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Endocrinology. Author manuscript; available in PMC 2005 September 29. Published in final edited form as: Published online 2005 May 19. doi: 10.1210/en.2005-0303. | PMCID: PMC1237031 NIHMSID: NIHMS3613 |
BMP Regulation of Early Osteoblast Genes in Human Marrow Stromal Cells is Mediated by ERK and PI3-K Signaling Anna M Osyczka and Phoebe S Leboy Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104 Bone marrow stromal cells (MSC) are the major source of osteoblasts for bone remodeling and repair in postnatal animals. Rodent MSC cultured with bone morphogenetic proteins (BMPs) differentiate into osteoblasts, but most human MSC show a poor osteogenic response to BMPs. Here we demonstrate that BMP-induced osteogenesis in poorly responsive human MSC requires modulation of ERK and PI3-kinase pathways. Either treating human MSC cultures with the MEK inhibitor PD98059, or transferring them to serum-free medium with insulin or IGF-1, permits BMP-dependent increases in expression of the early osteoblast-associated genes, alkaline phosphatase and osteopontin. Increased expression of these genes in BMP-treated serum-free cultures correlates with increased nuclear levels of activated Smads, while serum-free cultures of human MSC expressing constitutively active MEK (caMEK) show decreased expression of early osteoblast genes and decreased nuclear translocation of BMP-activated Smads. Inhibiting ERK activity in human MSC also elevates expression of Msx2, a transcription factor which is directly regulated by Smad binding elements in its promoter. Therefore, growth factor stimulation leading to high levels of ERK activity in human MSC results in suppressed BMP-induced transcription of several early osteoblast genes, probably because levels of BMP-activated nuclear Smads are decreased. In contrast, inhibiting the insulin/IGF-1 activated PI3-K/AKT pathway decreases BMP-induced alkaline phosphatase and osteopontin expression in serum-free cultures of human MSC but increases BMP activation of Smads; thus, PI3-K signaling is required for BMP-induced expression of early osteoblast genes in human MSC either downstream or independent of the BMP-activated Smad signaling pathway. Keywords: marrow stromal cells, bone morphogenetic protein, insulin, osteogenesis, alkaline phosphatase, osteopontin, ERK, PI3-K Repair of adult bone fracture involves bone marrow-derived mesenchymal cells, which serve as a source of osteochondral progenitors that invade the fracture site, proliferate and differentiate into cartilage and bone. These mesenchymal progenitors (also known as mesenchymal stem cells or marrow stromal cells, MSC 1) can be isolated from bone marrow and cultured under conditions that promote their in vitro differentiation into specific mesenchymal phenotypes such as chondrocytes or osteoblasts ( 1; 2). To achieve the osteoblastic phenotype, progenitor cells need to express several bone-type extracellular matrix proteins, such as collagen type I, osteopontin (OP), bone sialoprotein and osteocalcin, and produce high levels of the ecto-enzyme tissue-nonspecific alkaline phosphatase (ALP). High-level expression of ALP is required for mineralization of skeletal tissues ( 3; 4), and is induced early during osteoblast differentiation ( 5; 6). In both animal and human MSC cultures, ALP and OP serve as useful markers of early osteogenesis and the expression of these genes usually increases by the end of the first week of culture. Among many transcription factors expressed early in osteogenesis, runx2 (cbfa1) is noteworthy because it is required for bone formation and is an important early indicator of osteogenic capacity of cells ( 7; 8). Bone morphogenetic proteins (BMPs) are osteoinductive growth factors that can induce bone formation both in vivo and in vitro ( 9– 11). A number of clinical studies have assessed the efficacy of recombinant human BMPs in the healing of critical-size bone defects and the acceleration of bone fracture healing in humans ( 9; 12). Although the latter studies have been promising, relatively high doses of BMPs were required to induce adequate bone formation compared to animal models, and large variations in response among individual patients were observed ( 13). In rodent cell cultures, BMPs have shown a potent osteogenic activity ( 14– 17), but their ability to induce osteogenesis of human cells is less clear ( 8; 18). Consistent with these studies, we have recently reported major differences in BMP response of rodent and human MSC cultures. While addition of BMPs to rat MSC cultures resulted in a marked stimulation of ALP mRNA and enzyme activity, 90% of human MSC cultures stimulated with BMP did not show elevated ALP, despite transcriptional regulation of several other BMP-responsive genes ( 19; 20). Jorgenson et al ( 21) have recently reported similar data indicating that BMP-treated human MSC do not show elevated ALP. We have now identified culture conditions in which BMP treatment of human MSC results in high ALP expression and profound stimulation of osteopontin. The classic BMP signaling pathway operates by activation of the Smad family of transcription factors, and there is evidence that it can also act through a Smad-independent p38 mitogen activated protein kinase (MAPK) signaling pathway ( 22). There have also been reports suggesting that other kinase pathways such as ERK, JNK, PI3-K, Wnt and NF-kB may substitute for, activate, or modulate BMP signaling ( 23; 24). Here we present evidence that, in BMP-unresponsive human MSC cultures, growth factor signaling leading to high and sustained activity of extracellular signal regulated kinase (ERK) negatively regulates BMP-mediated Smad signaling. Furthermore, human MSC require PI3-K/AKT activation to achieve BMP-stimulated high-level expression of several genes associated with the early stages of osteoblast differentiation. Chemicals Unless otherwise stated, all chemical reagents came from Sigma (St. Louis, MA) and tissue culture solutions from Gibco/Invitrogen Corporation (Grand Island, N.Y). ITS+ Premix, composed of insulin, transferrin, selenious acid, linoleic acid and bovine serum albumin, was from BD Biosciences (Bedford, MA). Ascorbate-2-phosphate was obtained from Wako Chemicals, (Richmond, VA), and ERK inhibitor UO126 from BIOMOL Research Laboratories (Plymouth Meeting, PA). Proteinase inhibitor cocktails were purchased from CALBIOCHEM (San Diego, CA). PI3-K inhibitor Ly294002, primary rabbit polyclonal antibodies against p44/42 MAPK (ERK), phospho-p44/42 MAPK (p-ERK), phospho-Smad 1,5,8, AKT and phospho-AKT were purchased from Cell Signaling Technology (Beverly, MA). Primary rabbit anti-human MADR1/Smad1 antibodies (recognizing both human Smad1 and 5) were obtained from Upstate Biotechnology (Lake Placid, NY). Recombinant human bone morphogenetic protein type 2 (BMP-2) was kindly provided by Wyeth/ Genetics Institute (Cambridge, MA, USA). Adenovirus containing constitutively active MEK1 ( caMEK) was a gift from Dr. Janet Rubin, Emory University ( 25). Cell culture, inhibitor studies and adenoviral infections Human marrow samples were isolated from the proximal medullary cavities of femurs from patients undergoing total hip replacement. Primary cultures were established as described previously ( 19; 20) with a seeding density of 5x10 5cells/cm 2. Cells were grown in α-MEM supplemented with 10% pre-tested fetal bovine serum (FBS; premium select, Atlanta Biologicals, Oakbrook Drive, Norcross, GA), and antibiotics at 37°C in a humidified, 5% CO 2 atmosphere. Medium was changed initially at day 4, then every other day thereafter. At confluence, cells were detached using 0.25% trypsin in 1mM tetrasodium EDTA, centrifuged, re-suspended in fresh α -MEM/10% FBS and plated at 10 4cells /cm 2. All assays were carried out on first or second passage cell cultures. One day after plating the medium was exchanged for fresh medium supplemented with 0.35mM L-ascorbic acid-2-phosphate (ascorbate), 10−7M dexamethasone (Dex) or 100ng/ml BMP-2 as noted. At day 4, cells were washed with HBSS and medium was exchanged to one of the following: a) fresh α-MEM containing 10% FBS (SERUM; SC); b) α-MEM supplemented with either ITS+ Premix, insulin at 1.1 μM or IGF-I at 13. 1 nM (SF+I) or c) α-MEM supplemented only with 1.25mg/ml bovine serum albumin (SF−); osteogenic supplements were then replaced. Media with 10% FBS was estimated to contain 3-6nM insulin and, based on values in the literature, an additional 2nM IGF-I. ERK inhibitors PD98059 or UO126, PI3-K inhibitor Ly294002, and cycloheximide, or respective vehicle solution were added at day 4. Inhibitors were added 1-2h before addition of ascorbate, BMP-2 or Dex. For infections with adeno-caMEK or empty adenovirus, cells were switched to SF+I on day 4, infected on day 5 and analyzed by real-time PCR on day 6. Cells were infected using a multiplicity of infection (MOI) of 10. At this MOI, the proportion of adenovirus-infected MSC is usually between 40–70% without adenovirus-mediated cell toxicity. Alkaline phosphatase (ALP) assay Immediately prior to cell harvest for ALP assay, numbers of viable cells were estimated using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI). Cells in 24-well culture plates were washed once with HBSS and covered with 200μl of a solution prepared as a 1:10(v/v) dilution of the reagent containing the tetrazolium salt MTS and the electron coupling reagent phenazine ethosulfate in phenol red-free a-MEM. Cells were incubated for 15 min at 37°C in a humidified, 5% CO 2 atmosphere, MTS-containing media from