A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras

Store-Operated Ca²⁺ Entry (SOCE) Regulates Melanoma Proliferation and Cell Migration

<div><p>Store-operated Ca²⁺ entry (SOCE) is a major mechanism of Ca²⁺ import from extracellular to intracellular space, involving detection of Ca²⁺ store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca²⁺ channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.</p></div

Proposed mechanisms of SOCE-mediated cellular functions in melanoma.

<p>(<b>A</b>) The upper panel shows actin staining of SK-Mel-2 cells. Arrows indicate lamellipodia. The lower panel shows the number of SK-Mel-2 cells with more than one lamellipodium. **, <i>p</i><0.01, n = 331 for control shRNA, 850 for STIM1 shRNA, and 986 for Orai1 shRNA. (<b>B</b>) Calpain activity assay shows that thapsigargin increased calpain activity. Thapsigargin-induced elevation of calpain activity was inhibited by YM58483. *, <i>p</i><0.05, **, <i>p</i><0.01, N.S., not significant, n = 4. (<b>C</b>) Western blot analyses showed that thapsigargin (TG) increased cleavage of α-spectrin in SK-Mel-2 cells. Thapsigargin-induced cleavage of α-spectrin was inhibited by STIM1-knockdown. Representative data of 3 independent experiments are shown. (<b>D</b>) Densitometric analyses (bar graph) of western blots show that thapsigargin-induced cleavage of α-spectrin was inhibited by SOCE inhibitor YM58483, CaM inhibitor W5, MEK inhibitor PD03250901, and calpain inhibitor PD150606. *, <i>p</i><0.05; **, <i>p</i><0.01, n = 3.</p

SOCE was inhibited by STIM1- or by Orai1-knockdown in melanoma cell lines.

<p>(<b>A and B</b>) Western blot analyses of protein expressions of STIM1 (A) and Orai1 (B) in metastatic melanoma cell lines. shRNA transduction reduced expression of the target proteins. (<b>C</b>) Ca²⁺ peak amplitude of SOCE was reduced by STIM1- or Orai1-knockdown. **, <i>p</i><0.01, n = 6–10.</p

SOCE regulates proliferation of melanoma cells.

<p>(<b>A</b>) MTT assay of metastatic melanoma cell lines with or without STIM1- or Orai1-knockdown. **, <i>p</i><0.01, n = 8. Representative data of 3 independent experiments are shown. (<b>B</b>) MTT assay of the indicated cell lines in the presence or absence of YM58483. *, <i>p</i><0.05; **, <i>p</i><0.01, n = 8. Representative data of 3 independent experiments are shown.</p

SOCE regulates cell migration and metastasis of melanoma.

<p>(<b>A and B</b>) Boyden chamber assay showed that either STIM1- or Orai1-knockdown inhibited cell migration. **, <i>p</i><0.01, n = 4. (<b>C</b>) The scratch test showed that YM583483 (1 µM) inhibited cell migration. **, <i>p</i><0.01, n = 4. (<b>D</b>) Time-lapse video recording (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089292#pone.0089292.s002" target="_blank">Videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089292#pone.0089292.s003" target="_blank">S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089292#pone.0089292.s004" target="_blank">S3</a>) showed that STIM1- or Orai1-knockdown SK-Mel-2 cells exhibited a shorter migration distance than control SK-Mel-2 cells. **, <i>p</i><0.01, n = 10. (<b>E and F</b>) SK-Mel-2 cells with knockdown of either STIM1 or Orai1 were injected to the tail vein of Balb/c nu/nu mice. Three weeks later, the lungs were removed and fixed with picric acid. (<b>E</b>) Representative images of lung surface are shown. Arrows indicate metastatic melanoma colonies (white lesions). (<b>F</b>) The number of metastatic colonies in the lung surface was counted under a dissection microscope. **, <i>p</i><0.01, n = 8.</p

Expression of STIM1 and Orai1 in melanoma.

<p>(<b>A and B</b>) Western blot analyses of STIM1 (A) and Orai1 (B) expression in the indicated cell lines. (<b>C</b>) Immunohistochemical stainings of HE, MART1, STIM1 (the antibody from Abnova was used) and Orai1 (the antibody from Sigma was used) in a melanoma tissue microarray (original magnification, x400). Calibration bars represent 100 µm. (<b>D</b>) Analyses of the staining intensity of expression of STIM1 and Orai1. *, <i>p</i><0.05; **, <i>p</i><0.01, n = 8.</p

SOCE increases phosphorylation of ERK in melanoma.

<p>(<b>A</b>) Densitometric analyses (bar graph) of western blots show that thapsigargin increased phosphorylation of ERK1/2 in metastatic melanoma cell lines. STIM1-knockdown inhibited thapsigargin-induced phosphorylation of ERK1/2. *, <i>p</i><0.05; N.S., not significant, n = 3. (<b>B</b>) Densitometric analyses (bar graph) of western blots show that thapsigargin-induced phosphorylation of ERK1/2 was inhibited by SOCE inhibitor YM58483 and CaM inhibitor W5 in C8161 cells. *, <i>p</i><0.05, n = 3. (<b>C</b>) Thapsigargin-induced phosphorylation of ERK1/2 was inhibited by CaM inhibitor W5 and CaMKII inhibitor KN62 in C8161 cells. (<b>D</b>) Densitometric analyses (bar graph) of western blots show that thapsigargin-induced phosphorylation of ERK1/2 was inhibited by Raf-1 inhibitor GW5074, but not by Braf inhibitor GDC0879. *, <i>p</i><0.05; **, <i>p</i><0.01; N.S. not significant, n = 3. (<b>E</b>) Proposed signaling schema of SOCE-induced ERK activation.</p

SOCE occurs in melanoma.

<p>(<b>A</b>) Cytosolic Ca²⁺ levels in SK-Mel-2 cells. Extracellular Ca²⁺ (2 mM) was removed, followed by the addition of thapsigargin (TG) (2 µM) for Ca²⁺ depletion in the ER. Ca²⁺ (2 mM) was then added to the extracellular fluid, and SOCE-induced Ca²⁺ elevation was observed (SOCE phase). (<b>B</b>) Ca²⁺ peak amplitude in the SOCE phase was compared among melanoma and melanocyte cell lines. **, <i>p</i><0.01 (HEMA-LP), n = 6–8. (<b>C and D</b>) Cytosolic Ca²⁺ levels in SK-Mel-2 and SK-Mel-24 cells are shown as means ± SD (n = 6–10). SOCE was examined in the presence or absence of DMSO (1 µM) or YM58483 (1 µM) in SK-Mel-2 (<b>C</b>) and SK-Mel-24 cells (<b>D</b>).</p