| 1. |
- Axelsson, Lena, et al.
(författare)
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Clustering of β2-integrins on human neutrophils activates dual signaling pathways to Ptdlns 3-kinase
- 2000
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827. ; 256:1, s. 257-263
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Tidskriftsartikel (refereegranskat)abstract
- The β2-integrins on leukocytes can serve as a signaling unit during cell adhesion and locomotion, and to further clarify this important property we investigated the possible mechanisms of β2-integrin-induced activation of PtdIns 3-kinase. It has previously been demonstrated that clustering of β2-integrins activates p21(ras) by a tyrosine kinase-dependent pathway, and here we show that active p21(ras) interacts with its downstream target, PtdIns 3-kinase. Engagement of β2-integrins also activates the tyrosine kinases p58(c-fgr) and p59/61(hck) and causes them to associate with the p85 subunit of PtdIns 3-kinase. These findings suggest a mechanism whereby p58(c- fgr) and p59/61(hck) are directly involved in the activation of PtdIns 3- kinase. No coupling between p58(c-fgr) and p59/61(hck) could be detected; hence these kinases probably trigger independent but parallel signals to PtdIns 3-kinase. The effect of β2-integrin clustering on PtdIns 3-kinase activity was monitored as the activation of protein kinase B (PKB). Stimulation of PKB by β2-integrins was abolished by genistein and wortmannin but not by using methyl transferase inhibitors to abrogate the influence of p21(ras)-related proteins. Thus, even if PtdIns 3-kinase is not activated by p21(ras), it can maintain full enzyme activity due to the mentioned interaction with p58(c-fgr) or p59/61(hck). These tyrosine kinases apparently activate similar pathways that operate in parallel and therefore have the potential to substitute for each other in mediating adhesion and regulating cell locomotion. (C) 2000 Academic Press.
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| 2. |
- Burmakin, Mikhail, et al.
(författare)
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Imatinib increases oxygen delivery in extracellular matrix-rich but not in matrix-poor experimental carcinoma
- 2017
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Ingår i: Journal of Translational Medicine. - : BioMed Central. - 1479-5876. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Imatinib causes increased turnover of stromal collagen, reduces collagen fibril diameter, enhances extracellular fluid turnover and lowers interstitial fluid pressure (IFP) in the human colonic carcinoma KAT-4/HT-29 (KAT-4) xenograft model. Methods: We compared the effects of imatinib on oxygen levels, vascular morphology and IFP in three experimental tumor models differing in their content of a collagenous extracellular matrix. Results: Neither the KAT4 and CT-26 colonic carcinoma models, nor B16BB melanoma expressed PDGF beta-receptors in the malignant cells. KAT-4 tumors exhibited a well-developed ECM in contrast to the other two model systems. The collagen content was substantially higher in KAT-4 than in CT-26, while collagen was not detectable in B16BB tumors. The pO(2) was on average 5.4, 13.9 and 19.3 mmHg in KAT-4, CT-26 and B16BB tumors, respectively. Treatment with imatinib resulted in similar pO(2)-levels in all three tumor models but only in KAT-4 tumors did the increase reach statistical significance. It is likely that after imatinib treatment the increase in pO(2) in KAT-4 tumors is caused by increased blood flow due to reduced vascular resistance. This notion is supported by the significant reduction observed in IFP in KAT-4 tumors after imatinib treatment. Vessel area varied between 4.5 and 7% in the three tumor models and was not affected by imatinib treatment. Imatinib had no effect on the fraction of proliferating cells, whereas the fraction of apoptotic cells increased to a similar degree in all three tumor models. Conclusion: Our data suggest that the effects of imatinib on pO(2)-levels depend on a well-developed ECM and provide further support to the suggestion that imatinib acts by causing interstitial stroma cells to produce a less dense ECM, which would in turn allow for an increased blood flow. The potential of imatinib treatment to render solid tumors more accessible to conventional treatments would therefore depend on the degree of tumor desmoplasia.
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| 3. |
- Chiara, Federica, et al.
(författare)
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A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:41, s. 42516-42527
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Tidskriftsartikel (refereegranskat)abstract
- The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.
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| 4. |
- Essand, Magnus, et al.
