The authors have no conflict of interest.
Oxysterols Regulate Differentiation of Mesenchymal Stem Cells: Pro-Bone and Anti-Fat†
Article first published online: 12 JAN 2004
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 5, pages 830–840, May 2004
How to Cite
Kha, H. T., Basseri, B., Shouhed, D., Richardson, J., Tetradis, S., Hahn, T. J. and Parhami, F. (2004), Oxysterols Regulate Differentiation of Mesenchymal Stem Cells: Pro-Bone and Anti-Fat. J Bone Miner Res, 19: 830–840. doi: 10.1359/jbmr.040115
- Issue published online: 2 DEC 2009
- Article first published online: 12 JAN 2004
- Manuscript Accepted: 9 JAN 2004
- Manuscript Revised: 26 NOV 2003
- Manuscript Received: 15 SEP 2003
- stem cells;
- bone morphogenetic protein
Pluripotent mesenchymal stem cells can undergo lineage-specific differentiation in adult organisms. However, understanding of the factors and mechanisms that drive this differentiation is limited. We show the novel ability of specific oxysterols to regulate lineage-specific differentiation of mesenchymal stem cells into osteogenic cells while inhibiting their adipogenic differentiation. Such effects may have important implications for intervention with osteoporosis.
Introduction: Oxysterols are products of cholesterol oxidation and are formed in vivo by a variety of cells including osteoblasts. Novel pro-osteogenic and anti-adipogenic effects of specific oxysterols on pluripotent mesenchymal cells are demonstrated in this report. Aging and osteoporosis are associated with a decrease in the number and activity of osteoblastic cells and a parallel increase in the number of adipocytic cells.
Materials and Methods: The M2–10B4 pluripotent marrow stromal cell line, as well as several other mesenchymal cell lines and primary marrow stromal cells, was used to assess the effects of oxysterols. All results were analyzed for statistical significance using ANOVA.
Results and Conclusion: Pro-osteogenic and anti-adipogenic effects of specific oxysterols were assessed by the increase in early and late markers of osteogenic differentiation, including alkaline phosphatase activity, osteocalcin mRNA expression and mineralization, and the decrease in markers of adipogenic differentiation including lipoprotein lipase and adipocyte P2 mRNA expression and adipocyte formation. Complete osteogenic differentiation of M2 cells into cells expressing early and late markers of differentiation was achieved only when using combinations of specific oxysterols, whereas inhibition of adipogenesis could be achieved with individual oxysterols. Oxysterol effects were in part mediated by extracellular signal-regulated kinase and enzymes in the arachidonic acid metabolic pathway, i.e., cyclo-oxygenase and phospholipase A2. Furthermore, we show that these specific oxysterols act in synergy with bone morphogenetic protein 2 in inducing osteogenic differentiation. These findings suggest that oxysterols may play an important role in the differentiation of mesenchymal stem cells and may have significant, previously unrecognized, importance in stem cell biology and potential therapeutic interventions.
Mesenchymal tissue regeneration during adult life is highly dependent on a population of pluripotent stem cells commonly known as mesenchymal stem cells or marrow stromal cells (MSCs).(1,2) These cells are present in a variety of tissues and are prevalent in bone marrow stroma.(3,4) They can be readily isolated and expanded in culture, and on treatment with a variety of agonists, they can differentiate into lineage-specific cells including osteoblasts, chondrocytes, myocytes, adipocytes, and fibroblasts.(1–4) MSCs are thought to be an excellent potential tool for interventions in many diseases that result from reduced or impaired functioning of these cells.(5,6) Such interventions may include regeneration of defective extracellular matrix by systemic infusion of MSCs in individuals with generalized defects such as in osteogenesis imperfecta,(7,8) repair of damaged tissues such as bone and cartilage by localized injection into the affected areas,(9,10) and the therapeutic delivery of specific genes to diseased tissues.(11) All this, however, requires an understanding of the factors and molecular mechanisms involved in determining lineage-specific differentiation of MSCs and the identification of new strategies for driving lineage-specific differentiation in vitro and in vivo.
