NF-kappa B proteins are a family of transcription factors that are of central importance in inflammation and immunity. NF-kappa B also plays important roles in other processes, including development, cell growth and survival, and proliferation, and is involved in many pathological conditions. Reactive Oxygen Species (ROS) are created by a variety of cellular processes as part of cellular signaling events. While certain NF-kappa B-regulated genes play a major role in regulating the amount of ROS in the cell, ROS have various inhibitory or stimulatory roles in NF-kappa B signaling. Here we review the regulation of ROS levels by NF-kappa B targets and various ways in which ROS have been proposed to impact NF-kappa B signaling pathways.
Hepatocellular carcinoma (HCC), the major form of primary liver cancer, is one of the most deadly human cancers. The pathogenesis of HCC is frequently linked with continuous hepatocyte death, inflammatory cell infiltration and compensatory liver regeneration. Understanding the molecular signaling pathways driving or mediating these processes during liver tumorigenesis is important for the identification of novel therapeutic targets for this dreadful disease. The classical IKK beta-dependent NF-kappa B signaling pathway has been shown to promote hepatocyte survival in both developing and adult livers. In addition, it also plays a crucial role in liver inflammatory responses by controlling the expression of an array of growth factors and cytokines. One of these cytokines is IL-6, which is best known for its role in the liver acute phase response. IL-6 exerts many of its functions via activation of STAT3, a transcription factor found to be important for HCC development. This review will focus on recent studies on the roles of NF-kappa B and STAT3 in liver cancer. Interactions between the two pathways and their potential as therapeutic targets will also be discussed.
The non-canonical NF-kappa B pathway is an important arm of NF-kappa B signaling that predominantly targets activation of the p52/RelB NF-kappa B complex. This pathway depends on the inducible processing of p100, a molecule functioning as both the precursor of p52 and a RelB-specific inhibitor. A central signaling component of the non-canonical pathway is NF-kappa B-inducing kinase (NIK), which integrates signals from a subset of TNF receptor family members and activates a downstream kinase, I kappa B kinase-kappa (IKK alpha), for triggering p100 phosphorylation and processing. A unique mechanism of NIK regulation is through its fate control: the basal level of NIK is kept low by a TRAF-cIAP destruction complex and signal-induced non-canonical NF-kappa B signaling involves NIK stabilization. Tight control of the fate of NIK is important, since deregulated NIK accumulation is associated with lymphoid malignancies.
The complement system plays a crucial role in the innate defense against common pathogens. Activation of complement leads to robust and efficient proteolytic cascades, which terminate in opsonization and lysis of the pathogen as well as in the generation of the classical inflammatory response through the production of potent proinflammatory molecules. More recently, however, the role of complement in the immune response has been expanded due to observations that link complement activation to adaptive immune responses. It is now appreciated that complement is a functional bridge between innate and adaptive immune responses that allows an integrated host defense to pathogenic challenges. As such, a study of its functions allows insight into the molecular underpinnings of host-pathogen interactions as well as the organization and orchestration of the host immune response. This review attempts to summarize the roles that complement plays in both innate and adaptive immune responses and the consequences of these interactions on host defense.
CD4 T helper （Th） cells play critical roles in adaptive immune responses. They recruit and activate other immune cells including B cells, CD8 T cells, macrophages, mast cells, neutrophils, eosinophils and basophils. Based on their functions, their pattern of cytokine secretion and their expression of specific transcription factors, Th cells, differentiated from naive CD4 T cells, are classified into four major lineages, Thl, Th2, Th17 and T regulatory （Treg） cells, although other Th lineages may exist. Subsets of the same lineage may express different effector cytokines, reside at different locations or give rise to cells with different fates, whereas cells from different lineages may secrete common cytokines, such as IL-2, IL-9 and IL-10, resulting in massive heterogeneity of the Th cell population. In addition, the pattern of cytokine secretion may switch from that of one lineage toward another under certain circumstances, suggesting that Th cells are plastic. Tregs are also more heterogeneous and plastic than were originally thought. In this review, we summarize recent reports on heterogeneity and plasticity of Th cells, and discuss potential mechanisms and implications of such features that Th cells display.
