Johannes A Schmid - Academia.edu (original) (raw)

Papers by Johannes A Schmid

In a variety of cell types, the transcription factor nuclear factor kB (NF-kB) func- tions as a m... more In a variety of cell types, the transcription factor nuclear factor kB (NF-kB) func- tions as a mediator of stress and immune responses. In endothelial cells (ECs), it controls the expression of genes encod- ing, eg, cytokines, cell adhesion mol- ecules, and procoagulatory proteins. This study investigates the effect of NF-kB suppression on several pathophysiologic functions of ECs, including inflammation,

Abstract—A variety of pathophysiological situations that affect cells of the vasculature, includi... more Abstract—A variety of pathophysiological situations that affect cells of the vasculature, including endothelial and smooth muscle cells, leads to the expression of genes such as adhesion molecules and chemokines that are dependent on members,of the nuclear factor (NF)-kB family of transcription factors. The corresponding,gene products mediate important biological functions such as immune and inflammatory reactions, smooth muscle cell proliferation, and

Cytometry, 2004

Background: Specific signal detection has been a fundamental issue in fluorescence microscopy. In... more Background: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. Methods: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. Results: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. More-over, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. Conclusions: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.

Neoplasia (New York, N.Y.), 2011

In prostate cancers, epidemiological data suggest a link between prostate inflammation and subseq... more In prostate cancers, epidemiological data suggest a link between prostate inflammation and subsequent cancer development, but proof for this concept in a tumor model is lacking. A constitutively active version of IκB kinase 2 (IKK2), which is activated by many inflammatory stimuli, was expressed specifically in the prostate epithelium. Constitutive activation of the IKK2/nuclear factor κB axis was insufficient for prostate transformation. However, in combination with heterozygous loss of phosphatase and tensin homolog, IKK2 activation led to an increase in tumor size, formation of cribriform structures, and increase in fiber in the fibroblastic stroma. This phenotype was coupled with persistent inflammation evoked by chemokine expression in the epithelium and stroma. The hyperplastic and dysplastic epithelia correlated with changes evoked by decreased androgen receptor activation. Conversely, inflammation correlated with stromal changes highlighted by loss of smooth muscle cells aro...

Cancer research, Jan 15, 2002

In 85% of Ewing family tumors, the NH2 terminus of EWS is fused to the DNA-binding domain of FLI1... more In 85% of Ewing family tumors, the NH2 terminus of EWS is fused to the DNA-binding domain of FLI1, an ets transcription factor. The resulting chimeric protein is a strong transcriptional activator with transforming activity. We report that EWS and EWS-FLI1 interact via their common NH2 terminus with the COOH terminus of BARD1, a putative tumor suppressor, in vitro and in vivo. Because BARD1 associates via its NH2-terminal RING domain with the breast cancer susceptibility gene BRCA1 that provides a platform for interactions with proteins involved in DNA repair and checkpoint control, our results provide a link between the Ewing's sarcoma gene product and the genome surveillance complex.

Dermatology Practical & Conceptual, 2012

In recent years, in vivo skin imaging devices have been successfully implemented in skin research... more In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

PLOS ONE, 2015

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each ye... more Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.

Thrombosis and Haemostasis, 2013

The COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation of culli... more The COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation of cullin-RING-E3 ubiquitin ligases (CRLs), has emerged as a regulator of NF-κB signalling. As NF-κB drives the expression of pro-inflammatory and pro-atherosclerotic genes, we probed the yet unknown role of the CSN, in particular CSN5, on NF-κB-mediated atherogenic responses in endothelial cells. Co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs) revealed the presence of a super-complex between IKK and CSN, which dissociates upon TNF-α stimulation. Furthermore, CSN5 silencing enhanced TNF-α-induced IκB-α degradation and NF-κB activity in luciferase reporter assays. This was paralleled by an increased NF-κB-driven upregulation of atherogenic chemokines and adhesion molecules, as measured by qPCR and flow cytometry, and translated into an enhanced arrest of THP-1 monocytes on TNF-α-stimulated, CSN5-depleted HUVECs. Reverse effects on NF-κB activity and THP-1 arrest were seen upon CSN5 overexpression. Finally, double-immunostaining confirmed the expression of CSN subunits in the endothelium of human atherosclerotic lesions, and revealed an increased expression of CSN5 which correlated with atheroprogression. In conclusion, endothelial CSN5 attenuates NF-κB-dependent pro-inflammatory gene expression and monocyte arrest on stimulated endothelial cells in vitro, suggesting that CSN5 might serve as a negative regulator of atherogenesis.

The Journal of biological chemistry, Jan 23, 2014

The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have ... more The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C-terminus (PTS1) or close to the N-terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimu...

Thrombosis and Haemostasis, 2007

Theadventoffluorescent proteins has revolutionizedsignaling research,shifting focus from biochemi... more Theadventoffluorescent proteins has revolutionizedsignaling research,shifting focus from biochemical assays to analysis of live cells,organized tissuesand even entireorganisms.Modern applications of fluorescent proteins go beyond their usea ss pecific markersofcells or tissues,allowing the researchertovisualize intracellular translocations as well as biochemical reactions. In

FEBS Journal, 2013

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation... more Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre-and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamindependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4-and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery.

STEM CELLS, 2000

Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor ce... more Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor cells. We report here the establishment of two spontaneously immortalized adult non-tumorigenic human prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1 (CD133 high /CD44 high / OCT4 high /PTEN high ) was characterized as a putative progenitor cell, and BHPrE1 (p63 high /p53 high /p21(WAF1) high /RB high ) was characterized as a putative epithelial intermediate cell.

