Osteopontin is up-regulated and associated with neutrophil and macrophage infiltration in glioblastoma.

Osteopontin (OPN) is a glycophosphoprotein with multiple intracellular and extracellular functions. In vitro, OPN enhances migration of mouse neutrophils and macrophages. In cancer, extracellular OPN facilitates migration of cancer cells via its RGD sequence. The present study was designed to investigate whether osteopontin is responsible for neutrophil and macrophage infiltration in human cancer and in particular in glioblastoma. We found that in vitro mouse neutrophil migration was RGD-dependent. In silico, we found that the OPN gene was one of the 5% most highly expressed genes in 20 out of 35 cancer microarray data sets in comparison with normal tissue in at least 30% of cancer patients. In some types of cancer, such as ovarian cancer, lung cancer and melanoma, the OPN gene was one of those with the highest expression levels in at least 90% of cancer patients. In glioblastoma, the most invasive type of brain tumours/glioma, but not in lower grades of glioma it was one of the 5% highest expressed genes in 90% of patients. In situ, we found increased protein levels of OPN in human glioblastoma versus normal human brain confirming in silico results. OPN protein expression was co-localized with neutrophils and macrophages. In conclusion, OPN in tumours not only induces migration of cancer cells but also of leucocytes.

Osteopontin mediates Citrobacter rodentium-induced colonic epithelial cell hyperplasia and attaching-effacing lesions.

Although osteopontin (OPN) is up-regulated in inflammatory bowel diseases, its role in disease pathogenesis remains controversial. The objective of this study was to determine the role of OPN in host responses to a non-invasive bacterial pathogen, Citrobacter rodentium, which serves as a murine infectious model of colitis. OPN gene knockout and wild-type mice were infected orogastrically with either C. rodentium or Luria-Bertani (LB) broth. Mouse-derived OPN(+/+) and OPN(-/-) fibroblasts were incubated with C. rodentium and attaching-effacing lesions were demonstrated using transmission electron microscopy and immunofluorescence. Colonic expression of OPN was increased by C. rodentium infection of wild-type mice. Furthermore, colonic epithelial cell hyperplasia, the hallmark of C. rodentium infection, was reduced in OPN(-/-) mice, and spleen enlargement by infection was absent in OPN(-/-) mice. Rectal administration of OPN to OPN(-/-) mice restored these effects. There was an 8- to 17-fold reduction in bacterial colonization in OPN(-/-) mice, compared with wild-type mice, which was accompanied by reduced attaching-effacing lesions, both in infected OPN(-/-) mice and OPN(-/-) mouse fibroblasts. Moreover, adhesion pedestals were restored in OPN(-/-) cells complemented with human OPN. Therefore, lack of OPN results in decreased pedestal formation, colonization, and colonic epithelial cell hyperplasia responses to C. rodentium infection, indicating that OPN impacts disease pathogenesis through bacterial attachment and altered host immune responses.

Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin-1ß-mediated signaling pathways.

Osteoclasts are bone-resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic™ slides pre-coated with 0.01-20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate-resistant acid phosphatase activity and pre-osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40-60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin-1ß (IL-1ß). IL-1ß production was inhibited using the NO-synthase inhibitor only on FN indicating a FN-specific cross-talk between NO and IL-1ß signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL-1ß signaling.

Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice.

Osteopontin (OPN) is a matricellular cytokine present in most tissues and body fluids; it is known to modulate immune responses. In previous studies using the dextran sulfate sodium (DSS) acute colitis model, we found exacerbated tissue destruction and reduced repair in OPN-null ((-/-)) mice compared with wild-type (WT) controls. As OPN is normally present in milk, we hypothesized that administration of OPN may protect the intestines from the adverse effects of experimental colitis. A volume of 20 or 2 microg/ml bovine milk OPN, dissolved in drinking water, was given to mice 24 h before, and during administration of DSS. Clinical parameters of colitis and neutrophil functions were analyzed as previously reported. Orally administered OPN was absorbed and detected in the colon mucosa by immunohistochemistry. The 20 microg/ml OPN- and DSS-treated WT mice showed 37% less weight loss and reduced colon shortening and spleen enlargements than control mice (P<0.05). OPN administration also reduced the disease activity index, improved red blood cell counts, and reduced gut neutrophil activity compared with the DSS-treated WT mice that were not administered OPN (P<0.05). Immunohistochemical detection of F4/80-labelled cells (macrophages) was also less frequent. The level of transforming growth factor beta1 (TGF-beta1) was increased and the levels of pro-inflammatory mediators decreased in colon tissue samples of OPN-treated mice analyzed by ELISA. The reversal of experimental colitis parameters by exogenous OPN was not as robust in the OPN(-/-) mice. Administration of prokaryotic-expressed recombinant OPN and bovine serum albumin were ineffective. This study shows that administration of a physiological concentration of milk OPN in drinking water ameliorates the destructive host response in DSS-induced acute colitis.

Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells.

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF.

Characterisation of the constitutive over-expression of AJ18 in a novel rat stromal bone marrow cell line (D8-SBMC).

OBJECTIVE: The elucidation of the molecular pathways involved in osteoblast proliferation and differentiation has been greatly enhanced by the availability of cell culture model systems. However, many of the current bone cell culture systems suffer from disadvantages such as the inability to generate mineralised bone-like nodules, a transformed genetic background, cell heterogeneity, and a relatively long time frame from cell seeding to mineralisation, often in the order of several weeks. Here we describe the establishment and characterisation of a novel bone cell line named D8-SBMC. As a first demonstration of their potential value, D8-SBMC was utilised to further support a role for AJ18 during osteogenesis. DESIGN: D8-SBMC was established from a single cell suspension of the previously characterised long term rat stromal bone marrow cells [Kotev-Emeth S, Pitaru S, Pri-Chen S, Savion N. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res 2002;43(4):606-12; Pitaru S, Kotev-Emeth S, Noff D, Kaffuler S, Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res 1993;8(8):919-29]. AJ18 was constitutively and stably over-expressed in D8-SBMC and analysed. RESULTS: D8-SBMC possesses the ability to form robust mineralised bone-like nodules within 8 days proceeding cell confluency. Interestingly, a cement line-like matrix is also generated between the culture dish and a basal monolayer of cells. Constitutive and stable over-expression of AJ18 resulted in an increase in cell proliferation and mineralisation. Expression of bone marker genes, such as bone sialoprotein, osteopontin, osteocalcin, collage type 1, and osteonectin, was up-regulated by AJ18 over-expression. CONCLUSION: A novel bone cell line, D8-SBMC, was established and characterised. D8-SBMC may be a valuable model system for biomineralisation studies. D8-SBMC was utilised to further understand the role of AJ18 in cell proliferation and differentiation during osteogenesis.

Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation.

BACKGROUND: A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the alphavbeta3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. METHODS: We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. RESULTS: Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-alphavbeta3 integrin interaction does not induce the expression of MT1-MMP. CONCLUSION: These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.

Osteopontin expression is required for myofibroblast differentiation.

Osteopontin (OPN) is a multifunctional cytokine that is strongly expressed in healing wounds and fibrotic lesions, both of which are characterized by the formation of myofibroblasts. We examined the role of OPN in myofibroblast differentiation induced by the profibrotic cytokine transforming growth factor-beta1. In cultured cardiac or dermal fibroblasts treated with transforming growth factor-beta1, there was a 2- to 5-fold increase in the expression of the myofibroblast markers alpha-smooth muscle actin and extradomain A fibronectin but no significant increase of these proteins in OPN-null fibroblasts. Phalloidin staining for actin filaments and immunostaining for alpha-smooth muscle actin and focal adhesion proteins showed reduced stress fibers, focal adhesions, and lamellipodia in OPN-null fibroblasts compared with wild-type cells. OPN-null fibroblasts exhibited 40% to 60% less spreading, 50% less resistance to detachment by shear force, and a approximately 3-fold reduction in collagen gel contraction. These defects were partially rescued by ectopic expression of OPN. Mass spectrometric analysis of proteins in focal adhesions formed on collagen type I beads revealed an enrichment of HMGB1 protein in wild-type cells, whereas HMGB1 was not detected in OPN-null cells. Treatment of wild-type cells with small interfering RNA to knock down OPN reduced transforming growth factor-beta1-induced alpha-smooth muscle actin and HMGB1 to levels observed in OPN-null cells. These studies demonstrate that OPN is required for the differentiation and activity of myofibroblasts formed in response to the profibrotic cytokine transforming growth factor-beta1.

