Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.
- bone morphogenetic proteins
- mucopolysaccharidosis I
- stem cells
determining how components of the extracellular matrix (ECM) such as proteoglycans influence cytokine-induced cell growth and differentiation in malignancies and other diseases is critical for understanding disease pathophysiology and for developing novel therapeutic strategies (42).
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily of extracellular signaling molecules that regulate the growth, differentiation, and apoptosis of cells in the brain, bone, bone marrow, and diverse tissues (2, 3). The >15 known BMPs are grouped into subfamilies based on homology within the mature domains, and BMP signaling is initiated by binding to specific type I and type II transmembrane serine/threonine kinase receptors (25). Type I receptors activated by ligand-bound type II receptors phosphorylate R-Smads (Smad-1, Smad-5, and presumably, Smad-8) anchored to the cell membrane, which then complex with Co-Smads (Smad-4), translocate to the nucleus, and regulate gene expression in cooperation with other transcription factors. The activity of R-Smads is further influenced by inhibitory Smads (I-Smads-6 and -7) (25).
It is increasingly recognized that ECM and cell surface heparan sulfate (HS) proteoglycans (HSPG) are critical determinants of the biological activity of BMPs and their endogenous antagonists in vivo and in vitro (6, 9, 11, 12, 27–29, 34, 37). As with other proteins, it is believed that specific sulfated residues in the glycosaminoglycan (GAG) side chains of HSPG bind to BMPs and their antagonists and thereby modulate receptor-mediated signaling, diffusion, and localization of these molecules.
BMPs and GAGs also play integral roles in the development of the nervous and skeletal systems. Hurler syndrome (mucopolysaccharidosis type I) is an inborn error of metabolism that causes accumulation of partially degraded HS and dermatan sulfate GAGs due to deficiency of α-l-iduronidase enzyme (IDUA) (26, 35, 41). Neurological dysfunction and skeletal abnormalities are among the most devastating manifestations of this disease. We recently (30) showed that HS in Hurler syndrome cells are structurally and functionally abnormal and have impaired capability to bind and mediate fibroblast growth factor-2 (FGF-2) signaling.
How, and to what extent, endogenous and exogenous GAGs influence the activity of different BMPs in various malignancies, and the structural features of GAGs responsible for modulation of BMP signaling, remain incompletely defined. It also remains to be determined whether, besides FGF-2, the signaling through other relevant morphogens is impaired by the abnormal HS in Hurler syndrome, which might then suggest a common mechanism underlying the pathophysiological manifestations of this disease.
We hypothesized that both endogenous and exogenous (soluble) GAGs directly modulate the biological activity of BMP-4 and that this effect is dependent on specific sulfate residues of GAGs. To test this hypothesis, we examined whether soluble heparin and a desulfated heparin, the endogenous HS present in malignant cells, and the structurally abnormal HS accumulated in Hurler cells influence BMP signaling and activity.
We demonstrate here that 1) components of the BMP signaling pathway are expressed and are active in malignant osteosarcoma cells and in progenitor cells in Hurler syndrome; 2) soluble heparin alters the time course of BMP-4-induced phosphorylation of nuclear Smad-1; 3) heparin directly inhibits the early phase of BMP-4-induced Smad-1 phosphorylation and also augments the inhibitory effect of chordin and that this effect is dependent partly on N-sulfation of glucosamine residues; 4) endogenous cell-associated sulfated GAGs themselves promote proliferation and block BMP-4-mediated differentiation of malignant cells; and 5) the abnormally accumulated GAGs in Hurler cells impair BMP-4 activity, which can be restored by in vitro enzyme correction of the cells that clears the accumulated GAGs. Thus both endogenous (cell or matrix associated) GAGs and exogenous (soluble) heparin, via N- and O-sulfated disaccharide residues, directly inhibit BMP-4 activity. These findings have implications for understanding the pathobiology of diverse diseases and for developing novel therapeutic agents that may restore BMP signaling and activity.
