Serines in the Intracellular Tail of Podoplanin (PDPN) Regulate Cell Motility

Podoplanin (PDPN) is a transmembrane receptor that affects the activities of Rho, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null Pdpn knock-out mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN-free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, whereas their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target

Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb

Background: The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs). Methods and results: By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5′ flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3′UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c. Conclusions: These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis. Keywords: microRNA Transcription factors Gene expression Lung epithelial cells Target

Genome-Wide Analyses of Nkx2-1 Binding to Transcriptional Target Genes Uncover Novel Regulatory Patterns Conserved in Lung Development and Tumors

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors

Spatial and temporal pattern of Nkx2-1 protein expression in developing mouse lung.

<p>Immunohistochemistry analyses of Nkx2-1 protein expression using the Nkx2-1 antibody (ab76013) in (<i>A</i>) E11.5 lung (bar = 100 µm)) and in (<i>B</i>) E19.5 lung (bar = 100 µm). Nkx2-1 is expressed in all lung epithelial cells at E11.5, but is restricted to bronchiolar and type II alveolar cells at E19.5. Confocal immunofluorescence co-localization analysis of Nkx2-1 (green) and Ki67 (red) proteins in (<i>C</i>) E11.5 (bar = 20 µm) and (<i>D</i>) E19.5 embryonic lung (bar = 20 µm) using the Nkx2-1 antibody (ab76013) and in (<i>E</i>) E11.5 (bar = 20 µm) and (<i>F</i>) E19.5 embryonic lung (bar = 20 µm) using the Nkx2-1 antibody (07-601). Nkx2-1 and Ki67 co-localized in most epithelial nuclei at E11.5 (<i>C,E</i> inserts) but are detected in different cells at E19.5 (<i>D,F</i> inserts). (<i>G</i>) Western blot analysis of Nkx2-1 protein expression using Nkx2-1 rabbit polyclonal antibody (07-601) in developing mouse lung at the indicated time points, and adult lung, thyroid and liver. MLE15 mouse epithelial cells and H441 human lung carcinoma cells were also analyzed. Actin was used as control. Two main bands between 40 and 50 kD are detected with a differential developmental pattern of expression (n = 3) (black arrows). Other minor bands of unknown identity are also detected (*) Lb (lung buds), V (blood vessels), Br (bronchioles), E (epithelium), M (mesenchyme), A (alveolus), green arrow.</p

NKX2-1 levels in human lung tumors significantly correlate with expression of developmental Nkx2-1 target genes.

<p>Heatmaps of human lung tumor genes identified in GSE12667 database showing gene expression levels of the human homologues of Nkx2-1 target genes identified in mouse lung development at E11.5 (upper panel) and E19.5 (lower panel); genes are organized according to their Pearson correlation value (y axis) to relative NKX2-1 expression level (x axis).</p

Downregulation of Nkx2-1 affects expression of proliferation-related genes in mouse lung epithelial cells.

<p>(<i>A</i>) Western blot analysis of Nkx2-1 downregulation by shRNA in MLE15 cells using the Nkx2-1 antibody 07-601 (Millipore-Upstate). Two bands between 40–50 kD are detected in mouse MLE15 cells and E17.5 lung. Densitometry analyses show a significant downregulation of Nkx2-1 major bands in shRNA transduced cells. Nkx2-1 downregulation by shRNA also reduces the level of minor bands of higher molecular weight (*, asterisks) (<i>B</i>) Representative flow cytometry analyses of DNA content and cell cycle progression in the same cells; n = 3. (<i>C</i>) Relative number of cells in each stage of the cell cycle. (D) Cell count analysis of MLE15 cells transduced with empty vector or Nkx2-1shRNA. 5000 cells were plated and grown in standard conditions for up to 4 days. Attached cells were trypzinized and counted; n = 6 (E) Real time RT-PCR analyses of Nkx2-1 mRNA expression levels in Nkx2-1 shRNA or empty vector transduced cells; n = 3; similar analyses were performed for selected targets; n = 6. Error bars represent s.e.m.; (*) p≤0.05.</p

Selected genes in non-redundant over-represented biological processes among genes bound by Nkx2-1.

<p>Selected genes in non-redundant over-represented biological processes among genes bound by Nkx2-1.</p

Human lung tumor data sets used in the correlation and GSEA studies.

<p>bold GSEA values <0.05.</p>*<p>Bound/Expressed.</p

Nkx2-1 binding patterns to selected target genes in lung development.

<p>(<i>A</i>) Binding patterns of Nkx2-1 to known lung target genes. Binding ratios (IP/Input) are shown at different chromosomal locations. Pink (binding in E11.5 lung), blue (binding in E19.5 lung). Transcripts are indicated by a black line. (<i>B</i>) Binding patterns of Nkx2-1 to newly identified targets involved in cell proliferation. (<i>C</i>) Chromatin immunoprecipitation-PCR validation of Nkx2-1 binding to proliferation-related target genes. Nkx2-1 IP DNA from E11.5 and E19.5 lungs, Input and IgG immunoprecipated control were used in PCR analyses. Oligonucleotides in the promoter region were used to analyze binding of Nkx2-1, and at exon regions of the same gene as control, (n = 2).</p

Selected GO categories over-represented within developmental genes correlated to NKX2-1 levels.

<p>GO level ≥4; Bayes Factor ≥0; p≤0.03; number of genes≥10.</p