Fetal Rat Calvaria Cells Research Articles

Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal Rat Calvaria Cells in an Oxidative Stress Environment.

Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up-regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide-induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for 4 days (D5-9) with 300 μM hydrogen peroxide resulted in fewer alkaline phosphatase-positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (P < 0.05). This suppression was partially blocked when cells were pretreated 12 h with 20 μM QRC. Hydrogen peroxide also produced sustained up-regulation of heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC), which was partially blocked in hydrogen peroxide-treated cells that first received QRC pretreatment. The alterations in the antioxidant stress response coincided with alterations in phosphorylated ERK1/2, but not Nrf2. These results suggest that QRC suppresses hydrogen peroxide-induced activation of the antioxidant response, and partially preserves osteoblast phenotypic development. J. Cell. Physiol. 231: 2779-2788, 2016. © 2016 Wiley Periodicals, Inc.

Quercetin protects osteoblast development in the presence of oxidative stress in fetal rat calvaria cells

Studies examining quercetin (QRC) effects on bone health are limited. The purpose of this study was to examine the extent that QRC impacts osteoblast cell development in the presence of oxidative stress, using osteoblast‐like cells isolated from fetal rat calvaria. At confluence, cells were pre‐treated for 12 hours with QRC aglycone (0 or 20 μM). Cells were then incubated for 4 days with QRC aglycone continued at the same dose as pre‐treatment (0, 20 μM) with or without hydrogen peroxide (H2O2; 0, 300 μM), a known inducer of oxidative stress. There were four groups: untreated Control, QRC alone, H2O2 alone, and QRC/H2O2. Expression of genes related to osteoblast phenotype development (runt‐related transcription factor 2, Runx2; alkaline phosphatase, ALP; type I collagen, COLL; bone sialoprotein, BSP; and osteocalcin, OCN) were analyzed using real‐time quantitative PCR. Additionally, alkaline phosphatase staining was used as an indicator of cells with osteoblastic potential. Results for Controls and QRC treated cells were similar. H2O2 alone suppressed ALP, BSP, and OCN gene expression (p &lt; 0.05) and alkaline phosphatase staining. QRC, in the presence of H2O2, partially prevented the H2O2‐ induced suppression (p &lt; 0.05). These results suggest that QRC is partially protective of osteoblast development in the presence of oxidative stress.Grant Funding Source: USDA Agricultural Research Services

The EP4-ERK-dependent pathway stimulates osteo-adipogenic progenitor proliferation resulting in increased adipogenesis in fetal rat calvaria cell cultures

We previously reported that fetal rat calvaria (RC) cells are osteo-adipogenic bipotential and that PGE2 receptors EP2 and EP4 are involved in bone nodule formation via both common and distinct MAPK pathways in RC cell cultures. Because PGE2 participates in multiple biological processes including adipogenesis, it is of interest to determine the additional role(s) of PGE2 in RC cells. PGE2 increased the number of adipocyte colonies when RC cells were treated during proliferation but not other development stages. Of four EP agonists tested, the EP4 agonist ONO-AE1-437 (EP4A) was the most effective in promoting adipogenesis. Concomitantly, EP4A increased the number of cells with BrdU labeling and gene expression of CCAAT/enhancer binding protein (C/EBP)δ and c-fos but not peroxisome proliferator-activated receptor γ2 and C/EBPα. Amongst MAPK inhibitors, U0126, an inhibitor of MEK1/2, abrogated the EP4A-dependent effects. Our results suggest that the PGE2–EP4-ERK pathway increases the number of osteo-adipogenic bipotential progenitor cells, with a resultant increase in adipogenesis in RC cell cultures.

Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis

Periodontitis is characterized by periodontal tissue inflammation and alveolar bone loss. The intermittent administration of parathyroid hormone (PTH), a major regulator of bone remodeling, has been demonstrated to stimulate osteoblastic activity. Although the systemic administration of PTH has been reported to protect against periodontitis-associated bone loss, the effect of the topical administration of PTH is unclear. In this study, the effect of intermittent administration of PTH on osteoblastic differentiation was examined in cultured calvaria cells and then the effect of topical and intermittent administration of PTH was determined by measuring the recovery of alveolar bone loss after inducing experimental periodontitis in rats. Alkaline phosphatase activity and bone nodule formation were measured in fetal rat calvaria cells. Experimental periodontitis was induced by placing nylon ligature around rat maxillary molars for 20 d. After ligature removal (day 0), PTH was topically injected into buccal gingiva three times a week for 10 wk. Micro-computed tomography analysis and histological examination were performed on days 35 and 70. Intermittent exposure of PTH in calvaria cells increased alkaline phosphatase activity and bone nodule formation by 1.4- and 2.4-fold, respectively. Ligature procedures induced marked alveolar bone loss around the molars on day 0 and greater bone recovery was observed in the PTH-treated rats on day 70. An increase in osteoid formation on the surface of alveolar bone was detected in the PTH-treated rats. Intermittent treatment with PTH stimulated osteoblastic differentiation in fetal rat calvaria cell cultures, and topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis.

Protein kinase D mediates the synergistic effects of BMP-7 and IGF-I on osteoblastic cell differentiation

We previously showed that exogenous insulin-like growth factor-I (IGF-I) and bone morphogenetic protein-7 (BMP-7) synergistically stimulated osteoblast differentiation in fetal rat calvaria (FRC) cells. We have now shown that BMP-7 alone and the BMP-7 and IGF-I combination synergistically stimulated protein kinase D (PKD) phosphorylation at Ser744/748 and Ser916. Transfection of FRC cells with a constitutively active PKD stimulated marker expression, while transfection with a catalytically inactive PKD did not. Moreover, Gö6976, which inhibits protein kinase C (PKC) α and β1, blocked PKD phosphorylation and the synergistic action of the BMP-7 and IGF-I combination on osteoblast differentiation, whereas Gö6983, which inhibits PKCα, β, γ, δ, and ζ, did not. Our results suggest that the FRC cell differentiation induced by BMP-7 and the BMP-7 and IGF-I combination requires stimulation of PKD activity. Our results are consistent with a novel mechanism in which combined BMP-7 and IGF-I signaling activates upstream novel PKC(s), which then phosphorylates and activates PKD, leading to enhanced osteoblast differentiation.

Iron chelator deferoxamine alters iron-regulatory genes and proteins and suppresses osteoblast phenotype in fetal rat calvaria cells

There are few studies describing the extent to which low iron status affects osteoblastogenesis, despite evidence that iron deficiency produces adverse effects on bone density. The purpose of this study was to evaluate alterations in intracellular iron status by measuring iron-regulated gene and protein expression and to describe development of osteoblast phenotype in primary cells treated with iron chelator deferoxamine (DFOM) during differentiation. Using the well-described fetal rat calvaria model, cells were incubated with 0–8µM DFOM throughout differentiation (confluence to day (D) 21), or only during early differentiation (confluence to D13–15) or late differentiation (D13–15 to D21). Changes in intracellular iron status were determined by measuring alterations in gene and protein expression of transferrin receptor and ferritin light chain and heavy chain. Development of osteoblast phenotype was monitored by measuring expression of genes that are known to be up-regulated during differentiation, analyzing the percentage of mineralized surface area, and counting the number of multi-layered bone nodules at the end of culture. Results indicate that treatment throughout differentiation with 8µM DFOM alters iron-regulated genes and proteins by mid-differentiation (D13–15) in a pattern consistent with iron deficiency with concomitant down-regulation of osteoblast phenotype genes, especially osteocalcin. Additionally, alkaline phosphatase staining was lower and there was about 70% less mineralized surface area (p<0.05) by D21 in wells treated throughout differentiation with 8µM DFOM compared to control. Down-regulation of osteocalcin and alkaline phosphatase mRNA (p<0.05) and suppressed mineralization (p<0.05) was also evident at D21 in cells treated only during early differentiation. In contrast, treatment during late differentiation did not alter osteoblastic outcomes by D21. In conclusion, it appears that iron is required for normal osteoblast phenotype development, and that early rather than late differentiation events may be more sensitive to iron availability.

