ORIGINAL RESEARCH ARTICLE
Journal of
Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear Factor-kappa B (RANK) Signaling DIANA GOLDEN, ELIZABETH A. SARIA,
AND
Cellular Physiology
MARC F. HANSEN*
Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut Bone remodeling requires osteoclast activation, resorption, and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF-kB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38MAPK, Akt, and p65NF-kB. However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL-induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL-induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65NF-kB pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK-mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis. J. Cell. Physiol. 230: 2951–2960, 2015. © 2015 Wiley Periodicals, Inc.
The skeleton undergoes constant remodeling to remove old bone and maintain its structure throughout life. Normal bone remodeling involves a balance of bone resorption by osteoclasts and bone synthesis by osteoblasts. Osteoblasts are derived from mesenchymal stem cells, while osteoclasts arise through differentiation of hematopoietic progenitors (Theill et al., 2002). Receptor Activator of NF-kB (RANK), its ligand (RANKL), and the decoy receptor Osteoprotegerin (OPG) belong to the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) superfamily and are key regulators of bone development (Hofbauer and Heufelder, 2001; Horowitz et al., 2001). Previous studies have led to a model that when bone remodeling is not taking place, RANKL and OPG, which are both expressed by the osteoblast, are bound together on the surface of the osteoblast, while RANK is expressed on the surface of circulating pre-osteoclasts. Bone remodeling is initiated when the RANKL on the osteoblasts binds to and activates RANK on the pre-osteoclasts (Anderson et al., 1997; Tsuda et al., 1997; Nakagawa et al., 1998). The RANK/RANKL interaction activates a signaling cascade in osteoclasts with downstream targets including the ERK, p38MAPK, Akt, and NF-kB signaling pathways (Teitelbaum and Ross, 2003; Kawamura et al., 2007). These signaling cascades are essential in promoting osteoclast activation, differentiation, and migration (Teitelbaum and Ross, 2003; Johnson and Lapadat, 2002). To date, RANK signaling function has been largely defined in osteoclasts where RANK is well known as a major factor in osteoclast differentiation, activation, and bone resorption (Teitelbaum, 2000; Teitelbaum and Ross, 2003). In contrast to osteoclasts, the role of RANK in osteoblasts has been much less defined. Transgenic mice overexpressing RANK or with deletions in the germline of RANK both have mature osteoblasts (Dougall et al., 1999; Li et al., 2000). This has led many to downplay the role of RANK in osteoblasts. However, studies have shown that RANK is overexpressed in osteosarcoma (Wittrant et al., 2006; Mori et al., 2007a,b), a mesenchymal tumor of bone of osteoblastic origin. This © 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .
suggests that RANK signaling may play a role in normal osteoblasts as well. Osteoblast migration is essential to the bone remodeling process. After osteoclast activation and bone resorption by
Abbreviations: NF-kB, nuclear factor kappa B; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; RANK, receptor activator of NF-kB; RANKL, receptor activator of NF-kB ligand; sRANKL or sRL, soluble receptor activator of NF-kB ligand; OPG, Osteoprotegerin; MEK1/2, MAP kinase 1/2; ERK, extracellular signal related kinase; p38MAPK, p38 mitogen-activated protein kinase; Akt, protein kinase B; PI3K, phosphoinositide 3-kinase; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; IL-13, interleukin 13; TGFb1, transforming growth factor beta 1; FGF, fibroblast growth factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Gab1, Grb2 associated-binding protein 1; Grb2, growth factor receptor bound protein 2; BMP, bone morphogenic protein; Ras, Rat sarcoma; s.e.m., standard error measurement. Contract grant sponsor: National Institute for Arthritis, Musculoskeletal and Skin Diseases; Contract grant number: AR47684. Contract grant sponsor: Department of DefenseCongressionally Directed Medical Research Program; Contract grant number: PR100793. *Correspondence to: Marc F. Hansen MS, PhD, Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, MC-3101, Farmington, CT 06030-3101. E-mail: [email protected] Manuscript Received: 9 April 2014 Manuscript Accepted: 16 April 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 20 April 2015. DOI: 10.1002/jcp.25024
