Thrombin-Mediated Formation of Globular Adiponectin Promotes an Increase in Adipose Tissue Mass.

A decrease in the circulating levels of adiponectin in obesity increases the risk of metabolic complications, but the role of globular adiponectin, a truncated form produced by proteolytic cleavage, has not been defined. The objective of this investigation was to determine how globular adiponectin is generated and to determine whether this process impacts obesity. The cleavage of recombinant full-length adiponectin into globular adiponectin by plasma in vitro was used to identify Gly-93 as the N-terminal residue after proteolytic processing. The amino acid sequence of the cleavage site suggested thrombin was the protease responsible for cleavage, and inhibitors confirmed its likely involvement. The proteolytic site was modified, and this thrombin-resistant mutant protein was infused for 4 weeks into obese adiponectin-knockout mice that had been on a high-fat diet for 8 weeks. The mutation of the cleavage site ensured that globular adiponectin was not generated, and thus did not confound the actions of the full-length adiponectin. Mice infused with the mutant adiponectin accumulated less fat and had smaller adipocytes compared to mice treated with globular adiponectin, and concurrently had elevated fasting glucose. The data demonstrate that generation of globular adiponectin through the action of thrombin increases both adipose tissue mass and adipocyte size, but it has no effect on fasting glucose levels in the context of obesity.

PDGF-BB-mediated activation of CREB in vascular smooth muscle cells alters cell cycling via Rb, FoxO1 and p27kip1.

INTRODUCTION & AIM: The vascular response to injury leads to the secretion of several factors, including platelet-derived growth factor (PDGF-BB). PDGF-BB stimulates smooth muscle cell (SMC) conversion to the synthetic phenotype, thereby enhancing proliferation and migration, and contributing to neointimal hyperplasia. Likewise, the cAMP response element binding protein (CREB) transcription factor has been shown to mediate SMC proliferation in response to various mitogens. We therefore investigated the contribution of CREB to PDGF-BB-dependent proliferation of SMCs with the intention of identifying signaling pathways involved both up and downstream of CREB activation. METHODS & RESULTS: Treatments were performed on vascular SMCs from a porcine coronary artery explant model. The role of CREB was examined via adenoviral expression of a dominant-negative CREB mutant (kCREB) as well as inhibition of CREB binding protein (CBP). Involvement of the p27kip1 pathway was determined using a constitutively expressing p27kip1 adenoviral vector. PDGF-BB stimulated transient CREB phosphorylation on Ser-133 via ERK1/2-, PI3-kinase- and Src-dependent pathways. Expression of kCREB decreased PDGF-BB-dependent cell proliferation. PCNA expression and Rb phosphorylation were also inhibited by kCREB. These cell cycle proteins are controlled via p27kip1 expression in response to CREB-dependent post-translational modification of FoxO1. kCREB had no effect on Cyclin D1 expression, but did prevent PDGF-BB-induced Cyclin D1 nuclear translocation. An interaction inhibitor of CBP confirmed that Cyclin D1 is downstream of PDGF-BB and CREB. CONCLUSION: CREB phosphorylation is required for SMC proliferation in response to PDGF-BB. This phenotypic change requires CBP and is mediated by Cyclin D1 and p27kip as a result of changes in FoxO1 activity.

Establishing the interchangeability of arterial stiffness but not endothelial function parameters in healthy individuals.

BACKGROUND: Development of instruments capable of detecting early stage vascular disease has increased interest in employing arterial stiffness (e.g. pulse wave velocity (PWV), augmentation index (AIx)) and endothelial dysfunction (e.g. reactive hyperemia index (RHI)) to diagnose atherosclerotic disease before occurrence of a cardiovascular event. However, amongst the equipment designed for this purpose, there is insufficient information regarding each of these parameters to establish appropriate cutoffs to distinguish between healthy and unhealthy blood vessels. To address these limitations, the study was designed to establish the upper arterial stiffness and endothelial function thresholds in a healthy population, by comparing the outputs from different instruments capable of measuring PWV, AIx and RHI. METHODS: A systematic comparison of PWV, AIx and RHI was conducted to determine the inter-relationships between these parameters of vascular functionality. Outputs were obtained non-invasively using three instruments, the VP-1000 (VP), SphygmoCor (SC), and EndoPAT (EP), in 40 apparently healthy males and females. RESULTS: Correlations were found between the brachial-ankle PWV and radial-ankle PWV (by VP and SC), and PWV (VP) with AIx (SC). The interchangeability of these outputs was demonstrated by the Bland Altman test, making it feasible to extrapolate cut-offs for radial-ankle PWV and AIx equivalent to brachial-ankle PWV that signify healthy vessels. In contrast, RHI showed no association with AIx, suggesting these endothelial and arterial parameters are functionally distinct. CONCLUSIONS: It was concluded that it is possible to compare the vascular function outputs of different instruments and identify healthy from unhealthy vessels, even though the approaches for quantifying the underlying physiological processes may differ. In this way, non-invasive determination of arterial function could be a new paradigm for detecting existing early stage asymptomatic atherosclerotic disease in individuals using techniques that are amenable to the clinical setting.

