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First published online 31 January 2006
doi: 10.1242/jcs.02775

Journal of Cell Science 119, 733-743 (2006)
Published by The Company of Biologists 2006
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Ca2+ oscillations induced by testosterone enhance neurite outgrowth

Manuel Estrada1,2, Per Uhlen1,2 and Barbara E. Ehrlich1,2,*

1 Departments of Pharmacology, Cell and Molecular Physiology, Yale University, New Haven, CT 06520, USA
2 Neurosciences Institute of the Marine Biological Laboratory, Woods Hole, MA 02543, USA


Figure 1
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  		Fig. 1. Single cell imaging of testosterone-induced intracellular Ca2+ oscillations. (A) Fluorescence sequence over the time period indicated (images in pseudocolor). The Ca2+ signal began at the extreme tip of the cell and propagated as a Ca2+ wave. A few seconds later a new Ca2+ wave was produced, generating Ca2+ oscillations. (B) Normalized Ca2+ changes induced by 10 nM testosterone. (C) Cells stimulated with 100 nM testosterone. The time of addition of the hormone is indicated (arrows in B and C). (D) Power spectral analysis shows that the dominant peak of testosterone-induced Ca2+ oscillations occurs at 22 mHz. (E) Concentration dependence of the amplitude of the testosterone-induced Ca2+ transient, with a maximum response produced at 100 nM testosterone. (F) 17ß-estradiol (red line) but not 17{alpha}-estradiol (green line) induces Ca2+ transients. Cell pre-incubated with 1 mM tamoxifen, an antagonist of estrogen receptor, did not modify the testosterone-induced Ca2+ oscillations, indicating that the estrogen pathway is not required and the response was specific for the testosterone effect. (G) The linescan mode of recording fluorescence intensity was used to monitor different cellular regions. The x axis shows time, read from left to right. The y axis is the distance within the cell shown on the far left. The dashed lines, labeled 1 to 3, are the representative regions plotted in panel H. (H) Transient rises in Ca2+ were observed at different times, showing a cytosolic Ca2+ wave that propagated towards the nucleus, into the nucleus, and often to the opposite end of the cell. *P<0.05.

 

Figure 2
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  		Fig. 2. The effect of targeted parvalbumin fusion proteins on testosterone-induced cytosolic and nuclear Ca2+ responses. Western blots of PV-DSR fusion proteins. Cells were transfected with expression vectors for PV-NLS (A), PV-NLS-CDEF (B) or PV-NES (C). Total cell lysates were separated by SDS-PAGE and parvalbumin protein was visualized with a monoclonal anti-PV antibody; ß-actin is shown as a loading control. In all three conditions the expression of PV was increased in the transfected cells, as compared with the untreated cells. Experiments are representative of three independent experiments. The subcellular distribution of the PV constructs was examined using confocal microscopy. Red indicates the location of DSR, blue indicates nuclear staining with TO-PRO3. (D) PV-NLS-DSR and (E) PV-NLS-CDEF-DSR (targeted to nucleus) are restricted to the nucleus. (F) Expression of PV-NES-DSR (targeted to cytosol) is uniformly distributed throughout the cytosol but excluded from the nucleus. (G) Cells transfected with the PV-NLS-DSR did not exhibit any nuclear Ca2+ increase after testosterone exposure, but the cells showed normal cytosolic Ca2+ oscillations. (H) In cells expressing PV-NLS-CDEF-DSR, a mutated PV that does not bind Ca2+, testosterone induced cytosolic and nuclear Ca2+ oscillations. (I) Cells expressing PV-NES-DSR did not exhibit any cytosolic or nuclear Ca2+ increase.

