Information about cholesterol subcellular localization and transport pathways inside cells is essential for understanding and treatment of cholesterol-related diseases. However, there is a lack of reliable tools to monitor it. This work follows the fate of Sterolight, a BODIPY-labelled sterol, within the cell and demonstrates it as a suitable probe for visualization of sterol/lipid trafficking. Sterolight enters cells through an energy-independent process and knockdown experiments suggest caveolin-1 as its potential cellular carrier. Intracellular transport of Sterolight is a rapid process, and transfer from ER and mitochondria to lysosomes and later to lipid droplets requires the participation of active microtubules, as it can be inhibited by the microtubule disruptor nocodazole. Excess of the probe is actively exported from cells, in addition to being stored in lipid droplets, to re-establish the sterol balance. Efflux occurs through a mechanism requiring energy and may be selectively poisoned with verapamil or blocked in cells with mutated cholesterol transporter NPC1. Sterolight is efficiently transferred within and between different cell populations, making it suitable for monitoring numerous aspects of sterol biology, including the live tracking and visualization of intracellular and intercellular transport.
A number of human autoinflammatory diseases manifest with severe inflammatory bone destruction. Mouse models of these diseases represent valuable tools that help us to understand molecular mechanisms triggering this bone autoinflammation. The Pstpip2cmo mouse strain is among the best characterized of these; it harbors a mutation resulting in the loss of adaptor protein PSTPIP2 and development of autoinflammatory osteomyelitis. In Pstpip2cmo mice, overproduction of interleukin-1β (IL-1β) and reactive oxygen species by neutrophil granulocytes leads to spontaneous inflammation of the bones and surrounding soft tissues. However, the upstream signaling events leading to this overproduction are poorly characterized. Here, we show that Pstpip2cmo mice deficient in major regulator of Src-family kinases (SFKs) receptor-type protein tyrosine phosphatase CD45 display delayed onset and lower severity of the disease, while the development of autoinflammation is not affected by deficiencies in Toll-like receptor signaling. Our data also show deregulation of pro-IL-1β production by Pstpip2cmo neutrophils that are attenuated by CD45 deficiency. These data suggest a role for SFKs in autoinflammation. Together with previously published work on the involvement of protein tyrosine kinase spleen tyrosine kinase, they point to the role of receptors containing immunoreceptor tyrosine-based activation motifs, which after phosphorylation by SFKs recruit spleen tyrosine kinase for further signal propagation. We propose that this class of receptors triggers the events resulting in increased pro-IL-1β synthesis and disease initiation and/or progression.
- MeSH
- adaptorové proteiny signální transdukční genetika imunologie MeSH
- antigeny CD45 genetika imunologie MeSH
- cytoskeletální proteiny genetika imunologie MeSH
- diabetes mellitus 1. typu genetika imunologie patologie MeSH
- interleukin-1beta genetika imunologie MeSH
- myši knockoutované MeSH
- myši MeSH
- neutrofily imunologie patologie MeSH
- osteomyelitida genetika imunologie patologie MeSH
- signální transdukce genetika imunologie MeSH
- stupeň závažnosti nemoci MeSH
- toll-like receptory genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1β by neutrophil granulocytes. In this study, we show that in addition to IL-1β, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1β levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- buněčné linie MeSH
- cytoskeletální proteiny genetika metabolismus MeSH
- interleukin-1beta imunologie metabolismus MeSH
- kosti a kostní tkáň imunologie patologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- mutace MeSH
- myši transgenní MeSH
- myši MeSH
- NADPH-oxidasa 2 genetika metabolismus MeSH
- neutrofily imunologie metabolismus MeSH
- osteomyelitida genetika imunologie patologie MeSH
- primární buněčná kultura MeSH
- signální transdukce genetika imunologie MeSH
- superoxidy imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cellular cholesterol plays fundamental and diverse roles in many biological processes and affects the pathology of various diseases. Comprehensive and detailed understanding of the cellular functions and characteristics of cholesterol requires visualization of its subcellular distribution, which can be achieved by fluorescence microscopy. Many attempts have been made to develop fluorescent cholesterol reporters, but so far, none of them seems to be ideal for studying all aspects of cholesterol management. To meet the requirements for the right probe remains a great challenge, and progress in this field continues. The main objective of this review is to not only present the current state of the art, but also critically evaluate the applicability of individual probes and for what purpose they can be used to obtain relevant data. Hence, the data obtained with different probes might provide complementary information to build an integrated picture about the cellular cholesterol.