each well transferred to 96-well plates, and the absorbance measured at 490nm. After washing twice with HBSS, cells were lysed in 10% Triton X-100, and stored at 4°C until ALP assay. ALP activity was determined kinetically as described previously ( 14). Isolation of total RNA, cDNA synthesis and real-time PCR analysis Total RNA was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH). Aliquots of total RNA (2μg) were reverse transcribed and cDNA samples corresponding to 50–250ng input RNA were amplified as described previously ( 19). The reaction mixtures contained 2–3μM of forward and reverse primer and 3–4mM MgCl 2. Table 1 lists the primer sequences and annealing temperatures individually optimized for each primer pair. A final melt curve from 60–95°C was performed to confirm the specificity of the PCR reaction and the identity of PCR products was confirmed by gel electrophoresis. For calculating mRNA levels, results from real-time RT-PCR were converted to arbitrary units of mRNA assuming a concentration-dependent straight line for a semi-log plot, with a value of 3.5 for the fold change in mRNA/cycle (slope), and the crossing point cycle number with no cDNA template as an estimate of y-intercept. | TABLE 1Oligonucleotide primers for real-time RT-PCR analyses |
Western blot analyses Whole cell extracts were obtained by lysing cells in Cell Lysis Buffer provided by Cell Signaling Tech. (Beverly, MA). Nuclear extracts were obtained by scraping cells in TBS, centrifuging and re-suspending pellets in ice-cold buffer composed of 10mM HEPES (pH 8.0), 10mM KCl, 1mM EDTA (pH 8.0), 1mM EGTA (pH 8.0), 1 mM DDT, 2mM phenyl-methylsulfonyl fluoride (PMSF), 0.5mM sodium orthovanadate, 1mM tosyl-L-phenylalanine and 10mM ethylmaleimide. After 15min incubation on ice, NP-40 was added and nuclei pelleted by centrifugation at 16000g. The nuclear pellets were re-suspended in cold buffer composed of 20mM HEPES (pH 8.0), 1mM EDTA, 1mM EGTA, 0.4 M NaCl, 1mM DTT, 2mM PMSF, 0.5mM sodium orthovanadate, 0.1mM N-tosyl-L-phenylaline chloromethyl ketone and 10mM N-ethyl maleimide. Protein concentration was determined with the MicroBCA Protein Assay Reagent kit (Pierce, Rockford, IL). 40–80mg of protein from whole cell extract or 10–20mg of protein from nuclear extracts were separated on NuPAGE 4–12%Bis-Tris gels (Invitrogen Life Tech, Carlsbad, CA) under reducing conditions using MOPS buffer and then transferred to Immuno-Blot® PVDF membranes (Bio-Rad, Hercules, CA). Membranes were incubated overnight with primary antibodies diluted 1:1000 and then exposed for 2h to horseradish peroxidase-linked anti-rabbit IgG (Amersham Biosciences Corp., Piscataway, NJ) diluted 1:1250. The peroxidase-based signal was detected using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Science, Boston, MA) using a Kodak Image Station 440CF. BMP induction of ALP activity in human MSC cultures requires serum withdrawal and addition of insulin or IGF-1. We have previously reported that the majority of human MSC isolates show a relatively poor osteogenic response to BMPs in vitro in comparison to similar rodent MSC cultures ( 20; 26). We have therefore examined whether altering culture conditions of the poorly responsive human MSC would elicit a more robust osteogenic response to BMP-2. The effects of BMP-2 was compared in the presence and absence of serum, with and without 1.1μM insulin or 13.1nM IGF-1. As shown in , BMP-2 increased ALP activity at day 7 approximately 4 fold when human MSC were cultured for the last three days in serum-free medium with IGF-1 or insulin, but not when they were maintained in serum-containing medium. Decreasing the serum concentration to 2% did not have any beneficial effect on BMP-2 stimulation of ALP activity (data not shown). Human MSC cultured for up to 14 days in serum-containing medium failed to show BMP-induced ALP increase with either BMP-2, BMP-4 or BMP-7, and with several sources of fetal bovine serum (data not shown). Dose dependency studies showed that at 6–11nM, insulin and IGF-1 had similar effects on human MSC in serum-free cultures, enhancing BMP-stimulated ALP activity approximately 2-4 fold ( Table 2). Addition of either insulin or IGF-1 slightly increased number of cells in serum-free human MSC cultures, but adding BMP-2 to insulin or IGF-1-treated cultures decreased cell number 10-20% ( Table 3). The net effect of insulin or IGF-1 in combination with BMP-2 was to increase ALP levels without a significant increase in proliferation. Therefore, the effect of adding insulin or IGF-1 to BMP-treated cultures was to increase ALP activity/cell, rather than produce greater numbers of ALP positive cells. Similar data were obtained using serum-free medium supplement ITS+ Premix®, which contains 1.1μM insulin. Unless stated otherwise, further studies were performed using serum-free medium in which insulin was supplied as ITS+ Premix. | TABLE 2ALP activity of human MSC continuously cultured in serum or after transfer to serum-free medium with insulin or IGF-1 |
