Niacin induces PPARγ expression and transcriptional activation in macrophages via HM74 and HM74a-mediated induction of prostaglandin synthesis pathways
Introduction
HM74 (GPR109B, GenBank accession no. NM_006018) and HM74a (GPR109A, GenBank accession no. NM_177551) are highly homologous Gi-G-protein-coupled receptors that were initially identified as receptors for niacin (nicotinic acid, Vitamin B3) [1], [2], [3]. HM74a, the human homologue of murine PUMA-G (protein upregulated in macrophages by IFN-γ[4]), is the higher affinity receptor with niacin binding and G-protein activation evident at 0.05–1 μM Kd/EC50[1], [2], [3]. HM74 has 96% nucleotide sequence identity and 89% amino acid identity with HM74a, encompassing 7 predicted amino acid changes and a 24 amino acid C-terminal extension [2], [5]. These changes are sufficient to give HM74 much reduced affinity for niacin, with ligand binding and G-protein activation generally described as non-saturable [1], [2].
Despite the significantly higher affinity of niacin for HM74a than HM74, it is unlikely that it represents the endogenous ligand for either receptor [6]. However, HM74a might mediate the pharmacological anti-lipolytic effects of niacin, which can be achieved at plasma concentrations ≥4 μM [6]. The mechanism of niacin's anti-lipolytic effect is thought to involve an inhibitory G-protein signal that reduces production of adipocyte cyclic AMP (cAMP) via adenylyl cyclase and so reduces hormone-sensitive lipase activity due to reduced protein kinase A activation [6], [7]. This inhibits release of free fatty acids (FFA) from adipose tissue into the circulation, resulting in a 15–35% increase in hepatic secretion of high density lipoprotein (HDL) and a 5–25% decrease in low density lipoprotein (LDL), the primary target for cholesterol-lowering treatment to reduce both the risk of and mortality due to coronary heart disease [7], [8]. That HM74a might mediate this effect is supported by niacin-induced reduction of intracellular cAMP in HM74a-transfected CHO-K1 [3], 293EBNA [1], HEK293 and 3T3L1 adipocytes [9]. Additionally, experiments in PUMA-G-deficient mice demonstrated 75% reduction in niacin binding to adipocyte membranes associated with loss of niacin-induced inhibition of FFA release [3].
The observation that the fatty acid-derived ketone body (d)-β-hydroxybutarate ((d)-β-OHB) mediates similar anti-lipolytic effects to niacin with respect to reduction of serum FFA [10], [11] and inhibition of adipocyte lipolysis [12] recently led to the identification of (d)-β-OHB as the first endogenous ligand for HM74a [13]. (d)-β-OHB suppressed FFA efflux from murine adipocytes in a PUMA-G-dependent manner and induced [35S]GTPγS binding to membranes from HM74a and PUMA-G transfectants with an EC50 of 0.8 mM [13]. This is within the serum (d)-β-OHB concentration achieved following 2 days starvation in man [14] and suggests that HM74a additionally mediates homeostatic negative feedback mechanisms to conserve fat stores during starvation [13].
A common side-effect of niacin administration is an intense prostaglandin-mediated skin flush due to vasodilation following release of prostaglandins PGD2 and PGE2 from resident dermal macrophages and/or epidermal cells [15], [16]. PGD2 is non-enzymatically dehydrated to form 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), the most potent endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ) [17]. Using the human differentiated monocytic cell line MonoMac6, it has been shown that niacin induces PPARγ expression and intracellular cAMP in monocytic cells, although the receptor(s) mediating this effect was not identified [18]. Niacin also enhanced transcription of both the oxidised LDL (oxLDL) scavenger receptor CD36 and of ABCA1, the key transporter for efflux of cellular cholesterol onto apolipoprotein A-I-containing HDL particles [18]. Niacin-induced effects on macrophage lipid regulation could present an additional mechanism whereby clinical administration of niacin might elevate HDL cholesterol levels and also reduce macrophage foam cell formation in atherogenesis [18], [8].
We have identified HM74 as a hypoxia-inducible gene from cDNA array analysis of normoxic and hypoxic monocytic cell lines. Interestingly, nicotinamide, the downstream conversion product of nicotinic acid and other prinicipal form of Vitamin B3, is central to the ARCON (accelerated radiotherapy with carbogen and nicotinamide) therapeutic strategy to overcome radiotherapy resistance due to hypoxia [19]. Nicotinamide operates by reducing intermittent vascular shut-down [20] to decrease perfusion-limited tumour hypoxia [21], in analogy to the effects of niacin on vascular parameters to increase local blood flow [15], [16]. Niacin and nicotinamide also have protective effects versus chemical and UV-induced carcinogenesis via a number of mechanisms including impaired immune surveillance [22], [23]. HM74 might therefore provide insight into mechanism(s) of vasodilation and a link between treatments for cancer and dyslipidaemia.
We have considered whether the receptor(s) mediating macrophage responses to niacin could be HM74a and/or HM74. We have determined that HM74 and HM74a mRNA is hypoxia-inducible in human monocytic cell lines and monocyte-derived macrophages. Additionally, we show that activation of both HM74 and HM74a by niacin stimulates the prostaglandin synthesis pathway resulting in elevated expression of PPARγ and enhanced PPARγ-dependent transcription activation. This demonstrates for the first time the effect of activation of the HM74 and HM74a GPCRs on macrophage gene expression and suggests an additional mechanism whereby HM74 and HM74a could mediate the clinical effects of niacin.
Section snippets
Cell culture
Cell lines were obtained from the Cancer Research UK cell service unless otherwise stated. Human monocytic cell lines THP1 (acute monocytic leukaemia) and U937 (lymphoma), 1397 and JB EBV-transformed lymphocytes (a gift from Dr. S. Holbrook, Weatherall Institute of Molecular Medicine, Oxford, UK), Jurkat (T cell acute lymphatic leukaemia), breast cancer cell lines MDA-MB-468 and MDA-MB-231, PC3 (prostate) and 293T (embryonic kidney) cells were maintained in RPMI 1640. HCT116 and HT29 (colon),
HM74 and HM74a are hypoxia-inducible in monocytic cells
We originally identified HM74 as a gene of interest in PMA-differentiated [25], [26] THP1 macrophages where, by cDNA array analysis, HM74 mRNA was upregulated 2.3-fold following 16 h hypoxic exposure (0.1% O2, data not shown). Ribonuclease protection assay (RPA) confirmed that hypoxic upregulation of HM74 mRNA was specific to PMA-treated cells in THP1 and U937 monocytic cell lines (Fig. 1A, top panel). As HM74 is also expressed in lymphocytes [2], we assayed 1397 and JB EBV-transformed
Discussion
The identification of HM74 and HM74a as niacin receptors has provided opportunity to determine the molecular mechanism(s) of niacin's clinical effects. It has recently been shown that niacin stimulates PPARγ- and cAMP-dependent mechanisms of reverse cholesterol transport in macrophages [18]. Our data now demonstrates that human macrophage cell lines and primary cells in culture express both HM74 and HM74a and that these receptors can directly mediate macrophage responses to niacin via
Acknowledgements
The authors are grateful to Prof. Peter J. Ratcliffe for many helpful discussions and to Cancer Research UK for funding.
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