Image1_The actin-bundling protein, PLS3, is part of the mechanoresponsive machinery that regulates osteoblast mineralization.jpeg

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.</p

Image3_The actin-bundling protein, PLS3, is part of the mechanoresponsive machinery that regulates osteoblast mineralization.jpeg

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.</p

Image4_The actin-bundling protein, PLS3, is part of the mechanoresponsive machinery that regulates osteoblast mineralization.jpeg

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.</p

Image2_The actin-bundling protein, PLS3, is part of the mechanoresponsive machinery that regulates osteoblast mineralization.jpeg

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.</p

Characterization of forced expression of autotaxin in human breast cancer MDA-B02 cells.

<p>(A) Cells transfected with bidirectional expression vectors pBiL-ATX or pBil-NPP1 were plated with (+) or without (-) doxycycline (Dox). Proteins from conditioned media (CM) or lysates of tumor cells (CL) of two stable clones (no. 30 and no. 38 to ATX, no. 10.5 and no. 42 to NPP1) were electrophorezed then immunoblotted with an anti-ATX antibody (Left panel) or anti-Myc antibody (Right panel). (B) Quantifications of luciferase activity (Left panel), lysoPLD activity (Middle panel) and PDE activity (Right panel) in each clone and parental MDA-B02 cells. (C) Cell proliferation was stimulated with LPC (10 µM) in absence or presence Ki16425 (10 µM). Results are expressed as the % of BrdU incorporation compared to unstimulated MDA-B02 parental cells. Data correspond to the mean ± SD of 6 replicates and are representative of at least 3 independent experiments. (D) Cell invasion was stimulated with 10% FBS used as chemoattractant. Results are the mean ± SD of cells of 3 replicates and are representative of at least 3 independent experiments. Data are expressed as the number of cells/mm². *, <i>P</i><0.05. **, <i>P</i><0.01</p

Distribution of ATX mRNA in different subsets of cases defined by classical prognostic parameters in primary tumors of metastastic patients with bone metastases and of non metastatic patients without metastasis recurrence.

<p>Data are expressed as the median of ATX/L32 mRNA ratio.</p>*<p><i>p</i> values were obtained using the non parametric Mann & Whitney test.</p>**<p>in ductal carcinomas only and <i>p</i> values were obtained using the non parametric Kruskall-Wallis test.</p

Characterization of silencing autotaxin expression in mouse breast carcinoma 4T1 cells.

<p>(A) RT-PCR amplification products for LPA receptors, LPA₁ (1), LPA₂ (2), LPA₃ (3), LPA₄ (4), LPA₅ (5), GPR87, P2Y5, and autotaxin (ATX) from 4T1 cells total RNAs were analyzed on a 2% agarose gel. MW, molecular weight marker. (B) Cell invasion was stimulated with increased LPA concentrations used as chemoattractant. Results are the mean ± SD of cells of 3 replicates and are representative of at least 3 independent experiments. Data are expressed as the number of cells/mm². (C) Autotaxin expression in 3 clones of 4T1 cells transfected with a pStrike vector coding for either irrelevant small hairpin RNAi (sbATX, clones no. 14, no. 16, no. 20) or specific small hairpin RNAi (siATX, clones no. 1, no. 17, no. 52). (Upper panel) Immunoblotting using anti-ATX polyclonal antibody or anti-?tubulin as loading control. (Lower panel) lysoPLD activity (pmol LPA/ml) measured in cell culture conditioned media. (D) Cell proliferation assessed by BrdU incorporation of 4T1 cells and a pool of three 4T1-sbATX clones (no. 14, no. 16, no. 20) or three 4T1-siATX clones (no. 1, no. 17, no. 52), in response to increased concentrations of LPC. Results are expressed in mean ± SD of 6 replicates and are representative of 3 separates experiments. (E) Invasion assay. Cells were placed in presence or absence of LPA (0.1–1 µM) in the upper chamber and FBS, used as chemoattractant, was placed in the lower chamber. Results are the mean ± SD of 3 replicates and are representative of at least 3 independent experiments. Data are expressed as the number of cells/mm². *, <i>P</i><0.05.</p

Effect of forced expression of autotaxin <i>in vivo</i> on MDA-B02 cells increased the formation osteoclasts at the bone metastatic site.

<p>(Upper left panels) Representative immunohistological examination of proximal tibia sections from metastatic animals 29 days after tumor cell inoculation, using the anti-ATX antibody 4F1. T indicates tumor cells. (Lower left panels) Representative histological examination of TRAP-stained proximal tibia sections from metastatic animals. T indicates tumor cells. Bone is stained in dark blue and osteoclats are stained in red (arrows). (Right panel) Quantification of active-osteoclast resorption surface per trabecular bone surface (Oc.S/BS). Results are the mean ± SE of 8–9 animals per group. *: <i>P</i><0.05. Scale bars: 200 µm.</p

Effect of autotaxin expression in orthotopic primary tumor growth and spontaneously metastasis dissemination of mouse 4T1 cells.

<p>4T1 parental cells, 4T1-sbATX clones and 4T1-siATX clones were injected in the mammary gland of normal syngenic female BALB/C mice. At day 14, primary tumors were resected, and weighed. (A) Box plots represent tumor weight (in mg). (B) Primary tumors were embedded in paraffin. Tumor tissue sections were analysed by mmunohistochemistry using a specific antibody directed against the nuclear ki-67 antigen. The mitotic index (numbers in each panel) was calculated as the percentage of nuclei positive for ki-67 (results are the mean ± SD, scale bar: 50 µm). (C) Animals were sacrificed 35 days after tumor cell injection and lungs were collected to quantify spontaneously metastasis formation of 4T1 cells. (Upper panels) representative photographs of lung tissue sections stained with eosin. (Lower panel) Quantification of lung metastasis foci. The number of metastatic foci was enumerated under microscope. P<0,05. T indicates metastatic foci. Scale bar: 200 µm.</p

Effect of forced expression of autotaxin on osteolytic bone metastasis formation of MDA-B02 cells.

<p>(A) (Left panels) Representative radiographs of hind limbs from metastatic mice bearing MDA-B02 cells or MDA-B02-ATX clone no. 30 or MDA-B02-NPP1 clone no. 42, 29 days after tumor cell inoculation. Osteolytic lesions are indicated by arrows (scale bar: 0.5 cm). (Right panel) Quantification of osteolytic lesion areas on radiographs in metastatic animals. Data correspond to the mean ± SE of two independent experiments of 7 to 10 animals per group. (B) (Left panels) Representative bone histology of Goldner's trichrome-stained tibial metaphysis from metastatic animals. Bone is stained in blue; bone marrow and tumor cells are stained in red. (scale bar: 1 mm). (Right panel) Histomorphometric analysis of metastatic hindlimbs using the bone volume/tissue volume ration (BV/TV, black bars and left axis) and the tumor volume/tissue volume ratio (TumV/TV, open bars and right axis) as referents. Values are the mean ± SE of 7–10 animals per group representative of two independent experiments. *, <i>P</i><0.05.</p