An emerging role for adenosine and its receptors in bone homeostasis.

Bone is continually being remodeled and defects in the processes involved lead to bone diseases. Many regulatory factors are known to influence remodeling but other mechanisms, hitherto unknown, may also be involved. Importantly, our understanding of these currently unknown mechanisms may lead to important new therapies for bone disease. It is accepted that purinergic signaling is involved in bone, and our knowledge of this area has increased significantly over the last 15 years, although most of the published work has studied the role of ATP and other signaling molecules via the P2 family of purinergic receptors. During the last few years, however, there has been increased interest within the bone field in the role of P1 receptors where adenosine is the primary signaling molecule. This review will bring together the current information available in relation to this expanding area of research.

Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes.

Osteoblasts and adipocytes differentiate from a common precursor cell, the mesenchymal stem cell (MSC). Adenosine is known to signal via four adenosine receptor subtypes, and significantly, recent findings indicate that these may play a role in MSC differentiation. We therefore investigated adenosine receptor expression and activation during the differentiation of MSCs to osteoblasts and adipocytes. The A(2B) R was dominant in MSCs, and its expression and activity were transiently upregulated at early stages of osteoblastic differentiation. Both activation and overexpression of A(2B) R induced the expression of osteoblast-related genes [Runx2 and alkaline phosphatase (ALP)], as well as ALP activity, and stimulation increased osteoblast mineralization. The expression of A(2A) R was upregulated during later stages of osteoblastic differentiation, when its activation stimulated ALP activity. Differentiation of MSCs to adipocytes was accompanied by significant increases in A(1) R and A(2A) R expression, and their activation was associated with increased adipogenesis. Enhanced A(2A) R expression was sufficient to promote expression of adipocyte-related genes (PPARγ and C/EBPα), and its activation resulted in increased adipocytic differentiation and lipid accumulation. In contrast, the A(1) R was involved mainly in lipogenic activity of adipocytes rather than in their differentiation. These results show that adenosine receptors are differentially expressed and involved in lineage-specific differentiation of MSCs. We conclude, therefore, that fruitful strategies for treating diseases associated with an imbalance in the differentiation and function of these lineages should include targeting adenosine receptor signal pathways. Specifically, these research avenues will be useful in preventing or treating conditions with insufficient bone or excessive adipocyte formation.

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland.

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.

Complement C5a receptors in the pituitary gland: expression and function.

Communication between the immune and endocrine system is important for the control of inflammation that is primarily mediated through the hypothalamic-pituitary-adrenal axis. The innate immune system rapidly responds to pathogens by releasing complement proteins that include the anaphylatoxins C3a and C5a. We previously reported the existence of C3a receptors in the anterior pituitary gland and now describe the presence of C5a receptors in the gland. C5a and its less active derivative (C5adR) can bind to its own receptor and to another receptor called C5L2. Using RT-PCR and immunocytochemistry, C5a receptors and C5L2 were demonstrated in the rat anterior pituitary gland and in several rodent anterior pituitary cell lines. Western blotting analysis showed that C5a stimulated the phosphorylation of MAPK and AKT but not p38; C5adR on the other hand, had no effect on any of the signal molecules investigated. The effects of C5a and C5adR on the secretion of the inflammatory molecule, macrophage migration inhibitory factor (MIF) were investigated by ELISA. Both compounds showed a dose-dependent inhibition of MIF release, 30-40% inhibition at around 35-70 nM agonist with IC50 values of around 20 nM. C5a and C5adR also stimulated ACTH secretion (up to 25%) from AtT-20DV16 cells. These data show that functional C5a receptors (C5a and C5L2) are present in the anterior pituitary gland and they may play a role in dampening down inflammation by inhibiting the release of MIF and stimulating the release of ACTH.

Adenosine stimulates connexin 43 expression and gap junctional communication in pituitary folliculostellate cells.

Adenosine is known to stimulate interleukin (IL)-6 and vascular endothelial growth factor (VEGF) secretion from pituitary TtT/GF folliculostellate [corrected] (FS) cells indicating that it is an important paracrine regulator of anterior pituitary function. This study demonstrates that rodent anterior pituitary cell lines produce extracellular adenosine that is able to increase intercellular gap junction communication in FS cells. Ecto-5'-nucleotidase (CD73), the enzyme that generates adenosine from AMP, was demonstrated by immunocytochemistry in approximately 20% of anterior pituitary cells, and some of these cells colocalized with prolactin and growth hormone. CD73 mRNA and protein were detected in GH3 and MMQ (somatotroph-lactotroph lineages) and TtT/GF cells, and enzyme activity was demonstrated by the conversion of exogenously added fluorescent ethenoAMP to ethenoadenosine. Adenosine production, as measured by HPLC, was detected in GH3 (1 microM/h) and MMQ (3 microM/h) but not in TtT/GF cells. Adenosine (EC50: 0.5 microM) and NECA (universal adenosine receptor agonist; EC50 0.1 microM) stimulated connexin 43 (Cx43) mRNA and protein expression within 1-2 h in TtT/GF cells. Adenosine and NECA also stimulated gap junctional intercellular communication (as assessed by transmission of Alexa Fluor 488) by 6- to 8-fold in comparison with untreated TtT/GF cells. In cocultures of MMQ and TtT/GF cells, Cx43 expression in TtT/GF cells increased in proportion to the number of MMQ cells plated out. These data suggest that adenosine, formed locally in the anterior pituitary gland can stimulate gap junction communication in FS cells.

Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin.