One situation in which MSCs might be used in human disease is in age-related and postmenopausal osteoporosis, where the decreased number and osteogenic activity of osteoprogenitor MSCs, in part, leads to decreased bone formation.(6,12-14) A reduction in bone formation by osteoblasts in the face of increased bone resorption by osteoclasts in these disorders results in decreased bone mass, increased susceptibility to fractures, and impaired fracture healing.(14) Therefore, the systemic and/or local application of osteoprogenitor MSCs, or factors that enhance their osteogenic differentiation, could be of great potential benefit in boosting bone forming capacity in osteoporosis. Future improved treatment of osteoporosis will likely require the use of bone anabolic agents that can enhance the osteogenic differentiation and bone-forming capacity of osteoblastic precursor cells, and hence increase bone mass and reduce fracture risk.(15–17) Several growth factors and hormones have been tested in this regard, including bone morphogenetic protein (BMP), growth hormone, and parathyroid hormone (PTH).(15,16) BMPs play critical roles in the differentiation of MSCs into osteoblasts both in vitro and in vivo.(18,19) BMP2 is the most potent known inducer of bone formation in vivo, and it enhances the differentiation of osteoprogenitor and non-osteoprogenitor precursors into cells with osteoblastic phenotype.(20) The use of BMPs in fracture healing is currently hampered by the large concentrations of the recombinant protein necessary to induce adequate bone formation.(20) Currently, only PTH, which on intermittent injection increases bone mass and reduces bone fracture incidence, has been approved by the Food and Drug Administration (FDA). However, because of its potential adverse side effects and cost, it is currently only used for severely osteoporotic patients. Hence identification of novel factors and strategies for enhancing bone formation is currently an area of intense investigation.
Because MSCs are the common progenitors for both osteoblasts and adipocytes,(1–4) reductions in the number of osteoblastic cells in aging and osteoporosis have been attributed, in part, to an increased differentiation of these common progenitor cells into adipocytes rather than osteoblasts.(14) It has been observed that the numbers of adipocytes in the bone marrow increases in parallel with a decrease in the number of osteoblasts in a variety of types of osteoporosis.(21) Moreover, the volume of adipose tissue in bone increases with age in normal subjects and is substantially elevated in age-related osteoporosis,(22) with the number of adipocytes adjacent to bone trabeculae increasing in parallel to the degree of trabecular bone loss.(23) Based on this and similar observations, it has been suggested that bone loss in age-related osteoporosis is at least in part caused by a shift in MSC differentiation from the osteoblastic to the adipocytic pathway.(14,21) If this is true, it is possible that intervening in this apparent shift in lineage-specific differentiation of MSCs could increase the number of cells capable of undergoing differentiation into functioning osteoblasts.(21)
Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in the circulation and in human and animal tissues.(24–26) These compounds may be formed either by auto-oxidation, as a secondary byproduct of lipid peroxidation, or by the action of specific mono-oxygenases, most of which are members of the cytochrome P450 family of enzymes.(27) In addition, oxysterols may be derived from dietary intake.(28) Oxysterols have potent effects in physiologic and pathological processes including cholesterol metabolism, inflammation, apoptosis, steroid production, and atherosclerosis.(24-26,29) Moreover, because of the abundance of cholesterol in living organisms, the pro-oxidant nature of our cellular metabolic processes, and the multitude of enzymatic and non-enzymatic pathways for oxysterol production, we speculate that oxysterols may play additional, as yet unidentified, roles in biological systems. Recently, several reports have noted the possible role of oxysterols in cellular differentiation.(30–32) For example, the oxysterols 22(R)- and 25-hydroxycholesterol induce the differentiation of human keratinocytes in vitro,(30,31) whereas monocyte differentiation is induced by the oxysterol 7-ketocholesterol.(32)
We previously reported that products of the cholesterol biosynthetic pathway are important for the proper osteogenic differentiation and activity of MSCs.(33) Because oxysterols may be a derivative of the endogenous cellular cholesterol biosynthetic pathway,(25–27) we hypothesized that the oxysterols generated by osteoprogenitor cells, as well as those derived from exogenous sources, may be involved in their osteogenic differentiation. In this report, we present the first line of evidence for osteogenic activity of specific oxysterols and their ability to inhibit adipogenic differentiation. In addition, we show the synergistic interaction of these oxysterols with BMP2.
