Prostaglandin E2 stimulates collagen and non-collagen protein synthesis and prolyl hydroxylase activity in osteoblastic clone MC3T3-E1 cells

We investigated the stimulative effect of prostaglandin E2 (PGE2) on an osteoblastic cell line, clone MC3T3-E1, in serum-free medium. PGE2 elevated collagen and non-collagen protein syntheses in a dose-related fashion up to 2 micrograms/ml, the maximal increases being 2- and 3-fold, respectively, over that in the control. Its stimulative effect was evident as early as 12 h. PGE2 slightly increased DNA content, but its effect was less than that on collagen and non-collagen protein syntheses. Moreover, PGE stimulated an increase in prolyl hydroxylase activity with a maximal effect at 1-2 micrograms/ml, the activity being 15-fold over that of the control. These results strongly indicate that PGE2 directly enhances total protein synthesis including that of collagen in osteoblasts in vitro, suggesting its direct effect on bone formation in vivo as well.     

Bordetella bronchiseptica dermonecrotizing toxin stimulates protein synthesis in an osteoblastic clone, MC3T3-E1 cells

Abstract The effects of Bordetella bronchiseptica dermonecrotizing toxin on protein synthesis in an osteoblastic clone, MC3T3-E1 cells, were investigated. The rate of protein synthesis in the serum-starved cells was increased by the toxin after a latent period of about 4 h, and reached 2.5 times that of the control 24 h after addition of toxin. The toxin raised the level of protein synthesis even in actively proliferating cells. The stimulatory effect of the toxin on protein synthesis occurred earlier than other toxic events so far reported, such as the stimulation of DNA synthesis and the inhibition of osteoblastic differentiation, and was apparently dependent on the toxin concentrations over the range 0.05 ng ml⁻¹ to 6.0 ng ml⁻¹. Therefore, the stimulatory effect of the toxin on protein synthesis could be useful in determining the mode of action of the toxin.     

Cadmium stimulates prostaglandin E2 synthesis in osteoblast-like cells, MC3T3-E1

The effect of Cd on prostaglandin E2 production in osteoblasts was studied using cloned osteoblast-like cells, MC3T3-E1, which were established from new-born mouse calvaria. Treatment of the cells with Cd caused a dose- (0-10 microM) and time- (0-24 h) dependent increase in the release of prostaglandin E2 from the cells into the culture medium. A lag time of 4 h was required for the onset of the phenomenon. The release of [14C]arachidonic acid from prelabeled cell membrane was little influenced by the Cd treatment, while conversion of [14C]arachidonic acid to prostaglandin E2 by the homogenate of the cells treated with Cd was enhanced as compared to that by untreated cells. The stimulatory effect of Cd on prostaglandin E2 production was abolished in the presence of cycloheximide (100 ng/ml). By Western blot analysis with polyclonal rabbit anti-cyclooxygenase antibody, it was revealed that Cd treatment augmented the amount of immunoreactive cyclooxygenase in the cells. These results strongly suggest that Cd stimulates prostaglandin E2 production through the induction of cyclooxygenase in MC3T3-E1 cells. This effect of Cd may be involved in the mechanisms of Cd-induced bone injury.     

Prostaglandin F2 alpha stimulates proliferation of clonal osteoblastic MC3T3-E1 cells by up-regulation of insulin-like growth factor I receptors.

