Tag Archives: in vitro

IP-6 Inhibits Mineralization of Mouse Osteoblasts Cultures

Abstract

Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures.

Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6 to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin gene expression.

Addison WN, McKee MD
Bone Apr 2010
PMID: 20079473

Iron Overload Inhibits Osteoblasts via Oxidative Stress

Abstract

Iron overload inhibits osteoblast biological activity through oxidative stress.

Iron overload has recently been connected with bone mineral density in osteoporosis. However, to date, the effect of iron overload on osteoblasts remains poorly understood. The purpose of this study is to examine osteoblast biological activity under iron overload. The osteoblast cells (hFOB1.19) were cultured in a medium supplemented with different concentrations (50, 100, and 200 μM) of ferric ammonium citrate as a donor of ferric ion. Intracellular iron was measured with a confocal laser scanning microscope. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescin diacetate fluorophotometry. Osteoblast biological activities were evaluated by measuring the activity of alkaline phosphatase (ALP) and mineralization function. Results indicated that iron overload could consequently increase intracellular iron concentration and intracellular ROS levels in a concentration-dependent manner. Additionally, ALP activity was suppressed, and a decline in the number of mineralized nodules was observed in in vitro cultured osteoblast cells. According to these results, it seems that iron overload probably inhibits osteoblast function through higher oxidative stress following increased intracellular iron concentrations.

He YF, Ma Y, Gao C, Zhao GY…
Biol Trace Elem Res May 2013
PMID: 23334864

Mild Low Iron Promotes Osteoblasts; Excess or Serious Low Iron Bad In Vitro

Abstract

A comparison of the biological activities of human osteoblast hFOB1.19 between iron excess and iron deficiency.

Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0-200 μmol/L ferric ammonium citrate and 0-20 μmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.

Zhao GY, Zhao LP, He YF, Li GF…
Biol Trace Elem Res Dec 2012
PMID: 23054865

Iron Overload Inhibits Osteoblasts in Mouse and Rat Cells

Abstract

Excess iron inhibits osteoblast metabolism.

Hemochromatosis is an iron overload disorder associated with osteopenia and osteoporosis. To learn more about the effects of iron on bone cells, we examined the effects of ferric ion on the proliferation, differentiation, and mineralization of two types of cultured osteoblasts, the cell line MC3T3-E1 and rat calvarial osteoblast-like (ROB) cells. We used ferric ammonium citrate (FAC) as a donor of ferric ion, and FAC inhibited the proliferation of MC3T3-E1 cells in a dose-dependent manner. FAC (0.1-1 microg/ml) inhibited indices of osteoblast differentiation, such as the expression of type I collagen (mRNA and protein), the activity of alkaline phosphatase, and the deposition of calcium by osteoblasts. These results suggest that iron overload might give rise to osteoporosis by inhibiting osteoblast proliferation and differentiation.

Yamasaki K, Hagiwara H
Toxicol. Lett. Dec 2009
PMID: 19735707

Zinc Acexamate Stimulates Fracture Healing in Rats

Abstract

Increase in bone protein components with healing rat fractures: enhancement by zinc treatment.

The alteration in bone components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed 7 and 14 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many protein molecules were induced in the diaphyseal tissues with fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 and 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Also, the culture of the diaphyseal tissues with fracture healing caused a significant increase in bone alkaline phosphatase activity and deoxyribonucleic acid (DNA) content. Meanwhile, the presence of zinc acexamate (10-5 and 10-4 M), a stimulator of bone formation, in a culture medium induced a significant elevation of protein content and alkaline phosphatase activity in the diaphyseal tissues with fracture healing. Such an effect was completely abolished by the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis. The present study suggests that fracture healing induces a newly synthesized bone protein component including stimulatory factor(s) for bone formation. Zinc supplementation may stimulate the healing of femoral fracture.