MATERIALS AND METHODS
Oxysterols, β-glycerophosphate (βGP), silver nitrate, and Oil red O were obtained from Sigma (St Louis, MO, USA); RPMI 1640, α-MEM, and DMEM were from Irvine Scientific (Santa Ana, CA, USA); and FBS was from Hyclone (Logan, UT, USA). PD98059 was purchased from BIOMOL Research Labs (Plymouth Meeting, PA, USA); TO-901317, SC-560, NS-398, Ibuprofen, and Flurbiprofen were from Cayman Chemical (Ann Arbor, MI, USA); ACA and AACOCF3 were from Calbiochem (La Jolla, CA, USA); and recombinant human BMP2 was from R&D Systems (Minneapolis, MN, USA). Antibodies to phosphorylated and native extracellular signal-regulated kinases (ERKs) were obtained from New England Biolabs (Beverly, MA, USA) and troglitazone was from Sankyo (Tokyo, Japan).
The M2-10B4 mouse marrow stromal cell line obtained from American Type Culture Collection (Rockville, MD, USA) was derived from bone marrow stromal cells of a (C57BL/6J × C3H/HeJ) F1 mouse and supports human and murine myelopoiesis in long-term cultures (as per ATCC). These cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS and supplemented with 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 U/ml streptomycin (all from Irvine Scientific). The MC3T3-E1 mouse preosteoblastic cell line was purchased from ATCC and cultured in α-MEM containing 10% heat-inactivated FBS and supplements as described above. C3H-10T1/2 mouse pluripotent embryonic fibroblast cells were kindly provided by Dr Kristina Bostrom (UCLA) and were cultured in DMEM containing 10% heat-inactivated FBS and supplements as described above. Primary mouse marrow stromal cells were isolated from male 4- to 6-month-old C57BL/6J mice and cultured and propagated as previously reported.(34)
Alkaline phosphatase activity assay
Colorimetric alkaline phosphatase (ALP) activity assay on whole cell extracts was performed as previously described.(34)
von Kossa and Oil red O staining
45Ca incorporation assay
Matrix mineralization in cell monolayers was quantified using the45Ca incorporation assay as previously described.(35)
Western blot analysis
RNA isolation and Northern blot analysis
After treatment of cells under appropriate experimental conditions, total RNA was isolated using the RNA isolation kit from Stratagene (La Jolla, CA, USA). Total RNA (10 mg) was run on a 1% agarose/formaldehyde gel and transferred to Duralon-UV membranes (Strategene) and cross-linked with ultraviolet light. The membranes were hybridized overnight at 60°C with a32P-labeled mouse osteocalcin cDNA probe,(36) mouse lipoprotein lipase (LPL), mouse adipocyte protein 2 (aP2) PCR-generated probes, and human 28S and 18S rRNA probes obtained from Geneka Biotechnology (Montreal, Quebec, Canada) and Maxim Biotech (San Francisco, CA, USA), respectively. The PCR products were generated using primer sets synthesized by Invitrogen (Carlsbad, CA, USA) with the following specifications: mouse aP2 gene (accession no. M13261); sense (75-95) 5′-CCAGGGAGAACCAAAGTTGA-3′, antisense (362-383) 5′-CAGCACTCACCCACTTCTTTC-3′, generating a PCR product of 309 bp. Mouse LPL (accession no. XM_134193); sense (1038-1058) 5′-GAATGAAGAAAACCCCAGCA-3′, antisense (1816-1836) 5′-TGGGCCATTAGATTCCTCAC-3′, generating a PCR product of 799 bp. The PCR products were gel-purified and sequenced by the UCLA sequencing core, showing the highest similarity to their respective GenBank entries. After hybridization, the blots were washed twice at room temperature with 2× SSC and 0.1%SDS and twice at 60°C with 0.5× SSC and 0.1% SDS and exposed to X-ray film. The extent of gene induction was determined by densitometry.
Conditioned medium from M2 cells cultured in 24-well plates was prepared after treatment with oxysterols or control buffer for 24 or 48 h, followed by rinsing the cells to remove the treatment media and replacing it with 0.5 ml RPMI containing 0.1% bovine serum albumin (BSA)/well. After 24 h, the conditioned medium was collected, centrifuged to remove debris, and used in the BMP2 ELISA immunoassay according to the manufacturer's instructions (R&D Systems).
