Rafts - the current picture

Although evidences that cell membrane contains microdomains are accumulating, the exact properties, diversity and levels of organization of small lipid patches built mainly of cholesterol and sphingomyelin, termed rafts, remain to be elucidated. Our understanding of the cell membrane is increasing with each new raft feature discovered. Nowadays rafts are suggested to act as sites of cell signaling events, to be a part of protein sorting machinery but also they are used by several pathogens as gates into the cells. It is still unclear how rafts are connected to the membrane skeleton and cytoskeleton and with how many different types of rafts are we actually dealing with. This review summarizes some of the most recent discoveries trying to make a view of the complex raft properties

PKC-θ is a negative regulator of TRAIL-induced and FADD-mediated apoptotic spectrin aggregation

Introduction. During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Material and methods. For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging. Results. TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down of PRKCQ in Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of the β-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene, FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane. Conclusions. In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ

Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

Macropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown.. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably.Based on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions

Myocyte-Specific Overexpressing HDAC4 Promotes Myocardial Ischemia/Reperfusion Injury

Background: Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. Methods: In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. Results: Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. Conclusions: Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury

Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling, human myelofibrosis

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPNlike phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34 + hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF

Molecular targeting in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a heterogenous disease associated with distinct genetic and molecular abnormalities. Somatic mutations result in dysregulation of intracellular signaling pathways, epigenetics, and apoptosis of the leukemia cells. Understanding the basis for the dysregulated processes provides the platform for the design of novel targeted therapy for AML patients. The effort to devise new targeted therapy has been helped by recent advances in methods for high-throughput genomic screening and the availability of computer-assisted techniques for the design of novel agents that are predicted to specifically inhibit the mutant molecules involved in these intracellular events. In this review, we will provide the scientific basis for targeting the dysregulated molecular mechanisms and discuss the agents currently being investigated, alone or in combination with chemotherapy, for treating patients with AML. Successes in molecular targeting will ultimately change the treatment paradigm for the disease

NF-κB signaling in neoplastic transition from epithelial to mesenchymal phenotype

Abstract NF-κB transcription factors are critical regulators of innate and adaptive immunity and major mediators of inflammatory signaling. The NF-κB signaling is dysregulated in a significant number of cancers and drives malignant transformation through maintenance of constitutive pro-survival signaling and downregulation of apoptosis. Overactive NF-κB signaling results in overexpression of pro-inflammatory cytokines, chemokines and/or growth factors leading to accumulation of proliferative signals together with activation of innate and select adaptive immune cells. This state of chronic inflammation is now thought to be linked to induction of malignant transformation, angiogenesis, metastasis, subversion of adaptive immunity, and therapy resistance. Moreover, accumulating evidence indicates the involvement of NF-κB signaling in induction and maintenance of invasive phenotypes linked to epithelial to mesenchymal transition (EMT) and metastasis. In this review we summarize reported links of NF-κB signaling to sequential steps of transition from epithelial to mesenchymal phenotypes. Understanding the involvement of NF-κB in EMT regulation may contribute to formulating optimized therapeutic strategies in cancer. Video Abstrac