Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

Melatonin Modulates lipid Metabolism in HepG2 Cells Cultured in High Concentrations of Oleic Acid: AMPK Pathway Activation may Play an Important Role

Melatonin exists as an active ingredient in several foods and has been reported to inhibit fatty liver disease in animals; however, its molecular mechanisms are not well elucidated. Herein, we explored effects of melatonin on lipid accumulation induced by oleic acid in HepG2 cells and characterized the underlying molecular mechanisms. Pretreatment with melatonin (0.1–0.3?mM) significantly inhibited accumulation of triglyceride and cholesterol induced by incubating HepG2 cells with high concentrations of oleic acid (oleic acid overload) (p?<?0.05). Melatonin pretreatment induced phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), causing their activation and inactivation, respectively. Expression levels of peroxisome proliferator activated receptor-α (PPARα) and its target gene carnitine palmitoyl-CoA transferase 1 (CPT1), which are associated with lipolysis, were upregulated by melatonin, whereas expression of sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD1), which are associated with lipogenesis, were downregulated. Melatonin did not change expression of genes involved in cholesterol metabolism, including 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and SREBP-2. Melatonin inhibits lipid accumulation induced by oleic acid overload in HepG2 cells. The phosphorylation and activation of AMPK may have important roles in inactivating lipid anabolic pathways and activating triglyceride catabolic pathways.     

Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2K^b-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10⁻⁷ M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells. © 1996 Wiley-Liss, Inc.     

Effect of sterols and fatty acids on growth and triglyceride accumulation in 3T3-L1 cells

Epidemiologic studies suggest a role of dietary fat in the development of obesity. Populations that consume Western diets have a higher incidence of obesity than do those that consume a vegetarian type diet such as Asians. Because dietary fats are made up mostly of triglyceride with minor lipids such as sterols, the objective of this study was to examine the effect of different fatty acids, the main component of triglycerides, and sterols on cell growth and triglyceride accumulation in 3T3-L1 cells. These cells are being used as an in vitro model for studying obesity because upon differentiation in culture they accumulate triglycerides. Cells were seeded at 5,000 cells/cm(2) and supplemented with 0, 3, 10, or 30 microM of oleic acid, elaidic acid, or docosahexaenoic acid (DHA). Similarly, cells were supplemented with 0, 2, 8, or 16 microM of cholesterol, beta-sitosterol (SIT), or campesterol. Cell growth was measured by cell counting. Cellular triglycerides were measured by the Oil Red O method. In some experiments, fatty acids were combined with sterols and growth and triglyceride content were assessed as described. Both DHA and SIT had inhibitory effects on 3T3-L1 cell growth. However, SIT was more potent than DHA in this regard. The combination of SIT and oleic acid was the most potent in inhibiting cell growth and increasing cellular triglyceride content. It is concluded that cell growth and triglyceride accumulation in 3T3-L1 cells is influenced by fatty acid and sterols. When used alone, DHA and SIT inhibit cell growth. SIT was more effective in this process than was DHA. There was an interaction between fatty acids and sterols. The most effective combination inhibiting cell growth and triglyceride concentration was the combination of SIT and oleic acid. This combination reduced cell growth and increased triglyceride accumulation. These data suggest that diets rich in both monounsaturated fatty acids and phytosterols may play a role in controlling obesity.     

Genotoxicity of paraquat: micronuclei induced in bone marrow and peripheral blood are inhibited by melatonin

The ability of melatonin to influence paraquat-induced genotoxicity was tested using micronucleated polychromatic erythrocytes as an index of damage in both bone marrow and peripheral blood cells of mice. Melatonin (10 mg/kg) or an equal volume of saline were administered intraperitoneally (ip) to mice 30 min prior to an ip injection of paraquat (20 mg/kgx2), and thereafter at 6-h intervals until the conclusion of the study (72 h). The number of the micronucleated polychromatic erythrocytes increased after paraquat administration both in peripheral blood and bone marrow cells. Melatonin administration to paraquat-treated mice significantly reduced micronuclei formation in both peripheral blood and bone marrow cells; these differences were apparent at 24, 48 and 72 h after paraquat administration. The induction of micronuclei was time-dependent with peak values occurring at 24 and 48 h. The reduction in paraquat-related genotoxicity by melatonin is likely due in part to the antioxidant activity of the indole. We did not observe effects of melatonin over paraquat in paraquat+melatonin groups incubated at 0, 60 and 120 min. Mitomycin C, which was used as a positive control, also caused the expected large rises in micronuclei in both bone marrow and peripheral blood cells at 24, 48 and 72 h after its administration.     

BMP-2 and sonic hedgehog have contrary effects on adipocyte-like differentiation of C3H10T1/2 cells

The signaling pathways of bone morphogenic protein 2 (BMP-2) and Sonic hedgehog (Shh) are related during embryogenesis. Both proteins have been implicated as important components during osteogenic differentiation; e.g., considering their in vitro effects in the pluripotent C3H10T/1/2 cell system. Also, BMP-2 has been frequently reported to stimulate adipogenesis as well as osteogenesis in these cells. We investigated the relative potencies of Shh and BMP-2 with regard to adipogenesis. We performed differentiation experiments by stimulating C3H10T1/2 cells with BMP-2, Shh, or a combination. We monitored adipocyte-like differentiation via gene expression analysis and cytologic staining. An adipocytic phenotype was observed in BMP-2-treated cells, as shown by upregulation of two adipocytic marker mRNAs, PPAR-gamma and aP2, and by staining of lipid-filled cell vesicles with Oil Red O. In contrast, no adipocyte-like differentiation could be detected either after treatment with Shh or after exposure to a combination of Shh and BMP-2. Our results demonstrate for the first time that Shh and BMP-2 have contrary effects on adipocyte-like differentiation. Whereas BMP-2 promotes the adipocytic lineage, Shh suppresses the expression of the BMP-2-induced fat-cell phenotype.     

Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement and Oil Red O stain and measurement were employed to assess the effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts. The results showed that except for individual concentrations of zinc ion there was no effect on the proliferation of MSCs and osteoblasts. Zinc ion inhibited the osteogenic differentiation of MSCs at all the concentrations tested. It also inhibited adipogenic differentiation at all concentrations tested except 10(-9)mol/L. Both of the inhibition effects were attenuated with time increasing. Zinc ion depressed adipocytic trans-differentiation of osteoblasts at concentrations of 10(-11) and 10(-10)mol/L, but the effect could be reversed to promote or even be removed when concentration was increased. It suggests that the influence of zinc ion on osteogenic, adipogenic differentiation of MSCs and adipocytic trans-differentiation of osteoblasts depends on zinc ion concentrations and incubation time. The protective effects of zinc ion on bone may be mediated by modulating differentiation of MSCs away from the adipocytes and inhibiting adipocytic trans-differentiation of osteoblasts. This may in turn promote osteoblast formation and reduce secretion of cytokines which may inhibit osteoclast formation and activation. These findings may be valuable for better understanding the mechanism of the effect of zinc ion on bone.     

Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37 degrees C, 7% CO2, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro.     

Shared oxidative pathways in response to gravity-dependent loading and gamma-irradiation of bone marrow-derived skeletal cell progenitors

Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

Summary A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37°C, 7% CO₂, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro. Supported by National Cancer Institute Virus Cancer Program Contract NCI NO1-7-1051.     

Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(+) cells

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.     

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²⁺

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.     

Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability of BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0mMSr(2+)) under osteogenic or adipogenic medium for 1 and 2weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPARγ2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPARγ in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.     

Melatonin enhances hydrogen peroxide-induced apoptosis in human promyelocytic leukaemia HL-60 cells

Melatonin is an indoleamine secreted by the pineal gland that shows multiple tasks. This ubiquitously acting free radical scavenger has recently been shown to stimulate the production of reactive oxygen species (ROS) in tumour cells, making them undergo apoptosis, whilst it prevents apoptosis in healthy cells. The mechanisms by which melatonin exerts these dual actions are, however, not yet clearly understood. Thus, the aim of this study was to further investigate how melatonin can enhance oxidative stress-induced apoptosis in a leukaemia cell line. The results show that melatonin increased the apoptotic effects of H(2)O(2) in human myeloid HL-60 cells as assessed by cellular viability, mitochondrial permeability transition induction, mitochondrial membrane depolarization, ROS generation, caspases 3, 8 and 9 activity, phosphatidylserine externalization, and DNA fragmentation techniques. When healthy leucocytes were exposed to H(2)O(2), melatonin increased the viability of the cells. Taken together, the findings indicate that melatonin is a potential physiological tool capable of protecting healthy cells from chemotherapy-induced ROS production as well as inducing tumour cell death. Because cancer cells manifest increased oxidative stress as a result of their elevated metabolism, the use of melatonin may be useful in impairing their ROS buffering capacity.     

LIGHT regulates the adipogenic differentiation of mesenchymal stem cells

LIGHT is a cytokine belonging to the TNF family. This cytokine has been extensively defined in its role on T‐cell regulation and dendritic cell maturation. It also exhibits the role in liver regeneration. We recently identified its role in regulation of hematopoietic stem cell differentiation. However, the question whether this cytokine regulates mesenchymal stem cells (MSCs) proliferation and/or differentiation remains unknown. In this study, we observed that MSCs express LT‐βR but not HVEM. PCR analysis show LIGHT mRNA is undectable in MSCs. LIGHT did promote neither MSCs proliferation nor migration. However, LIGHT promoted MSCs differentiation into adipocyte which was confirmed by Oil Red O Staining Assay. Since either MSCs or adipocytes are the major cell population in bone marrow niche, we then suggest that LIGHT regulate bone marrow niche, such as MSCs differentiation. J. Cell. Biochem. 114: 346–353, 2013. © 2012 Wiley Periodicals, Inc.     

Melatonin and lipid uptake by murine fibroblasts: clinical implications

The current study was undertaken to uncover the role of melatonin in lipid metabolism in the murine fibroblasts. The results show melatonin in vitro enhances lipid accumulation and lipid droplet formation in this cell line. Using oil red O staining, it was found that when oleic acid was present in the culture media, melatonin at doses of 0.1-2mM, significantly increased the lipid concentrations in the cells. However, low levels of melatonin, with or without oleic acid, did not influence lipid metabolism in the cultured fibroblasts. When a non-specific melatonin receptor antagonist, luzindole 10 microM was co-incubated with 1mM melatonin, the stimulatory effects of melatonin on lipid accumulation in these cells was significantly reduced. It appears that the effects of melatonin on lipid metabolism in murine fibroblasts is mediated by melatonin membrane receptors.