Computer-assisted statistical analyses were performed using the StatView 4.5 program. All p values were calculated using ANOVA and Fisher's projected least significant difference (PLSD) significance test. A value of p < 0.05 was considered significant.
Novel osteogenic activities of oxysterols
To begin testing our hypothesis, we examined the effects of oxysterols on indices of osteoblastic differentiation in in vitro cultures of MSCs. In cultures of MSCs in vitro, stimulation of ALP activity, osteocalcin gene expression, and mineralization of cell colonies are indices of increased differentiation into osteoblast phenotype.(37,38) We found that specific oxysterols, namely 22(R)-hydroxycholesterol (22R), 20(S)-hydroxycholesterol (20S), and 22(S)-hydroxycholesterol (22S), induced ALP activity, an early marker of osteogenic differentiation, in pluripotent M2-10B4 murine MSCs (M2; Fig. 1A). This effect was specific, because other oxysterols, including 7-ketocholesterol (7K), did not induce ALP activity in these cells (Fig. 1A). The induction of ALP activity was both dose- and time-dependent at concentrations between 0.5 and 10 μM, and showed a relative potency of 20S > 22S > 22R. A 4-h exposure to these oxysterols followed by replacement with osteogenic medium without oxysterols was sufficient to induce ALP activity in M2 cells, measured after 4 days in culture.
Although induction of ALP activity is an early marker of osteogenic differentiation and plays an important role in both the differentiation and eventual mineralization processes,(39) this response was not sufficient for the induction of mineralization in M2 cells by individual oxysterols. Individual oxysterols (22R, 20S, and 22S) at concentrations between 0.5 and 10 μM were unable to induce mineralization after as many as 14 days of exposure, despite their ability to cause large increases in ALP activity measured 4 days after treatment (data not shown). The individual oxysterols also had minimal to no effect on osteocalcin gene expression after as many as 14 days of treatment (data not shown). However, ALP activity (Fig. 1B), robust mineralization (Figs. 1C and 1D), and osteocalcin gene expression (Figs. 1E and 1F) were all induced in M2 cultures by a combination of the 22R + 20S or 22S + 20S oxysterols. Other combinations of oxysterols including 22R + 22S, or combinations of 22R or 22S with 7K, did not induce mineralization in M2 cell cultures (data not shown). The combination of 20S with either 22R or 22S also produced osteogenic effects in the mouse pluripotent embryonic fibroblast C3H10T1/2 cells (Fig. 1G), in murine calvarial pre-osteoblastic MC3T3-E1 cells, and in primary mouse MSCs (Fig. 1H), as assessed by stimulation of ALP activity and mineralization. Thus, we concluded that the combination of 20S with either S or R stereoisomers of 22-hydroxycholesterol has osteogenic effects on osteoblast precursor cells. Although stimulation of MSCs by BMP2 can enhance their osteogenic differentiation,(37) the osteogenic effects of the oxysterols were not caused by the induction of BMP2 expression in M2 cells. RT-PCR analysis of BMP2 mRNA expression in M2 cells treated for 4 or 8 days with 22R + 20S oxysterols (5 μM) showed no induction by oxysterol treatment (data not shown). In addition, ELISA assay using conditioned media from M2 cells treated with 22R + 20S (5 μM) for 24 and 48 h also did not show any induction of BMP2 protein expression (data not shown). Interestingly, the BMP inhibitor noggin (Ng) at 200 ng/ml caused a 40% inhibition in 22R + 20S (RS, 2.5 μM)-induced ALP activity and a 90% inhibition of that induced by rhBMP2 (100 ng/ml; Control = 4 ± 2; RS = 75 ± 7; RS + Ng = 42 ± 5; BMP2 = 120 ± 11; BMP2 + Ng = 10 ± 2 activity units/mg protein; p < 0.05 for Control versus RS and BMP2 and for RS and BMP2 in the presence and absence of Ng). We speculate that in light of our RT-PCR and ELISA data that did not show any induction of BMP2 by oxysterols, the inhibition of response to oxysterols by noggin might be caused by inhibition of synergism between oxysterols and BMP2 present in FBS. Alternatively, oxysterols may induce the expression of other members of the BMP family that can interact with noggin, such as BMP4 and BMP7, the expression of which would not be detected by our BMP2-specific ELISA assay.
