BMP-2/4 and BMP-6/7 Differentially Utilize Cell Surface Receptors to Induce Osteoblastic Differentiation of Human Bone Marrow-derived Mesenchymal Stem Cells
2008; Elsevier BV; Volume: 283; Issue: 30 Linguagem: Inglês
10.1074/jbc.m800850200
ISSN1083-351X
AutoresKaren Lavery, Pamela Swain, Dean Falb, Moulay Hicham Alaoui-Ismaili,
Tópico(s)Connective Tissue Growth Factor Research
ResumoBone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily of growth factors and are used clinically to induce new bone formation. The purpose of this study was to evaluate receptor utilization by BMP-2, BMP-4, BMP-6, and BMP-7 in primary human mesenchymal stem cells (hMSC), a physiologically relevant cell type that probably mediates the in vivo effects of BMPs. RNA interference-mediated gene knockdown revealed that osteoinductive BMP activities in hMSC are elicited through the type I receptors ACVR1A and BMPR1A and the type II receptors ACVR2A and BMPR2. BMPR1B and ACVR2B were expressed at low levels and were not found to play a significant role in signaling by any of the BMPs evaluated in this study. Type II receptor utilization differed significantly between BMP-2/4 and BMP-6/7. A greater reliance on BMPR2 was observed for BMP-2/4 relative to BMP-6/7, whereas ACVR2A was more critical to signaling by BMP-6/7 than BMP-2/4. Significant differences were also observed for the type I receptors. Although BMP-2/4 used predominantly BMPR1A for signaling, ACVR1A was the preferred type I receptor for BMP-6/7. Signaling by both BMP-2/4 and BMP-6/7 was mediated by homodimers of ACVR1A or BMPR1A. A portion of BMP-2/4 signaling also required concurrent BMPR1A and ACVR1A expression, suggesting that BMP-2/4 signal in part through ACVR1A/BMPR1A heterodimers. The capacity of ACVR1A and BMPR1A to form homodimers and heterodimers was confirmed by bioluminescence resonance energy transfer analyses. These results suggest different mechanisms for BMP-2/4- and BMP-6/7-induced osteoblastic differentiation in primary hMSC. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily of growth factors and are used clinically to induce new bone formation. The purpose of this study was to evaluate receptor utilization by BMP-2, BMP-4, BMP-6, and BMP-7 in primary human mesenchymal stem cells (hMSC), a physiologically relevant cell type that probably mediates the in vivo effects of BMPs. RNA interference-mediated gene knockdown revealed that osteoinductive BMP activities in hMSC are elicited through the type I receptors ACVR1A and BMPR1A and the type II receptors ACVR2A and BMPR2. BMPR1B and ACVR2B were expressed at low levels and were not found to play a significant role in signaling by any of the BMPs evaluated in this study. Type II receptor utilization differed significantly between BMP-2/4 and BMP-6/7. A greater reliance on BMPR2 was observed for BMP-2/4 relative to BMP-6/7, whereas ACVR2A was more critical to signaling by BMP-6/7 than BMP-2/4. Significant differences were also observed for the type I receptors. Although BMP-2/4 used predominantly BMPR1A for signaling, ACVR1A was the preferred type I receptor for BMP-6/7. Signaling by both BMP-2/4 and BMP-6/7 was mediated by homodimers of ACVR1A or BMPR1A. A portion of BMP-2/4 signaling also required concurrent BMPR1A and ACVR1A expression, suggesting that BMP-2/4 signal in part through ACVR1A/BMPR1A heterodimers. The capacity of ACVR1A and BMPR1A to form homodimers and heterodimers was confirmed by bioluminescence resonance energy transfer analyses. These results suggest different mechanisms for BMP-2/4- and BMP-6/7-induced osteoblastic differentiation in primary hMSC. Bone morphogenetic proteins (BMPs) 2The abbreviations used are: BMP, bone morphogenetic protein; MSC, mesenchymal stem cell(s); hMSC, human MSC; MSCGM, mesenchymal stem cell growth medium; ODM, osteogenic differentiation medium; RNAi, RNA interference; siRNA, small interfering RNA; RT, reverse transcription; qPCR, quantitative PCR; Rluc, Renilla luciferase; GFP2, green fluorescent protein; BRET2, bioluminescence resonance energy transfer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: BMP, bone morphogenetic protein; MSC, mesenchymal stem cell(s); hMSC, human MSC; MSCGM, mesenchymal stem cell