| 1. |
- Dondalska, Aleksandra, et al.
(författare)
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Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide
- 2020
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37in vitroandin vivo. We examined the effect of ssON on MRGPRX2 activationin vitroby measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activationin vivoin two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.
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| 2. |
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| 3. |
- Franck, Marina Christina Mikaela, et al.
(författare)
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Urocortin3-expressing neurons in sensory transmission
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Urocortin 3 (UCN3) is a neuropeptide involved in mechanosensation and stress regulation, and Ucn3-Cre neurons have been assigned a role in mechanical itch. Here, we show that Ucn3 marks a population of excitatory neurons in the mouse dorsal horn, divided into two non-overlapping subpopulations expressing protein kinase C g or calretinin/calbindin 2. Chemogenetic activation of spinal Ucn3-Cre neurons evoked a targeted biting/licking behavior towards the corresponding dermatome. Genetic deletion of vesicular glutamate transporter 2 (VGLUT2) in Ucn3-Cre neurons removed the phenotype, showing that the biting/licking behavior is VGLUT2-dependent. Conditional deletion of VGLUT2 did not affect acute thermal or mechanical withdrawal responses, nor thermal withdrawal responses after nerve growth factor-induced hypersensitivity or the prurifensive response to 48/80 or von Frey stimuli applied in nape. Instead, we found that a group of spinal Ucn3 neurons were activated in response to artificial scratching or 48/80-induced itch. Electrophysiological experiments showed that spinal Ucn3 neurons received both glycinergic and GABAergic tonic inhibition, and monosynaptic inputs from both Aβ and C fibers, which could be confirmed by rabies tracing. Spinal Ucn3/Ucn3-Cre neurons thus represent a mechanically sensitive population with several roles in the itch-scratch cycle.
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| 4. |
- Gao, Tianle, et al.
(författare)
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The Neuropeptide Y System Regulates Both Mechanical and Histaminergic Itch
- 2018
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Ingår i: Journal of Investigative Dermatology. - : ELSEVIER SCIENCE INC. - 0022-202X .- 1523-1747. ; 138:11, s. 2405-2411
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Tidskriftsartikel (refereegranskat)abstract
- Itch is a somatosensory modality that serves to alert an organism to harmful elements removable by scratching, such as parasites and chemical irritants. Recently, ablation or silencing of neuropeptide Y (NPY)-expressing spinal interneurons was reported to selectively enhance mechanical itch, whereas chemical itch was unaffected. We examined the effect of activating the NPY/Y-1 receptor system on scratch behavior in mice. We found that intrathecal administration of the Y-1 agonist [Leu(31), Pro(34)]-NPY (LP-NPY) attenuated itch behavior induced by application of 0.07 g von Frey filament in the nape of the neck compared with saline treatment, indicating that activation of the spinal NPY/Y-1 system dampens mechanical itch. However, intrathecal administration of LP-NPY also attenuated chemically induced scratching provoked by intradermal application of histamine or the mast cell degranulator 48/80 (histaminergic itch), and the latter effect could be reversed by administration of the Y-1 antagonist BIBO3304. Intrathecal application of the native nonselective agonist NPY also attenuated histamine or 48/80-induced scratching. Our analyses emphasize the importance of including additional quantitative parameters to characterize the full spectrum of itch behavior and show that the NPY/Y-1 system dampens both mechanically and chemically induced scratching and hence is shared by the two submodalities of itch.
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| 5. |
- Jakobsson, Jon E. T., et al.
(författare)
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Neuropeptide Y in itch regulation
- 2019
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Ingår i: Neuropeptides. - : Elsevier BV. - 0143-4179 .- 1532-2785. ; 78
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Forskningsöversikt (refereegranskat)abstract
- Itch is a somatosensory sensation that informs the organism about the presence of potentially harmful substances or parasites, and initiates scratching to remove the threat. Itch-inducing (pruritogenic) substances activate primary afferent neurons in the skin through interactions with specific receptors that converts the stimulus into an electrical signal. These signals are conveyed to the dorsal horn of the spinal cord through the release of neurotransmitters such as natriuretic polypeptide b and somatostatin, leading to an integrated response within a complex spinal inteneuronal network. A large sub-population of somatostatin-expressing spinal interneurons also carry the Neuropeptide Y (NPY) Y1 receptor, indicating that NPY and somatostatin partly regulate the same neuronal pathway. This review focuses on recent findings regarding the role of the NPY/Y1 and somatostatin/SST2A receptor in itch, and also presents data integrating the two neurotransmitter systems.
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| 6. |
- Lennartsson, Johan, et al.
(författare)
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The Fer Tyrosine Kinase Is Important for Platelet-derived Growth Factor-BB-induced Signal Transducer and Activator of Transcription 3 (STAT3) Protein Phosphorylation, Colony Formation in Soft Agar, and Tumor Growth in Vivo.
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:22, s. 15736-15744
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Tidskriftsartikel (refereegranskat)abstract
- Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF β-receptor (PDGFRβ) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRβ kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.
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| 7. |
- Lennartsson, Johan, et al.
(författare)
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The Fer tyrosine kinase is necessary for platelet-derived growth factor-BB-induced Stat3 phosphorylation and colony formation in soft agar
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF b-receptor (PDGFRb) through multiple autophosphorylation sites, i.e. Tyr579, Tyr581, Tyr740 and Tyr1021. Using low molecular weight inhibitors we found that PDGF-BB-induced Fer activation is dependent on PDGFRb kinase activity, but not on the enzymatic activity of Src or Jak kinases. To elucidate the function of Fer downstream of PDGFRb, we downregulated the expression using siRNA; under conditions with reduced Fer expression, PDGF-BB was unable to induce phosphorylation of Stat3, whereas Stat5, Erk1/2 and Akt were unaffected. In addition, PDGFRb autophosphorylation was partially dependent on Fer expression. On a functional level, we could demonstrate that expression of Fer is dispensable for PDGF-BB-induced proliferation and migration. However, Fer was found to be essential for colony formation in soft agar, consistent with its critical role in Stat3 activation, which has frequently been implicated in cell transformation.