Igarashi A, Yamaguchi M
Int. J. Mol. Med. Dec 1999
PMID: 10567672

Zinc Acexamate Increases Effect of IGF-1 and TGF-β in Healing Bone Fractures in Rats

Abstract

Increase in bone growth factors with healing rat fractures: the enhancing effect of zinc.

The effect of zinc, a stimulator of bone formation, on bone protein components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed between 1 and 7 days after the femoral fracture, and the diaphyseal tissues were cultured in a serum-free Dulbecco’s modified Eagle’s medium for 24 h. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. The amount of protein in the medium cultured with the diaphyseal tissues obtained from fracture healing rats was markedly elevated as compared with that of normal rats, indicating that bone protein components were secreted into culture medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) showed that many protein molecules were secreted from the diaphyseal tissues with fracture healing. Especially, protein molecule of about 66 kDa was markedly secreted by fracture healing. The presence of zinc acexamate (10(-5) and 10(-4) M) in culture medium induced a significant elevation of medium protein content; the zinc effect was enhanced by culture with the diaphyseal tissues of fracture healing rats. Also, the culture of diaphyseal tissues with fracture healing caused a significant increase in insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in culture medium. The production of IGF-I and TGF-beta1 from bone tissues with fracture healing was significantly enhanced in the presence of zinc acexamate (10(-6)-10(-4) M). Moreover, the addition of IGF-I (10(-8) M) or TGF-beta1 (10(-10) M) in a culture medium caused a significant elevation of protein content in the medium cultured with the femoral-diaphyseal tissues from normal and fracture healing rats. The effect of IGF-I or TGF-beta1 was significantly enhanced in the presence of zinc acexamate (10(-4) M). Also, deoxyribonucleic acid (DNA) content in the diaphyseal tissues from normal and fracture healing rats was significantly raised by the culture with IGF-I or TGF-beta1. The effect of IGF-I was significantly enhanced by zinc. The present study demonstrates that fracture healing increases production of bone IGF-I and TGF-beta1, and that this elevation is enhanced by zinc treatment.

Igarashi A, Yamaguchi M
Int. J. Mol. Med. Oct 2001
PMID: 11562784

Zinc Acexamate Increases Bone Stimulating Factors in Rat Tissue

Abstract

Enhancement of albumin expression in bone tissues with healing rat fractures.

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.

Yamaguchi M, Igarashi A, Misawa H, Tsurusaki Y
J. Cell. Biochem. May 2003
PMID: 12704798

Zinc Acexamate Can Restore Bone in Diabetic Rats

Abstract

Alteration in serum and bone component findings induced in streptozotocin-diabetic rats is restored by zinc acexamate.

The effect of zinc acexamate in streptozotocin (STZ)-induced diabetic rats was investigated. Rats received a single subcutaneous administration of STZ (6.0 mg/100 g body weight), and the animals were orally administered once daily for 14 days with zinc acexamate (2.5, 5 or 10 mg/100 g body weight). The administration of STZ caused a significant increase in serum glucose, triglyceride and calcium levels and a significant decrease in body weight, serum zinc and inorganic phosphorus levels, indicating diabetic condition. Moreover, calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the femoral-diaphyseal and -metaphyseal tissues were significantly reduced in STZ-diabetic rats. The change in these serum and bone components of STZ-diabetic rats was significantly restored by the oral administration of zinc acexamate (2.5, 5 or 10 mg Zn/100 g body weight). The restoration of bone components was not seen by the oral administration of zinc sulfate (2.5 mg Zn/100 g) for 14 days. Moreover, when the femoral-diaphyseal and -metaphyseal tissues obtained at 14 days after STZ administration were cultured for 48 h in a medium containing either vehicle or zinc acexamate (10(-5) M), the femoral calcium content and alkaline phosphatase activity were significantly increased in vitro. The effect of zinc acexamate was completely abolished in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. The present study demonstrates that the oral administration of zinc acexamate has a preventive effect on STZ-induced diabetic condition in rats, and that it can restorate bone loss of STZ-induced diabetes in vivo.