growth medium; ODM, osteogenic differentiation medium; RNAi, RNA interference; siRNA, small interfering RNA; RT, reverse transcription; qPCR, quantitative PCR; Rluc, Renilla luciferase; GFP2, green fluorescent protein; BRET2, bioluminescence resonance energy transfer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. are members of the transforming growth factor-β superfamily of growth factors. BMPs are key regulators of cellular growth and differentiation and regulate tissue formation in both developing and mature organisms. To date, ∼20 unique BMP ligands have been identified and categorized into numerous subclasses based on amino acid sequence similarity (1Wordinger R.J. Clark A.F. Exp. Biol. Med. 2007; 232: 979-992Crossref PubMed Scopus (86) Google Scholar, 2Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar). BMP-7 (osteogenic protein-1) and BMP-2 are well studied members of this family of growth factors and are now being used clinically to induce new bone formation in spine fusions and long bone nonunion fractures (3Gautschi O.P. Frey S.P. Zellweger R. ANZ J. Surg. 2007; 77: 626-631Crossref PubMed Scopus (332) Google Scholar, 4Garrison K.R. Donell S. Ryder J. Shemilt I. Mugford M. Harvey I. Song F. Health Technol. Assess. 2007; 11: 1-168Crossref PubMed Scopus (253) Google Scholar). BMP-2 and BMP-7 belong to two closely related BMP subclasses, namely the BMP-2/4 subclass and the BMP-5/6/7 subclass (1Wordinger R.J. Clark A.F. Exp. Biol. Med. 2007; 232: 979-992Crossref PubMed Scopus (86) Google Scholar). The capacity of ligands from both BMP subclasses to induce osteoblastic differentiation has been rigorously demonstrated (5Canalis E. Economides A.N. Gazzerro E. Endocr. Rev. 2003; 24: 218-235Crossref PubMed Scopus (723) Google Scholar). However, a thorough understanding of the mechanism through which distinct BMP ligands affect target cells is lacking. Such information is central to realizing the potential of individual BMPs as therapeutic agents and for the rational targeting of a specific BMP to the appropriate clinical indication.BMP activities are mediated by tetramers of serine/threonine kinase receptors, consisting of two type I and two type II receptors. Three type I receptors (BMPR1A (ALK-3), BMPR1B (ALK-6), and ACVR1A (ALK-2)) and three type II receptors (BMPR2, ACVR2A, and ACVR2B) have been identified (6Goumans M.J. Mummery C. Int. J. Dev. Biol. 2000; 44: 253-265PubMed Google Scholar). Receptor co-patching studies have revealed the presence of both preformed and BMP-induced type I/type II receptor oligomers (7Nohe A. Hassel S. Ehrlich M. Neubauer F. Sebald W. Henis Y.I. Knaus P. J. Biol. Chem. 2002; 277: 5330-5338Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Binding of BMP ligands to receptor complexes leads to phosphorylation of the type I receptors by constitutively active type II receptors (8Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2094) Google Scholar). BMP-activated type I receptors phosphorylate intracellular signaling proteins, including the receptor-regulated Smads, Smad-1, -5, and -8 (9Miyazawa K. Shinozaki M. Hara T. Furuya T. Miyazono K. Genes Cells. 2002; 7: 1191-1204Crossref PubMed Scopus (577) Google Scholar), which form heteromeric complexes with the common mediator Smad, Smad-4. Activated Smad complexes then translocate to the nucleus and act as transcription factors to induce the expression of BMP-responsive genes. Other BMP signaling pathways have also been identified and shown to mediate the osteoinductive signals of BMPs. These include the Smad-independent p38 mitogen-activated protein kinase pathway (7Nohe A. Hassel S. Ehrlich M. Neubauer F. Sebald W. Henis Y.I. Knaus P. J. Biol. Chem. 2002; 277: 5330-5338Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 10Guicheux J. Lemonnier J. Ghayor C. Suzuki A. Palmer G. Caverzasio J. J Bone Miner. Res. 2003; 18: 2060-2068Crossref PubMed Scopus (265) Google Scholar) and the phosphatidylinositol 3-kinase/AKT pathway (11Osyczka A.M. Leboy P.S. Endocrinology. 