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osteoclasts, osteoblasts migrate into the bone pit and lay down the matrix to form solid bone, completing the bone remodeling process (Masi and Brandi, 2001). Osteoblast chemotaxis is induced by various chemoattractants such as PDGF, VEGF, and IGF1 (Mayr-Wohlfart et al., 2002; Mehrotra et al., 2004; Nakasaki et al., 2008). RANKL, produced by osteoblasts, acts a chemoattractant for monocytic cells (Breuil et al., 2003) and osteoclasts (Henriksen et al., 2003). RANKL-induced migration activates Src and the PI3K/Akt pathway (Breuil et al., 2003; Henriksen et al., 2003). RANKL released into the bone microenvironment not only induces osteoclast activity but also has been shown to have effects on cancer cells that express RANK. RANK expression and a migration response to RANKL have been shown in tumor cells with a tendency to metastasize to bone such as in breast cancer cells (Jones et al., 2006), prostate cancer cells (Armstrong et al., 2008), melanoma cells (Jones et al., 2006), as well as osteosarcoma (Wittrant et al., 2006; Mori et al., 2007a,b; Akiyama et al., 2010). This led to our hypothesis that sRANKL may serve as a chemotactic factor for osteoblasts during normal bone remodeling. We found that sRANKL induced a significant migratory response in osteoblasts through the PI3K/AKT signaling pathway when MEK1/2 is inhibited. These findings suggest that sRANKL may play a role in osteoblast migration during the bone remodeling process. Materials and Methods Cell culture The human osteoblast cell line hFOB 1.19 used was obtained from the American Tissue Type Collection. This cell line has a temperature sensitive mutant (tsA58) of SV 40 T-antigen that is permissive for proliferation at 33.5°C and the gene coding for G418 resistance (Harris et al., 1995). When the culture temperature is raised to the restrictive temperature, 39.5°C, the cells differentiate along the osteoblastic lineage and undergo mineralization in approximately 14 days (Harris et al., 1995). Cells were cultured as Harris et al. (1995). The medium was changed every third day. Bone marrow-derived human mesenchymal stem cells (hMSCs) and normal human osteoblasts (NHOsts) (Lonza Walkersville Inc., Walkersville, MD) were also used. These primary cell cultures were maintained in proper proliferation medium or osteogenic differentiation medium supplied by Lonza according to the manufacturer’s conditions. hMSCs and NHOsts cells have a manufacturer’s recommended finite life span in vitro and were used for only 6–8 population doublings. Osteosarcoma tumor cell lines SAOS2 and MG63 were obtained from the American Type Culture Collection and were grown and treated as previously reported (Mori et al., 2007a). The pre-osteoclastic cell line RAW 264.7 was a generous gift from Dr. Joseph Lorenzo and maintained according to standard media conditions (Hotokezaka et al., 2002). Western blotting hFOB and hMSC cells were induced to differentiate by temperature shift or transfer to differentiation media respectively and harvested at days 0, 3, 6, and 12 following induction of differentiation. NHOst cells growing in growth media, RAW 264.7, SAOS2, and MG63 cells were also harvested as controls. The harvested cells were lysed and 60 mg of the supernatants were mixed with SDS-PAGE sample buffer, denatured for 2 min at 95°C and separated on 4–15% gradient SDS gels (Biorad, Hercules, CA). The proteins were electrophoretically transferred to PVDF membrane in transfer buffer containing 20% methanol for immunodetection. The blot was blocked in 5% non-fat milk in 1x PBST blocking buffer for 1 h at room temperature and then incubated with primary RANK antibody in 5% BSA/PBST (1:500; Abcam, Cambridge, MA) overnight at 4°C with gentle agitation on a platform shaker. GAPDH was visualized using a 1:5,000 dilution JOURNAL OF CELLULAR PHYSIOLOGY