The PPARgamma-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations

BackgroundOsteoblasts and adipocytes are derived from a common mesenchymal progenitor and an inverse relationship between expression of the two lineages is seen with certain experimental manipulations and in certain diseases, i.e., osteoporosis, but the cellular pathway(s) and developmental stages underlying the inverse relationship is still under active investigation. To determine which precursor mesenchymal cell types can differentiate into adipocytes, we compared the effects of BRL-49653 (BRL), a selective ligand for peroxisome proliferators-activated receptor (PPAR)γ, a master transcription factor of adipogenesis, on osteo/adipogeneis in two different osteoblast culture models: the rat bone marrow (RBM) versus the fetal rat calvaria (RC) cell system.ResultsBRL increased the number of adipocytes and corresponding marker expression, such as lipoprotein lipase, fatty acid-binding protein (aP2), and adipsin, in both culture models, but affected osteoblastogenesis only in RBM cultures, where a reciprocal decrease in bone nodule formation and osteoblast markers, e.g., osteopontin, alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin was seen, and not in RC cell cultures. Even though adipocytes were histologically undetectable in RC cultures not treated with BRL, RC cells expressed PPAR and CCAAT/enhancer binding protein (C/EBP) mRNAs throughout osteoblast development and their expression was increased by BRL. Some single cell-derived BRL-treated osteogenic RC colonies were stained not only with ALP/von Kossa but also with oil red O and co-expressed the mature adipocyte marker adipsin and the mature osteoblast marker OCN, as well as PPAR and C/EBP mRNAs.ConclusionThe data show that there are clear differences in the capacity of BRL to alter the fate choices of precursor cells in stromal (RBM) versus calvarial (RC) cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte pool, multi-/bipotential osteo-adipoprogenitor pool and conversion of osteoprogenitor cells or osteoblasts into adipocytes (transdifferentiation or plasticity)). They also show that mechanisms beyond activation of PPARγ by its ligand are required for changing the fate of committed osteoprogenitor cells and/or osteoblasts into adipocytes.

Overexpression of Fibroblast Growth Factor 23 Suppresses Osteoblast Differentiation and Matrix Mineralization In Vitro

Fibroblast growth factor (FGF)23 is produced primarily in bone and acts on kidney as a systemic phosphaturic factor; high levels result in rickets and osteomalacia. However, it remains unclear whether FGF23 acts locally and directly on bone formation. We overexpressed human FGF23 in a stage-specific manner during osteoblast development in fetal rat calvaria (RC) cell cultures by using the adenoviral overexpression system and analyzed its effects on osteoprogenitor proliferation, osteoid nodule formation, and mineralization. Bone formation was also measured by calcein labeling in parietal bone organ cultures. Finally, we addressed the role of tyrosine phosphorylation of FGF receptor (FGFR) in mineralized nodule formation. Nodule formation and mineralization, but not osteoprogenitor proliferation, were independently suppressed by overexpression of FGF23 in RC cells. Increased FGF23 levels also suppressed bone formation in the parietal bone organ culture model. FGF23 overexpression enhanced phosphorylation of FGFR, whereas the impairment of mineralized nodule formation by FGF23 overexpression was abrogated by SU5402, an inhibitor of FGFR1 tyrosine kinase activity. These studies suggest that FGF23 overexpression suppresses not only osteoblast differentiation but also matrix mineralization independently of its systemic effects on Pi homeostasis.

LIF Inhibits Osteoblast Differentiation at Least in Part by Regulation of HAS2 and Its Product Hyaluronan

LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix. Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage. To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome. Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures slightly increased bone nodule formation. Treatment with hyaluronidase, on the other hand, stimulated bone nodule formation in early cultures but caused a small dose-dependent inhibition of osteoblast differentiation in the LIF- and HA-sensitive late time window. Together the data suggest that osteoblast differentiation is acutely sensitive to HA levels and that LIF inhibits osteoblast development at least in part by stimulating high molecular weight HA synthesis through HAS2.