2005; 146: 3428-3437Crossref PubMed Scopus (163) Google Scholar).Several studies have demonstrated that BMP ligands discriminate among individual type I and type II receptors. In COS-7 cells co-transfected with different type I receptor cDNAs and the C. elegans Type II receptor Daf-4, BMP-7 bound more efficiently to ACVR1A and BMPR1A than to BMPR1B, whereas BMP-4 bound only to BMPR1A and BMPR1B (12ten Dijke P. Yamashita H. Sampath T.K. Reddi A.H. Estevez M. Riddle D.L. Ichijo H. Heldin C.H. Miyazono K. J. Biol. Chem. 1994; 269: 16985-16988Abstract Full Text PDF PubMed Google Scholar). In contrast, when co-transfected with ACVR2A or ACVR2B, BMP-7 bound BMPR1B and ACVR1A more efficiently than BMPR1A (13Yamashita H. ten Dijke P. Huylebroeck D. Sampath T.K. Andries M. Smith J.C. Heldin C.H. Miyazono K. J. Cell Biol. 1995; 130: 217-226Crossref PubMed Scopus (458) Google Scholar). The association of BMP-7 with ACVR1A in the presence of ACVR2A or ACVR2B was also observed in P19 embryonic carcinoma cells (14Macias-Silva M. Hoodless P.A. Tang S.J. Buchwald M. Wrana J.L. J. Biol. Chem. 1998; 273: 25628-25636Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). In a similar study, differences in type II receptor affinities were observed between BMP-7 and BMP-4, with BMPR2 binding BMP-7 more effectively than BMP-4 (15Rosenzweig B.L. Imamura T. Okadome T. Cox G.N. Yamashita H. ten Dijke P. Heldin C.H. Miyazono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7632-7636Crossref PubMed Scopus (474) Google Scholar).Mesenchymal stem cells (MSC) are a multipotent cell type that can differentiate down the osteoblastic, chondrogenic, myogenic, or adipogenic lineages. Primary human bone marrow-derived mesenchymal stem cell (hMSC) differentiation is an important model for BMP bioactivity, since it is likely that this cell type contributes to healing and bone formation following the clinical administration of BMPs. The present study was designed to evaluate BMP receptor utilization by osteoinductive BMPs, including BMP-2, BMP-4, BMP-6, and BMP-7, during the osteoblastic differentiation of primary hMSC. A model was developed to systematically knock down all type I or type II BMP receptors, alone or in combination, to elucidate receptor utilization by each ligand. The results obtained reveal significant differences in type I and type II receptor usage among the four BMPs evaluated. Interestingly, this work also suggests distinct type I receptor dimerization patterns within receptor complexes utilized by BMP-2/4 and BMP-5/6/7 subclass members. The model employed in these studies could be broadly applied to better understand BMP signaling potentials in other clinically relevant cell and tissue types.EXPERIMENTAL PROCEDURESCell Culture and Culture Media—Primary hMSC and hMSC culture media, including mesenchymal stem cell growth medium (MSCGM) and osteogenic differentiation medium (ODM), were purchased from Lonza (Walkersville, MD). Cells were expanded in vitro and used for experimentation within five passages of the initial thaw. HEK-293 cells were purchased from ATCC (Manassas, VA) and cultured in minimal essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 1.5 g/liter NaHCO3, 2 mm l-glutamine, 1 mm sodium pyruvate and penicillin/streptomycin.BMP Treatment—Recombinant BMP-2, BMP-6, and BMP-7 were produced in Chinese hamster ovary cells and are available at Stryker Biotech (16Sampath T.K. Maliakal J.C. Hauschka P.V. Jones W.K. Sasak H. Tucker R.F. White K.H. Coughlin J.E. Tucker M.M. Pang R.H. J. Biol. Chem. 1992; 267: 20352-20362Abstract Full Text PDF PubMed Google Scholar). BMP-4 was obtained from R&D Systems (Minneapolis, MN). ODM was prepared according to the manufacturer's instructions using the provided supplements of ascorbic acid and β-glycerophosphate but excluding the dexamethasone. Unless stated otherwise, the concentration of fetal bovine serum in ODM was ∼10%. BMPs were diluted in ODM to the indicated concentrations.Alizarin Red Staining—hMSC were seeded into 48-well dishes at 1.0 × 104 cells/well in MSCGM. Twenty-four hours later, MSCGM was replaced with ODM alone or ODM containing the indicated concentration of BMP. Media changes were performed every 3–4 days. Alizarin red staining was performed on day 17 using an osteogenesis quantitation kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions.Quantification of Gene Expression—RNA was isolated using the TurboCapture 96 