| TABLE 3Numbers of viable human MSC continuously cultured in serum or transferred for 3 days to serum-free medium with insulin or IGF-1. |
Switching human MSC cultures to SF+I promotes BMP-2 induction of alkaline phosphatase, osteopontin and Msx2 mRNAs Comparisons of gene expression in 7-day cultures grown for the last 3 days in SF+I or continuously cultured in serum-containing medium are presented in . Transfer to SF+I had a marked effect on BMP-2 stimulation of genes expressed early in osteoblast differentiation. ALP mRNA, which was unaffected by BMP treatment in the presence of serum, was increased by BMP-2 if cultures were grown for the last 3 days in SF+I. Previous studies ( 26) demonstrated that serum-containing cultures of human MSC showed BMP-induced increases in mRNA for the matrix protein osteopontin and the transcription factor Msx2. Studies with cyclohexamide treatment of human MSC cultures indicated that new protein synthesis was required for BMP-2 induction of ALP and osteopontin mRNAs, but not for Msx2 mRNA (data not shown). This is consistent with the assumption that BMP induction of Msx2 expression in human MSC is a direct result of Smad activation. In the present work, both osteopontin and Msx2 mRNA levels were modestly stimulated with BMP-2 in serum-containing MSC cultures, but BMP-2 had a far greater effect if cells were switched to SF+I. Analyses at increasing times after transfer to SF+I revealed that levels of ALP mRNA further increased with longer exposure to SF+I and that at least 10 h of exposure to BMP-2 was required to see enhanced ALP mRNA expression in SF+I. This relatively long exposure time is consistent with evidence from cyclohexamide studies that BMP does not regulate ALP simply by binding of BMP-activated Smads to a regulatory region of the ALP gene. Analyses of mRNA levels of other early osteoblast genes revealed that changing from serum-containing to serum-free conditions did not affect collagen type I mRNA expression (data not shown), while levels of Runx-2 mRNA were increased 2-3 fold by BMP-2, both in serum-containing and SF+I cultures. Analyses of late differentiation markers indicated that bone sialoprotein mRNA was increased by BMP-2 in serum-containing cultures, but transfer to SF+I medium significantly decreased BSP mRNA expression with and without inducers. BMP-2 treatment did not alter osteocalcin mRNA levels regardless of whether serum was present or not; however, it should be noted that vitamin D3 was not added ( 27). Dexamethasone (DEX), which increased ALP expression of human MSC cultured in serum, did not increase ALP mRNA in SF+I conditions. High ERK activity negatively regulates BMP-2 stimulation of ALP, osteopontin and Msx2 expression Because serum-free medium lacks growth factors that activate the extracellular signal regulated kinase (ERK) pathway, we examined whether sustained activation of ERK would prevent BMP stimulation of gene expression in serum-free insulin-containing (SF+I) cultures of human MSC, using a constitutively active MEK1 (caMEK) adenoviral construct that phosphorylates ERK. As shown in , expressing caMEK in SF+I cultures markedly decreased ALP, osteopontin and Msx2 mRNA levels. Conversely, inhibiting ERK signaling with 50μM PD98059 significantly increased ALP, osteopontin and Msx2 mRNA expression in BMP-stimulated serum-containing cultures (). Although addition of insulin or IGF-1 to serum-containing human MSC cultures did not permit BMP stimulation of ALP (), adding either insulin or IGF-1 in the presence of 50μM PD98059 slightly enhanced BMP induction of ALP activity in these cultures (data not shown). Western blot assays for phosphorylated ERK, using whole cell extracts harvested at 5,15,45 and 90 min, showed that ERK activation peaked 15 minutes after media change in both serum-containing and serum-free culture conditions. At this time point, ERK activation was the highest in human MSC cultured in serum-containing medium without ERK inhibitor (), and BMP had no significant effect on ERK activation in these cultures. In serum-free cultures, addition of either insulin or BMP-2 caused a slight increase in ERK activation at 15 minutes, but the levels of phosphorylated ERK in these culture conditions were markedly lower than those in serum-containing cultures. Similar results were obtained using an in vitro kinase assay to measure 32P incorporation into the ERK substrate Elk-1 (data not shown). Human MSC with low ERK activity have high levels of phospho-Smad 1,5,8 and greater Smad nuclear localization To obtain further insights into whether serum-free