of an anti-GAPDH antibody (Abcam). After a final washing with 1 PBST 3 times for 5 min, protein bands were revealed using horseradish peroxidase-coupled secondary antibodies (Biorad) and chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Phosphorylation assays For phosphorylation studies, hFOB 1.19 cells were cultured at the permissive temperature to 80% confluence and then serum-starved for 24 h. Total p65NF-kB, phospho-p65NF-kB (Ser536), total Akt, phospho-Akt (Ser473), total p38MAPK, phospho-p38MAPK, total ERK, and phospho-ERK were detected using antibodies at dilutions of 1:1,000 (Cell Signaling Technology, Beverly, MA, USA). Protein bands were revealed using horseradish peroxidase-coupled secondary antibodies (1:2000; Cell Signaling Technology) and Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology). Modified Boyden chamber assay (chemotaxis) Because the cells do not adhere to the membranes without fibronectin coating, the 8-mm pore membranes were prepared prior to assay by incubating the bottom side of the membrane overnight at 4°C with a standard concentration of fibronectin 20 mg/ml (Fiedler et al., 2006; Mishima and Lotz, 2008). The fibronectin solution (Sigma, Carlsbad, CA, USA) was then aspirated off and the membranes were extensively washed to remove unbound fibronectin. The membranes were then dried and used the same day. Chemotaxis reagents, sRANKL or IGF1 (Peprotech, Rocky Hill, NJ) to be tested were placed in the lower chamber using 30 ml/well. 12,500 serum-starved hFOB 1.19 cells growing at the permissive temperature were treated with DMSO as the vehicle control or with 40 mM U0126 (MEK1/2 inhibitor), 20 mM SB203508 (p38MAPK inhibitor) and/or 50 mM LY294002 (PI3K inhibitor) (Sigma) for 2 h. The treated cells were then added to the upper chamber after chamber assembly. After 16 h incubation, the cell suspension was swabbed off gently and the membrane was removed without causing any suction below. The membrane was soaked in ice-cold 100% methanol for 15 min and the bottom side was stained in 0.2% solution of crystal violet for 20 min before being washed 3 times in distilled water and dried. Images for counting were acquired using an inverted microscope with a 20 objective (Olympus, Center Valley, PA). The cells on the membrane were individually counted by viewing three random fields within the well. Each experiment was performed independently three times with four replicates on each plate. Wound healing assay (migration) The cells were plated onto the pre-marked 35-mm dishes to create a monolayer with near 90% confluence and incubated at the permissive temperature for 6 h to allow cells to adhere and spread on the dishes completely. Serum-free media was added to the cells to serum-starve overnight. Inhibitors were added 2 h prior to wounding. Scratches of approximately similar size (500 mm) were made, to minimize any possible variation caused by the difference in the width of the scratches, with a p200 pipette tip. The debris was removed by a PBS rinse followed by the addition of the individual treatment conditions to be compared. Images were acquired using an inverted microscope with a phase-contrast 10x objective (Olympus), at 0 and 16 h. For each image, distances between one side of scratch and the other were measured using OpenLab software (Improvision, Waltham, MA). Oris migration chambers Cell migration was assessed using 96-well Oris migration chambers (OrisTM Universal Cell Migration Assembly kit; Platypus
OSTEOBLAST MIGRATION INVOLVES RANK SIGNALING
Technologies, Madison, WI). hFOB 1.19, MG63 or SAOS2 cells were (1.2 105) were seeded into each test well and incubated at 37°C to permit cell attachment. After 24 h, all well inserts were removed, and the medium was refreshed. The cells were treated and incubated for 16 h to permit cell migration and then were stained with Calcein AM. p65NF-kB peptide inhibitor (Ser529/ Ser536) was provided by Novus (Novus USA, Littleton, CO). Cell migration was quantitated by measuring the fluorescence of cells migrated into the area using fluorescent plate reader. Statistics All values expressed as means and s.e.m. Differences between groups were analyzed using the Student’s t-test or one-way ANOVA (Prism Software Corporation, Irvine, CA). P values of
Journal of
Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear Factor-kappa B (RANK) Signaling DIANA GOLDEN, ELIZABETH A. SARIA,
AND
Cellular Physiology
MARC F. HANSEN*
Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut Bone remodeling requires osteoclast activation, resorption, and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF-kB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38MAPK, Akt, and p65NF-kB. However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL-induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL-induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65NF-kB pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK-mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis. J. Cell. Physiol. 230: 2951–2960, 2015. © 2015 Wiley Periodicals, Inc.
