N-linked sialyated sugar receptors support haematopoietic cell-osteoblast adhesions.

Haematopoietic progenitor cells proliferate and develop predominantly when they adhere to bone marrow stromal cells such as osteoblasts. Therefore, changes in adhesion may be a common mechanism by which stem cells survive, mature and properly traffic between the bone marrow and the circulation. To characterize these adhesion molecules, we reduced the bone marrow cavity to a simple adhesion assay between KG1a (a CD34+ haematopoietic cell line) and osteosarcoma monolayers (MG-63 or SaOS-2). The data demonstrated that adhesion was mediated by cell-to-cell rather than cell-to-matrix contact, was sensitive to trypsin, calcium chelators and glycosylation inhibitors. Selective pretreatment attributed the constitutive binding to N-linked glycans on KG1a. When carboprocessing was inhibited later at the high mannose intermediate (via deoxymannojirimycin), adhesion was retained. Surprisingly, binding of KG1a to SaOS-2 increased past constitutive levels as doses of tunicamycin or deoxymannojirimycin dropped. Selective pretreatment suggested that this 'inducible' binding resides with molecule(s) on SaOS-2. If the terminal sialic acid was digested (via neuraminidase), this induced response was duplicated. These data, verified in primary cells, suggest that the initial tethering between blood and bone cells in this model is probably due to heavily glycosylated, rapidly processed protein(s) on both cell types.

p21-activated kinase-1 (PAK1) inhibition of the human scavenger receptor class B, type I promoter in macrophages is independent of PAK1 kinase activity, but requires the GTPase-binding domain.

Scavenger receptor class B, type I (SR-BI), is a high density lipoprotein receptor that mediates the flux of cholesterol between high density lipoprotein and cells. Recent evidence suggests that SR-BI plays a role in atherosclerosis and that inflammatory mediators down-regulate SR-BI in the macrophage. The purpose of this study was to evaluate the ability of lipopolysaccharide (LPS) to down-regulate the activity of the human SR-BI promoter in the macrophage and to delineate the mechanisms involved. Experiments with cultured cells and in vivo derived macrophages showed that LPS has a powerful suppressive effect on SR-BI expression both in vitro and in vivo. Transient transfection studies demonstrated that LPS represses SR-BI promoter activity in the macrophage cell line RAW 264.7. Cotransfection with either a constitutively active p21-activated protein kinase-1 (PAK1) construct (T423E) or a kinase-deficient PAK1 construct (K299R) resulted in repression of the SR-BI promoter, similar to LPS. These results demonstrate that PAK1-mediated down-regulation of the SR-BI promoter is independent of PAK1 kinase activity and suggest that PAK1 mediates the LPS-induced decrease in promoter activity. Cotransfection with constitutively active Cdc42 or Rac expression constructs also resulted in down-regulation of the promoter; whereas the dominant-negative Cdc42 and Rac constructs elevated basal promoter activity and blunted the LPS response. Cotransfection of PAK1 constructs containing mutations in both the kinase domain and the Cdc42/Rac-binding domain attenuated the PAK1-mediated down-regulation of the promoter, suggesting that Rac and Cdc42 are required for PAK1-mediated decreases in SR-BI promoter activity. 5'-Deletion analysis and gel shift data suggest that LPS inhibits binding of a novel transcription factor to a myeloid zing finger protein-1-like element (-476 to -456) in the human SR-BI promoter. These results demonstrate that the PAK1 pathway down-regulates the SR-BI promoter and suggest that activation of this pathway may play an important role in cholesterol trafficking in the vessel wall.

TGFbeta and BMP-2 activation of the OPN promoter: roles of smad- and hox-binding elements.

Members of the transforming growth factor superfamily are known to transduce signals via the activation of Smad proteins. Ligand binding to transmembrane cell surface receptors triggers the phosphorylation of pathway-specific Smads. These Smads then complex with Smad 4 and are translocated to the nucleus where they effect gene transcription. Smads 1 and 4 were recently demonstrated to mediate BMP activation of the OPN promoter by inhibiting the interaction of Hoxc-8 protein with a Hox-binding element. While previous studies have indicated that specific DNA sequences are recognized by Smad complexes in several promoters, the role of Smad-binding elements (SBEs) in activation of the OPN promoter by members of the TGFbeta superfamily has not been previously evaluated. In this study we tested the hypothesis that a putative Smad-binding region containing the sequence AGACTGTCTGGAC is involved in the activation of the OPN promoter by members of the TGFbeta superfamily. Functional analyses demonstrated that the both the HBE- and Smad-binding region were involved in BMP-2-induced activation of the promoter, whereas, the HBE appeared to be the primary region involved in activation by TGFbeta. Deletion of the first 9 bases in the Smad-binding region substantially reduced BMP-2-mediated activation of the promoter. These results strongly suggest that both the Hox- and the Smad-binding regions play a role in BMP-2-induced activation of the OPN promoter.

