TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.     

Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts

We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO2 from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO2 was reduced further to 0.2%, bone nodule formation was almost abolished. The inhibitory effect of hypoxia on bone formation was partly due to decreased osteoblast proliferation, as measured by 3H-thymidine incorporation. Hypoxia also sharply reduced osteoblast alkaline phosphatase (ALP) activity and expression of mRNAs for ALP and osteocalcin, suggesting inhibition of differentiation to the osteogenic phenotype. Hypoxia did not increase the apoptosis of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O2 were less organized and much less abundant than in 20% O2 cultures. Furthermore, collagen produced by hypoxic osteoblasts contained a lower percentage of hydroxylysine residues and exhibited an increased sensitivity to pepsin degradation. These data demonstrate the absolute oxygen requirement of osteoblasts for successful bone formation and emphasize the importance of the vasculature in maintaining bone health. We recently showed that hypoxia also acts in a reciprocal manner as a powerful stimulator of osteoclast formation. Considered together, our results help to explain the bone loss that occurs at the sites of fracture, tumors, inflammation and infection, and in individuals with vascular disease or anemia.     

Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation

Mutations in fibroblast growth factor receptors (Fgfrs) 1-3 cause skeletal disease syndromes in humans. Although these Fgfrs are expressed at various stages of chondrocyte and osteoblast development, their function in specific skeletal cell types is poorly understood. Using conditional inactivation of Fgfr1 in osteo-chondrocyte progenitor cells and in differentiated osteoblasts, we provide evidence that FGFR1 signaling is important for different stages of osteoblast maturation. Examination of osteogenic markers showed that inactivation of FGFR1 in osteo-chondro-progenitor cells delayed osteoblast differentiation, but that inactivation of FGFR1 in differentiated osteoblasts accelerated differentiation. In vitro osteoblast cultures recapitulated the in vivo effect of FGFR1 on stage-specific osteoblast maturation. In immature osteoblasts, FGFR1 deficiency increased proliferation and delayed differentiation and matrix mineralization, whereas in differentiated osteoblasts, FGFR1 deficiency enhanced mineralization. Furthermore, FGFR1 deficiency in differentiated osteoblasts resulted in increased expression of Fgfr3, a molecule that regulates the activity of differentiated osteoblasts. Mice lacking Fgfr1, either in progenitor cells or in differentiated osteoblasts, showed increased bone mass as adults. These data demonstrate that signaling through FGFR1 in osteoblasts is necessary to maintain the balance between bone formation and remodeling through a direct effect on osteoblast maturation.     

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.     

Isomer-specific effects of conjugated linoleic acid on mineralized bone nodule formation from human osteoblast-like cells

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 microM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formation of mineralized bone nodules using bone cells of human origin, and provide evidence for isomer-specific effects of CLA on bone health.     

Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: Altered expression of collagenase,cell growth-related,and mineralization-associated genes

Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1–10), addition of acidic FGF (100 μg/ml) to actively proliferating cells increased (P < 0.05) ³H-thymidine uptake (2,515 ± 137, mean ± SEM vs. 5,884 ± 818 cpm/10⁴ cells). During the second stage of maturation (days 10–15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16–29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14–29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7–8) enhanced histone H4, osteopontin, type 1 collagen, and TGF-β mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14–15) reactivated H4, osteopontin, type I collagen, and TGF-β gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 μg/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-β and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence. © 1996 Wiley-Liss, Inc.     