(författare)
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Identification and characterization of a novel splicing variant of vesicular monoamine transporter 1
- 2005
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Ingår i: Journal of Molecular Endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 35:3, s. 489-501
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Tidskriftsartikel (refereegranskat)abstract
- Vesicular monoamine transporter 1 (VMAT1) is an integral protein in the membrane of secretory vesicles of neuroendocrine and endocrine cells that allows the transport of biogenic monoamines, such as serotonin, from the cytoplasm into the secretory vesicles. The full-length VMAT1 transcript is produced from 16 exons. We have identified and characterized an alternatively spliced form of VMAT1 that lacks exon 15, the next to last exon of VMAT1. The new form was therefore denoted VMAT1Delta15. Exon 15 does not contain an even multiple of three nucleotides. As a consequence, there is a shift of reading frame, and exon 16 is translated in an alternative reading frame, yielding a novel protein with a shorter and unrelated C-terminus compared with the native VMAT1 protein. VMAT1 and VMAT1Delta15 mRNAs are simultaneously expressed in normal and neoplastic neuroendocrine cells of the GI tract. However, VMAT1 expression is always higher than VMAT1Delta15 expression. We prove that VMAT1Delta15 is not localized in large, dense core vesicles as the native form but in the endoplasmic reticulum. Furthermore, while VMAT1 can take up serotonin, VMAT1Delta15 cannot, indicating different functions for the two forms of VMAT1.
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| 5. |
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| 6. |
- Hasumi, Yoko, et al.
(författare)
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Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling
- 2007
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 121:12, s. 2606-2614
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Tidskriftsartikel (refereegranskat)abstract
- The aim of our study was to further explore the use of anti-angiogenic therapy targeting the vascular endothelial growth factor receptor (VEGFR) on endothelial cells while simultaneously targeting platelet-derived growth factor receptors (PDGFRs) on adjacent pericytes. B16 mouse melanoma tumors exogenously expressing PDGF-BB (B16/PDGF-BB) display higher pericyte coverage on the vasculature compared to the parental B16 tumors (B16/mock). These models were used to investigate the effects of combination therapy targeting VEGFR and PDGFR signaling on size-matched tumors. Combination therapy using 25 mg/kg/day of the VEGFR inhibitor PTK787 and 100 mg/kg/day of the PDGFR inhibitor STI571 decreased the tumor growth rate of both tumor types, but the inhibition was only significant in the B16/PDGF-BB tumors. Combination therapy induced vessel remodeling, primarily by reducing the vessel density in B16/mock tumors, and by reducing the vessel size in B16/PDGF-BB tumors. When analyzing the effects of combination therapy on tumor vessel pericytes, it was found to primarily reduce the subpopulation of alpha-smooth muscle actin and PDGFRbeta-positive pericytes partly detached from the tumor vessels, without affecting the number of pericytes closely attached to the endothelium, which also express desmin. Taken together, these data demonstrate an increased benefit of targeting both VEGFR and PDGFR pathways in B16/PDGF-BB tumors, and demonstrates that the increased tumor growth inhibition in this model is accompanied by a reduction in a specific subset of pericytes, characterized by being loosely attached to endothelial cells and negative for the pericyte marker desmin.
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| 7. |
- Hayashi, Makoto, et al.
(författare)
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VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4, s. 1672-
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.
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| 8. |
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| 9. |
- Hellberg, Carina, et al.
(författare)
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Activation of Protein Kinase C α Is Necessary for Sorting the PDGF β-Receptor to Rab4a-dependent Recycling
- 2009
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 20:12, s. 2856-2863
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) β-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) α as the critical signaling component that regulates the sorting of the PDGF β-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKCα, using myristoylated inhibitory peptides or by knockdown of PKCα with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF β-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF β-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF β-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF β-receptors on early endosomes is regulated by sequential activation of PKCα and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.
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| 10. |
- Hellberg, Carina, et al.
(författare)
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Disruption of β2-integrin-cytoskeleton coupling abolishes the signaling capacity of these integrins on granulocytes
- 1999
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 265:1, s. 164-169
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Tidskriftsartikel (refereegranskat)abstract
- Integrin dependent adhesion and dynamic modulations of the actin network are prerequisites for normal cell locomotion. To investigate whether the actin microfilamentous system does play a role in regulation of β2-integrin-induced signalling, we pretreated granulocytes with staurosporine, a well-known protein kinase inhibitor that has also been shown to disrupt the cytoskeleton of intact cells. Pretreatment with staurosporine completely inhibited the β2-integrin-induced Ca2+ signal and also its ability to trigger actin polymerisation. This inhibition was not related to phosphorylation of the CD18-chain of the β2-integrin, nor to inhibition of protein kinases. Instead, association of β2-integrins with the cortical cytoskeleton, which was observed in untreated cells, was abolished after exposure to staurosporine, indicating that β2-integrin signalling depends on integrin-cytoskeleton interaction. These results suggest not only that the actin network provides an adhesive link to the extracellular matrix and a driving force for the locomotory response, but also that it participates in regulation of β2-integrin signalling during granulocyte locomotion.
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