Shannon H. Carroll

A2B Adenosine Receptor Promotes Mesenchymal Stem Cell Differentiation to Osteoblasts and Bone Formation in Vivo

Background: The A2BAR signals via cAMP. Cyclic AMP signaling has been shown to regulate MSC differentiation. Results: A2BAR KO mice have reduced differentiation of osteoblasts, a mild osteopenic phenotype, and impaired fracture physiology. A2BAR activation increases the differentiation of osteoblasts. Conclusion: The A2BAR regulates bone homeostasis. Significance: A2BAR signaling is a component of bone homeostasis, particularly after injury. The differentiation of osteoblasts from their precursors, mesenchymal stem cells, is an important component of bone homeostasis as well as fracture healing. The A2B adenosine receptor (A2BAR) is a Gαs/αq-protein-coupled receptor that signals via cAMP. cAMP-mediated signaling has been demonstrated to regulate the differentiation of mesenchymal stem cells (MSCs) into various skeletal tissue lineages. Here, we studied the role of this receptor in the differentiation of MSCs to osteoblasts. In vitro differentiation of bone marrow-derived MSCs from A2BAR KO mice resulted in lower expression of osteoblast differentiation transcription factors and the development of fewer mineralized nodules, as compared with WT mice. The mechanism of effect involves, at least partially, cAMP as indicated by experiments involving activation of the A2BAR or addition of a cAMP analog during differentiation. Intriguingly, in vivo, microcomputed tomography analysis of adult femurs showed lower bone density in A2BAR KO mice as compared with WT. Furthermore, A2BAR KO mice display a delay in normal fracture physiology with lower expression of osteoblast differentiation genes. Thus, our study identified the A2BAR as a new regulator of osteoblast differentiation, bone formation, and fracture repair.

A new role for the A2b adenosine receptor in regulating platelet function

Summary.  Background: Activation of platelets is a critical component of atherothrombosis and plays a central role in the progression of unstable cardiovascular syndromes. Adenosine, acting through adenosine receptors, increases intracellular cAMP levels and inhibits platelet aggregation. The A2a adenosine receptor has already been recognized as a mediator of adenosine‐dependent effects on platelet aggregation, and here we present a new role for the A2b adenosine receptor (A2bAR) in this process. Methods and Results: As compared with platelets from wild‐type controls, platelets derived from A2bAR knockout mice have significantly greater ADP receptor activation‐induced aggregation. Although mouse megakaryocytes and platelets express low levels of the A2bAR transcript, this gene is highly upregulated following injury and systemic inflammation in vivo. Under these conditions, A2bAR‐mediated inhibition of platelet aggregation significantly increases. Our studies also identify a novel mechanism by which the A2bAR could regulate platelet aggregation; namely, ablation of the A2bAR leads to upregulated expression of the P2Y1 ADP receptor, whereas A2bAR‐mediated or direct elevation of cAMP has the opposite effect. Thus, the A2bAR regulates platelet function beyond mediating the immediate effect of adenosine on aggregation. Conclusions: Taken together, these investigations show for the first time that the platelet A2bAR is upregulated under stress in vivo, plays a significant role in regulating ADP receptor expression, and inhibits agonist‐induced platelet aggregation.

Mechanisms of induction of adenosine receptor genes and its functional significance.

Adenosine is a metabolite generated and released from cells, particularly under injury or stress. It elicits protective or damaging responses via signaling through the adenosine receptors, including the adenylyl cyclase inhibitory A1 and A3, and the adenylyl cyclase stimulatory A2A and A2B. Multiple adenosine receptor types, including stimulatory and inhibitory, can be found in the same cell, suggesting that a careful balance of adenosine receptor expression in a particular cell is necessary for a specific adenosine‐induced response. This balance could be controlled by differential expression of the adenosine receptor genes under different stimuli. Here, we have reviewed an array of studies that have characterized basal or induced expression of the adenosine receptors and common as well as distinct mechanisms of effect, in hopes that ongoing studies on this topic will further elucidate detailed mechanisms of adenosine receptor regulation, leading to potential therapeutic applications. J. Cell. Physiol. 218: 35–44, 2009.

Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.

TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-alpha) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A(2B) adenosine receptor (A(2B)AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-alpha. Here, we show a regulatory loop by which TNF-alpha upregulates the A(2B)AR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A(2B)AR, and Nox inhibitors dampen the effect of TNF-alpha. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A(2B)AR.

Activation of the macrophage A2b adenosine receptor regulates tumor necrosis factor–α levels following vascular injury

OBJECTIVE The control of expression of tumor necrosis factor-alpha (TNF-alpha) impacts a variety of processes during a stress response. Macrophages are a major source of TNF-alpha, the level of which is known to be regulated by adenosine. Previous studies highlighted the role of the A2a adenosine receptor in this process, while the role of the A2b adenosine receptor (A2bAR) has not been clearly identified. Here, we examined the contribution of the A2bAR to TNF-alpha regulation by macrophages at baseline and under vascular stress. MATERIALS AND METHODS We employed a newer A2bAR-selective ligand, BAY 60-6583 in vitro and in vivo, and an A2bAR antagonist CVT-6883, as well as examined macrophages derived from control or A2bAR knockout mice. RESULTS We found that the expression of the A2bAR is upregulated in macrophages derived from wild-type mice subjected to arterial injury, and this receptor activity controls the level of TNF-alpha released from macrophages. CONCLUSION We identified a significant role for the A2bAR in the regulation of TNF-alpha, which would contribute to the anti-inflammatory actions of adenosine under vascular stress. This conclusion could focus attention on this receptor as a therapeutic target.

