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First published online 12 July 2005
doi: 10.1242/jcs.02436

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A novel protein kinase C -dependent signal to ERK1/2 activated by _Vß₃ integrin in osteoclasts and in Chinese hamster ovary (CHO) cells

Nadia Rucci^1,*, Claudia DiGiacinto^1,*, Luigi Orrù¹, Danilo Millimaggi¹, Roland Baron^2,3 and Anna Teti^1,

¹ Department of Experimental Medicine, University of L'Aquila, via Vetoio - Coppito 2, 67100, L'Aquila, Italy
² Department of Cell Biology and Orthopedics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA
³ ProSkelia SAS, 102 route de Noisy, 93230 Romainville, France

View larger version (66K): [in a new window]   Fig. 1. Integrin expression profile and adhesion to LM609. (A) Parental CHO cells (CHOV) and clonal CHOVß3 cells were subjected to (a) western blot analysis for V and ß3 subunits (primary antibodies diluted 1:300), and (b) to FACS analysis for Vß3, ß1, Vß5 and Vß6 integrin expression profile. Grey shadows, fluorescence background; black traces, integrin-specific fluorescence intensity. (B) CHOV and CHOVß3 cells were allowed to adhere to immobilized LM609 monoclonal antibody or FBS for the time indicated in graphs (left panels, crystal violet staining), or for 30 minutes for morphological analysis (middle and right panels, phase contrast microscopy). Original magnification 10x. (C) Osteoclast cultures (including TRAcP-positive mature multinucleated cells and putative mononuclear precursors) were obtained from bone marrow as described in Materials and Methods, gently lifted in 0.02% EDTA-containing buffer and allowed to attach to immobilized LM609 or FBS for the time indicated in graph (left panel) or for 30 minutes for morphological analysis (middle and right panels, staining for TRAcP). Original magnification 20x.

View larger version (54K): [in a new window]   Fig. 2. PKC isoform expression and subcellular redistribution. (A) CHOVß3 cells were serum-starved, detached and kept in suspension for 1 hour, then replated in LM609-coated wells for the indicated times. Aliquots of cells were left untreated (suspension) for baseline detection, or treated with 10-7 TPA for 5 minutes to activate classical and novel PKCs. Cell proteins were fractionated as described in Materials and Methods, resolved by SDS-PAGE and western blotted with the indicated anti-PKC (diluted 1:800), or with anti-actin (diluted 1:1000) antibodies for normalization. (B) Osteoclasts were lifted, replated in LM609-coated wells for 30 minutes and processed for PKC or actin detection as described in A. C, cytosolic; M, membrane; I, Triton-X-100-insoluble fractions; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. Similar results were obtained in three independent experiments.

View larger version (45K): [in a new window]   Fig. 3. Detection of total and phosphorylated MAPKs. (A) CHOVß3 cells and (B) osteoclast cultures were prepared as described in Fig. 2 and plated in LM609-coated wells for 30 minutes. An aliquot of suspended cells was also treated for 5 minutes with TPA (10-7 M) to show control positive assay for phosphorylated JNK. Total cell lysates were processed by SDS-PAGE and western blot for detection of total and phosphorylated fractions of ERK1/2 (primary antibodies diluted 1:500), p38 (primary antibodies diluted 1:800) and JNK (primary antibodies diluted 1:500). (C) Aliquots of CHOVß3 cells were maintained in suspension or plated in LM609-coated wells for the indicated times, with T0 representing cells cultured in standard conditions, starved overnight in serum-free medium. Cells were processed as described above and total and phosphorylated ERK1/2 were determined. (D) Fractionated CHOVß3 cell and (E) osteoclast lysates, obtained as described in Fig. 2, were probed with anti-total or anti-phosphorylated ERK, and anti-ß3 integrin antibodies (diluted 1:400) C, cytosolic; M, membrane; I, Triton-X-100-insoluble fractions; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. Similar results were obtained in three independent experiments.

View larger version (40K): [in a new window]   Fig. 4. Immunoprecipitation assays. (A) CHOVß3 cells and (B) osteoclast cultures were treated as described in Fig. 2, immunoprecipitated with the indicated antibodies and western blotted for PKC (diluted 1:800), FAK (diluted 1:1000), PYK2 (diluted 1:500), ERK-2 and ß3 detection (left and middle panels). Total cell lysates were also subjected to SDS-PAGE and western blotted for total FAK and PYK2 detection in CHOVß3 cells (A, right panels) and osteoclasts (B, right panels). Similar results were obtained in three independent preparations. IP, immunoprecipitation; PI, preimmune serum; TCL, total cell lysate; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. (C) An osteoclast plated in an LM609-coated well was immunostained with anti-PKC (diluted 1:10) and V (diluted 1:10) antibodies detected by conventional immunofluorescence using TRITC- (diluted 1:200) and FITC- (diluted 1:160) conjugated secondary antibody, respectively. Note that the osteoclast was allowed to adhere for 30 minutes, a time insufficient to permit actin ring formation. Scale bars: 20 µm (upper panels); 5 µm (lower panels). Brackets in the upper panels delineate the area showed at higher magnification in the lower panels. Arrows indicate areas of overlapping red (PKC) and green (V) fluorescent labels.

View larger version (54K): [in a new window]   Fig. 5. Recruitment of Shc and Grb2. (A) CHOVß3 cells and (B) osteoclast cultures were treated as described in Fig. 2, immunoprecipitated with the indicated antibodies and western blotted to detect PKC, Shc (diluted 1:200), Grb2 (diluted 1:1000) and ß3 integrin (left and middle panels), or to detect total tyrosine-phosphorylated proteins using the PY99 (diluted 1:1000) antibody (right panels). Similar results were obtained in three independent experiments. IP, immunoprecipitation; TCL, total cell lysate; WB, western blot analysis; Susp, cells in suspension. LM609, adhesion to LM609 for 30 minutes.