mRNA Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Reverse transcription was performed using 40 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) in a buffer containing 20 mm Tris-HCl, 50 mm KCl, 5 mm MgCl2, 500 μm each dNTP (Invitrogen, Carlsbad, CA), and 5 ng/μl random primers (Promega, Madison, WI). Reverse transcription was carried out at 23 °C for 10 min and 42 °C for 50 min followed by a 5-min inactivation step at 85 °C. All reagents and instrumentation for gene expression analysis were obtained from Applied Biosystems (Foster City, CA). Quantitative PCR was carried out using a 7900HT fast real time PCR system and predesigned TaqMan gene expression assays according to the manufacturer's specifications. Reference numbers for assays used in this study are as follows: GAPDH (Hs99999905_m1), cyclophilin (Hs99999904_ m1), BMPR1A (Hs00831730_s1), BMPR1B (Hs00176144_m1), BMPR2 (Hs00176148_m1), ACVR1A (Hs00153836_m1), ACVR2A (Hs00155658_m1), ACVR2B (Hs00609603_m1), ID-1 (Hs00357821_g1), NOGGIN (Hs00271352_s1), PTHR1 (Hs00174895_m1), IBSP (bone sialoprotein) (Hs00173720_m1), and DLX-5 (Hs00193291_m1).The analysis of osteoblast marker gene expression and siRNA-mediated receptor knockdown was performed using the standard curve method of relative quantification, according to the procedure recommended by Applied Biosystems. The analysis of BMP receptor expression in hMSC and tissue cDNAs was performed using the absolute standard curve method. Briefly, DNA plasmids containing the human sequences of each BMP receptor, GAPDH or cyclophilin, were used as templates in PCRs to amplify target DNA for standard curve preparation. TrueClone cDNAs encoding BMPR1A (accession number NM_004329.2), BMPR1B (accession number NM_001203.1), BMPR2 (accession number NM_ 001204.5), GAPDH (accession number NM_002046.3), and Cyclophilin A (accession number NM_021130.3) were obtained from OriGene (Rockville, MD). cDNAs for ACVR1A (accession number NM_001105), ACVR2A (accession number NM_001616.3), and ACVR2B (accession number NM_ 001106.3) were obtained from GenScript (Piscataway, NJ). DNA primers (IDT, Coralville, IA) were designed to flank the relevant TaqMan amplicon. Primer sequences are shown in supplemental Table 1. 10 ng of each DNA were exposed to 25 cycles of PCR according to the following thermal profile: denaturation at 95 °C for 20 s, annealing at 55 °C for 10 s, and extension at 70 °C for 15 s. PCR products were gel-purified using a Qiaquick gel extraction kit (Qiagen, Valencia, CA) and quantified by spectrophotometry. Standard curves were run using serial 1:10 dilutions of target DNA from 3 × 106 to 30 copies. The number of expressed molecules of each target gene in experimental samples was quantified against the appropriate standard curve and normalized to an arbitrary copy number (1000) of either GAPDH or cyclophilin from the same sample.Transient Gene Knockdown—Stealth RNAi DuoPaks (Invitrogen) containing two unique, prevalidated siRNA sequences per gene, were used to target the type I BMP receptors ACVR1A, BMPR1A, and BMPR1B and the type II receptors BMPR2, ACVR2A, and ACVR2B. Two Stealth RNAi negative controls (LO and Medium GC content) were utilized as controls to confirm the specificity of each targeted knockdown. Phenotypic results for ACVR1A and ACVR2A were additionally confirmed with a third pre-designed siRNA (Dharmacon, Lafayette, CO), using a chemistry-matched negative control from the same vendor.hMSC were transfected with siRNA using a Nucleofector II (Amaxa Biosystems, Gaithersburg, MD) and employing the manufacturer's hMSC kit. A total of 6 μg of siRNA was delivered to 5 × 105 hMSC, and transfected cells were cultured for 48 h in MSCGM to allow down-regulation of gene targets. Cells were then stimulated with 100 ng/ml of either BMP-2, -4, -6, or -7 for 24 h in MSCGM with 0.2% FBS or 96 h in ODM with 5% fetal bovine serum. BMP receptor and osteoblastic marker gene expression was measured by qPCR. To quantify the phenotypic effect of receptor knockdown, ID-1, IBSP, NOGGIN, and DLX-5 qPCR data were expressed as the percentage of inhibition of BMP-mediated induction of each gene in the targeted siRNA treatments relative to the control siRNA treatments, according