culture conditions promote BMP-mediated Smad signaling, Smad 1,5,8 activation was analyzed in whole cell and nuclear extracts using a polyclonal antibody that recognizes Smad 1, 5 and 8 phosphorylated at the C-terminal BMP activation domain. Western blot analysis of whole cell extracts showed that BMP-2 increased phosphorylation of Smad 1,5,8 in both serum-containing and serum-free culture conditions, but the activation of BMP-regulated Smads was markedly greater in serum-free cultures (). Switching cells to serum-free conditions also increased levels of activated Smads 1,5,8 in the nucleus (), but adding insulin had no further effect on amounts of activated Smads. These results suggested that Smad activation was promoted by serum removal and the insulin/IGF-1 requirement was unrelated to Smad activation. Increasing ERK signaling in SF+I cultures by over-expressing caMEK did not affect the whole cell levels of activated Smads (), but decreased the levels of activated Smads in the nucleus (). PI3-kinase activation is necessary for expression of early osteoblast genes, but not Msx2 The finding that insulin or IGF-1 was necessary for BMP induction of ALP in serum-free cultures suggested that the PI3-K/AKT pathway might be implicated in BMP-stimulated osteogenesis. The PI3-K inhibitor Ly294002, at 5-10μM, decreased both BMP-stimulated ALP activity and ALP mRNA expression in SF+I cultures (). A similar inhibitory effect of Ly294002 was seen for BMP-stimulated OP mRNA expression (), but not for Msx2 mRNA expression (). The efficacy of the Ly294002 inhibitor was confirmed by Western blot analysis of AKT phosphorylation, which is mediated by PI3-K (). To determine whether PI3-K inhibitor affects BMP-mediated Smad signaling in SF+I cultures, activation of Smad 1,5,8 was analyzed in whole cell extracts from SF+I cultures treated with Ly294002. As shown in , activation of Smad 1,5,8 increased with BMP-2 and was further elevated when Ly29004 inhibitor was added. We have previously reported differences in the BMP-mediated osteogenic response of human and rodent bone marrow-derived mesenchymal stem cells ( 19; 20; 26). Among 26 bone marrow cell samples obtained from the femoral cavity of adult human patients, 90% of isolates failed to increase alkaline phosphatase expression in response to 100ng/ml BMP-2. In contrast, rat and mouse MSC cultures significantly increase expression of ALP and other osteoblast-related genes with as little as 30 ng/ml of BMP-2 ( 5; 6; 17; 26; 28). Here we show that these poorly responsive human MSC markedly elevate both ALP and osteopontin expression when cultures are stimulated with BMP-2 in the presence of ERK inhibitor or in serum-free medium containing either insulin or IGF-1. Our examination of signaling pathways implicated in BMP regulation of human MSC osteogenesis indicates not only a negative role of ERK but also a positive role of PI3-K/AKT signaling in the regulation of ALP and osteopontin. Analyses of other osteoblast-related markers suggest that these kinase pathways may modulate transcription of some genes expressed early in osteoblast differentiation without affecting other osteoblast phenotypic markers. This is consistent with the assumption that osteoblast differentiation is not regulated by a single set of factors coordinately controlling all osteoblast genes and that it is not necessary for progenitor cells to express all genes seen early in osteoblast differentiation in order to up-regulate some late osteoblast markers ( 29). The role of ERK activation in osteoblast differentiation has been the subject of many studies, but no clear picture has emerged. In BMP-treated human and mouse osteoblastic cells, ERK activation is required for increased expression of the late osteoblast gene, osteocalcin ( 23; 30); however, activated ERK decreases ALP expression ( 31– 33). This is consistent with our results indicating that high levels of ERK activation have a negative effect on BMP induction of early osteoblast genes in human MSC. In C3H10T1/2 cells, an embryonic murine cell line frequently considered equivalent to MSC, BMP stimulates both ERK expression and ERK activity, and reducing ERK activation decreased BMP-induced ALP activity ( 24). In contrast, our studies with human MSC showed no significant BMP-2 effect on total ERK levels either in the presence of serum or after transfer of cultures to SF+I. In serum-free cultures BMP-2, insulin, or both, slightly increased phosphorylation of ERK but the major factor increasing ERK phosphorylation was the presence of serum. These results, combined with the observed effects of caMEK in serum-free cultures and ERK inhibitors in the presence of serum, argue that in human MSC lower levels of activated ERK are associated with improved BMP-2 stimulation of ALP and OP