Role of osteopontin in neutrophil function.

Osteopontin (OPN) is important for the function of fibroblasts, macrophages and lymphocytes during inflammation and wound healing. In recent studies of experimental colitis we demonstrated exacerbated tissue destruction in OPN-null mice, associated with reduced tumour necrosis factor-alpha expression and increased myeloperoxidase activity. The objective of this investigation therefore was to determine the importance of OPN expression in neutrophil function. Although, in contrast to macrophages, neutrophils expressed low levels of OPN with little or no association with the CD44 receptor, intraperitoneal recruitment of neutrophils in OPN-null mice was impaired in response to sodium periodate. The importance of exogenous OPN for neutrophil recruitment was demonstrated by a robust increase in peritoneal infiltration of PMNs in response to injections of native or recombinant OPN. In vitro, OPN(-/-) neutrophils exhibited reduced chemokinesis and chemotaxis towards N-formyl methionyl leucyl phenylalanine (fMLP), reflecting a reduction in migration speed and polarization. Exogenous OPN, which was chemotactic for the neutrophils, rescued the defects in polarization and migration speed of the OPN(-/-) neutrophils. In contrast, the defensive and cytocidal activities of OPN(-/-) neutrophils, measured by assays for phagocytosis, generation of reactive oxygen species, cytokine production and matrix metalloproteinase-9, were not impaired. These studies demonstrate that, while exogenous OPN may be important for the recruitment and migration of neutrophils, expression of OPN by neutrophils is not required for their destructive capabilities.

Methylglyoxal inhibits the binding step of collagen phagocytosis.

Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues. The extent of MGO-induced modifications was authenticated by amino acid analysis, solubility, and cross-linking. Cells were incubated with fluorescent beads coated with collagen, and the percentage of phagocytic cells was estimated by flow cytometry. MGO inhibited collagen binding (20% of control for 10 mm MGO) in a time- and concentration-dependent manner. MGO-induced inhibition of binding was prevented by aminoguanidine, which blocks the formation of collagen cross-links. MGO reduced collagen binding strength and blocked intracellular calcium signaling. MGO modified the Arg residue in the critical alpha2beta1 integrin-binding recognition sequence of triple helical collagen peptides, whereas MGO-induced cross-linking of Lys residues played only a small role in binding inhibition. Thus, MGO modifications of Arg residues in collagen could be a key factor in the impaired degradation of collagen that promotes fibrosis in chronic infections, such as periodontitis.

A critical role for the membrane-type 1 matrix metalloproteinase in collagen phagocytosis.

Degradation of collagen is important for the physiological remodeling of connective tissues during growth and development as well as in wound healing, inflammatory diseases, and cancer cell invasion. In remodeling adult tissues, degradation of collagen occurs primarily through a phagocytic pathway. However, although various steps in the phagocytic pathway have been characterized, the enzyme required to initially fragment collagen fibrils for subsequent phagocytosis has not been identified. We have used laser confocal microscopy, transmission electron microscopy, and biochemical assays to show that human fibroblasts initiate degradation of collagen through the collagenase activity of the membrane-bound metalloproteinase MT1-MMP. Degradation of natural and reconstituted collagen substrates correlated with the expression of MT1-MMP, which was localized at sites of collagen cleavage at the surface of the cells and also within the cells, whereas collagen degradation was abrogated when MT1-MMP expression was blocked by small interfering RNA treatment. In contrast to MT1-MMP, the gelatinolytic activity of MMP-2 was not required for collagen phagocytosis. These studies demonstrate a pivotal role of catalytically active MT1-MMP in preparing collagen fibrils for phagocytic degradation.

Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-alpha expression and non-programmed cell death.