MATERIALS AND METHODS
DMEM (low glucose), MEM (with nonessential amino acids and without l-glutamate), penicillin, streptomycin, lipid concentrate, Alexa Fluor 488 goat anti-rabbit IgG, and One Shot TOP10 Chemically Competent Escherichia coli were obtained from Invitrogen Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was from HyClone (Logan, UT). HEPES, EDTA, phenylmethylsulfonyl fluoride, Nonidet-40, Triton X-100, p-nitrophenylphosphate, p-nitrophenol, MgCl2, Tris, glycine, anti-goat IgG-horseradish peroxidase (HRP) (catalog no. A-5420), recombinant human epidermal growth factor (rhEGF), insulin-transferrin-selenium (ITS), ascorbic acid 2-phosphate, MCDB-201, and 2-nitrophenyl-β-d-galactopyranoside were from Sigma-Aldrich (St. Louis, MO). Tween 20 was from Fisher Scientific. The SaOS-2 cell line and McCoys 5A medium were purchased from ATCC (Manassas, VA). Heparin and N-desulfated, N-reacetylated heparin (NDSNAc) were from the Chemically Modified Heparin kit (Seikagaku America, Falmouth, MA). Recombinant human BMP-4, recombinant mouse chordin, recombinant human platelet-derived growth factor-BB (rhPDGF-BB), human BMP-4 polyclonal antibody (catalog no. AF-757), and mouse chordin polyclonal antibody (catalog no. AF-758) were from R&D Systems (Minneapolis, MN). Anti-rabbit IgG-HRP (catalog no. SC-2004) and anti-β-actin (catalog no. SC-1616) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Protease inhibitor cocktail and FuGENE-6 Transfection Reagent (catalog no. 11814443001) were from Roche Diagnostic (Indianapolis, IN). Preimmune rabbit serum was from Jackson ImmunoResearch Laboratories (West Grove, PA). Prestained SDS-PAGE markers, polyvinyldifluoride membrane, and dry milk were from Bio-Rad (Hercules, CA). ECL Advance Western Blotting Detection Kit and high performance chemiluminescence film were from Amersham Biosciences (Piscataway, NJ). Luciferase assay system (catalog no. E1501) and pSV-β-galactosidase (catalog no. E108A) control vector were from Promega (Madison, WI). All other chemicals were analytical grade. Recombinant human IDUA was kindly provided by Merry Passage (LA Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA), the rabbit polyclonal anti-p-Smad-1 antibody was provided by Dr. C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden), and the BMP-specific response element (BRE)2-luciferase vector (21) was provided by Dr. Peter ten Dijke (Leiden University Medical Center, Leiden, The Netherlands) with approval from the Netherlands Cancer Institute (NKI-AVL; Amsterdam, The Netherlands).
Culture of SaOS-2 Cells
SaOS-2 cells were grown and maintained in McCoys 5a medium with 15% FBS and 1× penicillin and streptomycin at 37°C in 5% CO2 per instructions from ATCC. Cells were maintained by passaging at 80–90% confluency.
Preparation of Conditioned Medium From SaOS-2 Cells
SaOS-2 cells were grown in McCoys 5a medium with 15% FBS without penicillin or streptomycin at 37°C in 5% CO2. After 3 days, medium was replaced by McCoys 5a medium with 1% BSA and 1× ITS overnight, and the conditioned medium was collected, centrifuged to remove any suspended cells, sterile filtered, and kept at 4°C until use.
RNA Isolation and Quantitative Real-Time RT-PCR