View larger version (46K): [in a new window]   Fig. 6. Ras/Raf-1/MEK1/2 activation. CHOVß3 cells were treated as described in Fig. 2 and probed for detection of (A) GTP-Ras (diluted 1:800), (B) serine- (diluted 1:500; left panels) and (C) tyrosine- (diluted 1:1000; right panels) phosphorylated Raf-1, co-precipitated PKC (left panels) and c-Src (right panels). An aliquot of cells in standard culture conditions was serum-starved overnight, then treated with 20% FBS for 30 minutes to show the integrity of the Raf-1 pathway (B,C). In A, the GTP analogue, GTPS, and GDP were added in the assay for positive and negative control, respectively. (D) CHOVß3 and (E) osteoclasts were treated as described above in the presence of the Raf-1 inhibitor 5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone (Raf-1 in; 15 µM, 30 minutes) prior to treatment with 20% FBS or plating on LM609-coated wells for 30 minutes. Cells were then processed for SDS-PAGE and western blot for detection of total and phosphorylated Raf-1 and ERK1/2. (F) CHOVß3 cells and (G) osteoclasts were treated as above but in the absence of the inhibitor, lysed and tested for Raf-1 kinase activity using the Upstate Raf-1 kinase cascade assay kit. (H,I) Lysates from (H) CHOVß3 cells and (I) osteoclasts were also tested using the MEK1/2 immunoprecipitation kinase assay kit for detection of MEK1/2 kinase activity. Similar results were obtained in three independent experiments. IP, immunoprecipitation; TCL, total cell lysate; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. For F-I results are the mean ± s.e.m. of three independent experiments, with *P<0.0001.

View larger version (49K): [in a new window]   Fig. 7. MEK1/2 activation and effect of MEK1/2 inhibitors. (A) CHOVß3 cells were plated on immobilized LM609 substrate for the indicated times, lysed and total and phosphorylated MEK1/2 was determined. In the same gel, total and phosphorylated ERK1/2 were also detected (shown in Fig. 3C). (B) CHOVß3 cells and (C) osteoclasts were plated on immobilized LM609 substrate. Prior to plating, aliquots of cells were pretreated with the MEK1/2 inhibitors PD98059 (50 µM, 30 minutes) or U0126 (10 µM, 30 minutes). Lysates were subjected to western blot analysis and probed for detection of total and phosphorylated MEK1/2 and ERK1/2. (D)Lysates of (a) CHOVß3 cells and (b) osteoclasts treated as described above were also subjected to cytosol/nuclear fractionation and probed for detection of total and phosphorylated ERK1/2. (E) Osteoclasts cultured in standard conditions were starved in 1% FBS overnight, then stimulated with 20% FBS for 30 minutes. Total cell lysates (left panels) and cytosol/nuclear fractions (right panels) were then probed for detection of total and phosphorylated MEK1/2 and ERK1/2. Similar results were obtained in three independent experiments. Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes; C, cytosolic fraction; N, nuclear fraction.

View larger version (41K): [in a new window]   Fig. 8. The role of intracellular Ca2+. (A) CHOVß3 cells and (B) osteoclasts were pretreated with the intracellular Ca2+ chelator BAPTA-AM (50 µM, 30 minutes) in the presence of 2 mM EGTA prior to adhesion to immobilized LM609 and protein fractionation (upper panels), immunoprecipitation with LM609 antibody (lower left panels) or western blotting (lower right panels). Lysates were then western blotted with the indicated antibodies. Similar results were obtained in three independent experiments. IP, immunoprecipitation; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes.

View larger version (56K): [in a new window]   Fig. 9. PKC-dependent ERK1/2 phosphorylation. (A) CHOVß3 cells and (B) osteoclast cultures were pretreated with the PKC inhibitors (Gö6976, 6 nM for CHOVß3 and 2 µM for osteoclasts, 30 minutes; TPA 10-7 M, overnight) prior to plating on LM609 substrate and western blotting with the indicated antibodies. Left panels in A show down-regulation of PKC upon overnight treatment with TPA. (C) CHOVß33 cells and (D) osteoclast cultures were pretreated with the PKC or c-Src (PP2, 50 µM, 30 minutes) inhibitors prior to plating on LM609 substrate and western blotting for total and phosphorylated ERKs. (E) CHOVß3 cells and (F) osteoclast cultures were pretreated with the PKC or c-Src inhibitors prior to plating on LM609 substrate, followed by immunoprecipitation with anti-PKC antibody and western blotting with the indicated antibodies to detect total and p-Y416 (activated) c-Src (diluted 1:1000). An aliquot of cells in standard culture conditions were serum-starved and then treated with 20% FBS for 30 minutes as positive control. (G) CHOVß3 cells and (H) osteoclast cultures were pretreated with the c-Src inhibitor prior to plating on LM609 substrate, followed by immunoprecipitation with LM609 antibody and western blotting with the indicated antibodies. Similar results were obtained in three independent experiments. IP, immunoprecipitation; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes.

View larger version (24K): [in a new window]   Fig. 10. Functional role of PKC. CHOVß3 cells (left panels) and osteoclast cultures (right panels) were treated with Gö6976 as described in the Materials and Methods and investigated for (A,B) adhesion to LM609, (C,D) migration through LM609, (E) invasion through Matrigel and (F) bone resorption. Results are the mean ± s.e.m. from three independent experiments. *P<0.005. The times of each assay are as follows: (A,B) 30 minutes; (C,E) 4 hours; (D) 12 hours; (F) 48 hours.

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