Biomedical subjects

Rolf Stricker

Publications and source records attributed to Rolf Stricker.

7 recordsLinked to original sources

PubMed2006

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1.

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1beta and tumour necrosis factor-alpha secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C(2)-ceramide-induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.

Animals↗

PubMed2006

Jab1, a novel protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced activation of activator protein-1.

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.

Animals↗

PubMed2006

Interaction of the brain-specific protein p42IP4/centaurin-alpha1 with the peptidase nardilysin is regulated by the cognate ligands of p42IP4, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, with stereospecificity.

The brain-specific protein p42IP4, also called centaurin-alpha1, specifically binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Here, we investigate the interaction of p42IP4/centaurin-alpha1 with nardilysin (NRDc), a member of the M16 family of zinc metalloendopeptidases. Members of this peptidase family exhibit enzymatic activity and also act as receptors for other proteins. We found that p42IP4/centaurin-alpha1 binds specifically to NRDc from rat brain. We further detected that centaurin-alpha2, a protein that is highly homologous to p42IP4/centaurin-alpha1 and expressed ubiquitously, also binds to NRDc. In vivo interaction was demonstrated by co-immunoprecipitation of p42IP4/centaurin-alpha1 with NRDc from rat brain. The acidic domain of NRDc (NRDc-AD), which does not participate in catalysis, is sufficient for the protein interaction with p42IP4. Interestingly, preincubation of p42IP4 with its cognate ligands D-Ins(1,3,4,5)P4 and the lipid diC8PtdIns(3,4,5)P3 negatively modulates the interaction between the two proteins. D-Ins(1,3,4,5)P4 and diC8PtdIns(3,4,5)P3 suppress the interaction with virtually identical concentration dependencies. This inhibition is highly ligand specific. The enantiomer L-Ins(1,3,4,5)P4 is not effective. Similarly, the phosphoinositides diC8PtdIns(3,4)P2, diC8PtdIns(3,5)P2 and diC8PtdIns(4,5)P2 all have no influence on the interaction. Further experiments revealed that endogenous p42IP4 from rat brain binds to glutathione-S-transferase (GST)-NRDc-AD. The proteins dissociate from each other when incubated with D-Ins(1,3,4,5)P4, but not with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In summary, we demonstrate that p42IP4 binds to NRDc via the NRDc-AD, and that this interaction is controlled by the cognate cellular ligands of p42IP4/centaurin-alpha1. Thus, specific ligands of p42IP4 can modulate the recruitment of proteins, which are docked to p42IP4, to specific cellular compartments.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Identification of gene structure and subcellular localization of human centaurin alpha 2, and p42IP4, a family of two highly homologous, Ins 1,3,4,5-P4-/PtdIns 3,4,5-P3-binding, adapter proteins.

Proteins which recognize the two messengers phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3), a membrane lipid, and inositol 1,3,4,5-tetrakisphosphate (InsP4), a water-soluble ligand, play important roles by integrating external stimuli, which lead to differentiation, cell death or survival. p42IP4, a PtdInsP3/InsP4-binding protein, is predominantly expressed in brain. The recently described centaurin alpha2 of similar molecular mass which is 58% identical and 75% homologous to the human p42IP4 orthologue, is expressed rather ubiquitously in many tissues. Here, elucidating the gene structure for both proteins, we found the human gene for centaurin alpha2 located on chromosome 17, position 17q11.2, near to the NF1 locus, and human p42IP4 on chromosome 7, position 7p22.3. The two isoforms, which both have 11 exons and conserved exon/intron transitions, seem to result from gene duplication. Furthermore, we studied binding of the two second messengers, PtdInsP3 and InsP4, and subcellular localization of the two proteins. Using recombinant baculovirus we expressed centaurin alpha2 and p42IP4 in Sf9 cells and purified the proteins to homogeneity. Recombinant centaurin alpha2 bound both InsP4 and PtdInsP3 equally well in vitro. Furthermore, fusion proteins of centaurin alpha2 and p42IP4, respectively, with the green fluorescent protein (GFP) were expressed in HEK 293 cells to visualize subcellular distribution. In contrast to p42IP4, which was distributed throughout the cell, centaurin alpha2 was concentrated at the plasma membrane already in unstimulated cells. The protein centaurin alpha2 was released from the membrane upon addition of wortmannin, which inhibits PI3-kinase. p42IP4, however, translocated to plasma membrane upon growth factor stimulation. Thus, in spite of the high homology between centaurin alpha2 and p42IP4 and comparable affinities for InsP4 and PtdInsP3, both proteins showed clear differences in subcellular distribution. We suggest a model, which is based on the difference in phosphoinositide binding stoichiometry of the two proteins, to account for the difference in subcellular localization.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Oligomerization controls in tissue-specific manner ligand binding of native, affinity-purified p42(IP4)/centaurin alpha1 and cytohesins-proteins with high affinity for the messengers D-inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4,5-trisphosphate.

