistent mTOR activation, ROS formation, HIF1α accumulation and HIF1α target gene expression, while pharmacologic mTOR inhibition or siRNA mediated inhibition of the mTORC1 subunit Raptor prevented ROS formation and proinflammatory priming. mTOR dependent ROS formation was associated with increased expression of NAPDH oxidases and necessary for the emergence of the primed phenotype as antioxidant treatment blocked oxLDL priming. Inhibition of cytosolic ROS formation could also block mTOR activation and HIF1α accumulation suggesting a positive feedback loop between mTOR and cytosolic ROS. Although mitochondrial ROS scavenging did not block HIF1α-accumulation at an early time point (24 h), it was persistently reduced on day 6. Therefore, mitochondrial ROS formation appears to occur initially downstream of the mTOR-cytoROS-HIF1α feedback loop but seems to be a crucial factor that controls the long-term activation of the mTOR-HIF1α-axis. Conclusion: In summary, our data demonstrate that mTOR dependent ROS production controls the oxLDL-induced trained innate immunity phenotype in human monocyte derived macrophages. Pharmacologic modulation of these pathways might provide a potential approach to modulate inflammation, associated with aberrant monocyte activation, during atherosclerosis development.

High REDOX RESPONSIVE TRANSCRIPTION FACTOR1 Levels Result in Accumulation of Reactive Oxygen Species in Arabidopsis thaliana Shoots and Roots.

Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H2O2, as well as biotic- and abiotic-induced redox signals. RRTF1 is highly conserved in angiosperms, but its physiological role remains elusive. Here we show that inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Transgenic lines overexpressing RRTF1 are impaired in root and shoot development, light sensitive, and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica, which reduces ROS accumulation locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors that scavenge ROS. More than 800 genes were detected in mature leaves and seedlings of transgenic lines overexpressing RRTF1; ∼ 40% of them have stress-, redox-, ROS-regulated-, ROS-scavenging-, defense-, cell death- and senescence-related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box-like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli and H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains a GCC-box-like sequence in its promoter, but transgenic lines overexpressing RAP2.6 do not accumulate higher ROS levels. RRTF1 also stimulates systemic ROS accumulation in distal non-stressed leaves. We conclude that the elevated levels of the highly conserved RRTF1 induce ROS accumulation in response to ROS and ROS-producing abiotic and biotic stress signals.

Human cytomegalovirus infection impairs endothelial cell chemotaxis by disturbing VEGF signalling and actin polymerization.

AIMS: Human cytomegalovirus (HCMV) infection has been linked to the pathogenesis of vasculopathies; however, its pathogenic relevance remains to be established. A prerequisite for vascular repair is endothelial cell migration. We evaluated the influence of HCMV on chemokinesis and chemotactic response of human coronary artery endothelial cells (HCAEC) towards vascular endothelial growth factor (VEGF). METHODS AND RESULTS: A virus dose-dependent reduction in chemokinesis and VEGF-dependent chemotaxis was observed (P < 0.05). UV-inactivated virus did not inhibit chemotaxis or chemokinesis, indicating that viral gene expression is mandatory. We identified two HCMV-induced mechanisms explaining the reduction of chemotaxis: first, a non-ambiguous reduction of VEGFR-2 protein was observed, due to decreased transcription. This protein down-modulation could not be inhibited by Ganciclovir. The remaining VEGFR-2 expressed on infected HCAEC was able to stimulate cell activation. Second, HCMV infection influences actin polymerization in HCAEC as shown by FACS analysis: actin polymerization was significantly reduced to 53 and 51% (P < 0.05) compared with non-infected HCAEC at 24 and 72 h p.i., respectively. Genetically and pharmacologically eliminated VEGFR-2 function resulted in a significant (P < 0.05) reduction of VEGF-induced activation of actin polymerization. CONCLUSION: We demonstrated a significant reduction of the chemotactic mobility of HCMV-infected HCAEC mediated by down-modulation of the VEGFR-2 and by inhibition of actin polymerization. This VEGF resistance of HCMV-infected endothelial cells is likely to promote atherogenesis.

Regulation of protein tyrosine phosphatase oxidation in cell adhesion and migration.

