Wnt signaling acts and is regulated in a human osteoblast differentiation dependent manner.

The Wnt signaling pathway is an important regulator of cellular differentiation in a variety of cell types including osteoblasts. In this study, we investigated the impact of Wnt signaling on the function of human osteoblasts in relation to the stage of differentiation. Differentiating osteoblasts were created upon glucocorticoid (GC) treatment, whereas nondifferentiating osteoblasts were created by excluding GCs from the culture medium. GC-induced differentiation suppressed endogenous beta-catenin levels and transcriptional activity. During GC-induced osteoblast differentiation, activation of Wnt signaling slightly decreased alkaline phosphatase activity, but strongly suppressed matrix mineralization. In addition, mRNA expression of several Wnt signaling related genes was strongly regulated during GC-induced osteoblast differentiation, including frizzled homolog 8, dickkopf homolog 1, and secreted frizzled-related protein 1. In contrast, in the absence of GC-induced differentiation, Wnt signaling acted positively by stimulating basal alkaline phosphatase activity. Interestingly, pre-stimulation of Wnt signaling in early osteoblasts enhanced their differentiation capacity later on during the GC-induced differentiation process. In conclusion, we showed a differentiation-dependent effect of Wnt signaling on osteoblasts. Wnt signaling stimulated early osteoblasts in their capacity to differentiate, whereas mature osteoblasts were strongly inhibited in their capacity to induce mineralization. Moreover, osteoblast differentiation suppressed endogenous Wnt signaling and changed the expression of multiple Wnt signaling related genes.

Long-term fluoxetine administration does not result in major changes in bone architecture and strength in growing rats.

Many studies have indicated that serotonin and its transporter play a role in bone metabolism. In this study we investigated the effect of selective serotonin re-uptake inhibitor (SSRI), fluoxetine (Prozac) on bone architecture and quality in growing female rats. We therefore administrated rats with clinically relevant doses of fluoxetine for a period of 6 months. DXA scans were performed during the treatment period in order to follow parameters as body weight, fat percentage and BMD. After 6 months of treatment, femurs were used to analyze bone architecture and bone strength, by means of microCT scans and three-point bending assays, respectively. We found a slightly diminished bone quality, reflected in a lower bone tissue strength, which was compensated by changes in bone geometry. As leptin and adiponectin could be possible factors in the serotonergic regulation of bone metabolism, we also determined the levels of these factors in plasma samples of all animals. Leptin and adiponectin levels were not different between the control group and fluoxetine-treated group, indicating that these factors were not involved in the observed changes in bone geometry and quality.

Serotonin and fluoxetine modulate bone cell function in vitro.

Recent studies have proposed a role for serotonin and its transporter in regulation of bone cell function. In the present study, we examined the in vitro effects of serotonin and the serotonin transporter inhibitor fluoxetine "Prozac" on osteoblasts and osteoclasts. Human mononuclear cells were differentiated into osteoclasts in the presence of serotonin or fluoxetine. Both compounds affected the total number of differentiated osteoclasts as well as bone resorption in a bell-shaped manner. RT-PCR on the human osteoclasts demonstrated several serotonin receptors, the serotonin transporter, and the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 1 (Tph1). Tph1 expression was also found in murine osteoblasts and osteoclasts, indicating an ability to produce serotonin. In murine pre-osteoclasts (RAW264.7), serotonin as well as fluoxetine affected proliferation and NFkappaB activity in a biphasic manner. Proliferation of human mesenchymal stem cells (MSC) and primary osteoblasts (NHO), and 5-HT2A receptor expression was enhanced by serotonin. Fluoxetine stimulated proliferation of MSC and murine preosteoblasts (MC3T3-E1) in nM concentrations, microM concentrations were inhibitory. The effect of fluoxetine seemed direct, probably through 5-HT2 receptors. Serotonin-induced proliferation of MC3T3-E1 cells was inhibited by the PKC inhibitor (GF109203) and was also markedly reduced when antagonists of the serotonin receptors 5-HT2B/C or 5-HT2A/C were added. Serotonin increased osteoprotegerin (OPG) and decreased receptor activator of NF-kappaB ligand (RANKL) secretion from osteoblasts, suggesting a role in osteoblast-induced inhibition of osteoclast differentiation, whereas fluoxetine had the opposite effect. This study further describes possible mechanisms by which serotonin and the serotonin transporter can affect bone cell function.

Stretch-induced phosphorylation of ERK1/2 depends on differentiation stage of osteoblasts.

The goal of this study was to investigate the effect of mechanical loading on osteoblasts and extracellular signal-regulated kinase (ERK1/2) signaling in relation to osteoblast differentiation and mineralization. A human osteoblast cell line (SV-HFO) was triggered to differentiate to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and beta-glycerophosphate. Osteoblasts were subjected to cyclic, equibiaxial stretch for 5, 15, or 60 min at different stages of differentiation (days 7, 14, and 21). Baseline (static) phosphorylated ERK1/2 and total ERK1/2 levels gradually increased during osteoblast differentiation. Cyclic stretch induced a rapid increase in ERK1/2 phosphorylation with a maximum between 5 and 15 min. Prolonged stretching for 60 min resulted in a decrease of phosphorylated ERK1/2 towards baseline level, suggesting a desensitization mechanism. The effect of stretch on ERK1/2 phosphorylation was strongest at later stages of differentiation (days 14 and 21). At day 21, the increase of ERK1/2 phosphorylation in response to stretch was significantly lower in non-differentiating than in differentiating osteoblasts. This could not be explained by differences in cell density, but did correlate with the formation of extracellular matrix, collagen fibrils. Mineralization of the extracellular matrix did not lead to a further increase of ERK1/2 phosphorylation. In conclusion, the current study demonstrates that the extent of activation of the ERK1/2 pathway is dependent on the differentiation or functional stage of the osteoblast. The presence of an extracellular matrix, but not per se mineralization, seems to be the predominant determinant of osteoblastic response to strain.

Expression of serotonin receptors in bone.

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.

Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow.

Mechanical stress produces flow of fluid in the osteocytic lacunar-canalicular network, which is likely the physiological signal for the adaptive response of bone. We compared the induction of prostaglandin G/H synthase-2 (PGHS-2) by pulsating fluid flow (PFF) and serum in osteocytes, osteoblasts, and periosteal fibroblasts, isolated from 18-day-old fetal chicken calvariae. A serum-deprived mixed population of primarily osteocytes and osteoblasts responded to serum with a two- to threefold induction of PGHS-2 mRNA. Serum stimulated PGHS-2-derived PGE(2) release from osteoblasts and osteocytes but not from periosteal fibroblasts as NS-398, a PGHS-2 blocker, inhibited PGE(2) release from osteocytes and osteoblasts with 65%, but not that from periosteal fibroblasts. On the other hand PFF (0.7 Pa, 5 Hz) stimulated (3 fold) PGHS-2 mRNA only in OCY. The related PGE(2) response could be completely inhibited by NS-398. We conclude that osteocytes have a higher intrinsic sensitivity for loading-derived fluid flow than osteoblasts or periosteal fibroblasts.