Prostaglandin F2 alpha (PGF2 alpha) stimulates proliferation of clonal osteoblastic MC3T3-E1 cells mainly via the stimulation of phospholipase C. These cells constitutively produced and secreted insulin-like growth factor I (IGF-I). In addition, a neutralizing anti-IGF-I antibody completely abolished DNA synthesis stimulated by PGF2 alpha in MC3T3-E1 cells, suggesting that IGF-I indeed mediates the PGF2 alpha effect. However, PGF2 alpha decreased the expression of IGF-I mRNA and the secretion of immunoreactive IGF-I into the medium, whereas progression activity in the conditioned medium was not affected by PGF2 alpha. Although IGF-I alone did not stimulate DNA synthesis in MC3T3-E1 cells, when PGF2 alpha was added to the cultures, IGF-I stimulated their proliferation. Thus, PGF2 alpha may potentiate the action of IGF-I. At the same time, PGF2 alpha increased the number of high affinity binding sites (molecular mass of 130 kDa) for IGF-I in a dose-dependent manner. The increase in IGF-I-binding site number preceded the elevation of DNA synthesis by approximately 3 h. Furthermore, MC3T3-E1 cells secreted at least three species of IGF-binding proteins (IGFBPs) with molecular masses of 24, 30, and 34 kDa. In the early period of PGF2 alpha exposure, PGF2 alpha attenuated the secretion of all of these IGFBPs, whereas thereafter, it markedly increased their secretion, especially that of the 34-kDa IGFBP, suggesting a modulation of metabolism and action of IGF-I. These effects of PGF2 alpha on IGF-I receptor number and IGFBP secretion may play a role in the synergism between PGF2 alpha and IGF-I that results in the stimulation of DNA synthesis in MC3T3-E1 cells.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.     

Prostaglandin E2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 cells in a PKA-dependent manner

The effect of prostaglandin E2（PGE2） on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regu- lates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteo- blasts in the presence of fluid shear stress （FSS）, which could better uncover the anabolic effect of PGEz in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A （PKA） pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 rain significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells＇ calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the ＇reset＇ process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone forma- tion as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases.     

Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.     

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.     

Effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity and collagen synthesis in osteoblastic cells, clone MC3T3-E1

A stimulative effect of 1,25-dihydroxyvitamin D3 was tested on osteoblastic cells, clone MC3T3-E1, cultured in serum-free medium with 0.1% bovine serum albumin. This steroid increased alkaline phosphatase activity in a dose-related fashion. The steroid also stimulated dose-dependently collagen and non-collagen protein syntheses, their maximal effects being observed at 12 and 24 h, respectively. The incorporation of [3H]-proline into collagen or non-collagen protein in cells exposed to this steroid for 12 h was 2.9 or 1.9-fold over that of control cultures, respectively. These results strongly indicate the stimulative effects of 1,25-dihydroxyvitamin D3 on the differentiation of osteoblasts in vitro.     

Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells

Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10(-7) M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

High mobility group-like protein in bovine milk stimulates the proliferation of osteoblastic MC3T3-E1 cells.

The active component in bovine milk on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified. Growth-promoting activity was measured by [(3)H]thymidine incorporation on the cell. The molecular weight of the purified protein was 10 kDa. The amino-terminal sequence of this 10-kDa protein was identical to bovine high mobility group protein (HMG) 1. This 10-kDa protein is suggested to be a basic protein and to have an HMG box, a consensus sequence motif among the HMG family. From these results, we named this protein HMG-like protein. HMG is a ubiquitous nonhistone component of chromatin and considered to be implicated in DNA replication. We found this protein in milk, and it showed a growth-promoting activity. We propose the possibility that HMG-like protein existed in milk and plays an important role for neonate in bone formation by activating osteoblasts.     

1,25(OH)2D3 increases cytosolic Ca++ concentration of osteoblastic cells, clone MC3T3-E1

Effect of 1,25(OH)2D3 in vitro on cytosolic Ca++ concentration of osteoblastic cells (MC3T3-E1) was studied. Marked but transient increase of cytosolic Ca++ concentration of osteoblastic cells was observed following the addition of 10 pg/ml of 1,25(OH)2D3, but not with 10 pg/ml of 24,25(OH)2D3. The increase of cytosolic Ca++ concentration of osteoblastic cells by 1,25(OH)2D3 was not observed when the cells were incubated in Ca++ free medium. Therefore, it was concluded that 1,25(OH)2D3 increased cytosolic Ca++ concentration of osteoblastic cells through the increase of Ca++ influx into the cells.     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.