Smad7: not only a regulator, but also a cross-talk mediator of TGF-β signalling

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation.     

β-Subunit of the Ostα-Ostβ organic solute transporter is required not only for heterodimerization and trafficking but also for function

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostβ required for heterodimerization with Ostα, trafficking of the Ostα-Ostβ complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostβ transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [(3)H]taurocholate transport activity. Deletion of the single TM domain of Ostβ abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostβ to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostβ resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostβ abolished [(3)H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct N(exo)/C(cyt) topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostβ is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostβ critical for these processes.     

Understanding the mechanism of IL-1β secretion

The cytokine interleukin-1β (IL-1β) is a key mediator of the inflammatory response. Essential for the host-response and resistance to pathogens, it also exacerbates damage during chronic disease and acute tissue injury. It is not surprising therefore that there is a huge level of interest in how this protein is produced and exported from cells. However, the mechanism of IL-1β release has proven to be elusive. It does not follow the conventional ER-Golgi route of secretion. A literature full of disparate observations arising from numerous experimental systems, has contributed to a complicated mix of diverse proposals. Here we summarise these observations and propose that secretion of IL-1β occurs on a continuum, dependent upon stimulus strength and the extracellular IL-1β requirement.     

Increased Aβ production prompts the onset of glucose intolerance and insulin resistance

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD.     

Heterochromatin protein (HP)1γ is not only in the nucleus but also in the cytoplasm interacting with actin in both cell compartments

Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization.     

Rosiglitazone, not only potentially protects myocardium against complications of type 2 diabetes mellitus but also induces myocardium infarction: Possible mechanisms?

Rosiglitazone is widely used to improve diabetes mellitus, but its adverse cardiovascular effect is recently recognized. The exact mechanism is still unknown. In this paper, we predict that rosiglitazone probably regulates the insulin gene expression, which cause complications in the long term use.     

Role of miRNAs in the pathogenesis of T2DM,insulin secretion,insulin resistance,and β cell dysfunction: the story so far

Journal of Physiology and Biochemistry - Diabetes, the most common endocrine disorder, also known as a silent killer disease, is characterized by uncontrolled hyperglycemia. According to the...     

MSU Crystals induce sterile IL-1β secretion via P2X7 receptor activation and HMGB1 release

BackgroudThe mechanism by which monosodium urate (MSU) crystals induce inflammation is not completely understood. Few studies have shown that MSU is capable of stimulating the release of IL-1β in the absence of LPS treatment. The purinergic P2X7 receptor is involved in the release of IL-1β in inflammatory settings caused by crystals, as is the case in silicosis.MethodsWe investigated the role of P2X7 receptor in sterile MSU-induced inflammation by evaluating peritonitis and paw edema. In in vitro models, we performed the experiments using peritoneal macrophages and THP-1 cells. We measured inflammatory parameters using ELISA and immunoblotting. We measured cell recruitment using cell phenotypic identification and hemocytometer counts.ResultsOur in vivo data showed that animals without P2X7 receptors generated less paw edema, less cell recruitment, and lower levels of IL-1β release in a peritonitis model. In the in vitro model, we observed that MSU induced dye uptake by the P2X7 receptor. In the absence of the receptor, or when it was blocked, MSU crystals induced less IL-1β release and this effect corresponded to the concentration of extracellular ATP. Moreover, MSU treatment induced HMGB1 release; pre-treatment with P2X7 antagonist reduced the amount of HMGB1 in cell supernatants.ConclusionsIL-1β secretion induced by MSU depends on P2X7 receptor activation and involves HMGB1 release.General significanceWe propose that cell activation caused by MSU crystals induces peritoneal macrophages and THP-1 cells to release ATP and HMGB1, causing IL-1β secretion via P2X7 receptor activation.     

Commensal Bacteria and MAMPs Are Necessary for Stress-Induced Increases in IL-1β and IL-18 but Not IL-6, IL-10 or MCP-1

