Beta-catenin is not activated by downregulation of PTEN in osteoblasts

Selective knockdown of phosphatase and tensin homolog (PTEN) has been recently shown to increase life long accumulation of bone and its ability to increase osteoblast lifespan. In order to determine how loss of PTEN function affects osteoblast differentiation, we created cell lines with stable knockdown of PTEN expression using short hairpin RNA vectors and characterized several clones. The effect of deregulated PTEN in osteoblasts was studied in relationship to cell proliferation and differentiation. Downregulation of PTEN initially affected the cell’s attachment and spreading on plastic but cells recovered after a brief period of time. When cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we noticed a small but significant increase in growth rates with PTEN reduction. The size of individual cells appeared larger when compared to control cells. Differentiation properties of these osteoblasts were increased as evidenced by higher expression of several of the bone markers tested (alkaline phosphatase, osteocalcin, osterix, bone morphogenetic protein 2, Cbfa1, osteoprotegerin, and receptor activator of NF-kappaB ligand) and their mineralization capacity in culture. As stabilization of beta-catenin is known to be responsible for growth deregulation with PTEN loss in other cell types, we investigated the activation of the canonical Wnt pathway in our cell lines. Immunofluorescence staining, protein expression in subcellular fractions for beta-catenin, and assays for activation of the canonical Wnt/beta-catenin signaling were studied in the PTEN downregulated cells. There was an overall decrease in β-catenin expression in cells with PTEN knockdown. The distribution of β-catenin was more diffuse within the cell in the PTEN-reduced clones when compared to controls where they were mostly present in cell borders. Signaling through the canonical pathway was also reduced in the PTEN knockdown cells when compared to control. The results of this study suggest that while decreased PTEN augments cell proliferation and positively affects differentiation, there is a decrease in β-catenin levels and activity in osteoblasts. Therefore, at least in osteoblasts, β-catenin is not responsible for mediating the activation of osteoblast differentiation with reduction in PTEN function.     

Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system

Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo 3-AM. In all developmental stages mice, [Ca(2+)](i)-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca(2+)](i)-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca(2+) channel blocker, gadolinium. The increases of [Ca(2+)](i) in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons.     

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins.     

Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0–10.0 g/cm²) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca²⁺ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca²⁺ pathway.     

Activation of mechanical responses in leukocytes

Different kinds of leukocytes undergo cytoskeleton-dependent mechanical responses associated with their specific physiological functions. We have investigated cellular stiffening of several types of leukocytes using a method which measures the force resisting cellular indentation. We have found that lymphocytes stiffen in response to crosslinking cell surface antigens in a process associated with the much studied capping and patching processes. Further studies of myosin-deficient mutants of the ameba Dictyostelium discoideum suggest that this stiffening process results from a myosin dependent contractile process. Rat basophilic leukemia cells and pancreatic islet cells stiffen when triggered to secrete. The function of these cytoskeleton dependent processes is now unknown, but, at least in the islet cells, may be related to a regulation of the rate of secretion. Primary neutrophils stiffen in response to the chemotactic agent, fMet-Leu-Phe. This stiffening may be responsible for retention of these cells in the pulmonary microcirculation during response to inflammation. These observations pose the challenge of determining the structural basis, mechanism, and physiological function of each of these cellular responses.     

Expression and subcellular localization of mechano-growth factor in osteoblasts under mechanical stretch

Mechano-growth factor (MGF) is a stretch sensitive factor in myocytes, and it might also be produced by other mechanocytes under mechanical stimulation. In this study, both the mRNA and protein expression of MGF were detected in stretched osteoblasts. Quantitative analysis showed that a cyclic stretching stimulation caused a quick and sharp increase of MGF mRNA and protein expression from a low basal level under no stretch; the mRNA and protein levels respectively peaked in 6 and 12 h to 5 and 5.2 fold over the basal level and returned to normal by 24 h. The subcellular distribution of MGF protein was revealed by immunofluorescence analysis to be restricted to the nucleus. We concluded that cyclic stretching stimulation could induce MGF expression in osteoblasts in a pulsing fashion; and the nuclear distribution of MGF suggested that MGF might act in mechanocytes as an autocrine growth factor.     

Studies on the receptors mediating responses of osteoblasts to thrombin

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.     

Phthalate esters influence FGF-2 translocation in Py1a rat osteoblasts

Exposure of the Py1a rat osteoblastic cells to butyl benzyl phthalate (BBP) and dibutyl phthalate (DBP) showed that these endocrine disrupting chemicals (EDC) strongly and reversibly affect the cytoplasmic fibroblast growth factor-2 (FGF-2) translocation into the nucleus in a dose-dependent and time-related manner. Stimulation of cells with high concentrations of BBP or DBP for short timing gave results comparable to those of cells treated with low concentrations for long timing. By confocal laser scanning microscope (CLSM) analysis it was found that the first relevant effect resulted in an accumulation of FGF-2 near the nuclear envelope, sometimes in the shape of clusters; the growth factor was then translocated into the nucleus and, finally, after long periods of exposure, the basal nuclear and cytoplasmic binding, typical of unstimulated cells, was re-established. In addition it was found that phthalate esters did not affect the FGF receptor 2 (FGFR-2) but decreased Con A binding indicating a possible inhibition of collagen fiber assembly. The different concentrations and timing of exposure of BBP and DBP affected the FGF-2 modulation in a similar way. Noticeable cumulative effects of BBP and DBP were not observed.     

Differential responses of the floating-leaved aquatic plant Nymphoides peltata to gradual versus rapid increases in water levels

We compared the growth responses of the floating-leaved species Nymphoides peltata to gradual and rapid rising water levels under two nutrient concentrations (1 g and 12 g of slow released fertilizer (N-P-K: 16-8-12) per container filled with 8 kg washed sand), and predicted the population expansion after these floods. The results showed that the capacity for petiole elongation was dependent on leaf age, and only leaves that were no more than five days old had the capability to reach the water surface when the water level increased rapidly from 50 cm to 300 cm. Plants subjected to a gradual rising water level tracked the increase in water depth whose petioles elongated at 3.96 ± 1.70 cm per day and 4.80 ± 0.16 cm per day under low and high nutrient concentrations respectively throughout the experiment period. When water levels were rapidly raised, leaf petioles elongated rapidly at 25.48 ± 1.51 cm per day and 26.64 ± 2.24 cm per day under low and high nutrient concentrations respectively during the first ten days. Plants under a constant water level maintained highest mean leaf recruitment (mean 3.0 ± 0.33 leaves and 24.4 ± 5.87 leaves every ten days under low and high nutrient concentrations, respectively). Therefore, more young leaves existed in the canopy ensuring that when the water level increases, young leaves can rapidly emerge after submergence. Gradual water level rise did not significantly affect biomass and ramet production (4.75 ± 1.41 g and 5.50 ± 1.22 ramets in low nutrient; 48.49 ± 21.45 g and 35.67 ± 11.78 ramets in high nutrient), but rapid water level rise negatively affected ramet production in both nutrient concentrations (3.00 ± 1.26 ramets and 11.25 ± 4.19 ramets in low and high nutrients, respectively). The results indicated that continual leaf recruitment and rapid petiole elongation were both important ways in which N. peltata adapted to increasing water levels. Extreme flooding may be a disturbance factor that affects plant growth and the population expansion of N. peltata, while small gradual water level rise should not harm this species.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E¹) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E¹. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E¹ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was demonstrated in this study, with positive links to cytoskeletal changes. The estradiol associated changes in osteoblast mechanical properties could bear implications for bone remodeling and its mechanical integrity.