expression. A positive effect of ERK activation on osteoblast differentiation has been observed with studies using other osteogenic stimuli. In dexamethasone-treated human MSC cultures, there is sustained ERK activation throughout osteogenic differentiation ( 34). Oscillatory fluid flow increases osteopontin mRNA in MC3T3-E1 cells, and this stimulation is abolished with ERK inhibitors, suggesting that ERK activation plays a positive role in osteopontin induction ( 35). These reports suggest that the negative effect ERK activation has on expression of early osteoblast genes in human MSC may be limited to osteogenesis induced by BMP. In epithelial cells over-expressing Smad1, ERK directly regulates BMP-activated Smads by phosphorylation at serine residues within the Smad linker region; this phosphorylation inhibited both nuclear accumulation of Smad1 and its transcriptional activity ( 36). Recent evidence indicates that linker phosphorylation of Smad1 is important in vivo ( 37). Several other Smads can be phosphorylated by ERK in the linker region, and whether it results in positive or negative consequences for Smad activity appears to be cell-specific ( 38; 39). Using a phospho-Smad 1,5,8 antibody that detects only BMP-regulated phosphorylation, we have shown that increased ERK activation resulted in decreased nuclear levels of BMP-activated Smads. It is therefore plausible that, in human MSC, ERK phosphorylation of BMP-related Smads in the linker region limits the ability of BMP-activated Smads to function as transcription factors. This hypothesis is supported by the increased Msx2 mRNA levels seen with BMP-treated human MSC cultured either in serum-free conditions or with ERK inhibitors in serum. BMP-2 has been shown to directly regulate Msx2 expression by Smad binding to its promoter ( 40; 41), and we have found that BMP stimulation of Msx2 in human MSC does not require new protein synthesis. The fact that BMPs directly regulate the Msx2 promoter by activated Smads, together with our observation that BMP-stimulated Msx2 expression is modulated by ERK activation, implies that levels of activated ERK are modulating BMP-activated Smad function in human MSC. In contrast to ERK, activation of the PI3-K/AKT pathway by insulin or IGF-1 seems to positively regulate BMP-2 induced ALP and OP in human MSC cultures. Both insulin and IGF-1 are highly specific for their respective receptors, and 100 fold excess of the incorrect ligand is required to displace the correct one ( 42). The fact that either insulin or IGF-1, at roughly equimolar concentrations, is capable of permitting a BMP response implies that activation of PI3-K signaling by either ligand is sufficient. This assumption is reinforced by the data that blocking PI3-K signaling abolishes the BMP osteogenic response. These results support and extend previous data that PI3-K and AKT serine/threonine kinase are required for BMP-induced osteogenesis in 2T3 cells ( 43) and fetal rat calvaria cells ( 44). Blocking PI3-K/AKT signaling has been reported to also inhibit Runx2-induced enhancement of ALP activity and mineralization in several murine osteogenic cell lines ( 45). It is therefore likely that the insulin or IGF-1 stimulated PI3-K pathway is important for osteoblast differentiation regardless of the source of osteogenic cells, and that the levels present in FBS can activate the PI3-K pathway in serum-containing cultures of human MSC. Our results are consistent with the hypothesis that PI3-K activation is needed to regulate ALP and ostepontin expression in human MSC at a site downstream or independent of BMP-mediated Smads. This would be in contrast to studies using the murine BMP-responsive osteogenic cell line 2T3, which suggest a positive role for PI3-K/AKT in Smad signaling, with dominant-negative AKT abrogating Smad-dependent transcription of BMP-2.( 43) It is not obvious why the PI3-K inhibitor increased Smad activation but did not increase Msx-2 mRNA, a gene directly regulated by BMP-activated Smads. It may be that the rate of Msx-2 transcription was already maximal without the inhibitor, or that the increased Smad activation was too transient to affect Msx2 levels 24h later. Our data indicating a negative role of ERK signaling and requirement of insulin/IGF-1 signaling should prove useful in understanding the limitations in BMP-based therapies with human MSC ( 12; 46). They also suggest that human MSC may differ from several commonly studied osteogenic cell lines and rodent MSC culture models in their response to BMPs and protein kinase signaling. It is also noteworthy that MSC from human, rat, and mouse, when cultured in standard serum-containing medium, are dissimilar in their response to osteogenic inducers. In rat MSC, ALP is induced with either BMPs or dexamethasone ( 14; 26), in