Mineralized tissue cells are a principal source of FGF23

While fibroblast growth factor (FGF) 23 is known as a phosphaturic factor in inherited and/or acquired hypophosphatemic disorders, it also serves an endocrine role in normal phosphate homeostasis. FGF23 acts negatively on the NaPi2a cotransporter and 25-hydroxy D 3-1α-hydroxylase with a resultant decrease in renal phosphate (Pi) reabsorption, while osteoblasts appear to be a primary source of FGF23 whose expression is counter-upregulated by 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). Here we have shown the distribution of FGF23 in normal rat bone and tooth, and its expression profile in fetal rat calvaria (RC) cell cultures. FGF23 mRNA was detectable in multiple fetal and adult tissues but levels were much higher in adult calvaria, femur and incisor, compared to the other tissues tested. Immunoreactive FGF23 was predominantly localized to osteoblasts, cementoblasts, and odontoblasts, with sporadic labeling in some chondrocytes, osteocytes and cementocytes. Notably, osteoclasts were also found to be a possible source of FGF23. Fetal bone and tooth germ cells labeled much less intensely than young adult osteoblasts and odontoblasts. In the RC cell model, FGF23 was expressed during osteoblast development. During matrix mineralization induced by β-glycerophosphate (βGP), FGF23 expression was transiently upregulated and then decreased to levels lower than in their non-βGP-treated counterparts. 1,25(OH) 2D 3 markedly increased FGF23 expression concomitant with the inhibition of βGP-induced mineralization. Our data suggest that FGF23 expression in bone is closely correlated with bone formation in vitro and vivo, and points towards an important role(s) for FGF23 in young adult but not fetal mineralized tissues as a systemic factor for Pi homeostasis.

Leukemia Inhibitory Factor Influences the Fate Choice of Mesenchymal Progenitor Cells

Osteoblasts and adipocytes derive from a common mesenchymal precursor, and in at least some circumstances, differentiation along these two lineages is inversely related. For example, we have recently observed that concomitant with inhibition of osteoblast differentiation and bone nodule formation, leukemia inhibitory factor (LIF) induces genes regulating lipid metabolism in fetal rat calvaria (RC) cell cultures. In this study, we further investigated the adipogenic capacity of LIF-treated RC cells. Quantitative analyses revealed that LIF increased the adipocyte differentiation induced by the peroxisome proliferator-activated receptor gamma agonist BRL49653 (BRL) in RC cell populations. Gene expression profiling of individual RC cell colonies in untreated cells or cells treated with LIF, BRL, or combined LIF-BRL suggested that some adipocytes arose from bipotential or other primitive precursors, including osteoprogenitors, since many colonies co-expressed osteoblast and adipocyte differentiation markers, whereas some arose from other cell pools, most likely committed preadipocytes present in the population. These analyses further suggested that LIF and BRL do not act at the same stages of the mesenchymal hierarchy, but rather that LIF modifies differentiation of precursor cells, whereas BRL acts later to favor adipocyte differentiation. Taken together, our data suggest that LIF increased adipocyte differentiation at least in part by altering the fate of osteoblastic cells and their precursors.

Pleiotropic effects of the steroid hormone 1,25-dihydroxyvitamin D3 on the recruitment of mesenchymal lineage progenitors in fetal rat calvaria cell populations

The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) inhibits osteogenesis while stimulating adipogenesis in vitro. We hypothesized that 1,25(OH)(2)D(3) redirects the fate of osteoblast/adipocyte bipotential progenitors and other potential progenitors towards adipogenesis, a process possibly underlying the pathogenesis of osteopenic diseases such as osteoporosis. We therefore tested the global effects of 1,25(OH)(2)D(3) on the recruitment of mesenchymal progenitors including osteogenic, chondrogenic, adipogenic and myogenic lineages (colony forming cell (CFC)-osteoblast (CFC-O), CFC-chondrocyte (CFC-C), CFC-adipocyte (CFC-A), and CFC-myoblast (CFC-M) respectively) in rat calvaria (RC) cell populations using gene expression profiling of single cell-derived colonies. Based on expression of lineage specific transcripts, 86% of single cell-derived colonies in untreated cultures simultaneously co-expressed transcripts of two, three, or four of the mesenchymal lineages tested. The distribution of mesenchymal progenitors in 1,25(OH)(2)D(3)-treated cultures was significantly changed compared with the control group, i.e. CFC-O were reduced (from 6 to 0%) and CFC-O/A bipotential (0 to 8.2%), CFC-C (4 to 10.2%) and CFC-Fibroblast (CFC-F) (4 to 16%) were increased. 1,25(OH)(2)D(3) did not affect the frequency of tri- or tetra-lineage colonies. Single lineage CFC-A colonies were not detected in either the control or 1,25(OH)(2)D(3) treatment group under the conditions tested. Since the parietal bones used for cell isolation derive from neuroectoderm, we also analyzed for expression of the neural markers nestin and beta3 tubulin in these colonies. Surprisingly, 90% (45 of 50) of the colonies in the control group expressed neural markers, a frequency not changed by 1,25(OH)(2)D(3) treatment. The current studies demonstrate the global and developmental stage-specific effects of 1,25(OH)(2)D(3) on mesenchymal lineage progenitors, and suggest that the effects of 1,25(OH)(2)D(3) on osteogenesis and adipogenesis in RC populations are mediated, at least in part, by increased recruitment of CFC-O/A, but not CFC-A type precursors.

