Modulation of Osteoblastic Cell Efferocytosis by Bone Marrow Macrophages

Apoptosis occurs at an extraordinary rate in the human body and the effective clearance of dead cells (efferocytosis) is necessary to maintain homeostasis and promote healing, yet the contribution and impact of this process in bone is unclear. Bone formation requires that bone marrow stromal cells (BMSCs) differentiate into osteoblasts which direct matrix formation and either become osteocytes, bone lining cells, or undergo apoptosis. A series of experiments were performed to identify the regulators and consequences of macrophage efferocytosis of apoptotic BMSCs (apBMSCs). Bone marrow derived macrophages treated with the anti‐inflammatory cytokine interleukin‐10 (IL‐10) exhibited increased efferocytosis of apBMSCs compared to vehicle treated macrophages. Additionally, IL‐10 increased anti‐inflammatory M2‐like macrophages (CD206+), and further enhanced efferocytosis within the CD206+ population. Stattic, an inhibitor of STAT3 phosphorylation, reduced the IL‐10‐mediated shift in M2 macrophage polarization and diminished IL‐10‐directed efferocytosis of apBMSCs by macrophages implicating the STAT3 signaling pathway. Cell culture supernatants and RNA from macrophages co‐cultured with apoptotic bone cells showed increased secretion of monocyte chemotactic protein 1/chemokine (C‐C motif) ligand 2 (MCP‐1/CCL2) and transforming growth factor beta 1 (TGF‐β1) and increased ccl2 gene expression. In conclusion, IL‐10 increases M2 macrophage polarization and enhances macrophage‐mediated engulfment of apBMSCs in a STAT3 phosphorylation‐dependent manner. After engulfment of apoptotic bone cells, macrophages secrete TGF‐β1 and MCP‐1/CCL2, factors which fuel the remodeling process. A better understanding of the role of macrophage efferocytosis as it relates to normal and abnormal bone turnover will provide vital information for future therapeutic approaches to treat bone related diseases. J. Cell. Biochem. 117: 2697–2706, 2016. © 2016 Wiley Periodicals, Inc.The process of efferocytosis (clearance of apoptotic cells) has been characterized in various tissues but the role of efferocytosis in the bone microenvironment is unclear. Bone marrow macrophage efferocytosis of apoptotic osteoblastic cells was enhanced by interleukin‐10 in a STAT‐3 dependent manner and resulted in increased production of TGF‐β1 and CCL‐2. The process of efferocytosis is likely important in bone remodeling and osseous wound healing.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134491/1/jcb25567.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134491/2/jcb25567_am.pd

Transcription factors OVOL1 and OVOL2 induce the mesenchymal to epithelial transition in human cancer

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer

Axl and Tyro3 expression during experimental PCa progression.

<p>(<b>A</b>) Experimental model. Human PCa cell lines (PC3^Luc, DU145^Luc) were implanted <i>s.c.</i> into male SCID mice as a model of a primary (1°) tumor development, and removed after 1 month. At monthly intervals thereafter human PCa cells were identified by anti-HLA staining; proliferative status (Ki67 staining) and Axl or Tyro3 levels were evaluated by FACS. (<b>B</b>) When <i>s.c.</i> tumors were identified and removed, and the animals were reimaged 1 day later and at 2 months by BLI to determine if regrowth had occurred at the primary tumor bed. ROI = Region of interest (<b>C</b>) Percent expression of HLA by lineage depleted (Lin^-) marrow cells or by primary tumor cells at 1 month. (<b>D</b>) Percent expression of Ki67 by lineage depleted (Lin^-) marrow cells or by primary tumor cells at 1 month. Percent expression of Axl or Tyro3 by primary tumor cells established with (<b>E</b>) DU145 or (<b>F</b>) PC3 cells or by DTCs recovered from marrow over time. (<b>G</b>) At 5 months bone metastatic lesions were detected in animals initially implanted with <i>s.c.</i> DU145. Percent expression of HLA by DTCs isolated from the non-metastatic limb or in the metastatic lesion. (<b>H</b>) Expression of Ki67 by HLA expressing DTCs, or by PCa cells recovered from bone metastatic lesions. (<b>I</b>) Percent expression of Axl and Tyro3 by HLA expressing DTCs isolated from the non-metastatic limb or by cells recovered from the metastatic lesion. *<i>p</i><0.05 compared to expression of each receptor in the primary tumors.</p

GAS6 receptor expression by PCa cell lines.

