Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Immunology, Endocrine and Metabolic Agents) - Volume 9, Issue 3, 2009

Vitamin D and its metabolites have been the focus of attention recently, and as stated in the title, this issue highlights some of the newly recognized actions of these compounds. In the first paper, Sequeira et al. begin with the metabolism of vitamin D to other metabolites and the classical role of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the regulation of calcium homeostasis. The authors then relate the evidence that 1,25(OH)2D3 can be made in the skin following UV irradiation, and as widely known, UV irradiation also causes DNA damage and skin cancer. Of utmost importance however, is that 1,25(OH)2D3 is protective against skin cancer, and this appears to occur through a membrane-associated pathway rather than through gene regulation. Boyan and co-workers report on the progress they have made in studying the effects of the metabolite 24,25- dihydroxyvitamin D3 in growth plate chondrocytes. 24,25(OH)2D3, which was once thought to be inactive, rapidly alters phospholipase activities and protein kinase C. The latter is involved in matrix synthesis and chondrocyte activation. 24,25(OH)2D3 also has an anti-apoptotic function that is induced by inorganic phosphate. The receptor for this metabolite could be a new therapeutic target in regulating ossification. Our paper discusses newly recognized receptors for both 1,25(OH)2D3 and 24,25(OH)2D3. The 1,25D3-MARRS (membrane associated, rapid response, steroid binding) receptor has been shown to mediate the pregenomic responses in chick both by RNAi and a neutralizing antibody. The classical VDR was found to not be involved. By comparison, rat intestinal cells use both the 1,25D3-MARRS receptor and the VDR. 24,25(OH)2D3 acts as an endogenous inhibitor of the rapid actions of 1,25(OH)2D3 in chick intestine and kidney. The inhibitory steroid binds to cell surface catalase resulting in decreased enzyme activity. The resulting increase in hydrogen peroxide acts to oxidized the thioredoxin domains in the 1,25D3-MARRS receptor. This in turn decreases binding of 1,25(OH)2D3. In the same studies, no effect was found on binding of 1,25(OH)2D3 to the classical VDR. These studies indicate that the 1,25D3-MARRS receptor is a potential therapeutic target for modulating intestinal calcium and phosphate absorption. In the comprehensive review by Buitrago et al., the authors summarize evidence from their lab and others on the effects of 1,25(OH)2D3 and estradiol on skeletal muscle. Both the classical VDR and ER were found in plasma membrane and mitochondria. Both steroid hormones also use tyrosine receptor kinases for signaling, as well as an array of other signaling pathways. Both hormones stimulate muscle cell proliferation and inhibit apoptosis-a physiological effect that may prove useful in treating muscle weakening from ageing and myopathies.

Exposure to ultraviolet radiation is essential for the formation of vitamin D in skin. Importantly, the skin contains all the necessary enzymes to convert the inactive vitamin D into the active hormone, 1α,25 dihydroxyvitamin D (1,25D). The same UVB which produces vitamin D also causes DNA damage. Recent studies have implicated 1,25D in protection against skin carcinogenesis via a reduction in DNA damage and maintenance of immune responses following UVR exposure. Vitamin D compounds have the potential to act as a protective measure against UVR-initiated skin cancer.