to Equation 1, % inhibition=(1−(A−B)/(C−B))×100 (Eq. 1) where A represents the quantity of ID-1 mRNA expression following receptor knockdown and BMP treatment, B represents the base line quantity of ID-1 mRNA expression without BMP treatment, and C represents the quantity of ID-1 mRNA expression following control transfection and BMP treatment.Experiments were performed on three separate occasions using hMSC from multiple donors. The average treatment values from the three replicate experiments were analyzed by two-sample t-tests and two-way analysis of variance using a Bonferroni adjustment for multiple comparisons.Generation of Renilla Luciferase (Rluc) and Green Fluorescent Protein (GFP)2 Fusion Expression Constructs—TrueClone cDNA encoding FGFR1 (accession number NM_023106.1) was obtained from OriGene (Rockville, MD). cDNAs for ACVR1A and BMPR1A were obtained as described above. Using these constructs as template, the open reading frame corresponding to each gene was amplified by PCR using DNA primers (IDT, Coralville, IA) containing an appropriate restriction site for subsequent cloning into pRluc-N1 and pGFP2-N1 fusion protein expression vectors (PerkinElmer Life Sciences). Primer sequences are shown in supplemental Table 2.PCR products were digested with the appropriate restriction enzymes, gel-purified, and ligated in frame into pRluc-N1 and pGFP2-N1. In brief, pRluc-N1 and pGFP2-N1 were restriction-digested and dephosphorylated using Antarctic phosphatase (New England Biolabs, Ipswich, MA). PCR products were cloned at the amino terminus of Rluc or GFP2, such that the expressed proteins were tagged with Rluc or GFP2 at the carboxyl terminus. Untagged expression constructs for each receptor were then created by incorporating a stop codon at the end of the receptor coding sequence. Recombinant constructs were transformed into One Shot TOP10 E. coli (Invitrogen) and plated on agar containing kanamycin (pRluc vectors) or zeocin (pGFP2 vectors). Plasmid DNA was prepared using the EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA). All recombinant constructs were verified by sequencing the full-length open reading frames.Bioluminescence Resonance Energy Transfer (BRET)2 Assays—Unless stated otherwise, all reagents, materials, and instrumentation used in the BRET2 assay were purchased from PerkinElmer Life Sciences. HEK-293 cells were seeded into 6-well dishes at 6 × 105 cells/well. The following day, cells were co-transfected with a total of 2 μg of DNA consisting of the indicated amount of recombinant receptor fusion DNA constructs together with pcDNA as a filler. Transfections were carried out using 6 μl of FuGENE HD (Roche Applied Science). After 48 h, cells were detached using phosphate-buffered saline glucose (Invitrogen), centrifuged, and resuspended in phosphate-buffered saline with glucose. Cells were then distributed in parallel to 96-well white or black CulturPlates for analysis of luminescence and fluorescence emissions, respectively. BRET2 assays were performed using a VICTOR Light luminescence counter. Briefly, DeepBlueC substrate was added by autoinjection to a final concentration of 5 μm, and luminescence was read immediately at 515/30 nm and 410/80 nm. The BRET2 ratio was calculated as the difference of emission at 515 nm/410 nm between the co-transfected Rluc and GFP2 fusion proteins and the Rluc fusion protein alone (17Pfleger K.D. Seeber R.M. Eidne K.A. Nat. Protoc. 2006; 1: 337-345Crossref PubMed Scopus (163) Google Scholar). Results were expressed in milli-BRET2 units, where 1 milli-BRET2 unit corresponds to the BRET2 ratio values multiplied by 1000.For saturation assays, cells were transfected with 10 ng of the donor Rluc receptor fusion construct and increasing quantities (from 50 to 1600 ng) of the acceptor GFP2-receptor fusion construct. GFP2 expression was quantified using a SpectraMax M5 multimode reader (Molecular Devices, Sunnyvale, CA). Data were analyzed by nonlinear regression, and BRET2 ratio values were plotted against the ratio of GFP2 DNA to Rluc DNA. For competition assays, cells were transfected with 10 ng of the donor Rluc-receptor fusion construct, 400 ng of the acceptor GFP2-receptor fusion construct, and 0, 800, or 