Uchiyama S, Yamaguchi M
Int. J. Mol. Med. Dec 2003
PMID: 14612972

Yamaguchi M, Uchiyama S
Int. J. Mol. Med. Nov 2003
PMID: 14533005

Zinc Acexamate Anabolic Effects > Zinc-Carnosine, Zinc, or MK-4 in Rat Tissue

Abstract

Potent effect of zinc acexamate on bone components in the femoral-metaphyseal tissues of elderly female rats.

1. The effect of zinc compounds on bone components in the femoral-metaphyseal tissues from elderly female rats (50 weeks old) was investigated in vitro. Bone tissues were cultured for 24 hr in Dulbecco’s modified Eagle medium containing either vehicle or zinc compounds (10[-7] to 10[-5] M).
2. Zinc content, alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the metaphyseal tissues were significantly increased by the presence of zinc sulfate (10[-6] and 10[-5] M), beta-alanyl-L-histidinato zinc (AHZ; 10[-6] and 10[-5] M) and zinc acexamate (10[-7] to 10[-5] M). At 10[-5] M, the effect of zinc acexamate on the increase of bone components was more potent than that of zinc sulfate or AHZ.
3. The effect of zinc acexamate (10[-5] M) on the increase of alkaline phosphatase activity in the metaphyseal tissues was remarkable as compared with that of insulin (10[-8] M), estrogen (10[-9] M), insulin-like growth factor-I (10[-8] M), transforming growth factor-beta (10[-10] M), sodium fluoride (10[-3] M), dexamethasone (10[-7] M) and vitamin K2 (menaquinone-4; 10[-5] M) with an effective concentration.
4. The stimulatory effect of zinc acexamate (10[-5] M) on alkaline phosphatase activity and calcium content in the metaphyseal tissues was completely blocked by the presence of dipicolinate (10[-3] M), a chelator of zinc ion, and of cycloheximide (10[-6] M), an inhibitor of protein synthesis.
5. The present study demonstrates that zinc acexamate has a potent anabolic effect on bone components in the femoral-metaphyseal tissues from female elderly rats in vitro. The effect of zinc acexamate may be based in part on protein synthesis related to zinc ion in bone cells.

Yamaguchi M, Gao YH
Gen. Pharmacol. Mar 1998
PMID: 9510097

Zinc-Carnosine or Zinc Reduce IL-6 in Mouse Osteoblast-Like Cells

Abstract

Zinc suppresses IL-6 synthesis by prostaglandin F2alpha in osteoblasts: inhibition of phospholipase C and phospholipase D.

We previously reported that prostaglandin F2alpha (PGF2alpha) induces phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein, resulting in the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells and that PGF2alpha stimulates the synthesis of interleukin-6 (IL-6) via PKC-dependent p44/p42 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated whether zinc affects the PGF2alpha-induced IL-6 synthesis in these cells. Zinc complex of l-carnosine (l-CAZ) dose-dependently suppressed the PGF2alpha-stimulated IL-6 synthesis. In addition, zinc alone reduced the IL-6 synthesis. L-CAZ suppressed the PGF2alpha-induced p44/p42 MAP kinase phosphorylation. However, the p44/p42 MAP kinase phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, or NaF, a direct activator of GTP-binding protein, was not affected by l-CAZ. l-CAZ reduced the PGF2alpha-stimulated formation of inositol phosphates and choline. However, l-CAZ did not affect the formation of inositol phosphates or choline induced by NaF. These results strongly suggest that zinc reduces PGF2alpha-induced IL-6 synthesis via suppression of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblasts.

Hatakeyama D, Kozawa O, Otsuka T, Shibata T…
J. Cell. Biochem. 2002
PMID: 11968002

Osteoblasts secrete IL-6 to stimulate osteoclast formation. Reducing IL-6 should help reduce resorption.