Total cellular RNA from SaOS-2 cells was prepared using the RNeasy RNA isolation kit (Qiagen, Valencia, CA). An aliquot of RNA was used to determine RNA quantity and quality by using an Agilent 2600 bioanalyzer (Agilent, Palo Alto, CA). RNA (500 ng) was used in a cDNA synthesis reaction using Superscript III (Invitrogen) as per the manufacturer's instructions. Quantitative real-time RT-PCR (qRT-PCR) was performed on an ABI 9600 HT Sequence Detection System (ABI, Foster City, CA). cDNA (2.5 ng) was amplified for up to 50 cycles in a two-step PCR reaction [95°C, 15 s (denaturation), 60°C, 40 s (annealing/elongation)], including 200 nM gene-specific primers: BMP-2, 5′-ATC ATG CCA TTG TTC AGA CG-3′ and 5′-CTC GTC AAG GTA CAG CAT CG-3′; BMP-3, 5′-TTG AAG CCA TCA AAT CAT GC-3′ and 5′-GGT ACA CAG CAA GGC TCA GG-3′; BMP-4, 5′-ACA CCA CAC ACA CAC GTT CC-3′ and 5′-GGT CAA GGT GAA TGT TTA GGG-3′; BMP-5, 5′-TGC TCC AAC CAA ATT AAA TGC-3′ and 5′-CAG CCA CAT GAG CGT ACT ACC-3′; BMP-6, 5′-CCA CAG GGT TAG AAC CAA CG-3′ and 5′-TCT GAG CCT CAC TTT CTA CAG G-3′; BMP-7, 5′-ACG GAC TCG TTT CCA GAG G-3′ and 5′-TTC CTT TCG CAC AGA CAC C-3′; BMP receptor 1A, 5′-TGT CAA ATA TGT TCT GGA CAG C-3′ and 5′-TTT CAT TGT CTT CAC AAG TTA GGG-3′; BMP receptor 1B, 5′-AAG CCT TGA ACA TCG TCC TG-3′ and 5′-TCC TTC TGG GAG CTT CTC TG-3′; BMP receptor 2, 5′-TAA GCT GTC TGA AGC CTT GC-3′ and 5′-TCA GCT TTC ATA GTG GCA TCC-3′; activin receptor 1, 5′-CCA TTA CCC ACG TGA CAC C-3′ and 5′-CAG AGT TTA AAT GCA CGT AAT GG-3′; activin-like receptor 1, 5′-GAA GAA GGT GGT GTG TGT GG-3′ and 5′-TCT GAG CTA GGC CTG AGA GG-3′; activin receptor 2, 5′-GCA TCT TGA TTG AAC ATC ATT TAC C-3′ and 5′-GGG ATA TGG GTT GAG ACT GC-3′; activin receptor 2B, 5′-TGG TGC ACA AGA AGA TGA GG-3′ and 5′-ATC ATG GTC CCA GCA CTC C-3′; Smad-1, 5′-AAA TTG CCT ACA TGT TTC AAT ACC-3′ and 5′-AAA GCC TAT TTC TGT ACT GTA AAC TCC-3′; Smad-5, 5′-TCG AAG AGG ATT GTA ATC ATG G-3′ and 5′-CCT ACA GTG CAG CCA CTA GC-3′, and SYBR green PCR mix (Invitrogen), ROX dye, and H2O. Gene expression was normalized using β-actin (5′-TCC CCC AAC TTG AGA TGT ATG AAG-3′ and 5′-AAC TGG TCT CAA GTC AGT GTA CAG G-3′). Primers were tested using control cDNA and water blanks. Analysis of real-time PCR included examining dissociation curve of PCR product. After it was ensured that each amplified product was genuine (by analysis of dissociation temperatures, amplicon size, and sequence), relative gene expression was calculated and expressed as the difference in cycle threshold (ΔCT) compared with the β-actin control.
Assessment of BMP-4 Induced Phosphorylation of Nuclear Smad-1 in SaOS-2 Cells
SaOS-2 cells were grown to 80–90% confluence in 150-cm petri dishes and then serum starved (in serum-free medium: complete medium without FBS but containing 1% BSA) overnight. Cells were treated with BMP-4 (25 ng/ml) for 30 min at 37°C, then cooled immediately by keeping the petri dishes on ice. In other experiments, BMP-4 treatment was done for various periods of time (5–480 min) or for 30 min at different concentrations (1–50 ng/ml). To examine the effect of sulfated GAGs on BMP-4 signaling, cells were treated with vehicle, BMP-4 (25 ng/ml), chordin (500 ng/ml), or BMP-4 + chordin, in the absence or presence of 10 μg/ml of heparin or NDSNAc. We used a chordin concentration of 500 ng/ml, which is a dose shown in MC3T3-E1 osteoblast-like cells to be adequate for inhibition of ascorbic acid-induced mineralization and induction of alkaline phosphatase activity and also for inhibition of BMP-4 induced p-Smad-1 (31).
Medium was removed, and cells were washed with cold 1× PBS containing 1 mM PMSF, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Cells were scraped and centrifuged at 4°C. Nuclear and cytoplasmic extracts were made as described earlier (16). Equal amount of nuclear extracts were run on SDS-PAGE, and Western immunoblotting was performed as described below by using a p-Smad-1 antibody. The same blot was reprobed with anti-β-actin antibody.
