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1.
Adv Sci (Weinh) ; 8(24): e2102381, 2021 12.
Article in English | MEDLINE | ID: mdl-34713625

ABSTRACT

Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma-related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro-inflammatory cargo. These sEVs transfer transcripts for ICAM-1, VCAM-1, E-selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil-endothelium interactions, and destabilize barrier integrity. Inhibition of sEV-release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT-plasma-sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism.


Subject(s)
Endothelial Cells/immunology , Extracellular Vesicles/immunology , Inflammation/immunology , Inflammation/physiopathology , Multiple Trauma/complications , Acute Kidney Injury/etiology , Acute Kidney Injury/immunology , Acute Kidney Injury/physiopathology , Animals , Disease Models, Animal , Endothelial Cells/physiology , Extracellular Vesicles/physiology , Male , Mice , Mice, Inbred C57BL , Multiple Trauma/immunology , Neutrophil Infiltration/physiology , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/physiopathology , Sepsis/etiology , Sepsis/immunology , Sepsis/physiopathology
2.
J Leukoc Biol ; 104(3): 615-630, 2018 09.
Article in English | MEDLINE | ID: mdl-29656400

ABSTRACT

Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F-actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil-like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma.


Subject(s)
Actins/metabolism , Neutrophil Infiltration/immunology , Protein Kinase C/metabolism , Shock, Hemorrhagic/immunology , Transendothelial and Transepithelial Migration/immunology , Animals , Cell Line , Humans , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/immunology , Neutrophils/metabolism , Polymerization , Protein Kinase C/immunology , Signal Transduction/immunology , Swine
3.
Development ; 143(13): 2281-91, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27226325

ABSTRACT

TALE-homeodomain proteins function as components of heteromeric complexes that contain one member each of the PBC and MEIS/PREP subclasses. We recently showed that MEIS2 cooperates with the neurogenic transcription factor PAX6 in the control of adult subventricular zone (SVZ) neurogenesis in rodents. Expression of the PBC protein PBX1 in the SVZ has been reported, but its functional role(s) has not been investigated. Using a genetic loss-of-function mouse model, we now show that Pbx1 is an early regulator of SVZ neurogenesis. Targeted deletion of Pbx1 by retroviral transduction of Cre recombinase into Pbx2-deficient SVZ stem and progenitor cells carrying floxed alleles of Pbx1 significantly reduced the production of neurons and increased the generation of oligodendrocytes. Loss of Pbx1 expression in neuronally committed neuroblasts in the rostral migratory stream in a Pbx2 null background, by contrast, severely compromised cell survival. By chromatin immunoprecipitation from endogenous tissues or isolated cells, we further detected PBX1 binding to known regulatory regions of the neuron-specific genes Dcx and Th days or even weeks before the respective genes are expressed during the normal program of SVZ neurogenesis, suggesting that PBX1 might act as a priming factor to mark these genes for subsequent activation. Collectively, our results establish that PBX1 regulates adult neural cell fate determination in a manner beyond that of its heterodimerization partner MEIS2.


Subject(s)
Aging/metabolism , Homeodomain Proteins/metabolism , Lateral Ventricles/metabolism , Neurogenesis , Transcription Factors/metabolism , Animals , Base Sequence , Cell Differentiation , Cell Lineage , Cell Movement , Cell Survival , Cells, Cultured , Doublecortin Domain Proteins , Doublecortin Protein , Enhancer Elements, Genetic/genetics , Gene Deletion , Gene Expression Regulation, Developmental , Gene Targeting , Homeodomain Proteins/genetics , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Neurogenesis/genetics , Neuropeptides/genetics , Neuropeptides/metabolism , Olfactory Bulb/metabolism , Oligodendroglia/cytology , Oligodendroglia/metabolism , Pre-B-Cell Leukemia Transcription Factor 1 , Protein Binding , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Transcription Factors/genetics , Tyrosine 3-Monooxygenase/metabolism
4.
J Biol Chem ; 291(1): 462-77, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26507660

ABSTRACT

Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.


Subject(s)
Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 7/metabolism , Multiprotein Complexes/metabolism , Protein Kinases/metabolism , trans-Golgi Network/metabolism , ADP-Ribosylation Factor 1/metabolism , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Guanosine Triphosphate/metabolism , Humans , Isoenzymes , Membrane Proteins/metabolism , Mice, Inbred C57BL , Models, Biological , Protein Binding , Protein Kinase D2 , Protein Transport
5.
Bioarchitecture ; 4(3): 111-5, 2014.
Article in English | MEDLINE | ID: mdl-24847910

ABSTRACT

Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue.


Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Neoplasm Invasiveness/genetics , Pancreatic Neoplasms/pathology , Protein Kinase C/genetics , Protein Kinases/genetics , Humans
6.
Mol Biol Cell ; 25(3): 324-36, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24336522

ABSTRACT

Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix-bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies.


Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Neoplasm Invasiveness/genetics , Pancreatic Neoplasms/pathology , Protein Kinase C/genetics , Protein Kinases/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Chorioallantoic Membrane/cytology , Extracellular Matrix , HEK293 Cells , HeLa Cells , Humans , Matrix Metalloproteinase 7/biosynthesis , Matrix Metalloproteinase 7/genetics , Matrix Metalloproteinase 7/metabolism , Matrix Metalloproteinase 9/biosynthesis , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Neoplasm Metastasis/genetics , Neovascularization, Pathologic/genetics , Protein Kinase D2 , RNA Interference , RNA, Small Interfering , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/metabolism
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