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| 8. |
- Ma, Haisha, et al.
(författare)
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Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation
- 2015
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Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 27:11, s. 2209-2219
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Tidskriftsartikel (refereegranskat)abstract
- Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor beta (PDGFR beta) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFR beta in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRS. In contrast, ligand-induced internalization of PDGFR beta was unaffected by HD-FTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFR beta accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3 T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRS and the migration, proliferation and tumorigenicity of cells stimulated by PDGF.
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| 9. |
- Ma, Haisha, 1983-
(författare)
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Regulation of Platelet-Derived Growth Factor Receptor Signaling and its Targeting in Cancer Therapy
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth.We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a decreased level of PDGFR ubiquitination (paper II). Consequently, internalized PDGFR could not be sorted properly and escaped degradation. This resulted in enhanced activation of phospholipase C γ (PLCγ) and changed kinetics of signal transducer and activator of transcription (STAT) 3 signalling, which further increased colony formation of HD-PTP silenced cells in soft agar, indicating a tumor suppressor role of HD-PTP.Activation of PDGFR leads to stimulation of downstream pathways. We identified Fer kinase as a critical signal transducer downstream of PDGFR in a proteomic screen. We showed that Fer kinase is essential for PDGF-induced STAT3 activation; as a result (paper III), Fer depletion severely blunted the ability of PDGFR signalling to promote anchorage-independent growth in soft agar and delayed tumor initiation in a mouse model.The crosstalk between host and tumor plays a critical role in tumor progression. At present most anti-cancer drugs are targeting tumor cells; we were interested in how targeting tumor host cells affects the efficacy of anti-tumor therapy. We found that selective PDGFRβ inhibition in host cells exerted tumor inhibitory effects on growth and vascularization of tumors with autocrine PDGF signaling, whereas tumors lacking such stimulation show only minor response on tumor growth (paper IV). Meanwhile, we demonstrated that PDGF/PDGFRβ signalling promotes expression of NG2, a marker for pericytes.
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| 10. |
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| 11. |
- Ma, Haisha, et al.
(författare)
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The Neuropeptide Y Y-2 Receptor Is Coexpressed with Nppb in Primary Afferent Neurons and Y-2 Activation Reduces Histaminergic and IL-31-Induced Itch
- 2020
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Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS. - 0022-3565 .- 1521-0103. ; 372:1, s. 73-82
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Tidskriftsartikel (refereegranskat)abstract
- Itch stimuli are detected by specialized primary afferents that convey the signal to the spinal cord, but how itch transmission is regulated is still not completely known. Here, we investigated the roles of the neuropeptide Y (NPY)/Y-2 receptor system on scratch behavior. The inhibitory Y-2 receptor is expressed on mouse primary afferents, and intrathecal administration of the Y-2 agonist peptide YY (PYY)(3-36) reduced scratch episode frequency and duration induced by compound 48/80, an effect that could be reversed by intrathecal preadministration of the Y-2 antagonist BIIE0246. Also, scratch episode duration induced by histamine could be reduced by PYY3-36. In contrast, scratch behavior induced by alpha-methyl-5HT, protease-activated receptor-2-activating peptide SLIGRL, chloroquine, topical dust mite extract, or mechanical itch induced by von Frey filaments was unaffected by stimulation of Y2. Primary afferent neurons expressing the Npy2r gene were found to coexpress itch-associated markers such as natriuretic peptide precursor b, oncostatin M receptor, and interleukin (IL) 31 receptor A. Accordingly, intrathecal PYY3-36 reduced the scratch behavior induced by IL-31. Our findings imply that the NPY/Y-2 system reduces histaminergic and IL-31-associated itch through presynaptic inhibition of a subpopulation of itch-associated primary afferents. SIGNIFICANCE STATEMENT The spinal neuropeptide Y system dampens scratching behavior induced by histaminergic compounds and interleukin 31, a cytokine involved in atopic dermatitis, through interactions with the Y-2 receptor. The Y-2 receptor is expressed by primary afferent neurons that are rich in itch-associated neurotransmitters and receptors such as somatostatin, natriuretic peptide precursor b, and interleukin 31 receptors.
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| 12. |
- Schmees, Christian, et al.
(författare)
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Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V
- 2012
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 23:13, s. 2571-2582
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Tidskriftsartikel (refereegranskat)abstract
- Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. To what extent the endocytic trafficking routes can contribute to such RTK hyper-activation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation synergized in stimulating PI 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of phosphatidylinositol (PI) 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in non-transformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor (EGF) promoted macropinocytosis of both receptors and increased their activation in non-transformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.
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| 13. |
- Tsioumpekou, Maria, et al.
(författare)
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Specific targeting of PDGFR beta in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression
- 2020
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Ingår i: Theranostics. - : IVYSPRING INT PUBL. - 1838-7640. ; 10:3, s. 1122-1135
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Tidskriftsartikel (refereegranskat)abstract
- PDGF-BB/PDGFR beta signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFR beta signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFR beta ATP binding site that allows specific targeting of PDGFR beta using the compound 1-NaPP1. To evaluate the impact of selective PDGFR beta inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFR beta expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFR beta kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFR beta kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFR beta by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFR beta by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFR beta ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFR beta signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFR beta in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.
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| 14. |
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