Nucleoplasmic Calcium Is Required for Cell Proliferation

Abstract

Ca2؉ signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca 2؉ to determine that cell proliferation depends upon Ca 2؉ signals within the nucleus rather than in the cytoplasm. Nuclear Ca 2؉ signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca 2؉ signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca 2؉ signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth. Ca2ϩ is a ubiquitous second messenger that mediates a wide range of cellular responses such as contraction, fluid and electrolyte secretion, exocytosis, gene transcription, and apoptosis (1). This ability to simultaneously control multiple processes occurs by careful modulation of Ca 2ϩ signals, not only over time but in different subcellular regions as well (2). For example, polarized Ca 2ϩ waves direct apical secretion in epithelia (3), whereas presynaptic increases in Ca 2ϩ trigger neurotransmitter release (4) and mitochondrial increases in Ca 2ϩ regulate apoptosis (5, 6). Ca 2ϩ signals also can be regulated independently in the nucleoplasm relative to the cytoplasm (7,8), but the physiological significance of this aspect of spatial control is not entirely understood. Nucleoplasmic Ca 2ϩ signals have distinct effects on activation of transcription factors (9, 10) and kinases (11, 12), but it is not known whether nuclear Ca 2ϩ signals also regulate more global aspects of cell function. Because cell proliferation (13,14) and progression through the cell cycle (15, 16) are Ca 2ϩ -dependent, we investigated the relative roles of nuclear and cytoplasmic Ca 2ϩ on cell growth. EXPERIMENTAL PROCEDURESMaterials, Reagents, and Cell Lines-SKHep1, HepG2, and HEK-293 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were used for all experiments. The cells were grown at 37°C with 5% CO 2 :95% O 2 in Dulbecco's modified Eagle's medium supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum, all from Invitrogen. The cells were grown on glass coverslips overnight in the absence of serum before infection with each parvalbumin (PV) 2 construct. Generation of Parvalbumin Constructs and AdenoviralInfection-Constructs encoding red fluorescence protein (DsRed) from Clontech (Mountain View, CA) and targeted PV proteins (PV-NLS, PV-NES, and PV-NLS-CD) were PCR-amplified and subcloned into pShuttle-CMV (kindly provided by Bert Vogelstein, Johns Hopkins) by restriction digestion with XhoI and XbaI to generate pShuttle-CMV-PVNLS-DSR, pShuttle-CMV-PVNES-DSR, and pShuttle-CMV-PVNLS-CD-DSR. Recombinant adenoviruses were generated by transformation of pShuttle-CMV-PV-NLS-DSR into AdEasier-1 cells, a deri...

Calpain-1 Cleaves Rad21 To Promote Sister Chromatid Separation

“…However, the normal physiological circumstances under which the cell experiences an increased nuclear [Ca 2ϩ ], consequent calpain-1 activation, and Rad21 cleavage await elucidation. Importantly, nuclear Ca 2ϩ is reportedly essential for cell cycle passage through prophase (33), the stage when cohesion along chromosomal arms is resolved. It is notable that a transient increase in intracellular [Ca 2ϩ ] in anaphase has been implicated in sister chromatid separation in sea urchin embryos (12), suggesting that calcium can play crucial roles at distinct stages during the cell cycle.…”

An update on nuclear calcium signalling

“…Nathanson and colleagues employed this approach to examine the involvement of nuclear Ca 2+ in regulating the proliferation of hepatocyte cell lines (Rodrigues et al, 2007). They observed that targeting the Ca 2+ -binding protein parvalbumin to the nucleus significantly reduced cell proliferation and altered the proportions of cells that were in the S and G 2 -M phases of the cell cycle.…”

Potenciais alvos terapêuticos contra o câncer

“…Além disso, outros estudos têm demonstrado a atividade imunológica desses peptídeos que são estocados nos grânulos de neutrófilos, mastócitos e células NK (natural killer), embora este aspecto não tenha sido muito aprofundado (26;41;42). Assim, os PAMs também podem ser capazes de induzir seus efeitos direta ou indiretamente no sistema imune por modulação de citocinas (43;44 (48). Para abordar essa questão foi desenvolvida pelo grupo de pesquisa Leite e cols., uma construção adenoviral que expressa uma proteína quelante de Ca 2+ , parvalbumina (PV), associada a uma sequência de localização nuclear (NLS), construção esta (Ad-PV-NLS) capaz de tamponar Ca 2+ seletivamente no nucleoplasma.…”

“…Observou-se que o tamponamento do Ca 2+ nuclear promove a sincronização das células tumorais no ciclo celular, com parada no início da mitose, especificamente na prófase, reduzindo assim a taxa de crescimento tumoral, sem, contudo, induzir a morte celular por apoptose. Esses achados foram confirmados por ensaios in vivo utilizando implantes de tumores xenográficos de hepatocarcinoma em camundongos (48). Reduzir a taxa de proliferação do tumor e sincronizar as células tumorais na mesma fase do ciclo celular faz da utilização da construção adenoviral Ad-PV-NLS, uma atraente estratégia de terapia gênica a ser testada para o tratamento de doenças crônicas degenerativas, como o câncer.…”