<p>(A) FACS analysis for GAS6 receptors. The human PC3, VCaP, LNCaP, and DU145 PCa cell lines, human bone marrow endothelial cells (HBME), MG-63 human osteosarcoma cell line and primary human fetal osetoblasts (hFOB) were stained with antibodies targeting Axl, Tyro3 or Mer and the % expression were compared to IgG controls for each antibody for n = 3 samples. The data is expressed as avg. ± s.d. *<i>p</i><0.05 compared to expression of Axl for each cell type. (B). Western blot analysis for Tyro3. Since Tyro3 expression was not detected in (A) by FACS, validation by Western Blot of PCa cell lysates (30 µg) was performed. Lysates (1 µg) recovered from HEK293 cells which over expressed of human Tyro 3 (or vector control) were used as positive and negative controls for Tyro3 expression. Normalization of PCa cell extracts was evaluated by staining with antibody to ß-Actin (not shown).</p

Conceptual model of Axl and Tyro3 expression during PCa progression to metastasis.

<p>Conceptual model demonstrating for Axl and Tyro3 expression during prostate cancer progression to metastasis. When Axl expression by PCa cells predominates, GAS6 inhibits growth. When Tyro3 expression predominates or when Tyro3 and Axl expression is equivalent, proliferation appears to be the predominant response of PCa to GAS6.</p

OVOL expression correlates with the epithelial cell state in multiple human cancer cell lines.

<div><p>(A) qPCR: cDNAs from human primary prostate cancer tissues (n = 40; Origene) was analyzed for the expression of E-cad, OVOL1 and OVOL2. The results were normalized to β-actin and shown relative to the average of all cancer samples for each gene as: .</p> <p>log ((Value of Gene (X) for a Sample) / (Value of Gene (X) for Average Cancer)). The sample values are shown in the dot plots and the correlation of OVOL1 or OVOL2 expression with E-cad was calculated (r). The graph depicts a representative experiment out of two with similar results.</p> <p>(B) Venn diagram: Depicts the RNA-seq results of differentially expressed genes common in the OVOL expressing cells and PC3-Epi, relative to PC3-EMT14. The intersection of A and B represents a common epithelial transcriptional signature of 277 genes. The RNA-seq data was analyzed from at least two biological replicates for each cell line.</p> <p>(C) Dot plot: Expression correlation analyses of the 277 genes identified in the epithelial signature from panel (B). Correlation is depicted between PC3-Epi, PC3-EMT14-OVOL1 or PC3-EMT14-OVOL2.</p> <p>(D) Venn diagram: Compares the results from panel (B) with the genes that correlate with the expression of the OVOLs in 917 human cancer cell lines. From the 129 genes that correlated (r > 0.5) with the OVOLs expression in the 917 cancer cell lines, 67 genes are induced by the expression of both OVOL1 and OVOL2 in PC3-EMT14 (C intersection D).</p> <p>(E) Heat map: ConceptGen analysis of the 67 gene-signature from panel (D) revealed a list of 18 annotated genes with functions related to the epithelial state of the cells.</p> <p>(F) Table: 45 genes negatively correlated with OVOL2 expression (r < -0.5) across 917 cancer cell lines. Among these 45 genes, the 10 shown are also downregulated by OVOL2 expression in PC3-EMT14. Note the TF ZEB1 and the mesenchymal marker vimentin (VIM) are the top genes in this list.</p> <p>See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s005" target="_blank">Figure S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s007" target="_blank">Tables S2, S3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076773#pone.0076773.s009" target="_blank">S4</a>.</p></div

OVOL1 and OVOL2 induce MET in MDA-MB-231 breast cancer cells.