Synergistic osteogenic effects of oxysterols with BMP2
As with other osteoprogenitor cells, BMP2 is able to induce osteoblastic differentiation of M2 cells in vitro.(40) Interestingly, we found that osteogenic combination of 22R + 20S oxysterols acted synergistically with BMP2 in inducing ALP activity (Fig. 2A), osteocalcin mRNA expression (Figs. 2C and 2D), and mineralization by M2 cells (Fig. 2B). Although synergism in stimulating ALP activity was found when individual oxysterols 20S, 22S, and 22R were added with BMP2, synergy in induction of mineralization was only produced when oxysterols were added in combinations of 22R + 20S or 22S + 20S oxysterols with BMP2.
Novel anti-adipogenic activities of oxysterols
Adipogenesis of adipocyte progenitors including MSC is regulated by the transcription factor peroxisome proliferator activated receptor γ (PPARγ), which on activation by ligand-binding, regulates transcription of adipocyte-specific genes.(41) We previously reported that, as expected with MSCs, M2 cells have the ability to undergo adipogenic differentiation in response to the PPARγ activator, Troglitazone (Tro).(34) In M2 cells treated with Tro to induce adipogenesis, 20S, 22S, and 22R, alone or in combination, inhibited adipogenesis (Figs. 3A and 3B). The relative anti-adipogenic potency of these oxysterols was similar to their relative potency in stimulating ALP activity in M2 cells, with 20S ≥ 22S > 22R. Similar to its lack of osteogenic effect, 7K was also unable to inhibit adipogenesis in M2 cells (data not shown). Inhibition of adipogenesis was also assessed by an inhibition of the expression of the adipogenic genes LPL and aP2 by 20S (Figs. 3C and 3D). However, addition of the oxysterols to already formed adipocytes in M2 cell cultures did not reduce the number of adipocytes after 8 days of treatment (data not shown), suggesting that the oxysterols were active only at the early stages of adipogenesis. Inhibitory effects of these three oxysterols on adipogenesis were also demonstrated using C3H10T1/2 and primary mouse MSC, in which adipogenesis was induced either by Tro or a standard adipogenic cocktail consisting of dexamethasone and isobutylmethylxanthine (data not shown).
Mechanism of osteogenic activity of oxysterols
Mesenchymal cell differentiation into osteoblasts is regulated by cyclo-oxygenase (COX) activity.(42–44) We examined the possible role of COX in mediating the osteogenic effects of oxysterols on M2 cells. In the presence of FBS, which corresponds to our experimental conditions, M2 cells in culture express both COX-1 and COX-2 mRNA at all stages of osteogenic differentiation (data not shown). Consistent with the role of COX in osteogenesis, our studies showed that the COX-1 selective inhibitor SC-560, at 1-20 μM, significantly inhibited the osteogenic effects of the 22R + 20S and 22S + 20S oxysterol combinations. SC-560 inhibited oxysterol-induced ALP activity (Fig. 4A), mineralization (Fig. 4B), and osteocalcin gene expression (Figs. 4C and 4D). Although less effective than SC-560, the nonselective COX inhibitors, ibuprofen and flurbiprofen, at nontoxic doses of 1-10 μM, also significantly inhibited the osteogenic effects of 22R + 20S oxysterol combination by 25-30%. In contrast, the selective COX-2 inhibitor, NS-398, at the highest nontoxic dose of 20 μM, had only negligible inhibitory effects. SC-560 (10 μM) also inhibited the synergistic induction of ALP activity by oxysterols and BMP2 (Fig. 4G). Furthermore, the osteogenic effects of the oxysterol combination on ALP activity (Fig. 4E) and mineralization (Fig. 4F) were also inhibited by the general phospholipase A2 (PLA2) inhibitor ACA and by the selective cytosolic PLA2 inhibitor, AACOCF3 (AAC). Moreover, rescue experiments showed that the effects of the COX-1 and PLA2 inhibitors on oxysterol-induced ALP activity were reversed by the addition of 1 μM PGE2 (Fig. 4H) and 25 μM arachidonic acid (Fig. 4I), respectively.