The skeleton undergoes constant remodeling to remove old bone and maintain its structure throughout life. Normal bone remodeling involves a balance of bone resorption by osteoclasts and bone synthesis by osteoblasts. Osteoblasts are derived from mesenchymal stem cells, while osteoclasts arise through differentiation of hematopoietic progenitors (Theill et al., 2002). Receptor Activator of NF-kB (RANK), its ligand (RANKL), and the decoy receptor Osteoprotegerin (OPG) belong to the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) superfamily and are key regulators of bone development (Hofbauer and Heufelder, 2001; Horowitz et al., 2001). Previous studies have led to a model that when bone remodeling is not taking place, RANKL and OPG, which are both expressed by the osteoblast, are bound together on the surface of the osteoblast, while RANK is expressed on the surface of circulating pre-osteoclasts. Bone remodeling is initiated when the RANKL on the osteoblasts binds to and activates RANK on the pre-osteoclasts (Anderson et al., 1997; Tsuda et al., 1997; Nakagawa et al., 1998). The RANK/RANKL interaction activates a signaling cascade in osteoclasts with downstream targets including the ERK, p38MAPK, Akt, and NF-kB signaling pathways (Teitelbaum and Ross, 2003; Kawamura et al., 2007). These signaling cascades are essential in promoting osteoclast activation, differentiation, and migration (Teitelbaum and Ross, 2003; Johnson and Lapadat, 2002). To date, RANK signaling function has been largely defined in osteoclasts where RANK is well known as a major factor in osteoclast differentiation, activation, and bone resorption (Teitelbaum, 2000; Teitelbaum and Ross, 2003). In contrast to osteoclasts, the role of RANK in osteoblasts has been much less defined. Transgenic mice overexpressing RANK or with deletions in the germline of RANK both have mature osteoblasts (Dougall et al., 1999; Li et al., 2000). This has led many to downplay the role of RANK in osteoblasts. However, studies have shown that RANK is overexpressed in osteosarcoma (Wittrant et al., 2006; Mori et al., 2007a,b), a mesenchymal tumor of bone of osteoblastic origin. This © 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .
suggests that RANK signaling may play a role in normal osteoblasts as well. Osteoblast migration is essential to the bone remodeling process. After osteoclast activation and bone resorption by
Abbreviations: NF-kB, nuclear factor kappa B; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; RANK, receptor activator of NF-kB; RANKL, receptor activator of NF-kB ligand; sRANKL or sRL, soluble receptor activator of NF-kB ligand; OPG, Osteoprotegerin; MEK1/2, MAP kinase 1/2; ERK, extracellular signal related kinase; p38MAPK, p38 mitogen-activated protein kinase; Akt, protein kinase B; PI3K, phosphoinositide 3-kinase; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; IL-13, interleukin 13; TGFb1, transforming growth factor beta 1; FGF, fibroblast growth factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Gab1, Grb2 associated-binding protein 1; Grb2, growth factor receptor bound protein 2; BMP, bone morphogenic protein; Ras, Rat sarcoma; s.e.m., standard error measurement. Contract grant sponsor: National Institute for Arthritis, Musculoskeletal and Skin Diseases; Contract grant number: AR47684. Contract grant sponsor: Department of DefenseCongressionally Directed Medical Research Program; Contract grant number: PR100793. *Correspondence to: Marc F. Hansen MS, PhD, Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, MC-3101, Farmington, CT 06030-3101. E-mail: [email protected] Manuscript Received: 9 April 2014 Manuscript Accepted: 16 April 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 20 April 2015. DOI: 10.1002/jcp.25024
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GOLDEN ET AL.