Osteoblast-like cells of the hypophysectomized rat: a model of aberrant osteoblast development

In a previous work, we demonstrated that the osteoprogenitors derived from the marrow stroma of the hypophysectomized (HX) rat demonstrate enhanced proliferative and differentiation capacities when placed in an optimal microenvironment. In this study, we sought to investigate the potential of the trabecular osteoblast-like cells of the HX rat. These cells represent a more mature pool of osteoblasts than the progenitors derived from the marrow stroma. We examined all three stages of osteoblast development using trabecular osteoblast-like cells derived from age-matched intact rats as a control. Using thymidine incorporation and cell number as indicators of proliferation, we found that these cells, like the osteoprogenitors derived from the HX rat, demonstrate augmented proliferation when placed in culture. Additionally, type I collagen expression remained at significant levels past the end stages of proliferation, at which point it is expected to be downregulated. Matrix maturation markers, such as alkaline phosphatase activity and bone sialoprotein expression, however, were significantly lower than in the controls. Mineralization potential, as measured by mineralized nodule formation, Ca(2+) content, and OPN and OCN expression, was also significantly reduced. Our results have uncovered an aberrant model of osteoblast development in which proliferation is deregulated, resulting in a minimal capacity of these cells to develop into fully differentiated mineralizing osteoblasts.     

Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti-CTGF neutralizing antibody caused a dose-dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.     

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins,osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley-Liss, Inc.     

Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation — a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.     

Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro

The ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co-culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose-dependent manner. HPL-conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze-thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 10(-5) M indomethacin. Dose-dependent inhibiton of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10(-6) M) or F2 alpha (at 10(-7) M). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.     

Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro.

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.     

Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone

Osteoprogenitor cells present in single-cell suspensions prepared from fetal rat calvaria (RC) form discrete mineralized three-dimensional bone nodules when cultured long-term in the presence of ascorbic acid and beta-glycerophosphate. These cells (CFU-O) constitute less than 1% of the total cell population under standard culture conditions and their number is increased in the presence of dexamethasone. Using the formation of the bone nodule as a marker for CFU-O, we have now analyzed the proliferation and differentiation capacity of these CFU-O by redistribution and continuous subculture experiments in the presence and absence of dexamethasone. Cell redistribution experiments showed no increase in nodule number after one population doubling with either treatment. After 5.4 population doublings of the entire RC population, nodule number increased up to 2.0-fold in control cultures and 4.5-fold in cultures containing 10 nM dexamethasone. Continuous subculture experiments in which cultures were split 1:3 every 3 day for up to seven subcultures showed that nodule number decreased in parallel with the split ratio in the absence of dexamethasone, while with dexamethasone nodule number was elevated above the number present in primary cultures for 1 or 2 subcultures after which nodule number decreased with the split ratio. Bone nodules were present for up to 18 population doublings. Measurements of nodule area by automated image analysis showed that dexamethasone increased nodule size and that nodule size decreased from primary to 1st to 2nd subculture with or without dexamethasone. The data suggest that dexamethasone selectively stimulates the proliferation of osteoprogenitor cells and that these progenitor cells have a limited capacity for generating daughter cells capable of expressing the bone phenotype.     

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.     

Statins inhibit osteoblast migration by inhibiting Rac-Akt signaling

Cell migration is a key event in repair and remodeling of skeletal tissues, but the mechanism of osteoblast migration has not been resolved. Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, increase bone. However, the effect of statins on osteoblast migration remains to be clarified. We investigated the effect of fluvastatin and mevastatin on platelet-derived growth factor (PDGF)-induced migration of osteoblastic MC3T3-E1 cells. PDGF promoted osteoblast migration, while the statins inhibited PDGF-induced migration, and mevalonate and geranylgeranylpyrophosphate but not farnesylpyrophosphate abolished the effect of statins. Dominant-negative Rac severely inhibited PDGF-induced osteoblast migration and reduced Akt phosphorylation. Further, fluvastatin reduced Akt phosphorylation and dominant-negative Akt inhibited PDGF-induced osteoblast migration. These results demonstrate that statins inhibit PDGF-induced osteoblast migration and Rac-Akt signaling plays an important role in the osteoblast migration, and suggest that statins restrain Rac function by inhibiting geranylgeranylation of Rac, which leads to the reduction in Akt activation and osteoblast migration.