Expression of Enpp1 in human primary breast cancer and bone metastasis tissues.

<p>Enpp1 (<b>A</b>) mRNA and (<b>B–D</b>) protein expression in human normal mammary epithelium (N), primary breast tumor (T), and breast cancer bone metastasis (BBM) was determined by (A) qRT-PCR and (B–D) IHC analysis. (A) Data are representative of three independent experiments performed in triplicate (*<i>p</i><0.05 for T versus N). (B–D) Proteins were identified using DAB (brown). Sections were counterstained with hematoxylin and visualized by light microscopy (200X). N – Normal, T – Tumor.</p

Expression of Enpp1 in breast cancer cell lines.

<p>Enpp1 (<b>A, B</b>) mRNA and (<b>C</b>) protein expression was determined in (A) NT2.5 murine breast cancer cells and bone sublines, and (B, C) immortalized normal human mammary epithelial cell lines and human breast cancer cell lines by (A, B) qRT-PCR and (C) Western analysis. (A, B) Data are representative of three independent experiments performed in triplicate and expressed as the mean ± s.e.m (**p<0.01, ***p<0.001). (C) Protein expression was determined by Western analysis performed on equal amounts of protein from total cell lysates.</p

Effect of Enpp1 on the development of bone metastasis.

<p>MDA-MB-231 cells stably expressing Enpp1 or empty vector (EV) were injected into the (<b>A, B</b>) tibia (n = 9/group) and (<b>C, D</b>) left cardiac ventricle (n = 5/group) of athymic nude mice and digital radiographic imaging was performed at weekly intervals. (A, C) Osteolytic area and (B, D) tumor area were measured on radiographic images and histological sections, respectively (*<i>p</i><0.05, **<i>p<</i>0.01). Representative images are shown at 4 weeks following tumor cell administration. Black outlined areas on histological images indicate areas of tumor.</p

Generation of breast cancer cells with increased Enpp1 expression and enzymatic activity.

<p>MDA-MB-231 cells were stably transduced with Enpp1 or empty vector (EV). Enpp1 (<b>A</b>) protein expression was determined by Western analysis performed on equal amounts of protein from total cell lysates. (<b>B</b>) Nucleotide phosphodiesterase activity was determined using the nucleotide derivative p-nitrophenyl thymidine 5′-monophosphate (pNP-TMP) as a substrate. Data are representative of three independent experiments performed in triplicate and expressed as the mean ± s.e.m (**<i>p<</i>0.01).</p

MIP-1δ enhances RANKL-induced osteoclastogenesis in vitro.

<p>BMM or RAW 264.7 cells were treated with MIP-1δ (0.1 pg/ml) and RANKL (5 ng/ml) individually or in combination for 7 days. (<b>A</b>) OCL formation was assessed by counting the total number of OCL per well under light microscopy (<b>D</b>, 200X Magnification; Scale bars, 50 µm<b>)</b>. (<b>B, C</b>) Fusion index was determined by counting the number of nuclei per OCL in 15–20 random fields and is reported as (B) the average number of nuclei per OCL and (C) the number of OCL containing a specified range of nuclei per cell (BMM shown, similar results were observed for RAW 264.7). Data are representative of three independent experiments performed in quadruplicate, expressed as the mean (n  = 12) ± s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

MIP-1δ promotes osteoclastogenesis and bone resorption in vivo.

<p>Mice (5/group) were administered MIP-1δ (1 ng/ml) or PBS subcutaneously over the calvariae twice each day for 5 days. Calvariae were removed and paraffin-embedded sections were generated for histomorphometric analysis. (A) OCL were identified by TRAP staining, counted under light microscopy (right, 400X), and reported relative to mm of marrow cavity surface (left). (B) Resorption area (left) was determined by measuring the total area occupied by marrow cavities within each calvarial section (right, 100X). Data are representative of two independent experiments, expressed as the mean (n  = 10) ± s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. **, <i>p</i><0.01.</p

MIP-1δ induces expression and activity of c-fos and NFATc1 via stimulation of the PLCγ2 and NF-κB signaling pathways.

<p>BMM were treated with or without MIP-1δ (0.1 pg/ml). (A) c-fos and NFATc1 protein expression were determined by Western analysis performed on equal amounts of protein from total cell lysates. (B) Activation of AP-1 was determined 30 minutes following treatment with MIP-1δ by non-radioactive EMSA analysis of nuclear cell lysates using AP-1 oligonucleotides. Oct-1 served as a loading control and 200-fold excess unlabeled oligonucleotide (200X-U.O.) served as a competitive control. (C) NFATc1 protein expression was determined 60 minutes following treatment with MIP-1δ by Western analysis performed on equal amounts of protein from nuclear cell lysates. (D) CTSK, TRAP, and DC-STAMP mRNA expression were determined by qRT-PCR 24 hours following treatment with MIP-1δ. Data are representative of three independent experiments performed in triplicate, expressed as the mean (n  = 9) ± s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. <i>p</i><0.01; ***, <i>p</i><0.001. (E, F) BMM were treated with MIP-1δ (0.1 pg/ml) alone or in combination with the (E) NF-κB inhibitor IKK2 inhibitor VIII (IKK2-I) or the (F) PLC inhibitor U73122 and protein expression was determined 60 minutes (c-fos, NFATc1) or 30 minutes (IκBα, phospho-PLCγ2) following MIP-1δ treatment by Western analysis performed on equal amounts of protein from total cell lysates. (E) Densitometry values are provided below respective lanes for ease of interpretation. Data are representative of at least two independent experiments.</p