mouse MSC it is induced with BMPs but not dexamethasone ( 47; 48), and in most isolates of human MSC ALP is induced with dexamethasone but not BMPs ( 19; 20). These observations, together with our new findings on BMP regulation of early osteoblast genes in human MSC cultures, imply that generalizing about mechanisms of osteoblast differentiation should be done with great caution. We are grateful to Wyeth/Genetics Institute for providing rhBMP2, and Dr. Janet Rubin, Emory University, for her gift of adenovirus containing caMEK. We also thank Andrew Chen, Lauran Madden, Geeta Bhargave, and Eleanor Golden for their excellent technical assistance, and Drs. David L. Diefenderfer and Jonathan Garino for providing human bone marrow cells. 1. Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:11917–11923. [PubMed] 2. Phinney DG. Building a consensus regarding the nature and origin of mesenchymal stem cells. Journal of Cellular Biochemistry. 2002:7–12. [PubMed] 3. Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa S, Millán JL, MacGregor GR, Whyte MP. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. Journal of Bone and Mineral Research. 1999;14:2015–2026. [PubMed] 4. Anderson HC, Sipe JB, Hessle L, Dhamyamraju R, Atti E, Camacho NP, Millán JL. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. American Journal of Pathology. 2004;164:841–847. [PubMed] 5. Rickard DJ, Kassem M, Hefferan TE, Sarkar G, Spelsberg TC, Riggs BL. Isolation and characterization of osteoblast precursor cells from human bone marrow. Journal of Bone and Mineral Research. 1996;11:312–324. [PubMed] 6. Thies RS, Bauduy M, Ashton BA, Kurtzberg L, Wozney JM, Rosen V. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology. 1992;130:1318–1324. [PubMed] 7. Ducy P. Cbfa1: a molecular switch in osteoblast biology. Developmental Dynamics. 2000;219:461–471. [PubMed] 8. Gori F, Thomas T, Hicok KC, Spelsberg TC, Riggs BL. Differentiation of human marrow stromal precursor cells: BMP-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation. Journal of Bone and Mineral Research. 1999;14:1522–1535. [PubMed] 9. Valentin-Opran A, Wozney J, Csimma C, Lilly L, Riedel GE. Clinical evaluation of recombinant human bone morphogenetic protein-2. Clinical . Orthopaedics and Related Research. 2002:110–120. 10. Schmitt JM, Hwang K, Winn SR, Hollinger JO. Bone morphogenetic proteins: An update on basic biology and clinical relevance. Journal of Orthopaedic Research. 1999;17:269–278. [PubMed] 11. Li RH, Wozney JM. Delivering on the promise of bone morphogenetic proteins. Trends in Biotechnology. 2001;19:255–265. [PubMed] 12. Govender S, Csimma C, Genant HK, Valentin-Opran A, BESTT SG. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures - A prospective, controlled, randomized study of four hundred and fifty patients. Journal of Bone and Joint Surgery American Volume. 2002;84A:2123–2134. 13. Groeneveld EH, Burger EH. Bone morphogenetic proteins in human bone regeneration. European Journal of Endocrinology. 2000;142:9–21. [PubMed] 14. Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev Biol. 1994;161:218–228. [PubMed] 15. Yamaguchi A, Ishizuya T, Kintou N, Wada Y, Katagiri T, Wozney JM, Rosen V, Yoshiki S. Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochemical and Biophysical Research Communications. 1996;220:366–371. [PubMed] 16. Abe E, Yamamoto M, Taguchi Y, Lecka-Czernik B, O’Brien CA, Economides AN, Stahl N, Jilka RL, Manolagas SC. Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: Antagonism by noggin. Journal of Bone and Mineral Research. 2000;15:663–673. [PubMed] 17. Hanada K, Dennis JE, Caplan AI. Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Journal of Bone and Mineral Research. 1997;12:1606–1614. [PubMed] 18. Lecanda F, Avioli LV, Cheng SL. Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. Journal of Cellular Biochemistry. 1997;67:386–398. [PubMed] 19. Diefenderfer DL, Osyczka AM, Reilly GC, Leboy PS. BMP responsiveness in human mesenchymal stem cells. Connective Tissue Research. 2003;44:305–311. [PubMed] 20. Diefenderfer DL, Osyczka AM, Garino JP, Leboy PS. Regulation of BMP-induced transcription in cultured human bone marrow stromal cells. Journal of Bone and Joint Surgery American Volume. 2003;85A:19–29. 21. Jorgensen NR, Henriksen Z, Sorensen OH, Civitelli R. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids. 2004;69:219–226. [PubMed] 22. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature. 2003;425:577–584. [PubMed] 23. Xiao GZ, Gopalakrishnan R, Jiang D, Reith E, Benson MD, Franceschi RT. Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. Journal of Bone and Mineral Research. 2002;17:101–110. [PubMed] 24. Lou JR, Tu Y, Li S, Manske PR. Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2. Biochemical and Biophysical Research Communications. 