Effects of different types of fluid shear stress on endothelial cell proliferation and survival

We attempted to clarify the effect of different types of shear stress on endothelial cell (EC) proliferation and survival. Bovine aortic ECs were subjected to either steady laminar, 1 Hz pulsatile, or 1 Hz to and fro shear at 14 dyne/cm(2). % of BrdU positive EC was 14.3 +/- 1.6% in steady, 21.5 +/- 3.2% in pulsatile, and 11.4 +/- 2.4% in to and fro after 4 h, respectively (P < 0.05). Pulsatile shear compared with static control. Rapamycin reduced BrdU incorporation in all shear regimens (P < 0.001). However, it was still higher in EC exposed to pulsatile shear than the other regimens (P < 0.005). PD98059 completely abolished the increased BrdU incorporation in all shear regimens, including pulsatile shear. Pulsatile shear had significantly elevated ERK1/2 phosphorylation at 5 min compared with steady (P < 0.05) and to and fro shear (P < 0.01) while there was no significant difference in pp70(S6k) phosphorylation between any shear regimen. The ratio of apoptotic cells in serum deprived EC in the presence of steady laminar, pulsatile and to and fro shear for 4 h were 2.7 +/- 0.78%, 2.7 +/- 0.42%, and 2.9 +/- 0.62%, respectively while after the addition of serum for 4 h, it was 4.3 +/- 0.73%. All shear regimens phosphorylated AKT in a time-dependent manner with no significant difference between regimens. Our results demonstrate that different types of shear stress regimens have different effects on EC and may account for the variable response of EC to hemodynamics in the circulation.     

Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction

We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y(2) puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol, and glycosylphosphatidylinositol-anchored proteins (GPI-anchored proteins), to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl-β-cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3-E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of GPI-anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol-specific phospholipase D in MC3T3-E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3-E1 osteoblastic cells. Our findings suggest that cholesterol and GPI-anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK)

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24–72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites for AGEs, with both higher- and lower-affinity sites now being apparent. Medium-term (1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage.     

Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.     

Sphingolipids stimulate cell growth via MAP kinase activation in osteoblastic cells

Ceramide, ceramide-1-phosphate (C1P) sphingosine (SPH) and sphingosine-1-phosphate (S1P) effects on proliferation and extracellular-signal regulated kinases, ERKs (also known as MAPKs), activation were investigated in human and rat osteoblastic cells. MAPK activation was sphingolipid-specific in cells from both species. In human osteoblastic cells, S1P and C1P markedly stimulated ERK2 phosphorylation with a slight increase in phosphorylation of ERK1. SPH nor ceramide induced phosphorylation of either ERK isoform. In rat osteoblastic cells, SIP, ceramide and SPH stimulated phosphorylation of both isoforms. C1P did not induce phosphorylation of ERK1 but produced a mild increase in phosphorylation of ERK2. In human cells, only S1P significantly (P<0.05) increased osteoblastic cell proliferation, while in the rat cells all four sphingolipids significantly (P<0.05) induced proliferation. The calcium channel blocker verapamil blocked (P<0.05) these effects in both cell types. The MAPK inhibitor, PD98059, inhibited (P<0.05) the mitogenic effect of SIP in human cells. In rat cells, PD98059 effects were less substantial but significant for S1P and C1P. This study demonstrates that sphingolipids are mitogens for both human and rat osteoblastic cells with the MAPK pathway and calcium mediating in part these effects in a species specific manner.     

Advanced glycation endproducts interefere with integrin-mediated osteoblastic attachment to a type-I collagen matrix

The adhesion of osteoblasts to bone extracellular matrix, of which type-I collagen constitutes >85%, can modulate diverse aspects of their physiology such as growth, differentiation and mineralisation. In this study we examined the adhesion of UMR106 rat osteoblast-like cells either to a control (Col) or advanced-glycation-endproduct-modified (AGEs-Col) type I collagen matrix. We investigated the possible role of different integrin receptors in osteoblastic adhesion, by co-incubating these cells either with beta-peptide (conserved sequence 113-125 of the beta subunit of integrins) or with two other peptides, RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala), which are recognition sequences for the alpha-subunits of alpha(1,5)beta(1) and alpha(2)beta(1) integrins. Collagen glycation inhibited the adhesion of UMR106 osteoblasts to the matrix (40% reduction versus Col, P > 0.001). beta-Peptide showed a dose- and glycation-dependent inhibitory effect on adhesion, and at a concentration of 100 microM decreased the attachment of UMR106 cells to both matrices (42% to Col, P<0.001and 25% to AGEs-Col, P<0.01). The synthetic peptides RGD (1mM) and DGEA (5mM) inhibited the attachment of UMR106 cells to Col (30 and 20%, P > 0.01 and P< 0.001, respectively), but not to AGEs-Col. beta-Peptide induced an increase in UMR106 cell clumping and a decrease in cellular spreading, while DGEA increased spreading with cellular extensions in multiple directions. These results indicate that both alpha and beta integrin subunits participate in osteoblastic attachment to type-I collagen, probably through the alpha(1,5)beta(1) and alpha(2)beta(1) integrins. AGEs-modification of type-I collagen impairs the integrin-mediated adhesion of osteoblastic cells to the matrix, and could thus contribute to the pathogenesis of diabetic osteopenia.     

Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.     

Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin43

The effect of a temporal gradient in shear and steady shear on the activity of extracellular signal-regulated protein kinases 1 and 2 (ERK1/ERK2), c-fos, and connexin43 (Cx43) in human endothelial cells was investigated. Three laminar flow profiles (16 dyn/cm(2)), including impulse flow (shear stress abruptly applied for 3 s), ramp flow (shear stress smoothly transitioned at flow onset), and step flow (shear stress abruptly applied at flow onset) were utilized. Relative to static controls, impulse flow stimulated the phosphorylation of ERK1/ERK2 8.5- to 7.5-fold, respectively at 10 min, as well as the mRNA expression of c-fos 51-fold at 30 min, and Cx43 8-fold at 90 min. These high levels of mRNA expression were sustained for at least 4 h. In contrast, ramp flow was unable to significantly induce gene expression and even inhibited the activation of ERK1/ERK2. Step flow, which contains both a sharp temporal gradient in shear stress and a steady shear component, elicited only moderate and transient responses, indicating the distinct role of these fluid shear stimuli in endothelial signal transduction. The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 inhibited impulse flow-induced c-fos and Cx43 mRNA expression. Thus these findings implicate the involvement of ERK1/ERK2, c-fos, and Cx43 in the signaling pathway induced by the temporal gradient in shear.     

Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts

Osteopontin (OP) is a highly phosphorylated bone matrix protein and contains the RGD cell-binding motif, which mediates cell adhesion through integrin receptors that include α_vβ₃. Casein kinase 2 (CK2) is a factor-independent serine/threonine kinase, which may be the predominant physiologically relevant kinase for OP phosphorylation. This study was designed to examine the effects of unphosphorylated recombinant rat OP, and CK2-phosphorylated OP (P-OP), on the adhesion and function of mouse osteoclasts (OC) and osteoblast-like cells (UMR 201-10B and UMR 106-06) in vitro. OP significantly increased OC adhesion compared to plastic alone, and cell attachment was further increased at least twofold on OP phosphorylated with CK2. Attachment was dependent on the integrity of the RGD domain and was completely abolished in the presence of 1 mM RGD peptide. Neither CK2 phosphorylation of mutant OP, in which the RGD was converted to RGE or RAD, nor protein kinase C (PKC) phosphorylation of wild-type OP enhanced OC attachment. An antibody to the β₃ integrin subunit, but not anti-mouse CD44 antibody, specifically blocked the proportion of attachment due to phosphorylation of OP. Actin ring formation in OC was increased by plating cells onto OP, with no further increase by phosphorylation. Both OP and CK2-phosphorylated OP enhanced attachment of the two osteoblastic cell lines, compared to plastic, but in contrast to OCs, there was no significant difference with phosphorylation. Osteoblast attachment was totally blocked by 1 mM RGD peptide, but was not influenced by the β₃ integrin antibody. Plating of UMR 201-10B cells onto OP further increased retinoic acid-induced alkaline phosphatase expression. The results suggest that specific phosphorylation of OP is important for interaction with OCs, compared with osteoblastic cells, and that alternative integrins may be important in the interaction between osteoblastic cells and OP compared with OCs. J. Cell. Physiol. 176:179–187, 1998. © 1998 Wiley-Liss, Inc.     

