[show abstract][hide abstract] ABSTRACT: Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.
[show abstract][hide abstract] ABSTRACT: A drop of endoplasmic reticulum (ER) Ca(2+) concentration triggers its Ca(2+) sensor protein STIM1 to oligomerize and accumulate within ER-plasma membrane (PM) junctions where it activates Orai1 channels, providing store operated Ca(2+) entry (SOCE). To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of N131 and N171. STIM1 NN/DQ resulted in a strong gain-of-function. Patch-clamp, TIRF and FRAP analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to ER-PM junctions with a decrease in current latency. Surprisingly, coexpression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca(2+) homeostasis.
Journal of Biological Chemistry 12/2012; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract Store-operated Ca(2+) entry (SOCE) is a widespread mechanism in cells to raise cytosolic Ca(2+) and to refill Ca(2+) stores. T cells critically rely on SOCE mediated by stromal interaction molecules (STIM) and Orai molecules for their activation and regulation of gene transcription; cells such as muscle cells, neurons or melanocytes probably utilize SOCE for the transmission of inducible receptor-mediated function as well as for generalized Ca(2+) homeostasis mechanisms. Exposure to environmental or cell-intrinisic reactive oxygen species (ROS) can affect several components involved in Ca(2+) homeostasis and thus alter multiple pathways. While all cells have a capacity to produce intracellular ROS, exposure of immune and skin cells to extracellular oxidative stress is particularly high during inflammation and/or with UV exposure. This review briefly summarizes cell-intrinsic sources of ROS and focuses on current findings and controversies regarding the regulation of STIM and Orai by oxidative modifications. We also introduce melanocytes as a new model system to study the function of STIM and Orai isoforms under physiological conditions that include exposure to UV light as an activating stimulus.
The Journal of Physiology 05/2012; 590(Pt 17):4193-200. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: UV radiation of the skin triggers keratinocytes to secrete endothelin-1 (ET-1) that binds to endothelin receptors on neighboring melanocytes. Melanocytes respond with a prolonged increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which is necessary for proliferation and melanogenesis. A major fraction of the Ca(2+) signal is caused by entry through Ca(2+)-permeable channels of unknown identity in the plasma membrane. ORAI Ca(2+) channels are molecular determinants of Ca(2+) release-activated Ca(2+) (CRAC) channels and are expressed in many tissues. Here, we show that ORAI1-3 and their activating partners stromal interaction molecules 1 and 2 (STIM1 and STIM2) are expressed in human melanocytes. Although ORAI1 is the predominant ORAI isoform, STIM2 mRNA expression exceeds STIM1. Inhibition of ORAI1 by 2-aminoethoxydiphenyl borate (2-APB) or downregulation of ORAI1 by small interfering RNA (siRNA) reduced Ca(2+) entry and CRAC current amplitudes in activated melanocytes. In addition, suppression of ORAI1 caused reduction in the ET-1-induced cellular viability, melanin synthesis, and tyrosinase activity. Our results imply a role for ORAI1 channels in skin pigmentation and their potential involvement in UV-induced stress responses of the human skin.
Journal of Investigative Dermatology 02/2012; 132(5):1443-51. · 6.19 Impact Factor