Differentiation of mesenchymal stem cells to osteoblasts and chondrocytes: a focus on adenosine receptors.

Skeletogenesis, either during development, post-injury or for maintenance, is a carefully coordinated process reliant on the appropriate differentiation of mesenchymal stem cells. Some well described, as well as a new regulator of this process (adenosine receptors), are alike in that they signal via cyclic-AMP (cAMP). This review highlights the known contribution of cAMP signalling to mesenchymal stem cell differentiation to osteoblasts and to chondrocytes. Focus has been given to how these regulators influence the commitment of the osteochondroprogenitor to these separate lineages.

The Macrophage A2b Adenosine Receptor Regulates Tissue Insulin Sensitivity

High fat diet (HFD)-induced type 2 diabetes continues to be an epidemic with significant risk for various pathologies. Previously, we identified the A2b adenosine receptor (A2bAR), an established regulator of inflammation, as a regulator of HFD-induced insulin resistance. In particular, HFD was associated with vast upregulation of liver A2bAR in control mice, and while mice lacking this receptor showed augmented liver inflammation and tissue insulin resistance. As the A2bAR is expressed in different tissues, here, we provide the first lead to cellular mechanism by demonstrating that the receptors influence on tissue insulin sensitivity is mediated via its expression in macrophages. This was shown using a newly generated transgenic mouse model expressing the A2bAR gene in the macrophage lineage on an otherwise A2bAR null background. Reinstatement of macrophage A2bAR expression in A2bAR null mice fed HFD restored insulin tolerance and tissue insulin signaling to the level of control mice. The molecular mechanism for this effect involves A2bAR-mediated changes in cyclic adenosine monophosphate in macrophages, reducing the expression and release of inflammatory cytokines, which downregulate insulin receptor-2. Thus, our results illustrate that macrophage A2bAR signaling is needed and sufficient for relaying the protective effect of the A2bAR against HFD-induced tissue inflammation and insulin resistance in mice.

An Adenosine Receptor-Krüppel-like Factor 4 Protein Axis Inhibits Adipogenesis

Background: In adipogenesis, new adipocytes are generated from precursor cells and contribute to adipose tissue expansion. Results: The A2b adenosine receptor (A2bAR) inhibits adipogenesis via expression of Krüppel-like factor 4 (KLF4). Conclusion: A2bAR signaling regulates adipogenesis and is correlated tightly with KLF4. Significance: A2bAR signaling via KLF4 may play an important role in adipose tissue biology. Adipogenesis represents a key process in adipose tissue development and remodeling, including during obesity. Exploring the regulation of adipogenesis by extracellular ligands is fundamental to our understanding of this process. Adenosine, an extracellular nucleoside signaling molecule found in adipose tissue depots, acts on adenosine receptors. Here we report that, among these receptors, the A2b adenosine receptor (A2bAR) is highly expressed in adipocyte progenitors. Activation of the A2bAR potently inhibits differentiation of mouse stromal vascular cells into adipocytes, whereas A2bAR knockdown stimulates adipogenesis. The A2bAR inhibits differentiation through a novel signaling cascade involving sustained expression of Krüppel-like factor 4 (KLF4), a regulator of stem cell maintenance. Knockdown of KLF4 ablates the ability of the A2bAR to inhibit differentiation. A2bAR activation also inhibits adipogenesis in a human primary preadipocyte culture system. We analyzed the A2bAR-KLF4 axis in adipose tissue of obese subjects and, intriguingly, found a strong correlation between A2bAR and KLF4 expression in both subcutaneous and visceral human fat. Hence, our study implicates the A2bAR as a regulator of adipocyte differentiation and the A2bAR-KLF4 axis as a potentially significant modulator of adipose biology.

Diabetes regulates fructose absorption through thioredoxin-interacting protein

Metabolic studies suggest that the absorptive capacity of the small intestine for fructose is limited, though the molecular mechanisms controlling this process remain unknown. Here we demonstrate that thioredoxin-interacting protein (Txnip), which regulates glucose homeostasis in mammals, binds to fructose transporters and promotes fructose absorption by the small intestine. Deletion of Txnip in mice reduced fructose transport into the peripheral bloodstream and liver, as well as the severity of adverse metabolic outcomes resulting from long-term fructose consumption. We also demonstrate that fructose consumption induces expression of Txnip in the small intestine. Diabetic mice had increased expression of Txnip in the small intestine as well as enhanced fructose uptake and transport into the hepatic portal circulation. The deletion of Txnip in mice abolished the diabetes-induced increase in fructose absorption. Our results indicate that Txnip is a critical regulator of fructose metabolism and suggest that a diabetic state can promote fructose uptake. DOI: http://dx.doi.org/10.7554/eLife.18313.001