The ERK pathway is another major signal transduction pathway previously associated with osteoblastic differentiation of osteoprogenitor cells.(45,46) Interestingly, the 20S oxysterol used alone or in combination with 22R oxysterol caused a sustained activation of ERK1 and ERK2 in M2 cells (Figs. 5A and 5B). Inhibition of the ERK pathway by the inhibitor PD98059 inhibited oxysterol-induced mineralization (Fig. 5C) but not ALP activity or osteocalcin mRNA expression in M2 cell cultures (data not shown). These results suggest that sustained activation of ERK is important in regulating certain specific, but not all, osteogenic effects of oxysterols.
Liver X receptors (LXR) are nuclear hormone receptors that, in part, mediate certain cellular responses to oxysterols, including 22R and 20S, but not 22S.(47,48) LXRα is expressed in a tissue-specific manner, whereas LXRβ is ubiquitously expressed.(47,48) By Northern blot analysis, we demonstrated the expression of LXRβ, but not LXRα, in confluent cultures of M2 cells (data not shown). To assess the possible role of LXR in mediating the effects of osteogenic oxysterols, we examined whether activation of LXRβ by the pharmacologic LXR ligand TO-901317 (TO) had effects similar to those exerted by 22R and 20S in M2 cells. Interestingly, in contrast to 22R and 20S, TO at 1-10 μM caused a dose-dependent inhibition of ALP activity in M2 cells (control [C]: 18 ± 2; ligands used at 10 μM: 22R = 45 ± 5; 20S = 140 ± 12; and TO = 3 ± 0.5 activity units/mg protein; p < 0.01 for C versus all ligands). Furthermore, TO treatment did not induce osteocalcin gene expression or mineralization after 10 days (data not shown). Therefore, the osteogenic effects of the oxysterols on M2 cells seem to be independent of the LXR receptor, as suggested by the potent osteogenic activity of the non-LXR oxysterol ligand 22S and the lack of osteogenic effects in response to the LXR ligand TO.
Mechanism of anti-adipogenic activity of oxysterols
As noted above, ERKs are important in the proliferation and osteogenic differentiation of MSCs. In addition to positively regulating the osteogenic activity of osteoblast progenitor cells, ERK activation also negatively regulates the adipogenic differentiation of adipocyte progenitor cells, and inhibition of ERK enhances adipogenic differentiation.(49–51) Consistent with this effect of ERK, inhibition of oxysterol-induced ERK activation by ERK pathway inhibitor PD98059 completely abolished the anti-adipogenic effects of 20S and 22S on Tro-induced adipogenesis in M2 cells (Fig. 5D). Interestingly, PD98059 potentiated the adipogenic effects of Tro (Fig. 5D), suggesting that, similar to the situation in pre-adipocytes,(51) spontaneous activity of ERK is inhibitory to the adipogenic differentiation of MSC. In contrast, the selective inhibitors for COX-1 and COX-2, SC-560, and NS-398, respectively, were not able to abolish the anti-adipogenic effects of the oxysterols (data not shown). These results suggested that the anti-adipogenic activity of oxysterols is mediated through activation of the ERK pathway.
This is the first demonstration of the ability of oxysterols to regulate lineage-specific differentiation of MSCs in favor of osteoblastic and against adipogenic differentiation. This effect of the oxysterols is in part mediated through COX/PLA2- and ERK-dependent mechanisms (Fig. 6). COX-1 and COX-2 are both present in osteoblastic cells and seem to be primarily involved in bone homeostasis and repair, respectively.(42,52) Metabolism of arachidonic acid into prostaglandins, including prostaglandin E2 (PGE2), by the COXs mediates the osteogenic effects of these enzymes.(53) COX products and BMP2 have complementary and additive osteogenic effects.(44) Activation of PLA2 releases arachidonic acid from cellular phospholipids and makes it available for further metabolism by COX enzymes into prostaglandins.(54,55) Consistent with previous reports of oxysterol-stimulated metabolism of arachidonic acid,(56,57) the present results suggest that the osteogenic activity of the oxysterols in MSC are in part mediated by the activation of PLA2-induced arachidonic acid release and its metabolism into osteogenic prostanoids by the COX pathway. Furthermore, COX activity also seems to be important in regulating the synergism between the osteogenic oxysterols and BMP2, but not in the anti-adipogenic effects of the oxysterols, suggesting the specific role of COX/PLA2 pathway in mediating the oxysterol-induced lineage specific differentiation of M2 cells into osteogenic cells.