<div><p>(A) Bright-phase microscopy: morphology changes in the OVO-overexpressing MDA-MB-231 cells towards an epithelial phenotype. Breast cancer cells were transfected with OVOL1, OVOL2 or both TFs. Scales are 100 µm.</p> <p>(B) qPCR: Expression of the epithelial cell markers E-cad and ESRP1, and the EMT-inducing TFs ZEB1, ZEB2, Slug, Snail, and Twist1 in OVOL-overexpressing cells relative to control. Results were normalized to β-actin.</p> <p>(C) Immunoblot: Expression of EMT markers in MDA-MB-231 cells overexpressing OVOL1 and/or OVOL2 (represented by +/- in the table above the blot).</p> <p>(D) Invasion/migration assay: Representative images and graphs of cancer cell invasion using a Boyden chamber assay. Bar graphs depict the lower migratory and invasive potential of cells overexpressing the OVOL TFs. Percent invasion represents the ratio invading/migrating cells. The graph depicts a representative experiment out of three with similar results.</p> <p>(E) qPCR: miRNA expression in MDA-MB-231 cells overexpressing OVOL1, OVOL2, or both, relative to the control. Results were normalized to miR-U6.</p> <p>(F) qPCR: miRNA expression in PC3-Epi, PC3-EMT14-OVOL1 or PC3-EMT14-OVOL2 relative to the control (PC3-EMT14-EV). Results were normalized to miR-16.</p> <p>Graphs show mean +/- sem; p-values were calculated and represented as * p < 0.05; ** p < 0.01; *** p < 0.001. The qPCRs and immunoblots are representative of two independent experiments with similar results.</p></div

Proposed model of EMT/MET balance in human cancer.

<p>In human cancer cells the mesenchymal and epithelial states are induced and maintained by transcriptional and post-transcriptional (splicing) regulatory programs. These programs are controlled by the feedback regulation between the OVOL and the ZEB1 TFs, critical inducers of MET and EMT respectively. In addition these TFs control the expression of ESRP1, a key-splicing regulator activated in MET and repressed in EMT. Therefore high OVOL and low ZEB1 stabilize the epithelial state decreasing cancer cell invasion and metastasis, and vice versa for the mesenchymal state.</p

Mesenchymal cancer cell populations isolated from co-cultures of epithelial prostate cancer cells and human macrophages.

<div><p>(A) Bright-phase microscopy: morphology changes in the mesenchymal cells PC3-EMT1, PC3-EMT12 and PC3-EMT14 as compared to the epithelial PC3-Epi prostate cancer cells. Scale bars are 200 µm.</p> <p>(B) Immunoblot: Expression of EMT markers in mesenchymal cancer cell lines PC3-EMT1, -EMT2, -EMT12, -EMT14 and -EMT17 compared to epithelial PC3-Epi.</p> <p>(C) Microarray: Gene expression analyses comparing mesenchymal to epithelial cancer cell lines. Venn diagram-I (VD-I) depicts a common EMT-associated signature expressed in the mesenchymal cancer lines PC3-EMT1, -EMT12 and -EMT14 compared to epithelial PC3-Epi. VD-II – Gene signature from VD-I intersected with the signature of ZEB1 silenced cells PC3-EMT1 and -EMT14 using the shRNA-sh4, relative to scramble (Scr) control shRNA.</p> <p>(D) Bright-phase microscopy: PC3-EMT14 cells transfected with ZEB1-shRNAs: sh2, sh4, or Scr. Scale bars are 100 µm.</p> <p>(E) Immunoblot: Expression of EMT markers in PC3-EMT1 and -EMT14 transfected with ZEB1-shRNAs compared to Scr or non-transfected controls.</p> <p>(F) Heat map: 50 genes signature identified in VD-II (panel C). Upregulated genes are in red, downregulated in blue. Fold changes in mesenchymal cancer cells are relative to epithelial PC3-Epi, and in sh4-transfected cells are relative to the Scr control.</p> <p>The immunoblots shown are representative of two independent experiments with similar results. See also Figure S1 and Table S1.</p></div