RFP_（134）对UMR106细胞EGF受体酪氨酸蛋白激酶的调节作用

以大鼠成骨肉瘤细胞（ＵＭＲ１０６）为模型,研究了表皮生长因子（ＥＧＦ）对其受体酪氨酸蛋白激酶（ＴＰＫ）的调节作用。以本实验室从植物中提取纯化的二萜类活性物质（ＲＦＰ１３４）为诱导分化剂,观察了ＲＦＰ１３４对ＵＭＲ１０６细胞ＥＧＦ受体ＴＰＫ的活性和磷酸化作用的影响,并与ＲＡ和ＲＦＰ１３４＋ＲＡ处理细胞做了比较,结果显示ＥＧＦ与其受体结合后能激活ＴＰＫ,使ＴＰＫ活性增加２倍．ＲＦＰ１３４,ＲＡ,ＲＦＰ１３４＋ＲＡ处理细胞后,分别降低ＥＧＦ诱导的受体ＴＰＫ活性５０％,４３％,５５％,降低磷酸化ＴＰＫ含量５５％,３６％,５３％。从结果中发现无ＥＧＦ刺激的细胞也具有受体ＴＰＫ磷酸化作用,用ＲＦＰ１３４,ＲＡ,ＲＦＰ１３４＋ＲＡ处理细胞,分别降低受体磷酸化ＴＰＫ含量５９％,４０％,５７％,而且我们发现用ＥＧＦ诱导的细胞受体ＴＰＫ含量高于无ＥＧＦ作用的细胞．提示ＵＭＲ１０６细胞本身可能具有受体ＴＰＫ活性,能够引起细胞受体自动磷酸化,ＥＧＦ刺激后ＴＰＫ的磷酸化作用增强,可见ＲＦＰ１３４对ＥＧＦ诱导的ＴＰＫ磷酸化和无ＥＧＦ诱导的受体自动磷酸化都具有明显的抑制作用,（并强于ＲＡ）这可能与在第二信使水平上阻抑ＰＴＰＫ活性密切相关     

AGE-R3/galectin-3 expression in osteoblast-like cells: Regulation by AGEs

The accumulation of irreversible advanced glycation endproducts (AGEs) on long-lived proteins, and the interaction of AGEs with cellular receptors such as AGE-R3/galectin-3 and RAGE, are considered to be key events in the development of long-term complications of diabetes mellitus, Alzheimer's disease, uremia and ageing. The aim of this study was to investigate the expression and sub-cellular distribution of galectin-3, as well as its possible modulation by AGEs, in MC3T3E1 mouse calvaria-derived osteoblasts and in UMR 106 rat osteosarcoma cells. Both osteoblastic lines were cultured either with control bovine serum albumin (BSA) or with AGEs-BSA for 48 h. Cells were evaluated for galectin-3 expression by fixing and immunofluorescent microscopic analysis; or Western blot analysis of whole cell extracts, sub-cellular fractions and culture media. Both cell lines express 30 kDa (monomeric) galectin-3, although expression was about 15-fold lower in the UMR106 osteosarcoma cells. Dimeric (70 kDa) galectin-3 was additionally observed in the UMR106 cells. Immunofluorescent analysis of galectin-3 distribution showed a diffuse cytoplasmic and strong nuclear pattern in MC3T3E1 osteoblasts, and a patchy cytoplasmic pattern in UMR106 cells. Western blot analysis for both cell lines showed that galectin-3 was mainly found in the cytoplasm and in minor amounts in the microsomal fraction, while considerable amounts were secreted into the culture media. Exposure to 100-200 microg/mL AGEs-BSA increased the cellular content of 30 kDa galectin-3 (20-25% for MC3T3E1 and 35-70% for UMR106 versus control BSA, p < 0.05), and decreased the culture media levels of galectin-3 (10-20% for MC3T3E1 and for UMR106 versus control BSA, p < 0.05). These results confirm the expression of galectin-3 in osteoblastic cells, and suggest different levels and sub-cellular distribution of this protein in transformed versus non-transformed osteoblasts. Osteoblastic exposure to AGEs alters their expression and secretion of galectin-3, which could have significant consequences on osteoblast metabolism and thus on bone turnover.     

Local hemodynamics affect monocytic cell adhesion to a three-dimensional flow model coated with E-selectin.