Osteopontin (OPN), a pro-inflammatory mediator, is constitutively expressed in normal gut and is upregulated in inflammatory colitis. To determine the significance of OPN in inflammatory bowel disease, we studied the development of acute, experimental colitis induced by dextran sulfate sodium (DSS) in OPN-null and wild-type (WT) mice. OPN expression was markedly increased in WT diseased colons, while a higher disease activity index, including spleen enlargement, bowel shortening, and mucosal destruction, was observed in OPN-null mice. Although peripheral blood neutrophil numbers were lower in DSS-treated OPN-null mice, tissue myeloperoxidase levels, reflecting enhanced neutrophil activity, were increased in the diseased colons. In comparison, lymphocyte numbers in peripheral blood were increased earlier than in DSS-treated WT mice. Despite a significantly greater spleen enlargement, flow cytometric analysis of splenocytes from the DSS-treated OPN-null mice revealed lower numbers of differentiated macrophages and (CD4+ and CD8alpha+) lymphocytes. Whereas pro-inflammatory cytokines, including G-CSF, RANTES, MIP1alpha, and TNF-alpha, were increased < 10-fold in DSS-treated WT splenocytes, expression of these cytokines was dramatically suppressed in the DSS-treated OPN-null splenocytes as well as gut tissues. The suppressed TNF-alpha response in OPN-null mice was reflected in a marked increase in non-apoptotic cell death in diseased colons. Collectively, these studies demonstrate that OPN is required for mucosal protection in acute inflammatory colitis.

Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.

Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

Stereochemical analysis of the functional significance of the conserved inverted CCAAT and TATA elements in the rat bone sialoprotein gene promoter.

Basal transcription of the bone sialoprotein gene is mediated by highly conserved inverted CCAAT (ICE; ATTGG) and TATA elements (TTTATA) separated by precisely 21 nucleotides. Here we studied the importance of the relative position and orientation of the CCAAT and TATA elements in the proximal promoter by measuring the transcriptional activity of a series of mutated reporter constructs in transient transfection assays. Whereas inverting the TTTATA (wild type) to a TATAAA (consensus TATA) sequence increased transcription slightly, transcription was reduced when the flanking dinucleotides were also inverted. In contrast, reversing the ATTGG (wild type; ICE) to a CCAAT (RICE) sequence caused a marked reduction in transcription, whereas both transcription and NF-Y binding were progressively increased with the simultaneous inversion of flanking nucleotides (f-RICE-f). Reducing the distance between the ICE and TATA elements produced cyclical changes in transcriptional activity that correlated with progressive alterations in the relative positions of the CCAAT and TATA elements on the face of the DNA helix. Minimal transcription was observed after 5 nucleotides were deleted (equivalent to approximately one half turn of the helix), whereas transcription was fully restored after deleting 10 nucleotides (approximately one full turn of the DNA helix), transcriptional activity being progressively lost with deletions beyond 10 nucleotides. In comparison, when deletions were made with the ICE in the reversed (f-RICE-f) orientation transcriptional activity was progressively lost with no recovery. These results show that, although transcription can still occur when the CCAAT box is reversed and/or displaced relative to the TATA box, the activity is dependent upon the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with preinitiation complex proteins that bind to the TATA box. Thus, the precise location and orientation of the CCAAT element is necessary for optimizing basal transcription of the bone sialoprotein gene.

Fibroblast growth factor 2 and cyclic AMP synergistically regulate bone sialoprotein gene expression.

Bone sialoprotein (BSP) is a noncollagenous protein of the mineralized bone extracellular matrix. We here report that FGF2 and cAMP act synergistically to stimulate BSP gene expression. Treatment of ROS 17/2.8 cells with either 10 ng/ml FGF2 or 1 microM FSK for 6 h resulted in 5.4- and 8.2-fold increases, respectively, in the levels of BSP mRNA. However, in the presence of both FGF2 and forskolin (FGF/FSK), BSP mRNA levels were increased synergistically by 20.4-fold. Using a luciferase reporter construct, encompassing BSP promoter nucleotides -116 to +60, transcription was also increased synergistically by 15.0-fold with FGF/FSK, compared to stimulations of 2.6- and 5.3-fold, respectively, for FGF2 and FSK alone. Transcriptional stimulation by FGF/FSK abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, FRE and Pit-1 elements. Whereas the FRE-protein complex was increased by FGF2 and FGF/FSK, the Pit-1-protein complex was decreased by FSK and FGF/FSK. Notably, transcriptional activity induced by FGF/FSK was blocked by protein kinase A, tyrosine kinase and MEK inhibitors. These studies indicate that the combinatorial effects of FGF and FSK act through PKA, tyrosine kinase and MAP-kinase-dependent pathways, which target the inverted CCAAT, CRE, FRE and Pit-1 elements in the BSP gene to synergistically increase BSP expression.