In this review, we will summarize recent findings surrounding the physiological actions of 24R,25- dihydroxyvitamin D3 [24,25(OH)2D3], an active vitamin D3 metabolite that regulates endochondral ossification and bone fracture healing. 24,25(OH)2D3 exerts rapid effects in resting zone growth plate chondrocytes through activation of a membrane-associated receptor. One consequence of this activation is rapid inhibition of phospholipase A2, resulting in altered membrane fluidity and arachidonic acid turnover. In addition, phospholipase D activity is increased, leading to increased diacylglycerol and protein kinase C (PKC) activation, as well as PKC-dependent matrix synthesis and chondrocyte maturation. 24,25(OH)2D3 reduces production of acid matrix metalloproteinases (MMP), specifically stabilizing neutral MMPs in extracellular matrix vesicles. Recent work has shown that 24,25(OH)2D3 also has an anti-apoptotic function, acting to protect rat growth plate chondrocytes and mouse ATDC5 cells from apoptosis induced by inorganic phosphate (Pi). Studies using rat costochondral resting zone chondrocytes indicate that the mechanism involves 24,25(OH)2D3-mediated stimulation of lysophosphatidic acid (LPA) signaling. In bone, immature osteoblasts respond to 24,25(OH)2D3, whereas more mature osteoblasts do not. This implicates 24,25(OH)2D3 in the maintenance of bone and suggests that its actions are maturation stage-dependent. Collectively, these studies show that 24,25(OH)2D3 regulates less mature chondrocytes and osteoblasts through rapid activation of its membrane-associated receptor, establishing it as an important modulator of endochondral ossification.

Vitamin D plays a central role in modulating calcium and phosphate homeostasis in the body. While the effects of its principal stimulatory metabolite, 1,25(OH)2D3, have been studied extensively in many physiological and disease states, research results have now emerged suggesting the possible biological role for another metabolite, 24,25(OH)2D3. The actions of 1,25(OH)2D3 are believed to be mediated by both pregenomic and genomic pathways involving the classical vitamin D receptor (VDR) and the more recently identified 1,25D3-MARRS (Membrane associated, rapid response steroid binding) receptor, also known as PDIA3/ERp57/GRp58/ERp60/ERp61. The metabolite 24,25(OH)2D3 is made when an animal is vitamin D replete and provides an endocrine feedback loop to inhibit the rapid actions of 1,25(OH)2D3, which appear to be mediated by 24,25(OH)2D3 binding to a subset of catalase at the plasma membrane. Here we discuss these two recently identified putative receptors for vitamin D metabolites while also reviewing 25(OH)D3 as a functional metabolite.

The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates a variety of signaling pathways in health and disease via intracellular Ca2+. An emerging approach in treatment and prevention of cancer and obesity is induction of apoptotic cell death. Cellular Ca2+ signals have been implicated in triggering apoptosis, however vitamin D/Ca2+-dependent mediators involved in apoptotic signaling have not been identified. Our studies reviewed here demonstrate that 1,25(OH)2D3 induces Ca2+-mediated apoptotic death of cancer cells and adipocytes via activation of the novel targets, Ca2+-dependent μ-calpain and Ca2+/calpain-dependent caspase-12. In this pathway -- sustained increase in intracellular Ca2+ → μ-calpain activation → caspase-12 activation → apoptosis -- cellular Ca2+ acts as an apoptotic initiator and directly recruits Ca2+-dependent apoptotic effectors capable of executing apoptosis. Our findings provide a new rationale for evaluating the role of vitamin D in prevention and treatment of cancer and obesity.

1α,25(OH)2-vitamin D3 and 17β-estradiol regulate skeletal muscle cell proliferation, differentiation, apoptosis and contractility through receptor-mediated transcriptional and non-genomic mechanisms. This review focuses on recent advances on signal transduction pathways activated by both steroid hormones. Data are given on the participation of the VDR and ERs (α and β) in activation of MAPKs in muscle cells. Likewise, we describe novel evidence supporting non-classical localizations of the VDR in the plasma membrane and ER β in mitochondria. 1α,25(OH)2D3 promotes DNA synthesis in skeletal muscle cells implicating c-Src/ERK1/2, whereas 17β-estradiol inhibits apoptosis through ERK2 and p38 MAPKs. This study provides basis for the understanding of vitamin D- and estrogen-dependent myopathies.