2000;268:757–762. [PubMed] 25. Rubin J, Murphy TC, Zhu L, Roy E, Nanes MS, Fan X. Mechanical Strain Differentially Regulates Endothelial Nitric-oxide Synthase and Receptor Activator of Nuclear {kappa}B Ligand Expression via ERK1/2 MAPK. Journal of Biological Chemistry. 2003;278:34018–34025. [PubMed] 26. Osyczka AM, Diefenderfer DL, Bhargave G, Leboy PS. Different effects of BMP-2 on marrow stromal cells from human and rat bone. Cells Tissues Organs. 2004;176:109–119. [PubMed] 27. Thomas GP, Bourne A, Eisman JA, Gardiner EM. Species-divergent regulation of human and mouse osteocalcin genes by calciotropic hormones. Experimental Cell Research. 2000;258:395–402. [PubMed] 28. Pereira RC, Delany AM, Canalis E. Effects of cortisol and bone morphogenetic protein-2 on stromal cell differentiation: Correlation with CCAAT-enhancer binding protein expression. Bone. 2002;30:685–691. [PubMed] 29. Liu F, Malaval L, Aubin JE. Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation. Journal of Cell Science. 2003;116:1787–1796. [PubMed] 30. Suzawa M, Tamura Y, Fukumoto S, Miyazono K, Fujita T, Kato S, Takeuchi Y. Stimulation of Smad1 transcriptional activity by Ras-extracellular signal-regulated kinase pathway: A possible mechanism for collagen-dependent osteoblastic differentiation. Journal of Bone and Mineral Research. 2002;17:240–248. [PubMed] 31. Higuchi C, Myoui A, Hashimoto N, Kuriyama K, Yoshioka K, Yoshikawa H, Itoh K. Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix. Journal of Bone and Mineral Research. 2002;17:1785–1794. [PubMed] 32. Lai CF, Cheng SL. Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-β in normal human osteoblastic cells. Journal of Biological Chemistry. 2002;277:15514–15522. [PubMed] 33. Rodríguez JP, Ríos S, Fernández M, Santibañez JF. Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women. Journal of Cellular Biochemistry. 2004;92:745–754. [PubMed] 34. Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. Journal of Biological Chemistry. 2000;275:9645–9652. [PubMed] 35. You J, Reilly GC, Zhen XC, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR. Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts. Journal of Biological Chemistry. 2001;276:13365–13371. [PubMed] 36. Kretzschmar M, Doody J, Massagué J. Opposing BMP and EGF signalling pathways converge on the TGF-β family mediator Smad1. Nature. 1997;389:618–622. [PubMed] 37. Aubin J, Davy A, Soriano P. In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis. Genes and Development. 2004;18:1482–1494. [PubMed] 38. Funaba M, Zimmerman CM, Mathews LS. Modulation of Smad2-mediated signaling by extracellular signal-regulated kinase. Journal of Biological Chemistry. 2002;277:41361–41368. [PubMed] 39. Hayashida T, deCaestecker M, Schnaper HW 2003 Cross-talk between ERK MAP kinase and Smad-signaling pathways enhances TGF-β dependent responses in human mesangial cells. The FASEB Journal03-0037fje. 40. Hussein SM, Duff EK, Sirard C. Smad4 and β-catenin co-activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2. Journal of Biological Chemistry. 2003;278:48805–48814. [PubMed] 41. Brugger SM, Merrill AE, Torres-Vazquez J, Wu N, Ting MC, Cho JYM, Dobias SL, Yi SE, Lyons K, Bell JR, Arora K, Warrior R, Maxson R. A phylogenetically conserved cis-regulatory module in the Msx2 promoter is sufficient for BMP-dependent transcription in murine and Drosophila embryos. Development. 2004;131:5153–5165. [PubMed] 42. Gutierrez J, Parrizas M, Maestro MA, Navarro I, Plisetskaya EM. Insulin and IGF-I binding and tyrosine kinase activity in fish heart. J Endocrinol. 1995;146:35–44. [PubMed] 43. Ghosh-Choudhury N, Abboud SL, Nishimura R, Celeste A, Mahimainathan L, Choudhury GG. Requirement of BMP-2-induced phosphatidylinositol 3-kinase and Akt serine/threonine kinase in osteoblast differentiation and Smad-dependent BMP-2 gene transcription. Journal of Biological Chemistry. 2002;277:33361–33368. [PubMed] 44. Shoba LNN, Lee JC. Inhibition of phosphatidylinositol 3-kinase and p70S6 kinase blocks osteogenic protein-1 induction of alkaline phosphatase activity in fetal rat calvaria cells. Journal of Cellular Biochemistry. 2003;88:1247–1255. [PubMed] 45. Fujita T, Azuma Y, Fukuyama R, Hattori Y, Yoshida C, Koida M, Ogita K, Komori T. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. Journal of Cell Biology. 2004;166:85–95. [PubMed] 46. Einhorn TA. Clinical applications of recombinant human BMPs: Early experience and future development. Journal of Bone and Joint Surgery American Volume. 2003;85A:82–88. 47. Chen TL. Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment. Bone. 2004;35:83–95. [PubMed] 48. Bellows CG, Ciaccia A, Heersche JN. Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone. Bone. 1998;23:119–125. [PubMed]
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