Mesenchymal stem cells （MSCs） have great potential for treating various diseases, especially those related to tissue damage involving immune reactions. Various studies have demonstrated that MSCs are strongly immunosuppressive in vitro and in vivo. Our recent studies have shown that un-stimulated MSCs are indeed incapable of immunosuppression; they become potently immunosuppressive upon stimulation with the supernatant of activated lymphocytes, or with combinations of IFN-γ, with TNF-α, IL-1α or IL-1β. This observation revealed that under certain circumstances, inflammatory cytokines can actually become immunosuppressive. We showed that there is a species variation in the mechanisms of MSC-mediated immunosuppression： immunosuppression by cytokine-primed mouse MSCs is mediated by nitric oxide （NO）, whereas immunosuppression by cytokine-primed human MSCs is executed through indoleamine 2, 3-dioxygenase （IDO）. Additionally, upon stimulation with the inflammatory cytokines, both mouse and human MSCs secrete several leukocyte chemokines that apparently serve to attract immune cells into the proximity with MSCs, where NO or IDO is predicted to be most active. Therefore, immunosuppression by inflammatory cytokine-stimulated MSCs occurs via the concerted action of chemokines and immune-inhibitory NO or IDO produced by MSCs. Thus, our results provide novel information about the mechanisms of MSC-mediated immunosuppression and for better application of MSCs in treating tissue injuries induced by immune responses.
Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.
The I kappa B kinase/NF-kappa B signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-kappa B is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-kappa B activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-kappa B inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-kappa B inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-kappa B signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-kappa B has been predominantly observed in epithelial cells, indicating that NF-kappa B signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-kappa B displays two faces in chronic inflammation: on the one hand increased and sustained NF-kappa B activation induces inflammation and tissue damage, but on the other hand inhibition of NF-kappa B signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-kappa B signaling, focusing particularly on the role of NF-kappa B in the regulation of immune homeostasis and inflammation in the intestine and the skin.
Emerging evidence has shown the association of aberrantly expressed microRNAs （miRNAs） with tumor development and progression. However, little is known about the potential role of miRNAs in gastric carcinogenesis. Here, we performed miRNA microarray to screen miRNAs differentially expressed in the paired gastric cancer and their adjacent nontumor tissues and found that miR-375 was greatly downregulated in gastric cancer tissues. Quantitative real-time PCR analysis verified that miR-375 expression was significantly decreased in more than 90% of primary gastric cancers compared with their nontumor counterparts from patients undergoing gastric resection. Overexpression of miR-375 significantly inhibited gastric cancer cell proliferation in vitro and in vivo. Forced expression of miR-375 in gastric cancer cells significantly reduced the protein level of Janus kinase 2 （JAK2） and repressed the activity of a luciferase reporter carrying the 3＇-untranslated region of JAK2, which was abolished by mutation of the predicted miR-375-binding site, indicating that JAK2 may be a miR-375 target gene. Either inhibition of JAK2 activity by AG490 or silencing of JAK2 by RNAi suppressed gastric cancer cell proliferation resembling that of miR-375 overexpression. Moreover, ectopic expression of JAK2 can partially reverse the inhibition of cell proliferation caused by miR-375. Finally, we found a significant inverse correlation between miR-375 expression and JAK2 protein level in gastric cancer. Thus, these data suggest that miR-375 may function as a tumor suppressor to regulate gastric cancer cell proliferation potentially by targeting the JAK2 oncogene, implicating a role of miR-375 in the pathogenesis of gastric cancer.
The introduction of four transcription factors Oct4, Klf4, Sox2 and c-Myc by viral transduction can induce reprogramming of somatic ceils into induced pluripotent stem ceils （iPSCs）, but the use of iPSCs is hindered by the use of viral delivery systems. Chemicalinduced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was inde- pendent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.