osteoclasts, osteoblasts migrate into the bone pit and lay down the matrix to form solid bone, completing the bone remodeling process (Masi and Brandi, 2001). Osteoblast chemotaxis is induced by various chemoattractants such as PDGF, VEGF, and IGF1 (Mayr-Wohlfart et al., 2002; Mehrotra et al., 2004; Nakasaki et al., 2008). RANKL, produced by osteoblasts, acts a chemoattractant for monocytic cells (Breuil et al., 2003) and osteoclasts (Henriksen et al., 2003). RANKL-induced migration activates Src and the PI3K/Akt pathway (Breuil et al., 2003; Henriksen et al., 2003). RANKL released into the bone microenvironment not only induces osteoclast activity but also has been shown to have effects on cancer cells that express RANK. RANK expression and a migration response to RANKL have been shown in tumor cells with a tendency to metastasize to bone such as in breast cancer cells (Jones et al., 2006), prostate cancer cells (Armstrong et al., 2008), melanoma cells (Jones et al., 2006), as well as osteosarcoma (Wittrant et al., 2006; Mori et al., 2007a,b; Akiyama et al., 2010). This led to our hypothesis that sRANKL may serve as a chemotactic factor for osteoblasts during normal bone remodeling. We found that sRANKL induced a significant migratory response in osteoblasts through the PI3K/AKT signaling pathway when MEK1/2 is inhibited. These findings suggest that sRANKL may play a role in osteoblast migration during the bone remodeling process. Materials and Methods Cell culture The human osteoblast cell line hFOB 1.19 used was obtained from the American Tissue Type Collection. This cell line has a temperature sensitive mutant (tsA58) of SV 40 T-antigen that is permissive for proliferation at 33.5°C and the gene coding for G418 resistance (Harris et al., 1995). When the culture temperature is raised to the restrictive temperature, 39.5°C, the cells differentiate along the osteoblastic lineage and undergo mineralization in approximately 14 days (Harris et al., 1995). Cells were cultured as Harris et al. (1995). The medium was changed every third day. Bone marrow-derived human mesenchymal stem cells (hMSCs) and normal human osteoblasts (NHOsts) (Lonza Walkersville Inc., Walkersville, MD) were also used. These primary cell cultures were maintained in proper proliferation medium or osteogenic differentiation medium supplied by Lonza according to the manufacturer’s conditions. hMSCs and NHOsts cells have a manufacturer’s recommended finite life span in vitro and were used for only 6–8 population doublings. Osteosarcoma tumor cell lines SAOS2 and MG63 were obtained from the American Type Culture Collection and were grown and treated as previously reported (Mori et al., 2007a). The pre-osteoclastic cell line RAW 264.7 was a generous gift from Dr. Joseph Lorenzo and maintained according to standard media conditions (Hotokezaka et al., 2002). Western blotting hFOB and hMSC cells were induced to differentiate by temperature shift or transfer to differentiation media respectively and harvested at days 0, 3, 6, and 12 following induction of differentiation. NHOst cells growing in growth media, RAW 264.7, SAOS2, and MG63 cells were also harvested as controls. The harvested cells were lysed and 60 mg of the supernatants were mixed with SDS-PAGE sample buffer, denatured for 2 min at 95°C and separated on 4–15% gradient SDS gels (Biorad, Hercules, CA). The proteins were electrophoretically transferred to PVDF membrane in transfer buffer containing 20% methanol for immunodetection. The blot was blocked in 5% non-fat milk in 1x PBST blocking buffer for 1 h at room temperature and then incubated with primary RANK antibody in 5% BSA/PBST (1:500; Abcam, Cambridge, MA) overnight at 4°C with gentle agitation on a platform shaker. GAPDH was visualized using a 1:5,000 dilution JOURNAL OF CELLULAR PHYSIOLOGY