UNLABELLED: We showed that human osteoprogenitor cells produced adenosine and expressed ecto-5'-nucleotidase and all four adenosine receptor subtypes. Adenosine stimulated IL-6 but inhibited osteoprotegerin secretion, suggesting that adenosine is a newly described regulator of progenitor cell function. INTRODUCTION: Maintaining skeletal homeostasis relies on there being a balance between bone formation and resorption; an imbalance between these processes can lead to diseases such as osteoporosis and rheumatoid arthritis. Recent reports showed that locally produced ATP, acting through P2 receptors, has pronounced effects on bone formation. However, ATP can be enzymatically cleaved to adenosine that has little or no activity at P2 receptors but mediates its action through the P1 family of receptors. We studied whether adenosine may also have an important role in controlling bone cell differentiation and function. MATERIALS AND METHODS: Extracellular adenosine levels were analyzed by high-performance liquid chromatography in HCC1 and bone marrow stromal (BMS) cells. Ecto-5'-nucleotidase (CD73) expression and activity was determined by RT-PCR, immunocytochemistry, and the cleavage of etheno-AMP to ethenoadenosine. Adenosine receptor expression and activity were determined by RT-PCR and cAMP measurements. The effects of adenosine receptor agonists on IL-6, osteoprotegerin (OPG), and RANKL expression were determined by ELISA and QRT-PCR. RESULTS: HCC1 and BMS cells produce adenosine and express CD73 and all four adenosine receptor subtypes. The A2b receptor was shown to be functionally dominant in HCC1 cells, as determined by cAMP production and in its stimulation of IL-6 secretion. Adenosine receptor agonism also inhibited OPG secretion and OPG but not RANKL mRNA expression. CONCLUSIONS: Our findings show that HCC1 and primary BMS cells produce adenosine, express CD73 and all four adenosine receptor subtypes. In HCC1 cells, adenosine has a potent stimulatory action on IL-6 secretion but an inhibitory action on OPG expression. These data show for the first time that adenosine may be an important regulator of progenitor cell differentiation and hence an important local contributor to the regulation of bone formation and resorption.

Complement C3a receptors in the pituitary gland: a novel pathway by which an innate immune molecule releases hormones involved in the control of inflammation.

Two-way communication exists between the endocrine and immune systems using molecules such as hormones and cytokines. Here we describe a new pathway by which C3a, a complement-derived cytokine, stimulates anterior pituitary hormone release and activates the hypothalamic-pituitary-adrenal axis, a reflex central to the stress response and to the control of inflammation. We show that C3a receptors are expressed in pituitary hormone secreting and non-hormone secreting (folliculostellate) cells and that both C3a and C3adesArg (a non-inflammatory metabolite) stimulate pituitary cell cultures to release prolactin, growth hormone, and adrenocorticotropin. Serum levels of these hormones, together with adrenal corticosterone, increase dose dependently with recombinant C3a and C3adesArg administration in vivo. Pertussis toxin blocks the response to C3a but not C3adesArg, which indicates the presence of two receptors, only one of which is coupled to Galphai-proteins. We propose that the complement innate immune molecules (cytokines) modulate tissue-specific and systemic inflammatory responses through communication with the endocrine pituitary gland.

Adenosine-induced IL-6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK.

Activation of adenosine receptors in folliculostellate (FS) cells of the pituitary gland leads to the secretion of IL-6 and vascular endothelial growth factor (VEGF). We investigated the action of adenosine A2 receptor agonists on IL-6 and VEGF secretion in two murine FS cell lines (TtT/GF and Tpit/F1), and demonstrated a rank order of potency, 5'-N-ethylcarboxamidoadenosine (NECA)>2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine>adenosine, suggesting mediation via the A2b receptor. NECA-mediated IL-6 release was inhibited by the PLC inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-tiene-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione, the PI3 kinase inhibitor wortmannin and the PKC inhibitors bisindolylmaleimide 1 and bisindolymaleimide X1 HCl (Ro-32-0432). NECA-mediated IL-6 release was attenuated (<50%) by the extracellular signal-regulated kinase MAPK inhibitor 2'-amino-3'-methoxyflavone, and completely (>95%) inhibited by the p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole. NECA stimulates p38 MAPK phosphorylation that is inhibited by Ro-32-0432 but not by wortmannin. Dexamethasone inhibits NECA-stimulated IL-6 and VEGF secretion. These findings indicate that adenosine can stimulate IL-6 secretion in FS cells via the A2b receptor coupled principally to PLC/PKC and p38 MAPK; such an action may be important in the modulation of inflammatory response processes in the pituitary gland.

Novel insights into how purines regulate pituitary cell function.

Purine nucleosides and nucleotides are widely distributed substances that exhibit a diverse range of effects in a number of tissues, acting as important extracellular signalling molecules in addition to their more established roles in cellular metabolism. They mediate their effects via activation of distinct cell surface receptors, termed adenosine (or P1) and P2 purinergic receptors. Although roles for adenosine and adenine nucleotides have been described previously in the pituitary gland, the distribution of the receptor subtypes and the effects of their activation on pituitary function are not well defined. Recent evidence, however, has emerged to describe a complex signalling system for purines in the pituitary gland. Data from a variety of studies have shown that the expression pattern, number and affinity of adenosine and/or P2 receptors may be cell-type specific and that non-endocrine in addition to endocrine cells elaborate these receptors. These variations, along with the diverse range of signalling pathways activated, dictate the response of individual cell types to extracellular purines, with roles now emerging for these substances in the regulation of hormone release, pituitary cell proliferation and cytokine/growth factor expression. In this review, we discuss these advances and examine some implications for pituitary growth control and the response of the hypothalamic-pituitary-adrenal axis to stress and inflammation.