Two distinct nuclear factor kappa B (NF kappa B) signaling pathways have been described; the canonical pathway that mediates inflammatory responses, and the non-canonical pathway that is involved in immune cell differentiation and maturation and secondary lymphoid organogenesis. The former is dependent on the I kappa B kinase adaptor molecule NEMO, the latter is independent of it. Here, we review the molecular mechanisms of regulation in each signaling axis and attempt to relate the apparent regulatory logic to the physiological function. Further, we review the recent evidence for extensive cross-regulation between these two signaling axes and summarize them in a wiring diagram. These observations suggest that NEMO-dependent and -independent signaling should be viewed within the context of a single NF kappa B signaling system, which mediates signaling from both inflammatory and organogenic stimuli in an integrated manner. As in other regulatory biological systems, a systems approach including mathematical models that include quantitative and kinetic information will be necessary to characterize the network properties that mediate physiological function, and that may break down to cause or contribute to pathology.
Endocytosis occurs at the cell surface and involves internalization of the plasma membrane （PM） along with its constituent membrane proteins and lipids. Endocytosis is involved in sampling of the extracellular milieu and also serves to regulate various processes initiated at the cell surface. These include nutrient uptake, signaling from cell- surface receptors, and many other processes essential for cell and tissue functioning in metazoans. It is also central to the maintenance of PM lipid and protein homeostasis. There are multiple means of internalization that operate concurrently, at the cell surface. With advancement in high-resolution visualization techniques, it is now possible to track multiple endocytic cargo at the same time, revealing a remarkable diversity of endocytic processes in a single cell. A combination of live cell imaging and efficient genetic manipulations has also aided in understanding the functional hierarchy of molecular players in these mechanisms of internalization. Here we provide an account of various endocytic routes, their mechanisms of operation and occurrence across phyla.
The nuclear factor-kappa B (NF-kappa B) transcription factor plays a critical role in diverse cellular processes associated with proliferation, cell death, development, as well as innate and adaptive immune responses. NF-kappa B is normally sequestered in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF-kappa B (I kappa Bs). The signal pathways leading to the liberation and nuclear accumulation of NF-kappa B, which can be activated by a wide variety of stimuli, have been extensively studied in the past two decades. After gaining access to the nucleus, NF-kappa B must be actively regulated to execute its fundamental function as a transcription factor. Recent studies have highlighted the importance of nuclear signaling in the regulation of NF-kappa B transcriptional activity. A non-Rel subunit of NF-kappa B, ribosomal protein S3 (RPS3), and numerous other nuclear regulators of NF-kappa B, including Akirin, Nurr1, SIRT6, and others, have recently been identified, unveiling novel and exciting layers of regulatory specificity for NF-kappa B in the nucleus. Further insights into the nuclear events that govern NF-kappa B function will deepen our understanding of the elegant control of its transcriptional activity and better inform the potential rational design of therapeutics for NF-kappa B-associated diseases.
Recent baby formula milk powder contamination incidents have shown that the classic markers or standards in milk quality control are insufficient in identifying ＂manipulated＂ poor-quality milk. In the present study, we demonstrated for the first time that cow milk contains large amounts of microRNAs （miRNAs） and that the unique expression profile of milk-specific miRNAs can serve as a novel indicator and possible new standard for the quafity control of raw milk and milk-related commercial products, such as fluid milk and powdered formula milk. First, using Solexa sequencing, we systematically screened miRNA expression in raw milk and identified a total of 245 miR- NAs in raw milk. Unlike other classic biomarkers whose expression levels are nearly identical at different periods of lactation, individual miRNAs can be significantly altered during lactation process, implicating that miRNAs may be a more accurate indicator to reflect the quality alteration of milk. Second, using TaqMan probe-based miRNA quantitative RT-PCR, we further identified seven miRNAs that have a relatively consistent expression throughout the lactation process, and more importantly, the expression profile of these seven milk-specific miRNAs can serve as an ideal biomarker for discriminating poor-quality or ＂manipulated＂ milk from pure raw milk, as well as for the quality control of commercial milk products, such as fluid milk and powdered formula milk. Together, our findings provide a basis for understanding the physiological role of milk miRNAs and a new potential standard for determining the quality of raw milk or milk-related commercial products.