Nongenomic actions of nonpeptide hormones initiated at the cell surface have been described for estrogen [1, 2], dihydrotestosterone [3], vitamin D [4, 5] and thyroid hormone [6, 7]. Reviews of some of these actions have recently appeared in Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry [8, 9]. Quite specific membrane protein receptors have been defined for DHT [3], vitamin D [4] and iodothyronines [6]. The term, ‘receptor,’ is justified here because discrete downstream consequences of ligand-protein interactions are reported. The nature of the receptor(s) in extranuclear actions of estrogen has been controversial. Classical nuclear estrogen receptors (ERs) residing in cytoplasm or inserted in the plasma membrane have been described, but evidence also exists for estrogen-binding by a G protein-like receptor (GPR) or an STX-binding protein. Papers by Drs. Marino and Galluzzo and by Drs. Micevych and Mermelstein in this issue of the journal critically review the current state of our knowledge of these receptors. Marino and Galluzzo discuss the dynamic model of ERs shuttling among cellular compartments-plasma membrane-cytoplasm-nucleus-and how different cellular effects might be achieved with this model. Such models teach that functional distinctions between nongenomic and genomic actions of the hormone are blurred and less useful than once was conceived to be the case. Micevych and Mermelstein explore rapid-onset actions of estrogen in brain via ERs trafficked to the plasma membrane that activate G proteins to transduce the estrogen signal. Neuronal metabotropic glutamate receptors are involved in the transduction process. Biologic endpoint in these studies is regulation of luteinizing hormone release. The Editor appreciates the efforts of Dr. Boris Cheskis and Ellis Levin in soliciting these papers for the journal.

17β-Estradiol (E2) is a steroid hormone that regulates the expression of a variety of genes involved in distinct physiological processes, including development, metabolism, and reproduction. The effects start when the hormone binds to the specific intracellular receptors ERα and ERβ, which act via multiple mechanisms. E2 has profound, rapid effects on the conformation of ERs allowing receptor dimerization and translocation into the nucleus where they bind specific hormone response elements present in DNA. The ER-E2 complex can also function as a cytoplasmic signaling molecule eliciting other changes in cells, including modulation of ion fluxes across membranes and stimulation of kinase and phosphatase cascades which, in turn, may influence processes such as proliferation of various cell types. Such extranuclear signaling pathways are rapid and supposedly independent of transcription and require membrane localized ERs. The recent finding that ERs undergo palmitoylation provides new insights into E2 rapid signaling and raises several new concerns in the field of estrogen biology. S-palmitoylation allows ERα and ERβ localization at the plasma membrane, where they associate with caveolin-1. Upon E2 stimulation, ERα dissociates from caveolin-1, whereas ERβ:caveolin-1 association increases allowing the activation of specific rapid signals relevant for cell proliferation. Here recently disclosed information on membrane ERs, and the emerging field of membrane integration and nuclear receptor signaling will be highlighted.

Estrogens, like many other hormones, affect both the central and peripheral nervous system. Initially identified as initiator of gene expression through activation of transcription factors that directly bind DNA, estrogens have also been shown to alter intracellular signaling via membrane surface activation of G proteins. The idea that estrogens can rapidly signal via membrane receptors is well accepted, but the exact nature of the membrane estrogen receptor(s) (mER) remains controversial. Several putative mERs have been described, including ER-X, an STX-binding protein and GPR30. That said, the majority of estrogenic effects initiated at the cell surface appear to be due to membrane trafficking of the classical estrogen receptors, estrogen receptor- α (ERα) and estrogen receptor-β (ERβ). This paper will review the evidence that rapid estrogen actions through different putative mERs mediate sexual receptivity and the luteinizing hormone (LH) surge. Since a large number of rapid responses appear to be due to ERα and ERβ, the same proteins that regulate gene expression within the nucleus, we will focus on how the classical ERα and ERβ are trafficked to the membrane and initiate cell signaling. As ERα and ERβ are themselves not GPCRs, to activate G proteins they interact with metabotropic glutamate receptors (mGluRs) that function as intermediaries, to transduce the ligand activation of membrane ERα and ERβ spare into a cellular response. This ER/mGluR hypothesis provides a mechanism to explain the wide-range of rapid estrogen actions in the brain.