1600 ng of the competitor receptor construct. Competitor receptors consisted of untagged BMP receptors or FGFR1. For competition assays, treatment averages were compared with the control using two-sample t tests and one-way analysis of variance, applying Dunnett's method for multiple comparisons.RESULTSBMP-2, BMP-4, BMP-6, and BMP-7 Induce Osteoblastic Differentiation in Primary hMSC—The ability of BMP-2, BMP-4, BMP-6, and BMP-7 to drive the osteoblastic differentiation of primary hMSC was compared at both the molecular and cellular level. The expression of six osteoblast marker genes was evaluated by qPCR at either 24 or 72 h, following treatment with the four osteoinductive BMPs (Fig. 1A). Expression of the genes ID-1, DLX-5, and NOGGIN was up-regulated to equivalent levels over the controls by all four BMPs within 24 h of treatment. Likewise, the expression of AP (alkaline phosphatase), PTHR1 (parathyroid hormone receptor 1), and IBSP was increased after 3 days of treatment by all four ligands. The observed gene regulation is in keeping with the reported expression of osteoblast-associated genes during in vitro osteoblastic differentiation (18Friedman M.S. Long M.W. Hankenson K.D. J. Cell. Biochem. 2006; 98: 538-554Crossref PubMed Scopus (186) Google Scholar, 19Lee M.H. Kim Y.J. Kim H.J. Park H.D. Kang A.R. Kyung H.M. Sung J.H. Wozney J.M. Ryoo H.M. J. Biol. Chem. 2003; 278: 34387-34394Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 20Yeh L.C. Tsai A.D. Lee J.C. J. Cell. Biochem. 2002; 87: 292-304Crossref PubMed Scopus (67) Google Scholar, 21Gazzerro E. Gangji V. Canalis E. J. Clin. Invest. 1998; 102: 2106-2114Crossref PubMed Scopus (276) Google Scholar, 22Qi H. Aguiar D.J. Williams S.M. La Pean A. Pan W. Verfaillie C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3305-3310Crossref PubMed Scopus (193) Google Scholar) and suggests that all four BMPs induce a similar gene expression response in hMSC.We next evaluated the capacity of the four BMPs to induce matrix mineralization. Calcium deposits were detected in all BMP treatments by Alizarin red staining (Fig. 1B) but not in hMSC cultured in ODM alone. The observed mineralization was BMP dose-dependent. Collectively, these results demonstrate that all four BMPs induce robust osteoblastic differentiation of primary hMSC and, further, that the quality and magnitude of this differentiation is similar among all four ligands. A systematic investigation of receptor utilization in hMSC was next undertaken to determine whether the four BMPs were exerting their osteoinductive activities via the same or different cellular receptors.Characterization of BMP Receptor Expression Profiles in Primary hMSC and Human Tissue cDNAs— We first assessed the expression of six known BMP receptors in primary hMSC from three separate donors. The copy number of expressed mRNA encoding each receptor was quantified and normalized to GAPDH. Although some variability was observed among donors, the relative expression levels of the six receptors were comparable (Fig. 2A). ACVR1A and BMPR2 were the most abundantly expressed type I and type II receptors, respectively, with normalized expression levels ranging from ∼29 to 67 copies for ACVR1A and ∼24 to 44 copies for BMPR2. BMPR1A and ACVR2A were expressed at intermediate levels, with normalized levels ranging from ∼15 to 20 copies for BMPR1A and ∼7 to 10 copies for ACVR2A. Expression of BMPR1B and ACVR2B mRNA was consistently the lowest of the six BMP receptors, with levels ranging from 1 to 2 copies for BMPR1B and <1 copy for ACVR2B.FIGURE 2Characterization of BMP receptor expression profiles in primary hMSC and human tissue cDNAs. A–C, expression of BMP receptors and endogenous control mRNA was measured by RT-qPCR using a standard curve prepared with known quantities of the appropriate target DNA. Values shown represent the mean ± S.D. of triplicate measurements. A, receptor expression levels in untreated hMSC from three donors. Values are expressed as the number of copies of target mRNA per 1000 copies of GAPDH. B and C, expression of type I (B) and type II (C) BMP receptors in 10 human tissue cDNAs. Values are expressed as the number of copies of target mRNA per 1000 copies of