 

Figure 3
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  		Fig. 3. The effect of inhibition of intracellular androgen receptor on testosterone-induced Ca2+ oscillations. (A) Fluorescence images of iAR localization were superimposed on bright-field images of single cells. (Upper panel) In control cells, iAR was distributed throughout the cell, but there was a higher relative abundance in the nucleus. Note also the clusters of iAR in the neurites. (Lower panel) Upon testosterone (100 nM) stimulation for 1 hour, the cytoplasmic iAR translocated to the nucleus. (B,C) Cells were transiently transfected with AR-siRNA and the expression of the protein was reduced ~80% with respect to control conditions, *P<0.05 versus basal (n=4). (D,E) Neither iAR-siRNA nor cyproterone modify the ability of testosterone to induce Ca2+ oscillations. (F) To show that internalization of testosterone is not required, the plasma membrane-impermeable testosterone bound to albumin (T-BSA) was tested and it mimicked the effects of the free hormone.

 

Figure 4
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  		Fig. 4. The source of the intracellular Ca2+ signal induced by testosterone. Cells were stimulated with testosterone in the presence of intracellular Ca2+ release inhibitors. (A) Pre-incubation of cells with nifedipine did not alter the Ca2+ response. (B) In Ca2+-free medium (0.5 mM EGTA) only the first peak was produced and no oscillations were observed. (C) Incubation with 1 mM Gd3+ (a nonspecific Ca2+ channel blocker) mimicked the response seen in Ca2+-free medium. (D) Ryanodine had no effect, whereas U73122 and 2APB abolished the Ca2+ signal (E,F).

 

Figure 5
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  		Fig. 5. Effect of Ins(1,4,5)P3R type 1 knock-down on testosterone-induced Ca2+ oscillations. (A,B) Cells transiently transfected with Ins(1,4,5)P3R type 1 siRNA (InsP3R1), showed a significant reduction (~80%) of the immunosignal. (C) In Ins(1,4,5)P3R type 1-siRNA transfected cells two types of Ca2+ response to testosterone were observed. The magnitude of the transient was reduced by 68% compared with untransfected cells, or abolished. (D) Control trace in cells not expressing the Ins(1,4,5)P3R siRNA.

 

Figure 6
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  		Fig. 6. Ca2+ signals induced by testosterone involve a pertussis toxin-sensitive G protein. (A) Cells were incubated for 20 minutes with 50 µM genistein, a tyrosine kinase inhibitor, and then stimulated with testosterone (100 nM). The use of genistein did not modify the Ca2+ increases produced by the hormone. (B) Cells were permeabilized with saponin, the saponin was removed to allow the membrane to reseal, and then cells were stimulated with testosterone (dashed line); using this treatment neuroblastoma cells maintained their capacity to respond to the hormone. Permeabilization in the presence of GDPßS (100 nM; solid line) blocked the testosterone-induced Ca2+ increases. (C) When cells were incubated with PTX (1 µg/ml) for 6 hours and then stimulated with testosterone, the Ca2+ increases were blocked. The time of addition of the hormone is indicated (arrows).

 

Figure 7
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  		Fig. 7. Testosterone enhances neurite outgrowth. Morphological changes in neuroblastoma cells were monitored after 3 days of treatment with testosterone. Cells were incubated with Cell Tracker green and visualized by confocal microscopy. (A) Neuroblastoma cells grown under control conditions have short neurites. (B) Cells treated with testosterone exhibit an increase in the neurite outgrowth compared with non-stimulated control cells. Bar, 30 µm. (C) Neurite outgrowth was normalized by calculating the ratio of the neurite length to the soma length (neurite/soma). Testosterone-induced neurite outgrowth was ~2.5-fold higher than control cells. Neurite elongation induced by testosterone was inhibited in cells with the cytosolic Ca2+ buffered by the expression of PV-NES-DSR. Buffering the nuclear pool of Ca2+ with PV-NLS-DSR induced a smaller increase in the neurite outgrowth compared with non-transfected cells. Cells expressing the mutated form of the nuclear localized parvalbumin showed a response to testosterone similar to the response observed in control cells. (D) Neurite elongation was smaller after inhibition of the iAR pathway (siRNA-AR or cyproterone) or when using T-BSA compared with the response with testosterone alone. These results suggest that genomic and non-genomic mechanisms for testosterone-induced neurite outgrowth in neuroblastoma cells are inter-dependent. Values are mean ± s.e.m., *P<0.05; **P<0.01.

 

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© The Company of Biologists Ltd 2006