A Novel Role for Niemann-Pick Disease Type 2C Protein in Papillae Formation

BACKGROUND: Despite the presence of papillary structures and papillary tumors in humans, the mechanism of papillae formation is unknown. We describe herein a novel role for Niemann-Pick disease type 2C (NPC2) protein, a cholesterol binding protein in the lysosome, in papillae formation. METHODOLOGY/PRINCIPAL FINDING: We examined NPC2 protein expression in surgical samples of papillary tissues by immunohistochemical stain, and all papillary tissues expressed NPC2 protein in the epithelium. To examine our hypothesis of NPC2 protein-mediated papillae formation, we carried out xenograft experiments using wild H460 cells (large cell lung carcinoma cell line) that constitutively expressed abundant NPC2 protein and NPC2 protein-depleted H460 cells by NPC2 shRNA. The xenografts of wild H460 cells and empty shRNA vector cells showed distinct papillae formation, whereas NPC2 protein-depleted H460 cells displayed markedly reduced or no papillae. Since all papillary tissues have open spaces we examined whether NPC2 protein might also contribute to the creation of open spaces. The TUNEL assay in the xenografts of wild and empty shRNA vector H460 cells showed massive cell death, and NPC2 protein-depleted cells displayed minimal cell death. Measurement of caspase 3/7 activities in cultured H460 cells supported NPC2 protein-mediated apoptotic cell death. The presence of excess NPC2 protein, however, did not always produce papillae as seen in the xenografts of CHO cells that were stably transfected with NPC2. CONCLUSIONS/SIGNIFICANCE: The NPC2 protein of certain cells forms papillae coupled with apoptosis that creates open space. This protein may have future applications to modulate papillae formation and papillary growth in tumor tissues

NPC2 protein expression in cultured H460 cells.

<p>Immunohistochemical staining showed abundant NPC2 protein in wild and empty vector cells and markedly reduced NPC2 protein in <i>NPC2-shRNA</i>-containing cells.</p

Characterization of three types of cultured H460 cells.

<p>Cell morphology: Wild and empty vector cells showed the same cell morphology, whereas cells containing <i>NPC2-shRNA</i> displayed clearly separated individual cells. Cell proliferation activity in NPC2-rich and-depleted H460 cells: Cells (1000 cells/well) were plated into a 96-well plate. Cell proliferation activity on each day was measured by incubating cells with the reagents for 2 hours in a CO2 incubator. The results are mean ± SD of quadruplicate samples of one of the three experiments. *<i>p</i><0.01 when compared with the empty vector cells.</p

Papillae formation in xenografts of three types of H460 cells.

<p>A: Hematoxylin-eosin stain. B: Immunohistochemical stain for NPC2 protein. Distinct papillae formation was seen in wild and empty vector cells. No papilla was seen in <i>NPC2-shRNA</i> containing cells (A). NPC2 protein was localized in the epithelium of papillae (B).</p

TUNEL assay in xenografts NPC2 protein-rich and-depleted cells.

<p>Apoptotic ell death was seen between papillary structures (brown colored areas) in wild H460 cells. The transition from epithelial cells (blue colored cells) to apoptotic cells (brown colored cells) was seen in the outer border of epithelium (middle panel). NPC2 protein-depleted H460 cells showed only small areas of apoptotic cell death.</p

NPC2 protein expression in human papillary tissues.

<p>Immunohistochemical stain showed the presence of cytoplasmic NPC2 protein (brown color) in surgical samples of human papillary tissues. All papillary tissues have open spaces outside of papillary projections. The numbers indicate the degree of the magnification.</p

NPC2 protein expression in CHO cells.

<p>Immunohistochemical stain of NPC2 protein in wild CHO cell (a), CHO cells stably transfected with <i>NPC2</i> (b) and xenograft of CHO cells transfected with <i>NPC2</i> (c). No papilla was seen in the xenograft, despite abundant NPC2 protein expression.</p