cyclophilin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then evaluated the BMP receptor expression profile in 10 human tissue cDNA preparations. For each tissue cDNA, the mRNA copy number for each receptor was quantified and normalized to cyclophilin. ACVR1A was the most abundantly expressed type I receptor in the majority of tissues, including brain, heart, kidney, liver, lung, and ovary (Fig. 2B). The normalized ACVR1A copy number ranged from <1 in testis to 38 in skeletal muscle. BMPR1A tended to be more abundantly expressed than BMPR1B, with copy numbers ranging from <1 to 41 and from <1 to 11, respectively, across tissue types. Tissues with notable patterns of type I receptor expression include skeletal muscle, with highly abundant ACVR1A and BMPR1A expression, as well as prostate and spinal cord, which both demonstrated roughly equal expression of all three type I receptors.Either ACVR2B or BMPR2 was the most abundantly expressed type II receptor in all tissues, with normalized mRNA copy numbers ranging from ∼5 in lung to 82 in skeletal muscle for ACVR2B and from 4 in liver to 201 in lung for BMPR2 (Fig. 2C). The expression level of BMPR2 in lung was the highest among the tissues tested. Skeletal muscle was notable for an unusual receptor expression pattern, with a high level of expression of both BMPR2 and ACVR2B. ACVR2A exhibited a low level of expression in most tissues, with copy numbers ranging from 3 to 21 copies. These data reveal significant variability among human tissues with regard to overall expression levels of each receptor as well as the relative BMP receptor expression levels within each tissue. This diversity of receptor expression patterns could provide some insight into the broad array of bioactivities reported for BMP ligands.hMSC BMP Receptors Are Specifically and Potently Inhibited by Nucleoporation of Targeted siRNA—siRNAs targeting the six BMP receptors were delivered to hMSC by nucleoporation to down-regulate receptor expression prior to BMP treatment. Receptors were knocked down both individually and in all possible combinations of type II (Fig. 3A) or type I (Fig. 3B) receptors. Receptor knockdown was confirmed by qPCR at the time of phenotypic analysis, typically 72 h postnucleofection. Target knockdown for ACVR1A, BMPR1A, ACVR2A, and BMPR2 ranged from ∼70 to 99% in all experiments relative to control nucleoporations. Knockdown for BMPR1B and ACVR2B ranged from 65 to 83% and from 30 to 77%, respectively, in one experiment. Knockdown overall for both of these low abundance receptors was probably higher, since mRNA expression following targeted nucleoporation fell below quantifiable levels in the remaining two of three experiments. In most instances, nontargeted receptors were not affected by the inhibition of other receptors. However, BMPR1B mRNA appeared to be consistently up-regulated following the knockdown of the other type I receptors (Fig. 3B). It is possible that the observed up-regulation reflects a positive feedback response representing compensation for the loss of other type I receptors.FIGURE 3BMP type I and type II receptors are potently inhibited by nucleoporation of targeted siRNA. A and B, primary hMSC were nucleoporated with a total of 6 μg of siRNA targeting each of the six receptors individually or as all possible combinations of type II (A) or type I (B) receptors or control siRNA. Target mRNA for each of the receptors was measured by RT-qPCR (RQ) 3 days after nucleoporation, at the time of phenotypic analysis. Values are shown relative to the quantity of receptor expression in control nucleoporations. Control values were set to 1 by dividing all nucleoporation replicates in the qPCR analysis of each receptor by the mean value of the control treatment. Values represent the mean ± S.D. of triplicate measurements of ODM-treated cells from one representative experiment. +, the targeted siRNA was included in the nucleoporation treatment; -, the targeted siRNA was omitted from the nucleoporation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)BMP-2/4 and BMP-6/7 Differentially Utilize BMP Receptors to Stimulate Osteoblastic Differentiation of hMSC—The eff
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