SIGNIFICANCE: Redox-regulated control of protein tyrosine phosphatases (PTPs) through inhibitory reversible oxidation of their active site is emerging as a novel and general mechanism for control of cell surface receptor-activated signaling. This mechanism allows for a previously unrecognized crosstalk between redox regulators and signaling pathways, governed by, for example, receptor tyrosine kinases and integrins, which control cell proliferation and migration. RECENT ADVANCES: A large number of different molecules, in addition to hydrogen peroxide, have been found to induce PTP inactivation, including lipid peroxides, reactive nitrogen species, and hydrogen sulfide. Characterization of oxidized PTPs has identified different types of oxidative modifications that are likely to display differential sensitivity to various reducing systems. Accumulating evidence demonstrates that PTP oxidation occurs in a temporally and spatially restricted manner. Studies in cell and animal models indicate altered PTP oxidation in models of common diseases, such as cancer and metabolic/cardiovascular disease. Novel methods have appeared that allow characterization of global PTP oxidation. CRITICAL ISSUES: As the understanding of the molecular and cellular biology of PTP oxidation is developing, it will be important to establish experimental procedures that allow analyses of PTP oxidation, and its regulation, in physiological and pathophysiological settings. Future studies should also aim to establish specific connections between various oxidants, specific PTPs, and defined signaling contexts. FUTURE DIRECTIONS: Modulation of PTP activity still appears as a valid strategy for correction or inhibition of dys-regulated cell signaling. Continued studies on PTP oxidation might present yet unrecognized means to exploit this regulatory mechanism for pharmacological purposes.

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ.

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by protein-tyrosine phosphatases (PTPs). We recently identified the PTP DEP-1/CD148/PTPRJ as a novel negative regulator of FLT3. This study addressed the role of DEP-1 for regulation of the acute myeloid leukemia (AML)-related mutant FLT3 internal tandem duplication (ITD) protein. Our experiments revealed that DEP-1 was expressed but dysfunctional in cells transformed by FLT3 ITD. This was caused by enzymatic inactivation of DEP-1 through oxidation of the DEP-1 catalytic cysteine. In intact cells, including primary AML cells, FLT3 ITD kinase inhibition reactivated DEP-1. DEP-1 reactivation was also achieved by counteracting the high levels of reactive oxygen species (ROS) production detected in FLT3 ITD-expressing cell lines by inhibition of reduced NAD phosphate (NADPH)-oxidases, or by overexpression of catalase or peroxiredoxin-1 (Prx-1). Interference with ROS production in 32D cells inhibited cell transformation by FLT3 ITD in a DEP-1-dependent manner, because RNAi-mediated depletion of DEP-1 partially abrogated the inhibitory effect of ROS quenching. Reactivation of DEP-1 by stable overexpression of Prx-1 extended survival of mice in the 32D cell/C3H/HeJ mouse model of FLT3 ITD-driven myeloproliferative disease. The study thus uncovered DEP-1 oxidation as a novel event contributing to cell transformation by FLT3 ITD.

Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling.

Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation.

The structure of the 5'-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation.

Analysis of the human protein-tyrosine phosphatase (PTP) PTPRJ mRNA detected three in-frame AUGs at the 5'-end (starting at nt +14, +191 and +356) with no intervening stop codons. This tandem AUG arrangement is conserved between humans and the mouse and is unique among the genes of the classical PTPs. Until now it was assumed that the principal open reading frame (ORF) starts at AUG(356). Our experiments showed that: (i) translation of the mRNA synthesized under the PTPRJ promoter starts predominantly at AUG(191), leading to the generation of a 55 amino acid sequence preceding the signal peptide; (ii) the longer form is being likewise correctly processed into mature PTPRJ; (iii) the translation of the region between AUG(191) and AUG(356) inhibits the overall expression, a feature which depends on the sequence of the encoded peptide. Specifically, a sequence of 13 amino acids containing multiple arginine residues (RRTGWRRRRRRRR) confers the inhibition. In the absence of uORF these previously unrecognized characteristics of the 5'-end of the mRNA present a novel mechanism to suppress, and potentially to regulate translation.