These results also show another signaling pathway mediating M2 responses to oxysterols is the ERK pathway. Sustained activation of ERKs mediates the osteogenic differentiation of human MSCs,(45) and activation of ERKs in human osteoblastic cells results in upregulation of expression and DNA binding activity of Cbfa1, the master regulator of osteogenic differentiation.(46) Furthermore, ERK activation seems to be essential for growth, differentiation, and proper functioning of human osteoblastic cells.(58) In addition, the ERK pathway is a negative regulator of adipogenic differentiation of adipocyte progenitor cells.(49–51) Consistent with the role of the ERK pathway in both osteogenic and adipogenic differentiation, oxysterol-induced mineralization and inhibition of adipogenic differentiation were blocked by the inhibitor, PD98059, and the oxysterols induced a sustained activation of ERK. It must be noted that, because both individual and combinations of oxysterols activated ERK, whereas only the combinations of oxysterols were able to induce full osteogenic differentiation of M2 cells, ERK activation seems to be an essential but not sufficient step in mediating the osteogenic effects of the oxysterols.
We also show for the first time that the osteogenic oxysterols synergistically stimulate BMP2-induced osteogenic differentiation of MSCs. It is of interest that when BMPs were originally extracted from bone matrix, they were found to be associated with lipids.(59) These crudely characterized lipids potentiated the osteogenic effects of BMP, as evidenced by a great reduction in the bone forming activity of BMP on their removal.(59) This line of evidence and the previous demonstration of the presence of lipids in healing fracture callus(60) suggest that lipids such as oxysterols may play an important role in regulating osteogenesis and the effects of BMP on this process. These studies suggest that specific oxysterols may potentiate the osteogenic differentiation of osteoblast precursor cells and that this effect of oxysterols is in part mediated through a potentiation of the osteogenic activity of BMPs. Furthermore, the synergistic effects of osteogenic oxysterols with BMP2 may provide an exciting new strategy for using BMP2 in local stimulation of fracture healing. However, because bone turnover is regulated by a combination of osteoblastic bone formation and osteoclastic bone resorption, it is important to note that the putative in vivo osteogenic capacity of oxysterol combinations reported here will depend also on their effect on osteoclastic differentiation/activity and bone resorption.
The possibility of inducing lineage-specific differentiation of MSCs into designated cells and tissues has potential implications for the prevention and treatment of a number of disorders that result from age-related tissue deterioration or from genetic defects.(6) For example, as noted earlier, age-related osteoporosis seems to be, at least in part, caused by a decreased osteogenic differentiation of osteoprogenitor cells.(61–63) Accordingly, the focus of developing new therapeutic approaches that would positively impact osteoporosis has largely shifted from finding additional antiresorption agents to finding anabolic agents that can enhance bone formation.(15,16) The possible use of lipid-based treatments involving oxysterols, rather than protein- or peptide-based interventions as currently used, introduces a whole new strategy for creating therapeutics that would potentially intervene in osteoporosis. Further identification of the downstream targets of these molecules in MSCs holds great potential for discovering new and previously unsuspected ways to regulate the lineage-specific differentiation of key MSC populations. This could improve the potential for the use of autologous MSCs in treatment of a variety of connective tissue diseases, as well as in tissue engineering.(6) In addition, fat cells increase significantly in mesenchymal tissues with age, in parallel with a decline in osteoblasts, muscle cells, and other key derivatives of MSCs.(64) The basis of this apparent shift in MSC differentiation is unknown, but the ability to reverse this process could significantly affect the management of age-related disorders.
This work was supported by National Institute on Aging Pepper Center Grant IP60-AG10415 and National Institutes of Health Grant HL30568.
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