Interactions between the BCL-2 family proteins determine the cell＇s fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology （BH） 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide span- ning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-XL, MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.
Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells （hESCs） are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the panretinoic acid receptor antagonist BMS-189453 （RAi） significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin＋RAi-treated cultures with Noggin＋RA-treated cul- tures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin＋RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin＋RAi-treated cultures, but not in the cardiomyocytes of Noggin＋RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ±0.88% （mean ± s.e.m） cardiac differen- tiation efficiency, 83% of the cardiomyocytes in Noggin＋RAi-treated cultures had embryonic ventricular-llke action potentials （APs）. With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin＋RA- treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca^2＋ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.
The epithelial-mesenchymal transition （EMT） converts epithelial tumor cells into invasive and metastatic cancer cells, leading to mortality in cancer patients. Although TWIST is a master regulator of EMT and metastasis for breast and other cancers, the mechanisms responsible for TWIST-mediated gene transcription remain unknown. In this study, purification and characterization of the TWIST protein complex revealed that TWIST interacts with several components of the Mi2/nucleosome remodeling and deacetylase （Mi2/NuRD） complex, MTA2, RbAp46, Mi2 and HDAC2, and recruits them to the proximal regions of the E-cadherin promoter for transcriptional repression. Depletion of these TWIST complex components from cancer cell lines that depend on TWIST for metastasis efficiently suppresses cell migration and invasion in culture and lung metastasis in mice. These findings not only provide novel mechanistic and functional links between TWIST and the Mi2/NuRD complex but also establish new essential roles for the components of Mi2/NuRD complex in cancer metastasis.
Nuclear factor-kappa B (NF-kappa B) is a critical regulator of multiple biological functions including innate and adaptive immunity and cell survival. Activation of NF-kappa B is tightly regulated to preclude chronic signaling that may lead to persistent inflammation and cancer. Ubiquitination of key signaling molecules by E3 ubiquitin ligases has emerged as an important regulatory mechanism for NF-kappa B signaling. Deubiquitinases (DUBs) counteract E3 ligases and therefore play a prominent role in the downregulation of NF-kappa B signaling and homeostasis. Understanding the mechanisms of NF-kappa B downregulation by specific DUBs such as A20 and CYLD may provide therapeutic opportunities for the treatment of chronic inflammatory diseases and cancer.
Best known for its role in targeting protein degradation by the proteasome, ubiquitin modification has also emerged as an important mechanism that regulates cell signaling through proteasome-independent mechanisms. The role of ubiquitin as a versatile signaling tag is characteristically illustrated in the NF-kappa B pathways, which regulate a variety of physiological and pathological processes in response to diverse stimuli. Here, we review the role of ubiquitination in different steps of the NF-kappa B signaling cascades, focusing on recent advances in understanding the mechanisms of protein kinase activation by polyubiquitin chains in different pathways that converge on NF-kappa B.
Since the discovery that deletion of the NF-kappa B subunits p50 and p52 causes osteopetrosis in mice, there has been considerable interest in the role of NF-kappa B signaling in bone. NF-kappa B controls the differentiation or activity of the major skeletal cell types - osteoclasts, osteoblasts, osteocytes and chondrocytes. However, with five NF-kappa B subunits and two distinct activation pathways, not all NF-kappa B signals lead to the same physiologic responses. In this review, we will describe the roles of various NF-kappa B proteins in basal bone homeostasis and disease states, and explore how NF-kappa B inhibition might be utilized therapeutically.