Proteins were transferred to a polyvinydifluoride (PVDF) membrane overnight at 45 volts at 4°C. PVDF membranes were blocked with 5% dry skimmed milk in 1× PBS containing 0.1% Tween 20 (blocking buffer) for 1 h at room temperature. The membranes were then incubated with anti-p-Smad-1 (1:100,000 dilution) in blocking buffer for 1 h at room temperature, washed, and then incubated with either anti-goat IgG HRP (for detection of β-actin) or anti-rabbit IgG HRP (for detection of p-Smad-1) antibody (1:200,000 dilution) in blocking buffer for 1 h at room temperature. Membranes were washed and developed in ECL Advance solution per the manufacturer's instructions. The same blot was reprobed with anti-β-actin (1:200,000 dilution) to determine loading on the gel and normalize p-Smad-1 levels. Densitometric analysis of the bands was performed using a UMAX Powerlock 1120 densitometer (UMAX Technologies, Dallas, TX) and expressed as the ratio of p-Smad-1 to β-actin.
Transient Transfection and Id-1 Transcriptional Reporter Assay
SaOS-2 cells were plated at 5 × 104 cells/well in 6-well plates. After 24 h, cells were cotransfected for 30 h with (BRE)2-luciferase and β-galactosidase vectors using the FuGENE-6 Transfection Reagent kit per the manufacturer's instructions and changed to serum-free medium overnight. Cells were treated with BMP-4 (25 ng/ml) with or without heparin or NDSNAc (10 μg/ml) for up to 24 h, washed with chilled buffer, and lysed. Luciferase activity was measured using the Promega luciferase assay (15, 21). β-Galactosidase activity was measured as described by us previously (17, 39) and used to normalize transfection efficiency.
BMP-4 Induced Proliferation and Differentiation of SaOS-2 Cells
Proliferation of SaOS-2 cells.
Cell proliferation assays were performed in 96-well plates. Five thousand SaOS-2 cells were plated per well in 200 μl complete medium and allowed to adhere and grow for 24 h. Cells were then washed and the medium was replaced by serum-free medium supplemented with 1× ITS. After overnight serum starvation, 25 ng/ml BMP-4, 10 μg/ml GAGs (heparin or NDSNAc), or both BMP-4 and GAGs were added to triplicate wells in the same serum-free medium. Control cells received an equivalent amount of vehicle (4 mM HCl + 0.2% BSA buffer). After 3 days, cell number was determined by using a standard curve generated from known numbers of SaOS-2 cells in the CyQuant NF cell proliferation assay kit (Molecular Probes) per the manufacturer's instructions.
Differentiation of SaOS-2 cells.
Increase in alkaline phosphatase activity was used as an indicator of differentiation of SaOS-2 cells. Cells were grown for 3 days as for the proliferation assay described above, and alkaline phosphatase activity was measured as described previously (36). Briefly, the medium was removed and the cell layers were washed with PBS. To each well, 200 μl of enzyme assay solution containing 8 mM p-nitrophenyl phosphate, 12 mM MgCl2, and 0.1 mM ZnCl2 in 0.1 M glycine-NaOH buffer, pH 10.5, were added and the plates incubated at 37°C for 5 min. The reaction was stopped by addition of 50 μl of 0.5 M NaOH. Plates were read at 405 nm, and enzyme activity was calculated from p-nitrophenol standards (1 unit = the amount of enzyme required to convert 1 nmole of substrate to p-nitrophenol per minute). Enzyme activity was expressed as units of enzyme per 1,000 SaOS-2 cells.
Another set of experiments was performed to determine the effect of cellular and exogenously added soluble sulfated GAGs on BMP-4-induced cell proliferation and differentiation. For these experiments, 1,500–2,000 SaOS-2 cells were plated in 96-well plates in complete medium without penicillin and streptomycin and with either 30 mM sodium chlorate (to block GAG sulfation) or 30 mM NaCl (control) and allowed to grow for 72 h. Cells were washed and medium was replaced by either serum-free medium (McCoy's 5a medium + 1% BSA + 1 × ITS without penicillin or streptomycin) or SaOS-2-conditioned medium. Media were supplemented with BMP-4 (25 ng/ml), soluble sulfated GAGs (10 μg/ml heparin or NDSNAc), and either 30 mM sodium chlorate or 30 mM NaCl. After 3 days, cells were counted and alkaline phophatase activity was determined, as described above.
Isolation and Culture of Multipotent Adult Progenitor Cells
Multipotent adult progenitor cells (MAPCs) were obtained from the bone marrow of normal donors and patients with Hurler syndrome and expanded in culture in MAPC medium, as described by us previously (30, 33). These studies were approved by the institutional human subjects subcommittee.
