Latha Ramakrishnan

The endo-lysosomal system as an NAADP-sensitive acidic Ca2+ store: Role for the two-pore channels

Accumulating evidence suggests that the endo-lysosomal system provides a substantial store of Ca(2+) that is tapped by the Ca(2+)-mobilizing messenger, NAADP. In this article, we review evidence that NAADP-mediated Ca(2+) release from this acidic Ca(2+) store proceeds through activation of the newly described two-pore channels (TPCs). We discuss recent advances in defining the sub-cellular targeting, topology and biophysics of TPCs. We also discuss physiological roles and the evolution of this ubiquitous ion channel family.

Molecular characterization of a novel cell surface ADP-ribosyl cyclase from the sea urchin.

The sea urchin is an extensively used model system for the study of calcium signalling by the messenger molecules NAADP and cyclic ADP-ribose. Both are synthesized by ADP-ribosyl cyclases but our molecular understanding of these enzymes in the sea urchin is limited. We have recently reported the cloning of an extended family of sea urchin ADP-ribosyl cyclases and shown that one of these enzymes (SpARC1) is active within the endoplasmic reticulum lumen. These studies suggest that production of messengers is compartmentalized. Here we characterize the properties of SpARC2. SpARC2 catalyzed both NAADP and cyclic ADP-ribose production. Unusually, the NAD surrogate, NGD was a poor substrate. In contrast to SpARC1, heterologously expressed SpARC2 localized to the plasma membrane via a glycosylphosphatidylinositol (GPI)-anchor. Transcripts for SpARC2 were readily detectable in sea urchin eggs and a majority of the endogenous membrane bound activity was found to be GPI-anchored. Our data reveal striking differences in the properties of sea urchin ADP-ribosyl cyclases and provide further evidence that messenger production may occur outside of the cytosol.

Methemoglobin-induced signaling and chemokine responses in human alveolar epithelial cells

Diffuse alveolar hemorrhage is characterized by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron- and oxidant-mediated activation of the transcription factor NF-κB. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-κB pathway; additionally, IL-8 required ERK activation and MCP-1 required JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary, we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron- and redox-mediated signaling involving the activation of the NF-κB and MAPK pathways. Decompartmentalization of hemoglobin may be a significant proinflammatory stimulus in a variety of lung diseases.

A single residue in a novel ADP-ribosyl cyclase controls production of the calcium mobilizing messengers, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.

The Signaling Protein CD38 Is Essential for Early Embryonic Development

Background: CD38 is a multifunctional ADP-ribosyl cyclase involved in Ca2+ signaling. Results: Xenopus CD38 is developmentally regulated, responsible for all ADP-ribosyl cyclase activity in the early embryo, and required for muscle differentiation. Conclusion: CD38 plays an vital role during Xenopus development. Significance: This is the first demonstration that ADP-ribosyl cyclases are essential for embryonic development. CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca2+-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca2+ signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development.

Pulmonary Arterial Hypertension: Iron Matters

The interplay between iron and oxygen is longstanding and central to all aerobic life. Tight regulation of these interactions including homeostatic regulation of iron utilization ensures safe usage of this limited resource. However, when control is lost adverse events can ensue, which are known to contribute to an array of disease processes. Recently, associations between disrupted iron homeostasis and pulmonary artery hypertension (PAH) have been described with the suggestion that there is a contributory link with disease. This review provides a background for iron regulation in humans, describes PAH classifications, and discusses emerging literature, which suggests a role for disrupted iron homeostatic control in various sub-types of PAH, including a role for decompartmentalization of hemoglobin. Finally, the potential for therapeutic options to restore iron homeostatic balance in PAH are discussed.

P244 Haemoglobin mediated proliferation and il-6 release in human pulmonary artery endothelial cells: a role for cd163 and implications for pulmonary vascular remodelling

Introduction Pulmonary arterial hypertension (PAH) is characterised by vascular remodelling of pulmonary arterioles. Disrupted iron homeostasis as well as subclinical haemolysis are implicated in PAH, although exact mechanisms remain unknown. IL-6, a proinflamatory cytokine and regulator of iron homeostasis is elevated in PAH patients and also been implicated in pulmonary vascular remodelling in murine models. Objectives In this study we explored the influences of free haemoglobin (Hb) on proliferative responses and secondary mediator, IL-6 release in human pulmonary artery endothelial cells (hPAECs). Methods Cells were challenged with Hb (10 uM) and/or IL-6 (1–10 ng/mL). Transcriptional regulation was analysed by RT-PCR, protein expression by immunocytochemistry, secretion by ELISA and proliferation by BrdU incorporation. Results Novel findings demonstrate that Hb and IL-6 individually and in combination increased proliferation of hPAECs (by 32%, 47% and 63% respectively; p < 0.05). CD163, a Hb scavenger receptor, was basally expressed as mRNA and protein (cell surface) on hPAECs and further modulated by Hb or IL-6 exposure. Hb treatment also caused increased transcription (30%; p < 0.05) and release of IL-6 (107%; p < 0.01) from hPAECs. Conclusion This is the first report of Hb-mediated proliferation, CD163 expression and IL-6 release in hPAECs with potential implications for autocrine and paracrine signalling in pulmonary vasculature. Hb uptake may be facilitated via CD163. These studies may provide novel insights regarding mechanisms for haemoglobin driven proliferative and second messenger responses of relevance to PAH. Abstract P244 Figure 1 IL-6 relaese after 24h

ADP-Ribosyl Cyclases Regulate Early Development of the Sea Urchin

ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca2+ signalling such as cADPR and NAADP. Although Ca2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

S112 Conditioned media from human pulmonary arterial endothelial cells treated with hepcidin or haemoglobin cause proliferation and migration of human pulmonary artery smooth muscle cells

Introduction Pulmonary arterial hypertension (PAH) is characterised by vascular remodelling of pulmonary arterioles. Disrupted iron homeostasis including subclinical haemolysis are implicated in PAH, although exact mechanisms remain unknown. Interactions linked to altered iron handling consequent to release of mediators by human pulmonary artery endothelial cells (hPAECS) and/or human pulmonary artery smooth muscle cells (hPASMCs) may be important in this regard. Objectives This study explored proliferative and migratory responses in hPASMCs linked to the primary exposure of hPAECs to indices of dysregulated iron homeostasis i.e., hepcidin or haemoglobin. Methods hPAECs were challenged with haemoglobin (10 uM) or hepcidin (100–1000 ng/mL). Proliferation and migration of hPASMCs were analysed using xCELLigence RTCA instrument by measuring the changes in cell index/impedance. Results Novel findings demonstrate that media conditioned with hepcidin for 48 hour from hPAECs increased proliferation of hPASMCs by 24% (95% CI 0.256–47.9, p<0.05) after 48 hour of treatment. Additionally, 24 hour conditioned media from both hepcidin and haemoglobin treated hPAECs caused 3.27 and 2.28 fold increases in migration respectively in hPASMCs. Conclusion These findings highlight a direct role for variant iron homeostasis in hPAECs which is linked to subsequent functional responses in hPASMCs of importance to vascular remodelling. These studies may provide novel insights regarding mechanisms for haemoglobin and hepcidin driven proliferative and migratory responses of relevance to PAH.