Oncogene, 2003

In Ewing's sarcoma family tumors, the ets transcription factor gene FLI1 is rearranged with one E... more In Ewing's sarcoma family tumors, the ets transcription factor gene FLI1 is rearranged with one EWS allele resulting in coexpression of germline EWS and chimeric EWS-FLI1 proteins. Here, we investigated the potential of germline EWS, FLI1 and EWS-FLI1 to oligomerize. In two functional in vivo tests, fluorescence resonance energy transfer (FRET) and the mammalian two-hybrid (MTH) assay, self-association of EWS and EWS-FLI1, but not of FLI1 was detected. In addition, interaction of EWS-FLI1 with EWS and FLI1 was observed. GST pulldown assays and immunoprecipitation experiments largely confirmed these results. The EWS N-terminal domain present in both EWS and EWS-FLI1 was found to contribute to homotypic and heterotypic interactions of these proteins. However, in the context of germline EWS, the presence of the whole or part of the C-terminal RNAbinding domain greatly supported the self-association potential of the protein. Involvement of an RNA component in EWS oligomerization was confirmed by sensitivity of the corresponding GST pull-down assay to RNaseA treatment. In contrast, EWS-FLI1 was able to self-associate and also bind to FLI1 via its C-terminal domain, which comprises the FLI1 DNA-binding motif. Accordingly, the EWS-FLI1 interaction was not disrupted by RNaseA treatment. Despite its potential to oligomerize, EWS-FLI1 bound to a tandem ets-binding site of the TGFb type II receptor promoter as a monomer. Therefore, the functional consequences of homo-and heterooligomerization of EWS and EWS-FLI1 proteins remain to be elucidated.

Oncogene, 2008

Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as ... more Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as well as the activity of the transcription factors NF-κB, serum response factor and C/EBP. α-Catulin, an α-catenin-related protein that shares structural similarities with cytoskeletal linker proteins, facilitates Rho signalling by serving as a scaffold for the Rho-specific guanine nucleotide exchange factor Lbc. We report here that α-catulin also

Mutation Research/Reviews in Mutation Research, 1999

Recent progress in the identification and functional analysis of protein kinases and adapter mole... more Recent progress in the identification and functional analysis of protein kinases and adapter molecules that lead to activation of NF-kB family transcription factors has lead to a quite detailed understanding of one of the major signalling pathways that mediate a cell's response to environmental stress in a variety of host-defense situations. NF-kB is recognized as a key regulatory factor mediating the coordinate expression of genes which are part of the cellular machinery that functions to protect an organism against damage posed by physical, chemical or microbial noxae. In a wide variety of patho-physiological situations such as immune and inflammatory reactions, the expression of cytokines, interleukins and adhesion molecules in cells of the immune system including T and B cells, endothelial as well as phagocyticrantigen presenting cells is to a large extent regulated by NF-kB. Moreover, this transcription factor appears to play a central role in the regulation of apoptosis, an important cellular program that decides upon a cell's fate not only during embryonic development but also on its way from normal to the transformed phenotype. Thus, NF-kB has emerged also as an attractive target for therapeutic interference in a variety of pathological situations, including chronic inflammatory and autoimmune diseases, HIV infection and cancer. q

Molecular Cancer, 2013

The NF-κB family of transcription factors has an essential role in inflammation and innate immuni... more The NF-κB family of transcription factors has an essential role in inflammation and innate immunity. Furthermore, NF-κB is increasingly recognized as a crucial player in many steps of cancer initiation and progression. During these latter processes NF-κB cooperates with multiple other signaling molecules and pathways. Prominent nodes of crosstalk are mediated by other transcription factors such as STAT3 and p53 or the ETS related gene ERG. These transcription factors either directly interact with NF-κB subunits or affect NF-κB target genes. Crosstalk can also occur through different kinases, such as GSK3-β, p38, or PI3K, which modulate NF-κB transcriptional activity or affect upstream signaling pathways. Other classes of molecules that act as nodes of crosstalk are reactive oxygen species and miRNAs. In this review, we provide an overview of the most relevant modes of crosstalk and cooperativity between NF-κB and other signaling molecules during inflammation and cancer.

Journal of Experimental Medicine, 1998

By differential screening of tumor necrosis factor ␣ (TNF-␣ ) and lipopolysaccharide (LPS)activat... more By differential screening of tumor necrosis factor ␣ (TNF-␣ ) and lipopolysaccharide (LPS)activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis ( iap ) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap 1, hiap 2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-␣ , interleukin 1 ␤ , and LPS, reagents that lead to activation of the nuclear transcription factor B (NF-B). Indeed, overexpression of I B ␣ , an inhibitor of NF-B, suppresses the induced expression of iap genes and sensitizes ECs to TNF-␣ -induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome-linked iap ( xiap ), using recombinant adenovirus overrules the I B ␣ effect and protects ECs from TNF-␣induced apoptosis. We conclude that xiap represents one of the NF-B-regulated genes that counteracts the apoptotic signals caused by TNF-␣ and thereby prevents ECs from undergoing apoptosis during inflammation.

Journal of Biomedical Optics, 2011

Journal of Biological Chemistry, 2006

Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the G... more Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 ((566)RL(567)), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D ((733)DD(734)) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (733)DD(734) by (733)VN(734)), and mutation of (566)RL(567) to (566)AS(567) (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.