Monocyte adhesion to the endothelium depends on concentrations of receptors/ligands, local concentrations of chemoattractants, monocyte transport to the endothelial surface and hemodynamic forces. Monocyte adhesion to the inert surface of a three-dimensional perfusion model was shown to correlate inversely with wall shear stress, but was also affected by flow patterns which influenced the near-wall cell availability. We hypothesized that (a) under the same flow conditions, insolubilized E-selectin on the model's surface may mediate adhesive interactions at higher wall shear stresses, compared to an uncoated model, and (b) pulsatile flow may modify the adhesion profile obtained under steady flow. An axisymmetric flow model with a stenosis and a sudden expansion produced a range of wall shear stresses and a separated flow with recirculation and reattachment. Pre-activated U937 cells were perfused through the model under either steady (Re = 100, 140) or pulsatile (Remean = 107) flow. The velocity field was characterized through computational fluid dynamics and validated by inert particle tracking. Surface E-selectin greatly increased cell adhesion in all regions at Re = 100 and 140, compared to an uncoated model under the same flow conditions. In regions where the cells near the wall were abundant (taper and stenosis), adhesion to E-selectin correlated with the reciprocal of local wall shear stress when flow was steady. Pulsatile flow distributed the adherent cells more evenly throughout the coated model. Hence, characterizing both the local hemodynamics and the biological activity on the vessel wall is important in leukocyte adhesion.     

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: Attenuation of MAP kinase activation by cAMP,parathyroid hormone and forskolin

The mitogen-activated protein (MAP) kinases (p44^mapk and p42^mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44^mapk/ERK1 and p42^mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.     

Temporal gradients in shear, but not spatial gradients, stimulate ERK1/2 activation in human endothelial cells

We have previously demonstrated temporal gradients in shear stress stimulate endothelial cell proliferation, whereas spatial gradients do not. In the present study, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway was investigated as a possible mediator for the promitogenic effect of temporal gradients. The sudden expansion flow chamber (SEFC) model was used to differentiate the effect of temporal gradients in shear from that of spatial gradients on ERK1/2 activation in human umbilical vein endothelial cells (HUVEC). ERK1/2 activation in the SEFC was not significantly different from control when HUVEC were exposed to spatial gradients alone. When a single temporal impulse was superimposed on spatial gradients, ERK1/2 activation was stimulated 330% (relative to spatial alone) within the region of spatial gradients. Inhibition of the ERK1/2 pathway with U-0126 abolished all effects of temporal gradients. To further separate temporal and spatial gradients, a conventional parallel plate flow chamber was utilized. Acute exposure to oscillations in flow at a frequency of 1 Hz stimulated ERK1/2 activation 620 +/- 88% relative to control, whereas a single impulse of flow increased ERK1/2 activation 166 +/- 19%. Flow without the temporal component did not significantly activate ERK1/2. These results suggest that the ERK1/2 pathway directly mediates the promitogenic effects of temporal gradients in shear stress.     

Spectroscopic Characterization of an Oxovanadium(IV) Complex of Oxodiacetic Acid and o-Phenanthroline. Bioactivity on Osteoblast-Like Cells in Culture

The oxovanadium(IV) complex of oxodiacetic acid (H(2)ODA) and o-phenanthroline of stoichiometry [VO(ODA)(ophen)]·1.5H(2)O, which presents the interesting tridentate OOO coordination, was thoroughly characterized by infrared, Raman, and electronic spectroscopies. The biological activity of the complex on the cell proliferation was tested on osteoblast-like cells (MC3T3E1 osteoblastic mouse calvaria-derived cells and UMR106 rat osteosarcoma-derived cells) in culture. The complex caused inhibition of cellular proliferation in both osteoblast cell lines in culture, but the cytotoxicity was stronger in the normal (MC3T3E1) than in the tumoral (UMR106) osteoblasts.     

E-cadherins identified in osteoblastic cells: Effects of parathyroid hormone and extracellular calcium on localization

The presence and regulation of cadherin localization in osteoblastic cells were examined. Monoclonal antibody (ECCD-1) that interferes with E-cadherin function prevented cell adhesion in UMR 106-H5 rat osteosarcoma cells and non-tumorigenic mouse calvarial MC3T3-E1 cells, whereas CCL39 fibroblast adhesion was not affected. Immunofluorescent antibodies (ECCD-2 and polyclonal L-CAM P1) revealed cadherins are localized along the osteoblastic cell-cell boundaries. Exposure of UMR 106-H5 cells to bovine parathyroid hormone (1–84) (PTH; 10 ng/ml × 1 hr) or low calcium medium (1.0 – 0.025 Mn) produced cellular retraction accompanied by intense immunofluorescence for cadherins throughout cells with a corresponding loss of punctate localization at remaining cell-cell adhesion points. Western immunoblot analysis indicated 108 kd and 115 kd cadherins are present, with a smaller 29.5 kd band that became predominantly associated with the cytosolic fraction of cells treated with parathyroid hormone or lowered calcium. The results demonstrate E-like cadherins are present in osteoblastic cells and implicate a regulatory role for parathyroid hormone and calcium in cadherin function and localization.