- MeSH
- biologický transport fyziologie MeSH
- cholesterol metabolismus MeSH
- fluorescenční barviva * MeSH
- lidé MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.
- MeSH
- biologický transport MeSH
- buněčná membrána chemie metabolismus MeSH
- buněčné linie MeSH
- cholesterol analýza metabolismus MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- lyzozomální nemoci z ukládání diagnóza metabolismus MeSH
- pyridiny chemie MeSH
- sloučeniny boru chemie MeSH
- steroly chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The development of drug resistance is a major problem which often occurs during anticancer chemotherapies. Photodynamic therapy (PDT) has been studied as an alternative treatment modality for drug-resistant tumors, however the question of resistance to PDT and potential cross-resistance with chemotherapy has yet to be fully answered. To investigate the mechanism of resistance to PDT, we developed an in vitro experimental model system in a mouse mammary carcinoma cell line 4T1. We used two ethylene glycol derivatives of tetraphenylporphyrin, and tetraphenylchlorin derivative, temoporfin, as photosensitizers (PS). PDT-resistant clones were obtained by exposure to a set concentration of PS followed by irradiation with increasing light doses. PDT resistance to soluble glycol porphyrins was mediated mainly by increased drug efflux through ABCB1 (P-glycoprotein) as we demonstrated by specific ABCB1 knockdown experiments, which in turn rescued the sensitivity of resistant cells to PDT. In contrast, resistance raised to temoporfin, which is generally more lipophilic than glycol porphyrins, elicited mechanism based on sequestration of the drug to lysosomes. The resistance that is acquired from a particular PS could be overcome by using a different PS, which is not susceptible to the same mechanism(s) of resistance. Elucidation of the underlying mechanisms in various types of resistance might facilitate improvements in PDT treatment design.
- MeSH
- ABC transportér, podrodina B, člen 11 genetika MeSH
- chemorezistence genetika MeSH
- ethylenglykoly aplikace a dávkování chemie MeSH
- fotochemoterapie MeSH
- fotosenzibilizující látky aplikace a dávkování chemie MeSH
- genový knockdown MeSH
- glykoly chemie MeSH
- lidé MeSH
- mesoporfyriny aplikace a dávkování chemie MeSH
- MFC-7 buňky MeSH
- myši MeSH
- nádory mléčné žlázy u zvířat farmakoterapie genetika patologie MeSH
- paclitaxel škodlivé účinky MeSH
- porfyriny aplikace a dávkování chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transmembrane adaptor proteins are molecules specialized in recruiting cytoplasmic proteins to the proximity of the cell membrane as part of the signal transduction process. A member of this family, SLP65/SLP76, Csk-interacting membrane protein (SCIMP), recruits a complex of SLP65/SLP76 and Grb2 adaptor proteins, known to be involved in the activation of PLCγ1/2, Ras, and other pathways. SCIMP expression is restricted to antigen-presenting cells. In a previous cell line-based study, it was shown that, in B cells, SCIMP contributes to the reverse signaling in the immunological synapse, downstream of MHCII glycoproteins. There it mainly facilitates the activation of ERK MAP kinases. However, its importance for MHCII glycoprotein-dependent ERK signaling in primary B cells has not been analyzed. Moreover, its role in macrophages and dendritic cells has remained largely unknown. Here we present the results of our analysis of SCIMP-deficient mice. In these mice, we did not observe any defects in B cell signaling and B cell-dependent responses. On the other hand, we found that, in dendritic cells and macrophages, SCIMP expression is up-regulated after exposure to GM-CSF or the Dectin-1 agonist zymosan. Moreover, we found that SCIMP is strongly phosphorylated after Dectin-1 stimulation and that it participates in signal transduction downstream of this important pattern recognition receptor. Our analysis of SCIMP-deficient dendritic cells revealed that SCIMP specifically contributes to sustaining long-term MAP kinase signaling and cytokine production downstream of Dectin-1 because of an increased expression and sustained phosphorylation lasting at least 24 h after signal initiation.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- B-lymfocyty metabolismus MeSH
- buněčné linie MeSH
- dendritické buňky metabolismus MeSH
- fosfolipasa C gama genetika metabolismus MeSH
- lektiny typu C genetika metabolismus MeSH
- MAP kinasový signální systém fyziologie MeSH
- mutantní kmeny myší MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