Side population (SP) cells isolated from fetal rat calvaria are enriched for bone, cartilage, adipose tissue and neural progenitors

In some tissues, stem cells are enriched within the side population (SP) cells characterized by the efficient efflux of Hoechst 33342, but few data are yet available to address whether such is the case in bone tissue. When we Hoechst-stained and FACS-analyzed freshly isolated 20- or 21-day fetal rat calvaria (RC) cells, a small fraction of cells (0.15 ± 0.05%) comprised of a distinct SP. When SP, non-SP and total/unfractionated (Total) RC cells were plated at a density of 30 cells per microtiter well, the percentage of wells containing bone-forming progenitors (CFU-O) was significantly higher in the SP compared to the non-SP or Total populations (13 ± 4% vs. 1.8 ± 0.4% and 0.7 ± 0.4% respectively). The SP was also highly enriched for CFU-alkaline phosphatase (CFU-ALP) and CFU-fibroblast (CFU-F). While Dex increased the recruitment of CFU-O and CFU-F in the SP, it did not increase the frequency of CFU-ALP. Limiting dilution analysis showed a non-linear relationship between cell densities (1, 5, 10, 20 and 30 cells/microtiter well) and the frequency of readout CFU-O, CFU-ALP and CFU-F in all populations, suggesting a cell non-autonomous component to proliferation–differentiation of these progenitor types. When the developmental potential of SP cells for chondrocyte, adipocyte and neural lineages was assessed, SP cells were also found to be enriched for progenitors of all three lineages. These data demonstrate that Hoechst staining and SP sorting by flow cytometry are a useful strategy for the enrichment of CFU-O and possibly other precursors present in RC cell populations.

Co-transfection with the osteogenic protein (OP)-1 gene and the insulin-like growth factor (IGF)-I gene enhanced osteoblastic cell differentiation

Previous studies from this laboratory showed that the action of Osteogenic Protein-1 (OP-1, BMP-7) on osteoblastic cell differentiation could be enhanced by other protein factors, such as Insulin-like Growth Factor (IGF)-I. In the present study, we examined the effects of co-transfection with a combination of the OP-1 and the IGF-I gene on osteoblastic cell differentiation. The results first showed that fetal rat calvaria (FRC) cells transfected with the OP-1 gene under the control of the cytomegalovirus (CMV) promoter showed substantial production of the OP-1 protein. Transfected FRC cells also showed a DNA concentration-dependent increase in alkaline phosphatase (AP) activity, an osteoblastic cell differentiation marker. Von Kossa-positive nodules, a hallmark of bone formation in long-term cultures of bone-derived cells, were also observed in the transfected cells after 26 days in culture, whereas none were observed in control cells. Co-transfection of FRC cells with the combination of the OP-1 and the IGF-I gene resulted in a synergistic stimulation of AP activity. The increase was DNA dose-dependent. The current data show that transfection of OP-1 gene into osteoblastic cells stimulates osteoblastic cell differentiation in vitro. The study further demonstrates the feasibility of employing gene transfer of a second gene in combination with an OP-1 vector to synergistically enhance OP-1 activity.