Assessment of gene expression in MAPC using microarrays.
The goal of these experiments was to compare the expression of BMPs and BMP receptors, Smads (intracellular signaling intermediates), and IDs (target genes) in human MAPC cells from normal donors and patients with Hurler syndrome. Normal and Hurler MAPC cells (n = 3 each) were grown in T-175 tissue culture flasks, and total RNA was isolated from cell lysates using the RNeasy kit (Qiagen), according to the manufacturer's instructions. RNA quantity and quality was determined by spectrophotometry and formaldehyde gel electrophoresis. Microarray experiments were performed according to the GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com/support/technical/manual/expression_manual.affx) from Affymetrix, Santa Clara, CA. Ten micrograms of total RNA were used to synthesize double-stranded cDNA (Superscript Choice System; GIBCO-BRL Life Technologies, Rockville, MD). In vitro transcription was performed to produce biotin-labeled cRNA using a BioArray HighYield RNA Transcription Labeling Kit (Enzo Diagnostics) according to the manufacturer's instructions. Biotinylated cRNA was processed with a GeneChip Sample Cleanup Module kit (Qiagen, Chatsworth, CA) and fragmented to 50 to 200 nucleotides. Sample labeling, hybridization, detection, and data acquisition were performed at the Biomedical Imaging and Processing Laboratory at the University of Minnesota (http://www.bmgc.umn.edu/bmgc/facilities/microarray/home.html), using Affymetrix hardware (Fluidics stations and Genechip 3000 scanner), software (GeneChip Operating Software), and protocols. The quality of the labeled target cRNA was determined using the Affymetrix GeneChip Test3 Array before hybridization on the Affymetrix Human Genome U133 (HG-U133) Set. This set consists of two GeneChip arrays containing 45,000 probe sets representing >39,000 transcripts derived from about 33,000 human genes. Expression data were analyzed using the Affymetrix Microarray Suite v4.0 and Affymetrix Data Mining Tool v2.0.
Mean expression intensities for normal and Hurler MAPC were calculated for each feature, and fold differences in the means were obtained (see Table 2). Statistical differences in the means were assessed using two-sided T-test with Welch correction.
BMP-4 Signaling in Hurler MAPC
Fifteen thousand normal or Hurler MAPCs were plated on each sterile glass coverslip, placed in 6-well plates in MAPC medium, and allowed to adhere for 4–6 h. MAPC were serum-deprived by changing the medium to MEM (with nonessential amino acids and without l-glutamate) containing 1% BSA and 1× penicillin-streptomycin overnight, before the addition of BMP-4 for various time periods (15–120 min) to determine the time to maximal phosphorylation of nuclear Smad-1.
Hurler MAPC were treated without or with purified recombinant IDUA (10 ng/ml) in MAPC medium for 3 days. Medium was then changed to serum-free MEM medium (as above) supplemented without or with 10 ng/ml IDUA overnight. Cells were then treated without or with various concentrations of BMP-4 for 30 min, fixed, and immunostained for detection of p-Smad-1 as described below.
The coverslips were gently washed three times with chilled PBS containing 1 mM PMSF, 1 mM Na3VO4, and 100 mM NaF on ice, and the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 30 min at room temperature. Cells were blocked with 5% goat serum with 10 ng/ml protamine overnight at 4°C. The addition of protamine helped reduce nonspecific staining due to binding of antibodies to accumulated GAGs in the Hurler MAPC. The blocking solution was completely removed, and 50 μl of rabbit p-Smad-1 antibody (1:2,000 dilution in 1% BSA) were added to each coverslip and incubated for 2 h at room temperature in a water bath fully covered to provide humidity. Cells were washed and incubated with Alexa Fluor 488 goat anti-rabbit IgG (0.1 μg/ml dilution) and 10 ng/ml protamine for 1 h at room temperature. Coverslips were mounted on glass slides using aqueous mounting medium. Images were taken using an Olympus Fluoview confocal laser scanning microscope. For controls, preimmune normal rabbit serum (1:2,000 dilution in 1% BSA; Jackson Immunoresearch, West Grove, PA) and Alexa Fluor 488 goat anti-rabbit IgG (0.1 μg/ml dilution) secondary antibody were used.