Regular interactions between commensal bacteria and the enteric mucosal immune environment are necessary for normal immunity. Alterations of the commensal bacterial communities or mucosal barrier can disrupt immune function. Chronic stress interferes with bacterial community structure (specifically, α-diversity) and the integrity of the intestinal barrier. These interferences can contribute to chronic stress-induced increases in systemic IL-6 and TNF-α. Chronic stress, however, produces many physiological changes that could indirectly influence immune activity. In addition to IL-6 and TNF-α, exposure to acute stressors upregulates a plethora of inflammatory proteins, each having unique synthesis and release mechanisms. We therefore tested the hypothesis that acute stress-induced inflammatory protein responses are dependent on the commensal bacteria, and more specifically, lipopolysaccharide (LPS) shed from Gram-negative intestinal commensal bacteria. We present evidence that both reducing commensal bacteria using antibiotics and neutralizing LPS using endotoxin inhibitor (EI) attenuates increases in some (inflammasome dependent, IL-1 and IL-18), but not all (inflammasome independent, IL-6, IL-10, and MCP-1) inflammatory proteins in the blood of male F344 rats exposed to an acute tail shock stressor. Acute stress did not impact α- or β- diversity measured using 16S rRNA diversity analyses, but selectively reduced the relative abundance of Prevotella. These findings indicate that commensal bacteria contribute to acute stress-induced inflammatory protein responses, and support the presence of LPS-mediated signaling in stress-evoked cytokine and chemokine production. The selectivity of the commensal bacteria in stress-evoked IL-1β and IL-18 responses may implicate the inflammasome in this response.     

Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition?

The transposase of the bacterial insertion sequence IS1 is normally expressed by inefficient translational frameshifting between an upstream reading frame which itself specifies a transposition inhibitor, InsA, and a second consecutive reading frame located immediately downstream. A fused-frame mutant which carries an additional base pair inserted at the point of frameshifting was constructed. This mutant exhibits high transposition activity and should express the transposase, InsAB', constitutively without frameshifting. Unexpectedly, a second protein species was observed to be expressed from this mutant. We demonstrate here that this protein, InsA*, results from continued frameshifting on the modified frameshift motif. The protein retains the activities of the repressor InsA. Its elimination, by further modification of the frameshift motif, results in a further increase in various transposition activities of IS1. These results support the hypothesis that a single IS1-encoded protein, InsAB', is necessary for transposition.     

Brain insulin resistance accelerates Aβ fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes

Type 2 diabetes mellitus is thought to be a significant risk factor for Alzheimer's disease. Insulin resistance also affects the central nervous system by regulating key processes, such as neuronal survival and longevity, learning and memory. However, the mechanisms underlying these effects remain uncertain. To investigate whether insulin resistance is associated with the assembly of amyloid β-protein (Aβ) at the cell surface of neurons, we inhibited insulin-signalling pathways of primary neurons. The treatments of insulin receptor (IR)-knockdown and a phosphatidylinositol 3-kinase inhibitor (LY294002), but not an extracellular signal-regulated kinase inhibitor, induced an increase in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of the neurons. The aged db/db mouse brain exhibited reduction in IR expression and phosphorylation of Akt, which later induced an increase in the high-density GM1-clusters on synaptosomes. Neurons treated with IR knockdown or LY294002, and synaptosomes of the aged db/db mouse brains markedly accelerated an assembly of Aβs. These results suggest that ageing and peripheral insulin resistance induce brain insulin resistance, which accelerates the assembly of Aβs by increasing and clustering of GM1 in detergent-resistant membrane microdomains of neuronal membranes.     

TLR4 activation induces IL-1β release via an IPAF dependent but caspase 1/11/8 independent pathway in the lung

Background

The IL-1 family of cytokines is known to play an important role in inflammation therefore understanding the mechanism by which they are produced is paramount. Despite the recent plethora of publications dedicated to the study of these cytokines, the mechanism by which they are produced in the airway following endotoxin, Lipopolysaccharide (LPS), exposure is currently unclear. The aim was to determine the mechanism by which the IL-1 cytokines are produced after LPS inhaled challenge.

Methods

Mice were challenged with aerosolised LPS, and lung tissue and bronchiolar lavage fluid (BALF) collected. Targets were measured at the mRNA and protein level; caspase activity was determined using specific assays.

Results

BALF IL-1b/IL-18, but not IL-1a, was dependent on Ice Protease-Activating Factor (IPAF), and to a lesser extent Apoptosis-associated Speck-like protein containing a CARD (ASC). Interestingly, although we measured an increase in mRNA expression for caspase 1 and 11, we could not detect an increase in lung enzyme activity or a role for them in IL-1a/b production. Further investigations showed that whilst we could detect an increase in caspase 8 activity at later points in the time course (during resolution of inflammation), it appeared to play no role in the production of IL-1 cytokines in this model system.

Conclusions

TLR4 activation increases levels of BALF IL-1b/IL-18 via an IPAF dependent and caspase 1/11/8 independent pathway. Furthermore, it would appear that the presence of IL-1a in the BALF is independent of these pathways. This novel data sheds light on innate signalling pathways in the lung that control the production of these key inflammatory cytokines.     

Unchaining the beast; insights from structural and evolutionary studies on TGFβ secretion,sequestration, and activation

TGFβ is secreted in a latent state and must be “activated” by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFβ mobilisation in vivo. Here we review the process of TGFβ secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFβ is secreted, sequestered and released. This allows the separation of more ancient TGFβ activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms.