A Novel Transcriptional Factor <i>Nkapl</i> Is a Germ Cell-Specific Suppressor of Notch Signaling and Is Indispensable for Spermatogenesis

<div><p>Spermatogenesis is an elaborately regulated system dedicated to the continuous production of spermatozoa via the genesis of spermatogonia. In this process, a variety of genes are expressed that are relevant to the differentiation of germ cells at each stage. Although Notch signaling plays a critical role in germ cell development in <i>Drosophila</i> and <i>Caenorhabditis elegans</i>, its function and importance for spermatogenesis in mammals is controversial. We report that <i>Nkapl</i> is a novel germ cell-specific transcriptional suppressor in Notch signaling. It is also associated with several molecules of the Notch corepressor complex such as CIR, HDAC3, and CSL. It was expressed robustly in spermatogonia and early spermatocytes after the age of 3 weeks. <i>Nkapl</i>-deleted mice showed complete arrest at the level of pachytene spermatocytes. In addition, apoptosis was observed in this cell type. Overexpression of NKAPL in germline stem cells demonstrated that <i>Nkapl</i> induced changes in spermatogonial stem cell (SSC) markers and the reduction of differentiation factors through the Notch signaling pathway, whereas testes with <i>Nkapl</i> deleted showed inverse changes in those markers and factors. Therefore, <i>Nkapl</i> is indispensable because aberrantly elevated Notch signaling has negative effects on spermatogenesis, affecting SSC maintenance and differentiation factors. Notch signaling should be properly regulated through the transcriptional factor <i>Nkapl</i>.</p></div

Nuclear localization signal (NLS) analyses of NKAPL <i>in vitro</i>.

<p>(A) Immunofluorescence with GFP antibody after transfection of EGFP-tagged vectors into 293T cells. Upper row shows images of GFP expression, and lower row shows those merged with DAPI staining to visualize nuclei. White scale bars = 50 μm. (B) The upper bar represents the open reading frame (ORF) of the <i>Nkapl</i> gene, and the lower bars illustrate the fragmented sequences of the <i>Nkapl</i> gene, which were inserted into EGFP-tagged vectors. Numbers over the bars indicate the fragmented sites on the ORF. Black regions indicate the sequence including the putative NLS signal amino acids predicted by cNLS Mapper prediction program. (C) Immunofluorescence of GFP antibody after the transfection of EGFP-tagged fragmented <i>Nkapl</i>. Left columns show the immunofluorescent images with GFP antibody, and the right columns show those merged with DAPI staining.</p

Correlation between serum CAXII levels and patients' clinicopathological characteristics.

<p>(A) CAXII levels in sera from patients with ADs and SCCs with a focus on the stage and differentiation. In SCCs, CAXII levels were significantly higher in well- and moderately differentiated tumors than in poorly differentiated ones (***P = 0.0272). In ADs, no significant difference based on the differentiation extent was detected. (B) Smoking history in lung cancer patients. The median CAXII level in the sera from non-smokers was 1.56, and that in smokers was 1.54, showing no significant difference.</p

NKAPL expression and localization analyses.

<p>(A) RT-PCR analysis of systemic tissue. The targeted amplicon was 471 bp. (B) Immunoblotting analysis of systemic tissue protein. (C) RT-PCR analysis of testis by age. RT+ or RT- represents the presence or absence of reverse transcription, respectively. M: DNA size marker. (D) Immunoblotting of testis by age. (E) Immunohistochemistry of adult testis with Nkapl antibody. Black scale bar = 50 μm. (F) Immunofluorescence of adult testis with Nkapl, TRA98, E-cadherin, and c-kit antibodies. White arrows indicate E-cadherin-positive undifferentiated spermatogonia, and white arrowheads represent c-kit-positive differentiating spermatogonia. White scale bar = 50 μm.</p

NKAPL interacts with other Notch signaling co-repressors and suppresses downstream transcription of all Notch family receptors.

<p>(A-C) interactions between Nkapl and CIR (Corepressor interacting with RBPJ), HDAC3 or CSL (Cbf1/Rbp-jk) by immunoprecipitation (IP) and immunoblotting (WB) with whole-cell extract to confirm expression of co-transfected genes. Nkap was used as positive control. (A) Interaction of NKAP and NKAPL with CIR (Corepressor interacting with RBPJ). (B) Interaction of NKAP and NKAPL with HDAC3. (C) Interaction of NKAP and NKAPL with CSL (Cbf1/Rbp-jk). (D)The Luciferase assay of transcription levels with pEluc-CSL and pcDNA-Notch 1 to 4 co-transfected NIH3T3 cells. Mock represents co-transfection with no insertion of pcDNA vector. (E) The luciferase assay after co-transfection with pEluc-CSL and pcDNA-Notch1 to 4 and pCAGGS-Nkap or Nkapl vectors. Mock represents co-transfection with no insertion of pCAGGS vectors. Error bars show standard deviation (SD) from the means.</p

Expression of CAXII antibody in lung cancer cell lines and tissues.

<p>(A) Immunoblot analysis of CAXII in lung cancer cell lines. CAXII was detected as an approximately 40-kDa protein with A549 cells. (B) Immunostaining of CAXII in A549 cells (a), adenocarcinoma (b), and squamous cell carcinoma (c) of the lung, and each showed membranous staining of CAXII.</p

Clinicopathological characteristics of the patients.

a<p>Adenocarcinoma.</p>b<p>Squamous cell carcinoma.</p>c<p>Small cell lung carcinoma.</p>d<p>Large cell neuroendocrine carcinoma.</p

<i>Nkapl</i> deletion caused significant changes in spermatogonial stem cell (SSC) maintenance markers and increases in differentiation-related factors.

<p>(A) Transcriptional changes of SSC maintenance markers between testes of adult Nkapl^+/- and Nkapl^-/- mice by qRT-PCR. (B) Transcriptional changes of differentiation-related factors. Error bars indicate standard deviation from the means. *P<0.05.</p

<i>Nkapl</i> deletion caused significant apoptosis at the level of pachytene spermatocytes due to aberrant changes of meiosis-related genes.

<p>(A) Immunohistochemistry of cleaved caspase-3 antibody for testes of <i>Nkapl+/-</i> and <i>Nkapl-/-</i> mice to detect apoptotic cells. (B) The average counts of apoptotic cells per seminiferous tubule. One hundred seminiferous tubules were randomly selected, and cleaved caspase-3-positive germ cells were counted. Error bars are standard deviation (SD) from the means. (C) Immunohistochemistry of MCA in testes of <i>Nkapl+/-</i> and <i>Nkapl-/-</i> mice. (D) Transcriptional changes of meiosis-related genes between testes of Nkapl^+/- and Nkapl^-/- mice by qRT-PCR. Error bars indicate SD from the means. *P<0.05.</p

NKAPL suppresses the transcription of downstream target genes through Notch signaling and plays critical roles in spermatogonial stem cell (SSC) maintenance and differentiation.

<p>(A) Immunoblotting for proteins extracted from testis or GS cells. Black arrowheads represent the specific reaction for Nkapl antibody. Asterisks represent non-specific reactions. (B) Immunoblotting of GS and GS-Nkapl cells to confirm the expression of the lentiviral-induced <i>Nkapl</i> gene. The reaction with FLAG antibody demonstrates its expression. Black arrows represent the specific reactions by each of the antibodies. (C) The transcriptional changes of <i>Notch1</i> to <i>3</i> between GS and GS-Nkapl cells by qRT-PCR. (D) The transcriptional changes of <i>Hes1</i> and <i>Hes5</i>. (E) The transcriptional changes of SSC markers. (F) The transcriptional changes of differentiation-associated factors. Error bars indicate standard deviation from the means. *P<0.05.</p