All experiments were performed three to six separate times. GraphPad Prism 4 software (GraphPad Software, San Diego, CA) was used to calculate the means ± SE and to determine the significance of differences. Data are shown as means ± SE; differences with P < 0.05 were considered statistically significant.
The effects of heparin or NDSNAc ± chordin on BMP-4-induced phosphorylation of nuclear Smad-1 (Fig. 2) and of endogenous and exogenous GAGs on cell proliferation and differentiation (Fig. 4) were compared using the repeated measures one-way analysis of variance (ANOVA) with Dunnett's multiple comparison post test. The dose-response curves of BMP-4 signaling in uncorrected and corrected Hurler MAPC (Fig. 5C) were compared using the model I (fixed effects) two-way ANOVA test.
Expression of BMP-s and BMP Receptors in SaOS-2 Cells
We first examined whether various BMPs, Smads, and BMP and activin receptors were expressed by malignant SaOS-2 cells, using qRT-PCR (Table 1). Transcripts for BMPs 2–7, the listed receptors, and both Smad-1 and Smad-5 were expressed consistently and at easily detectable levels. Of the transcripts examined, only that of the ACVR-L1 receptor was not consistently detectable.
Effect of Heparin on BMP-4-Induced Smad-1 Phosphorylation in SaOS-2 Cells
We confirmed that the BMP-4 signaling pathway was functionally active in SaOS-2 cells by demonstrating a concentration-dependent increase in the signaling intermediate p-Smad-1 in the nuclear fraction of these cells and acquisition of alkaline phosphatase activity as a marker of osteogenic differentiation (both effects maximal at 25 ng/ml BMP-4; data not shown).
Since sulfated GAGs bind to and modulate the activity of BMPs, we examined whether highly sulfated heparin influences BMP-4 signaling. SaOS-2 cells were cultured with BMP-4 alone or in the presence of heparin. As shown in Fig. 1, BMP-4-induced phosphorylation of nuclear Smad-1 was maximal at 30 min, started declining by 1 h, and continued to decline till 24 h, the last time point examined. Heparin in soluble form in the medium of the cells inhibited the early peak in phosphorylation at 30 min (P = 0.02 compared with BMP-4 alone). Although phosphorylation in the presence of heparin appeared to persist until 24 h, this was not statistically significantly different when compared with BMP-4 alone.
At 30 min, heparin also demonstrated a dose-response effect on BMP-4-induced phosphorylation of Smad-1, starting at 1 μg/ml and reaching a maximal effect at 25 μg/ml and declining thereafter up to 100 μg/ml (not shown). Together, these data indicate that soluble heparin affects the cellular response to BMP-4.
Effect of Differentially Sulfated GAGs on BMP-4 Signaling in the Absence or Presence of Chordin
We next examined whether heparin and NDSNAc influence BMP-4 signaling in SaOS-2 cells at 30 min. As shown in Fig. 2A, heparin significantly inhibited the BMP-4-induced phosphorylation of nuclear Smad-1 (P < 0.05). As expected, chordin, an extracellular BMP antagonist, markedly inhibited BMP-4-induced signaling (P < 0.01). Together, chordin + heparin inhibited BMP-4 signaling by 85% (P < 0.01). This represents near total blockade of BMP-4 signaling since the amount of nuclear p-Smad-1 was nearly as low as in the absence of BMP-4 (lane 1 in Fig. 2A).
NDSNAc, the chemically modified heparin that is depleted of N-sulfation but retains O-sulfated residues, affected BMP-4 signaling in a fashion similar to that seen with heparin but to a quantitatively lesser extent (Fig. 2B). Whereas NDSNAc alone did not significantly inhibit BMP-4-induced phosphorylation of nuclear Smad-1 (P > 0.05), it did induce significant inhibition in the presence of chordin (P < 0.01). This is likely to represent an additive effect over that of chordin alone, since in this series of experiments, the inhibitory effect of chordin alone did not reach statistical significance (P > 0.05).
Effect of Differentially Sulfated GAGs on BMP-4 Mediated Activation of the Id-1 Promoter
We then examined the downstream effects of BMP-4 ± GAGs on p-SMAD-1-mediated transcription of its target gene Id-1 in SaOS-2 cells. As shown in Fig. 3A, BMP-4 alone induced maximal activation of the Id-1 promoter (as detected by the luciferase assay) at 16–24 h. This activation was significantly inhibited (P < 0.05) by heparin at both 16 and 24 h. These data indicate that the heparin-induced inhibition of peak phosphorylation of nuclear p-SMAD-1 (Fig. 1) results in reduced transcription of a target gene (Id-1) and validates the biological relevance of the former observation.