Atypical G(αi) signal transduction.

G protein signaling is an extremely complex event that is involved in almost every cellular process. As such, G protein-coupled receptors are the most commonly found type of transmembrane receptors used by cells to initiate intracellular signaling events. However, the widely accepted model of cyclical GDP-GTP exchange in response to ligand binding to 7TMRs, followed by dissociation of the G protein subunits and activation of intracellular signaling cascades, has repeatedly been challenged in recent years. Some of the exceptions that have been brought forth include signaling by a non-dissociated, rearranged heterotrimer and the existence of "reverse-mode", active G proteins that interact with active receptors. Here, we focus on G(αi/o), one of the common G(α) classes, and outline a major exception to the classical model, that of G protein coupling to RTKs. We then describe a novel concept in G(αi/o) signaling, namely that the pathways induced by agonist binding circumvent the typical signaling pathways responsive to decreases in the second messenger cAMP, via adenylyl cyclase inhibition.

The cyclic AMP response element-binding protein (CREB) mediates smooth muscle cell proliferation in response to angiotensin II.

The cAMP response element-binding protein (CREB) is a transcription factor that mediates the cellular response to metabolic and mitogenic signals. Whether CREB contributes to vascular function has received little attention, especially in relation to the processes associated with atherosclerotic disease progression and restenosis. This study examined the involvement of CREB in the mitogenic actions of angiotensin II (AngII), a growth factor that promotes neointimal hyperplasia in response to vascular injury. Treatments were performed on quiescent vascular smooth muscle cells (VSMCs) obtained from a porcine explant model. Organ culture was performed on porcine hearts subjected to angioplasty ex vivo. Stimulation of VSMCs with AngII resulted in transient CREB phosphorylation. Proliferation of smooth muscle cells in response to AngII was reduced by 90 % after infection with adenovirus expressing dominant-negative killer CREB (kCREB) mutant. Likewise, expression of kCREB prevented angioplasty-induced neointimal hyperplasia. AngII-induced CREB phosphorylation was independent of cAMP activation. Examination of putative CREB kinases revealed that MSK was responsible for phosphorylating CREB. In addition, inhibition of PKC revealed that this kinase operates upstream and activates MSK. These results indicate that activation of CREB via PKC and MSK is essential for SMC proliferation in response to AngII.

Identification of novel signalling roles and targets for G(α) and G(ßγ) downstream of the insulin-like growth factor 1 receptor in vascular smooth muscle cells.

Vascular dysfunction is the underlying cause of nearly 80% of heart disease cases, and its initiation and progression can be exacerbated by circulating factors, such as IGF-1 (insulin-like growth factor 1). IGF-1, which is highly homologous with insulin, elicits a response via a classical tyrosine kinase receptor, the IGF-1R (IGF-1 receptor). However, it has been suggested that the IGF-1R may also be coupled to a heterotrimeric G-protein and can thus modulate cellular processes via this alternate pathway. The objective of the present study was to investigate the structural aspects of IGF-1R coupling to a heterotrimeric G-protein in VSMCs [vascular SMCs (smooth muscle cells)], as well as examine the contribution of this pathway to cellular responses that are related to vascular disease. We found that the intracellular subunit of the IGF-1R precipitates with two G-protein subunits. The G(ßγ)-mediated pathway contributes to both proliferation and migration. We also show that IGF-1 specifically activates G(αi) and can directly interact with both G(αi1) and G(αi2). A phospho-screen using a novel specific G(αi)-peptide inhibitor reveals a number of potential downstream effectors of this pathway, although our results show that it is not essential for SMC proliferation or migration.

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor.

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gßγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.