C‐type natriuretic peptide enhances osteogenic protein‐1‐induced osteoblastic cell differentiation via Smad5 phosphorylation

In the present study, we examined the hypothesis that the C-type natriuretic peptide (CNP) enhances osteogenic protein-1 (OP-1) action in stimulating osteoblastic cell differentiation in primary cultures of fetal rat calvaria cell (FRC). CNP enhanced synergistically the OP-1-induced Alkaline Phosphatase (AP) activity and mineralized bone nodule formation in a dose- and time-dependent manner. To examine possible mechanism of the synergy between OP-1 and CNP, the expression levels of key BMP receptors and signaling molecules were examined. Western blot analysis showed that BMPR-IB and -II receptor protein expression was not affected by CNP alone, but was stimulated by OP-1 alone. The combination of OP-1 and CNP did not further increase their protein levels. The Runx2 protein expression level was not altered by CNP alone, but was elevated by OP-1 alone, and was slightly reduced by the combination. The Smad5 protein expression level was slightly decreased by CNP alone, but was stimulated by OP-1 alone, and was not further stimulated by the combination. Smad5 phosphorylation was not stimulated by CNP alone, but was stimulated significantly by OP-1 alone. The combination of OP-1 and CNP further stimulated the OP-1-induced Smad5 phosphorylation. Thus, one mechanism of the observed synergy between OP-1 and CNP involves enhancement of the Smad5 phosphorylation.

Endothelin-1 down-regulates the expression of vascular endothelial growth factor-A associated with osteoprogenitor proliferation and differentiation

Endothelin-1 (ET-1) is implicated in the signaling between vascular endothelial cells (VECs) and osteoblasts during bone development, remodeling and repair. Vascular endothelial growth factor (VEGF) also plays an important role in these intercellular interactions. Our objectives were to identify which specific VEGF isoforms were produced during osteoblastic proliferation and differentiation and to determine the effects of ET-1 on VEGF mRNA and protein production by osteoblastic cells. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and ELISA were used to evaluate VEGF mRNA isoform expression and protein synthesis at different stages of ET-1-induced osteoblastic differentiation in fetal rat calvaria (FRC) osteoblastic cells. Three VEGF mRNA isoforms were identified corresponding to VEGF 120, VEGF 164 and VEGF 188. Predominant isoforms VEGF 120 and VEGF 164 had a bimodal expression that increased in the early proliferation and late mineralization phases. ET-1 stimulated osteoblastic proliferation and differentiation, but surprisingly, ET-1 down-regulated VEGF mRNA and protein expression and sustained the down-regulation over time in long-term cultures. Time course studies showed that ET-1 inhibited VEGF mRNA expression after incubation for 3 h in 7- and 14-day FRC cell cultures. Similarly, ET-1 inhibited VEGF protein secretion by 5.8- and 2.8-fold in 7- and 14-day FRC cells, respectively. VEGF-A protein secretion was inhibited by ET-1 in a dose-dependent manner with a maximal effect at 10 −7 M. This study supports a novel inhibitory role for ET-1 on VEGF synthesis in osteoblastic cells as a feedback mechanism in the temporal and spatial coupling of angiogenesis to bone formation and resorption.

Stanniocalcin 1 stimulates osteoblast differentiation in rat calvaria cell cultures.

Stanniocalcin 1 (STC1) is a mammalian homolog of STC, the fish calcium/phosphate-regulating polypeptide whose functions are only beginning to be elucidated. Recently, we demonstrated that STC1 stimulates, in an autocrine/paracrine fashion, bone mineralization by increasing phosphate uptake in osteoblasts apparently via the functional activity of the sodium-dependent phosphate transporter, Pit1. We have now assessed the role of STC1 on osteoblast development in fetal rat calvaria (RC) cell cultures. STC1 mRNA and protein were differentially expressed over the time course of cultures, and dexamethasone, a potent stimulator of differentiation in this model, shifted peak STC1 expression levels to earlier times. Overexpression [recombinant human (rh) STC1] and underexpression (antisense oligonucleotides) of STC1 accelerated and retarded, respectively, osteogenic development as well as osteopontin and osteocalcin mRNA expression in mature osteoblast cultures, but not osteoprogenitor cell cultures. Dexamethasone shifted the effective doses required for these effects to higher and lower concentrations of antisense oligonucleotides and rhSTC1, respectively. Concomitantly, rhSTC1 increased both sodium-dependent phosphate uptake and Pit1 gene expression in nodule formation stages, but not in primitive progenitor stages of RC cell cultures. Thus, STC1 accelerates osteoblast development in an autocrine/paracrine manner in the RC cell culture model.

Fluorescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations.

Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone-related protein receptor (PTH1R) as osteoblast markers in dual-color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP(-)PTH1R(-), ALP(+)PTH1R(-), ALP(-)PTH1R(+), and ALP(+)PTH1R(+) populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities ( approximately 150-8,500 cell/cm(2); n = 3). Our results indicate that colony forming unit-osteoblast (CFU-O)/bone nodule-forming cells are found in all fractions, but the frequency of CFU-O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP(-)PTH1R(-), ALP(-)PTH1R(+), ALP(+)PTH1R(-), and ALP(+)PTH1R(+) cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU-O number in the four fractions, with the largest stimulation in the ALP(-) cell populations. However, there was no significant difference in the number or size distribution of CFU-F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non-autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP(-)PTH1R(-) population was capable of forming nodules at the clonal level, at approximately 3- or 12-times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity.

Parathyroid hormone-related protein(1-34) regulates Phex expression in osteoblasts through the protein kinase A pathway.

Phex (a phosphate-regulating gene with homologies to endopeptidases on the X chromosome) is expressed predominantly in bone in which it has been implicated in the mineralization process. Multiple factors and hormones, including PTHrP, regulate formation, development, and/or homeostasis of bone. The purpose of the present study was to determine whether PTHrP(1-34) regulates Phex expression and identify the signaling pathway used. Phex mRNA and protein levels were analyzed by RT-PCR and immunoblotting, respectively. In UMR-106 cells, PTHrP(1-34) caused a time- and concentration-dependent decrease in Phex expression. Forskolin, an adenylate cyclase activator, had the same effect. Dibutiryl cAMP also decreased Phex expression, and its effect was blocked by H89, a protein kinase A (PKA) inhibitor. In contrast, 12-O-tetradecanoyl phorbol-13-acetate, a protein kinase C (PKC) activator, increased Phex expression in a time- and dose-dependent manner. This effect was reversed by bisindolylmaleimide Iota, a PKC inhibitor. Bovine PTH(3-34), which activates PKC but not PKA, had no effect. On the contrary, human PTH(1-31), which activates PKA but not PKC, decreased Phex expression. H89 but not bisindolylmaleimide Iota blocked the effect of PTHrP(1-34). PTHrP(1-34) also decreased Phex expression in cultures of fetal rat calvaria cells at d 7 of culture but not at later stages. These data demonstrate that PTHrP(1-34), through PKA, down-regulates Phex expression in osteoblasts.

Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation.

Mesenchymal stem cells give rise to osteoprogenitors that proliferate and differentiate into identifiable preosteoblasts, osteoblasts, bone lining cells and osteocytes. To identify and establish a molecular profile for the more primitive and uncharacterized cells in the lineage, relatively rare (<1%) osteoprogenitors present in primary cultures of fetal rat calvaria cell populations were identified by a replica plating technique. Since the cell number was limited in each colony sampled, we used global amplification PCR to analyze the repertoire of genes expressed in osteoprogenitors. We established a molecular fingerprint and a developmental sequence based on simultaneous expression patterns for both known osteoblast-associated markers (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, PTH1R and osteocalcin) and potential regulatory molecules (i.e. FGFR1, PDGF-Ralpha and PTHrP). By analysis of 99 osteoprogenitor and osteoblast colonies captured by replica plating at different developmental stages, we found: (1) a recognizable cohort of cells considered more primitive than committed osteoprogenitors; (2) a cohort of early progenitors transiently expressing bone sialoprotein; and (3) that mRNAs for FGF-R1, PDGF-Ralpha and PTH1R were expressed earlier than other markers and tended to increase and decrease in relative concert with the osteoblast-specific markers. The observations suggest that within the osteoblast differentiation sequence both discrete stages and continua of changing marker expression levels occur with variation in expression for any given marker. This combined approach of replica plating and global amplification PCR allows molecular fingerprinting of definitive primitive osteoprogenitors and will aid in identifying novel developmental stages and novel differentiation stage-specific genes as these cells progress through their differentiation sequence.