Bone morphogenetic proteins secreted by breast cancer cells upregulate bone sialoprotein expression in preosteoblast cells.

It is well established that bone metastases comprise bone; however, the exact factors/mechanisms involved remain unknown. We hypothesized that tumor cells secreted factors capable of altering normal bone metabolism. The aims of the present study were to (1) determine the effects of secretory products isolated from HT-39 cells, a human breast cancer cell line, on osteoprogenitor cell (MC3T3-E1 cells) behavior, and (2) identify tumor-derived factor(s) that alters osteoblast activities. Conditioned media (CM) from HT-39 cells were collected following a 24-h serum-free culture. The ability of CM to alter gene expression in MC3T3-E1 cells was determined by Northern analysis. CM effects on cell proliferation and mineralization ability were determined using a Coulter counter and von Kossa stain, respectively. MC3T3-E1 cells were treated with CM plus noggin, a factor known to block bone morphogenic proteins (BMPs), to determine whether BMPs, shown to be present in CM, were linked with CM effects on MC3T3-E1 cell activity. In addition, inhibitors of MAP kinase kinase (MEK), protein kinase C (PKC), and protein kinase A were used to identify the intracellular signaling pathway(s) by which the active factors in CM regulated osteoblast behavior. CM treatment significantly enhanced BSP mRNA (2.5-fold over control), but had no effect on cell proliferation. Mineralization assay showed that CM enhanced mineral nodule formation compared to controls. Noggin inhibited CM-induced upregulation of BSP mRNA, suggesting that BMPs were responsible for upregulating BSP gene expression in MC3T3-E1 cells. The PKC inhibitor blocked CM-mediated upregulation of BSP, suggesting involvement of the PKC pathway in regulating BSP expression. BMPs secreted by HT-39 cells may be responsible for enhancing BSP expression in MC3T3-E1 cells. Continued studies targeted at determining the role of BMPs in regulating bone metabolism are important for understanding the pathogenesis of bone diseases.

Secretory products from PC-3 and MCF-7 tumor cell lines upregulate osteopontin in MC3T3-E1 cells.

Tumor cells frequently have pronounced effects on the skeleton including bone destruction, bone pain, hypercalcemia, and depletion of bone marrow cells. Despite the serious sequelae associated with skeletal metastasis, the mechanisms by which tumor cells alter bone homeostasis remain largely unknown. In this study, we tested the hypothesis that the disruption of bone homeostasis by tumor cells is due in part to the ability of tumor cells to upregulate osteopontin (OPN) mRNA in osteoblasts. Conditioned media were collected from tumor cells that elicit either osteolytic (MCF-7, PC-3) or osteoblastic responses (LNCaP) in animal models and their effects on OPN gene expression were compared using an osteoblast precursor cell line, MC3T3-E1 cells. Secretory products from osteolytic but not osteoblastic tumor cell lines were demonstrated to upregulate OPN in osteoblasts while inhibiting osteoblast proliferation and differentiation. Signal transduction studies revealed that regulation of OPN was dependent on both protein kinase C (PKC) and the mitogen-activated protein (MAP) kinase cascade. These results suggest that the upregulation of OPN may play a key role in the development of osteolytic lesions. Furthermore, these results suggest that drugs that prevent activation of the MAP kinase pathway may be efficacious in the treatment of osteolytic metastases.

Effect of bone proteins on human prostate cancer cell lines in vitro.