of an anti-GAPDH antibody (Abcam). After a final washing with 1 PBST 3 times for 5 min, protein bands were revealed using horseradish peroxidase-coupled secondary antibodies (Biorad) and chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Phosphorylation assays For phosphorylation studies, hFOB 1.19 cells were cultured at the permissive temperature to 80% confluence and then serum-starved for 24 h. Total p65NF-kB, phospho-p65NF-kB (Ser536), total Akt, phospho-Akt (Ser473), total p38MAPK, phospho-p38MAPK, total ERK, and phospho-ERK were detected using antibodies at dilutions of 1:1,000 (Cell Signaling Technology, Beverly, MA, USA). Protein bands were revealed using horseradish peroxidase-coupled secondary antibodies (1:2000; Cell Signaling Technology) and Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology). Modified Boyden chamber assay (chemotaxis) Because the cells do not adhere to the membranes without fibronectin coating, the 8-mm pore membranes were prepared prior to assay by incubating the bottom side of the membrane overnight at 4°C with a standard concentration of fibronectin 20 mg/ml (Fiedler et al., 2006; Mishima and Lotz, 2008). The fibronectin solution (Sigma, Carlsbad, CA, USA) was then aspirated off and the membranes were extensively washed to remove unbound fibronectin. The membranes were then dried and used the same day. Chemotaxis reagents, sRANKL or IGF1 (Peprotech, Rocky Hill, NJ) to be tested were placed in the lower chamber using 30 ml/well. 12,500 serum-starved hFOB 1.19 cells growing at the permissive temperature were treated with DMSO as the vehicle control or with 40 mM U0126 (MEK1/2 inhibitor), 20 mM SB203508 (p38MAPK inhibitor) and/or 50 mM LY294002 (PI3K inhibitor) (Sigma) for 2 h. The treated cells were then added to the upper chamber after chamber assembly. After 16 h incubation, the cell suspension was swabbed off gently and the membrane was removed without causing any suction below. The membrane was soaked in ice-cold 100% methanol for 15 min and the bottom side was stained in 0.2% solution of crystal violet for 20 min before being washed 3 times in distilled water and dried. Images for counting were acquired using an inverted microscope with a 20 objective (Olympus, Center Valley, PA). The cells on the membrane were individually counted by viewing three random fields within the well. Each experiment was performed independently three times with four replicates on each plate. Wound healing assay (migration) The cells were plated onto the pre-marked 35-mm dishes to create a monolayer with near 90% confluence and incubated at the permissive temperature for 6 h to allow cells to adhere and spread on the dishes completely. Serum-free media was added to the cells to serum-starve overnight. Inhibitors were added 2 h prior to wounding. Scratches of approximately similar size (500 mm) were made, to minimize any possible variation caused by the difference in the width of the scratches, with a p200 pipette tip. The debris was removed by a PBS rinse followed by the addition of the individual treatment conditions to be compared. Images were acquired using an inverted microscope with a phase-contrast 10x objective (Olympus), at 0 and 16 h. For each image, distances between one side of scratch and the other were measured using OpenLab software (Improvision, Waltham, MA). Oris migration chambers Cell migration was assessed using 96-well Oris migration chambers (OrisTM Universal Cell Migration Assembly kit; Platypus
OSTEOBLAST MIGRATION INVOLVES RANK SIGNALING
Technologies, Madison, WI). hFOB 1.19, MG63 or SAOS2 cells were (1.2 105) were seeded into each test well and incubated at 37°C to permit cell attachment. After 24 h, all well inserts were removed, and the medium was refreshed. The cells were treated and incubated for 16 h to permit cell migration and then were stained with Calcein AM. p65NF-kB peptide inhibitor (Ser529/ Ser536) was provided by Novus (Novus USA, Littleton, CO). Cell migration was quantitated by measuring the fluorescence of cells migrated into the area using fluorescent plate reader. Statistics All values expressed as means and s.e.m. Differences between groups were analyzed using the Student’s t-test or one-way ANOVA (Prism Software Corporation, Irvine, CA). P values of