Recruitment of nuclear factor Y to the inverted CCAAT element (ICE) by c-Jun and E1A stimulates basal transcription of the bone sialoprotein gene in osteosarcoma cells.

Bone sialoprotein (BSP), a major protein in the extracellular matrix of bone, is expressed almost exclusively by bone cells and by cancer cells that have a propensity to metastasize to bone. Previous studies have shown that v-src stimulates basal transcription of bsp in osteosarcoma (ROS 17/2.8) cells by targeting the inverted CCAAT element (ICE) in the proximal promoter. To identify possible downstream effectors of Src we studied the effects of the proto-oncogene c-jun, which functions downstream of Src, on basal transcription of bsp using transient transfection assays. Increased expression of endogenous c-Jun induced by the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate and ectopic expression of c-Jun increased basal transcription of chimeric reporter constructs encompassing the proximal promoter by 1.5-3-fold in ROS 17/2.8 osteosarcoma cells, with more modest effects in a normal bone cell line, RBMC-D8. The effects of c-Jun were abrogated by mutations in the ICE box and by co-expression of dominant negative nuclear factor Y, subunit A (NF-YA). The increase in bsp transcription did not require phosphorylation of c-Jun and was not altered by trichostatin treatment or by ectopic expression of p300/CREB-binding protein (CBP) or mutated forms lacking histone acetyltransferase (HAT) activity. Similarly, ectopic expression of p300/CBP-associated factor (P/CAF), which transduces p300/CBP effects, or of HAT-defective P/CAF did not influence the c-jun effects. Surprisingly, E1A, which competes with P/CAF binding to p300/CBP, also stimulated BSP transcription through NF-Y independently of c-jun, p300/CBP, and P/CAF. Collectively, these studies show that c-Jun and E1A regulate basal transcription of bsp in osteosarcoma cells by recruiting the NF-Y transcriptional complex to the ICE box in a mechanism that is independent of p300/CBP and P/CAF HAT activities.

Osteoclast responses to lipopolysaccharide, parathyroid hormone and bisphosphonates in neonatal murine calvaria analyzed by laser scanning confocal microscopy.

Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 microg/ml), parathyroid hormone (PTH; 10(-8) M), and bisphosphonates (BPs; 1-25 microM clodronate or 0.1-2.5 microM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the alphavbeta3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BP-treated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and beta3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that stained for alkaline phosphatase, collagen type I, and bone sialoprotein, regardless of the presence of BPs. These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclasts and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts.

An in vivo model to study osteogenic gene regulation: targeting an avian retroviral receptor (TVA) to bone with the bone sialoprotein (BSP) promoter.

UNLABELLED: To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. INTRODUCTION: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. MATERIALS AND METHODS: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. RESULTS AND CONCLUSION: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased approximately 15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.

Thrombin mediated migration of osteogenic cells.

Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.

Collagen phagocytosis by fibroblasts is regulated by decorin.

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts. Collagen beads bound to gingival cells by alpha2beta1 integrins. Binding and internalization of decorin/collagen-coated beads decreased dose-dependently with increasing decorin concentration (p < 0.001). Inhibition of binding was sustained over 5 h (p < 0.001) and was attributed to interactions between decorin and collagen and not to decorin-collagen receptor interactions. Both the non-glycosylated decorin core protein and the thermally denatured decorin significantly inhibited collagen bead binding (approximately 50 and 89%, respectively; p < 0.05). Mimetic peptides corresponding to leucine-rich repeats 1-3, encompassed by a collagen-binding approximately 11-kDa cyanogen bromide fragment of decorin and leucine-rich repeats 4 and 5, previously shown to bind to collagen, were tested for their ability to inhibit collagen bead binding. Although the synthetic peptide 3 alone exhibited saturable binding to collagen, neither peptides 3 nor 1 and 2 markedly inhibited phagocytosis. Leucine-rich repeat 3 bound to a triple helical peptide containing the alpha2 integrin-binding site of collagen. When collagen beads were co-incubated with peptides 3 and 4, inhibition of collagen phagocytosis (55%) was equivalent to intact native/recombinant core protein. Thus a novel collagen binding domain in decorin acts cooperatively with leucine-rich repeat 4 to mask the alpha2beta1 integrin-binding site on collagen, an important sequence for the phagocytosis of collagen fibrils.