BACKGROUND: Despite the high incidence and serious consequences of skeletal metastasis in prostate cancer patients, the mechanisms involved in establishing secondary lesions in bone are not well-understood. In this study, the role of the mineralized bone matrix in the process of skeletal metastasis was evaluated. METHODS: Attachment, migration, and proliferation responses of human prostate cancer cells to a crude bone protein extract (CBE) were studied. LNCaP and DU145 cells were utilized in 24-hr attachment assays. Boyden chamber chemotactic assays and cell proliferation assays utilized DU145 cells. RESULTS: CBE and fibronectin (FN) promoted attachment of DU145 cells, whereas only FN facilitated attachment of LNCaP cells. CBE-mediated adhesion of DU145 cells was reduced by 94% with cycloheximide, by 98% with RGD peptides, and by 94% with an antibody to alphavbeta3. Although DU145 cells migrated toward FN, CBE did not promote migration of DU145 cells. DU145 cells grown in the presence of CBE-containing media demonstrated a significant reduction in cell number by day 4. The antiproliferative effect of CBE was not due to cell toxicity. CONCLUSIONS: In conclusion, results from this study indicate that mineralized bone proteins promote the attachment of DU145 cells in vitro and suggest that bone proteins may play a key role in vivo during the development of metastatic prostate lesions in bone.

Temporal expression of c-fos mRNA following balloon injury in the rat common carotid artery.

OBJECTIVE: Restenosis is a common problem which limits the effectiveness of percutaneous transluminal coronary angioplasty (PTCA). The cellular mechanisms of restenosis appear to involve smooth muscle cell (SMC) migration to the neointima in response to mitogens and growth factors, resulting in proliferation and deposition of cells in the lumen of the vessel. An antibody directed against PDGF attenuates this response in the rat. Thus, signaling cascades induced by growth factors including PDGF may be important targets for therapeutic intervention. METHODS: Since a number of growth factors activate c-fos via the p21-ras signaling pathway, we examined c-fos expression in a time course experiment involving restenotic lesions in rat carotid arteries. Sections of arteries collected at 1, 3, 7, 14 and 28 days following balloon injury were hybridized using a fluorescein-labeled RNA probe to c-fos. Immunohistochemistry was performed with antibodies to proliferating cell nuclear antigen (PCNA) and alpha-smc actin to characterize cellular constituents of the neointima, and detect any correlation between fos expression and PCNA localization. RESULTS: Expression of c-fos was low at day 1. By day 3, the media and adventitia were positively stained. At days 7 and 14, most cells in the neointima were labeled. By day 28, c-fos was expressed mainly in scattered cells along the luminal surface. Control sections revealed little labeling and confirmed specific staining by the antisense strand, PCNA localization and c-fos expression were similar at days 1, 3, 7 and 28, but at day 14 c-fos was expressed throughout the lesion, with PCNA localized mainly along the luminal edge. The majority of the cells making up the neointima stained rather intensely for alpha-smc actin, identifying them as SMCs. CONCLUSIONS: Results of these experiments indicate that, while c-fos expression correlates with lesion formation, it may be associated with a cellular process distinct from proliferation in this model.

Inhibition of nitric oxide synthase prevents myocardial protection by ramiprilat.

The objective of this investigation was to determine the role of nitric oxide synthase in the action of the angiotensin-converting enzyme inhibitor, ramiprilat, to reduce myocardial ischemia/reperfusion injury. Ramiprilat, the nitric oxide synthase inhibitor NG-nitro-L-NAME (L-NAME), ramiprilat plus L-NAME, or saline (n = 8 each group), were administered i.v. in intact animal preparations of experimentally induced acute myocardial ischemia. Anesthetized, open-chest rabbits were instrumented for measurement of systemic hemodynamics and left ventricular pressure from which left ventricular +dP/dtmax was derived. Animals were subjected to 30 min of left main coronary artery occlusion (marginal branch) followed by 2 hr of reperfusion. Ramiprilat (50 micrograms/kg) or saline was administered 5 min before reperfusion, and those rabbits receiving L-NAME (100 micrograms/kg/min) were pretreated starting 30 min before occlusion throughout the remainder of the experiment. After reperfusion, myocardial infarct size (IS) was determined via tetrazolium staining and expressed as a percentage of area at risk (AR). IS/AR% was significantly reduced in rabbits administered ramiprilat (19 +/- 3%) compared to those receiving saline (39 +/- 2%), ramiprilat plus L-NAME (43 +/- 4%) or L-NAME alone (43 +/- 2%; mean +/- S.E.M.; P < .05). AR as a percent of total left ventricular mass was not different between any of the four treatment groups. Systemic hemodynamic effects were not significantly different between groups. The results indicate that the effect of ramiprilat to reduce infarct size is abolished by pretreatment with L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo validation of a transit-time ultrasonic volume flow meter.

