Vitamin D and prostate cancer: The role of membrane initiated signaling pathways in prostate cancer progression

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Abstract

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been demonstrated to mediate both genomic and non-genomic responses in prostate cancer (CaP) cells. Here, we give an overview of membrane initiated 1,25(OH)2D3 signaling in prostate cancer cell progression. The presence of PDIA3 was investigated and homologous modeling of the putative PDIA3 receptor complex was conducted. Furthermore, the cellular distribution of nVDR was analyzed. We could show that both nVDR and PDIA3 are expressed in the prostate cancer cell lines investigated. The homologous modeling of PDIA3 showed that the receptor complex exists in a trimer formation, which suggests for allosteric activity. Our findings support previous reports and suggest that 1,25(OH)2D3 is an important therapeutic agent in inhibiting prostate cancer progression. Furthermore, our data show that 1,25(OH)2D3 regulate prostate cell biology via multiple pathways and targeting specific pathways for 1,25(OH)2D3 might provide more effective therapies compared to the vitamin D therapies currently clinically tested.

Introduction

Prostate cancer (CaP) is one of the most common cancer types in men, and etiological factors such as age, ethnical and genetic background, diet and hormones are believed to be involved in both development and progression of the disease [1].

Androgens have been shown to be involved in the regulation of prostate growth and proliferation [2], but not all CaP patients have responded to inhibition of androgens [3]. Androgen independent prostate cancer (AIPC) is a very aggressive and metastatic form of CaP and does not correspond to any of today's therapies. However, the present assumption is that steroid hormones can activate mutated androgen receptors in prostate cancer cells and therefore affect cancer cell growth [4].

During the last 20 years, an increasing number of reports have demonstrated that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates CaP cell biology, through regulation of transcription and activation of key components governing cell growth, differentiation and apoptosis via the cytosolic/nuclear vitamin D receptor VDR [5], [6]. Recently, studies suggest a newly discovered protein, membrane-associated rapid response steroid specific for 1,25(OH)2D3 (called 1,25-MARRS or the correct gene and protein name protein disulfide isomerase family A, member 3 (PDIA3)), as a potential membrane receptor that binds 1,25(OH)2D3 [7], [8], [9]. This protein has been hypothesized to function as a membrane-associated scaffold molecule, when bound to 1,25(OH)2D3, the ligand–receptor complex mediates several rapid signal transductions [10]. However, other studies have demonstrated a localization of nVDR at the plasma membrane, and suggest that nVDR may be the mediator of the rapid membrane-associated effects [11], [12], [13], [14], [15].

Rapid effects of vitamin D include opening of ion channels, changed enzyme activities, or activation of signaling cascades, and was initially referred to as non-genomic response [9], [12], [13], [16]. However, rapid increases in intracellular calcium concentrations and activation of second messengers, such as PKC, PKA, and MAP kinases, have effects on gene expression, and constitute novel long-term regulatory pathways for vitamin D. The origin of membrane initiated signaling caused by metabolites of the vitamin D endocrine system is debated and several putative receptors have been suggested (Annexin II, PDIA3, Catalase, and nVDR) [13], [19], [20], [21], [22], [23].

The vitamin D receptor-mediated regulation of cell and organ physiology is complex. To fully understand the dynamics involved in this process, the kinetic and dynamic parameters integrated within bioinformatical and mathematical models need to be characterized [24]. By modeling ligand–receptor interactions (in silico docking), two distinct, but overlapping binding sites for the nVDR have been suggested [23]. To explain the initiations of the different signaling pathways, the potential differences in stabilities of cis- and trans-locked 1,25(OH)2D3 in binding to their respective receptor pocket should be studied.

In this paper, we present an overview of the ongoing vitamin D research at our lab with emphasis on membrane initiated vitamin D signaling in prostate cancer. The aim of the present work was also to investigate the presence and partially characterize the previously identified membrane receptor, PDIA3, in prostate cancer cells. Thus, expression on transcriptional and translational levels of PDIA3 was examined in LNCaP, PNT-2 and PC-3 cell lines and homologous modeling of the PDIA3 receptor complex was performed.

Section snippets

In vitro cell culture conditions

LNCaP cancer cells (clone FGC Ecacc Cat. 89110211 Sigma–Aldrich), PC-3 cancer cells (ECACC Cat. 90112714 Sigma–Aldrich) and PNT-2 normal cells (ECACC Cat. 95012613 Sigma–Aldrich) were cultured in RPMI 1640 (Cat. 31870-025 Gibco Invitrogen) supplemented with 10% FBS and antibiotics at 37 °C in an atmosphere of 95% humidity and 5% CO2.

RNA extraction

Total RNA were automatically extracted from the harvested cells of the different cell lines with a KingFisher mL Instrument (Thermo Electron Corporation, USA)

Results and discussion

The expression analysis showed that PDIA3 was expressed in all three prostate cancer cell lines at both transcriptional and translational level (Fig. 1). The immunocytochemistry analysis revealed that nVDR is localized in the nucleus, and with a high abundance in the cytosol, where a part of the receptor population is present in a close vicinity to the plasma membrane (Fig. 2). These results are in concert with earlier findings [7], [8], [9], [12], [13], [23] that have suggested PDIA3 and nVDR

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