Several distinct receptor proteins for the second messengers Ins(1,3,4,5)P(4) and PtdIns(3,4,5)P(3) are already known, such as the brain-specific p42(IP4), which we have previously cloned from different species, and cytohesins. However, it is still unclear whether proteins interacting with phosphoinositide and inositolpolyphosphate second messengers are regulated differently in different tissues. Here, we investigated these native proteins for comparison also from rat lung cytosol and purified them by PtdIns(3,4,5)P(3) affinity chromatography. Proteins selectively binding Ins(1,3,4,5)P(4) with high affinity also showed high affinity and specificity towards PtdIns(3,4,5)P(3). In lung cytosol, two prominent protein bands were found in the eluate from a PtdIns(3,4,5)P(3) affinity column. We identified these proteins by mass spectrometry as the cytohesin family of Arf guanosine nucleotide exchange factors (cytohesin 1, ARNO, GRP-1) and as Bruton's tyrosine kinase. Western blot analysis indicated that p42(IP4) was present in lung only at very low concentrations. Applying the affinity purification scheme established for rat lung cytosol to cytosol from rat brain, however, yielded only p42(IP4). We identified cytohesins in rat brain by Western blotting and PCR, but cytohesins surprisingly did not bind to the PtdIns(3,4,5)P(3)-affinity column. Gel filtration experiments of brain cytosol revealed that brain cytohesins are bound to large molecular weight complexes (150 to more than 500 kDa). Thus, we hypothesize that this finding explains why brain cytohesins apparently do not bind the inositolphosphate ligand. In lung cytosol, on the other hand, cytohesins occur as dimers. Gel filtration also showed that p42(IP4) in brain cytosol occurs as a monomer. Thus, oligomerization (homomeric or heteromeric) of InsP(4)/PtdInsP(3) binding proteins can modulate their function in a tissue-dependent manner because it can modify their ability to interact with the ligands.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Cellular expression and subcellular localization of the human Ins(1,3,4,5)P(4)-binding protein, p42(IP4), in human brain and in neuronal cells.

In this study we describe for the human inositol-(1,3,4,5)-tetrakisphosphate (InsP4)-binding protein, p42IP4, the cellular distribution and subcellular localization in human brain and in transfected neuronal cells. The cDNA sequence of the human p42IP4 containing a single open reading frame yields a peptide of 374 amino acids with a calculated molecular mass of 43.4 kDa with a zinc-finger motif at the N-terminus, followed by two pleckstrin homology (PH) domains. Using a peptide-specific antiserum, p42IP4 protein was localized in a majority of neuronal cells of human brain sections. In the hypothalamus a subpopulation of paraventricular and infundibular nucleus neurons were strongly immunoreactive for p42IP4. In cortical areas the protein was predominantly found in large pyramidal cells. Some immunoreactivity for p42IP4 was also observed in the pyramidal cells of the hippocampal formation. Functional expression of p42IP4 protein in neuronal (NG108-15) and non-neuronal (CHO-K1) cells stably transfected with GFP-p42IP4 was shown in all cell fractions (homogenate, cytosol and membranes) by specific [3H]Ins(1,3,4,5)P4 binding activity, which correlated with p42IP4 protein detection by Western blot analysis. Using confocal laser scanning microscopy we showed that in NG108-15 and CHO-K1 cells stably transfected with GFP-p42IP4 the full length p42IP4 protein was localized in the cytoplasm, at the plasma membrane and in the nucleus. A deletion mutant of p42IP4 lacking the zinc finger domain resulted in solely a cytosolic and membrane localization but was not found in the nucleus. Thus we can conclude that human p42IP4 shows a region-specific localization in the human brain and the zinc finger motif within the protein is responsible for the localization of the protein in the cell nucleus.

Aged↗

PubMed2002

Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways.

Protease-activated receptors (PARs), newly identified members of G protein-coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/(betagamma-subunits)-phosphatidylinositol 3-kinase branch, and 2) the G(q)-PLC-(InsP(3) receptor)/Ca2+ -PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation.

Animals↗