The objective of this investigation was to validate a transit-time ultrasound blood flow metering system in vivo. Implanted chronically and acutely on the ascending aorta of the dog, the transit-time flow probe determined varying flow rates simultaneously with measurements made by the electromagnetic flow metering method. The transit-time technique was also compared to two methods in which blood was collected volumetrically by either graduated cylinder (ascending aorta/dog) or pump withdrawal (abdominal aorta/cat). Statistical analysis of the results provided evidence that the transit-time ultrasound method measured in vivo blood flow rate no differently than the electromagnetic or pump withdrawal techniques, however, transit-time determinations of blood volume were 10% below that indicated by graduated cylinder collection. With transit time represented on the y-axis, three linear regressions of all paired blood flow measurements were calculated yielding the following slopes (delta y/delta x) and regression coefficients (r), respectively: electromagnetic (1.00, 0.98), graduated cylinder (0.85, 0.93), and pump withdrawal (0.93, 1.00). The results validate the transit-time ultrasound system used in the present investigation as an accurate method capable of measuring blood flow in both acutely and chronically instrumented animal preparations.

Reduction of myocardial infarct size in rabbits by ramiprilat: reversal by the bradykinin antagonist HOE 140.

We wished to determine, using a novel specific antagonist of BK2, HOE 140, (a) if the angiotensin-converting enzyme (ACE) inhibitor, ramiprilat, reduces myocardial infarct size in a well-established animal model of ischemia/reperfusion with minimal coronary collateralization, and (b) if the reduction in myocardial infarct size occurred through a bradykinin-dependent mechanism Saline vehicle, ramiprilat, HOE 140, or ramiprilat plus HOE 140 (n = 6 each group), was administered intravenously (i.v.) in intact animal preparations of experimentally induced acute myocardial ischemia. Anesthetized, open-chest rabbits were instrumented for measurement of systemic hemodynamics and left ventricular pressure (LVP), from which LV + dP/dtmax was derived. Animals were subjected to 30-min left main coronary artery occlusion (marginal branch) followed by 2-h reperfusion. Ramiprilat (50 micrograms/kg) or saline was administered before reperfusion, and rabbits receiving HOE 140 were pretreated before occlusion (1 microgram/kg). In separate duration of action experiments (n = 6 each group), the above doses of ramiprilat or HOE 140 had significant vascular antagonism of sufficient duration against serial challenge with angiotensin I (AI) or bradykinin, respectively. After reperfusion, myocardial infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of area at risk (AR). IS/AR% was significantly reduced in rabbits that received ramiprilat (20 +/- 6%, p < 0.05) as compared with those that received saline (41 +/- 6%), ramiprilat plus HOE 140 (47 +/- 2%), or HOE 140 alone (43 +/- 4%, mean +/- SEM). AR as a percentage of total LV mass was not different between any of the four treatment groups. Tachycardia was observed during early reperfusion in each group treated with ramiprilat.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of myocardial infarct size by ramiprilat is independent of angiotensin II synthesis inhibition.

The angiotensin-converting enzyme inhibitor ramiprilat, the angiotensin II receptor antagonist losartan, angiotensin II, ramiprilat plus angiotensin II, or saline (N = 6 each group), were administered i.v. in anesthetized, open-chest rabbit preparations of acute myocardial ischemia. Animals were instrumented for measurement of systemic hemodynamics and left ventricular +dP/dtmax, then subjected to 30 min of left anterior descending coronary artery occlusion (marginal branch) followed by 2 h of reperfusion. Ramiprilat (50 micrograms/kg), losartan (10 mg/kg), or saline were administered prior to reperfusion, and angiotensin II (2.5 ng/kg per min) was infused 15 min prior to occlusion and throughout the remainder of the experiment. Losartan was supplemented (10 mg/kg) after 1 h of reperfusion. These non-hypotensive doses of ramiprilat and losartan were demonstrated to significantly antagonize the systemic pressor effects of i.v. challenge with angiotensin I (15% of control, maximum) and II (5% of control, maximum), respectively, for the duration of the experiment. Systemic hemodynamic and +dP/dtmax changes due to occlusion/reperfusion or drug administration were similar between treatment groups. Infarct size was measured post-experimentally using tetrazolium staining and is reported as a percent of area at risk. Infarct size/area at risk (%) was significantly lower in rabbits administered ramiprilat only (20 +/- 6%*) or ramiprilat plus angiotensin II (26 +/- 5%*), compared to those receiving saline (41 +/- 6%), angiotensin II (51 +/- 4%), or losartan (52 +/- 4%, mean +/- S.E.M., * P < 0.05). These data indicate that direct angiotensin II receptor stimulation or receptor antagonism does not alter the degree of myocardial necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)