Additionally, we examined the effect of NDSNAc on Id-1 promoter activation using an identical experimental design (Fig. 3B). NDSNAc inhibited BMP-4-induced Id-1 promoter activation at 24 h (P < 0.003) but to a lesser extent than heparin, consistent with the effects of these GAGs on BMP-4-induced phosphorylation of nuclear SMAD-1.
Effect of Endogenous and Exogeneous GAGs on SaOS-2 Cell Proliferation and Differentiation
We then examined the impact of cellular and exogenously provided soluble sulfated GAGs on SaOS-2 cell proliferation and alkaline phosphatase activity (Fig. 4).
SaOS-2 cell proliferation.
cells with intact cellular gags.
In cells not treated with sodium chlorate, BMP-4 inhibited cell proliferation (Control vs. BMP-4: P < 0.01). Heparin or NDSNAc alone did not have any effect (Fig. 4A). However, the presence of heparin (but not NDSNAc) neutralized the inhibitory activity of BMP-4 (Control vs. BMP-4 + heparin: P > 0.05).
cells with unsulfated cellular gags.
Basal cell proliferation was reduced in cells grown in sodium chlorate, which impairs sulfation of cellular GAGs (Control without chlorate vs. Control with chlorate: P < 0.01). BMP-4 further inhibited proliferation (Control with chlorate vs. BMP-4 with chlorate: P < 0.05). As seen in cells with intact GAGs, heparin or NDSNAc alone did not have any effect. Neither heparin nor NDSNAc could neutralize the inhibitory effect of BMP-4 (Control with chlorate vs. BMP-4 + heparin or BMP-4 + NDSNAc: P < 0.01 and P < 0.05, respectively).
These data indicate that 1) exogenous BMP-4 inhibits the proliferation of malignant SaOS-2 cells, 2) cellular sulfated GAGs promote the growth of these malignant cells since their desulfation inhibits proliferation, 3) the inhibitory activity of BMP-4 does not require cellular sulfated GAGs, 4) the effect of desulfation of cellular GAGs cannot be restored by soluble heparin or NDSNAc alone, suggesting that cell proliferation specifically requires cellular sulfated GAGs, and 5) soluble heparin neutralizes the effect of BMP-4 but only in presence of cellular sulfated GAGs, and N-sulfation is required since NDSNAc does not have this activity.
SaOS-2 cell differentiation.
In contrast to its effect on cell proliferation, chlorate-induced undersulfation of cellular GAGs tended to induce a small increase in the basal level of cell differentiation (measured by expression of alkaline phosphatase) in the absence of exogenous cytokines (Control columns in Fig. 4, B and C). By themselves, exogenous soluble NDSNAc or heparin did not affect cell differentiation in absence of exogenous cytokines, either in presence or absence of cellular sulfated GAGs (NDSNAc and heparin columns in Fig. 4, B and C, respectively).
As expected, BMP-4 induced cell differentiation in chlorate-untreated cells (BMP-4 vs. Control columns in Fig. 4, B and C: P < 0.01 for both). This effect of BMP-4 was significantly enhanced by chlorate treatment (BMP-4, shaded vs. solid bars in Fig. 4, B and C: P < 0.01 and P < 0.05, respectively). These data indicate that cellular sulfated GAGs impair BMP-4-induced SaOS-2 cell differentiation.
Although exogenous soluble heparin appeared to inhibit BMP-4-induced differentiation in both the presence or absence of cellular sulfated GAGs (BMP-4 vs. BMP-4 + heparin columns in Fig. 4C), the difference was not statistically significant. NDSNAc had no effect on BMP-4-induced differentiation (BMP-4 vs. BMP-4 + NDSNAc columns in Fig. 4B).
Effect of GAGs shed by SaOS-2 cells on differentiation.
We also examined the effect of SaOS-2-conditioned medium on acquisition of alkaline phosphatase activity in the absence or presence of heparin, BMP-4, or both, in untreated as well as chlorate-treated cells (Fig. 4D). SaOS-2-conditioned medium had no significant effect on the differentiating activity of BMP-4 in untreated or chlorate-treated cells. Exogenous heparin also did not have any effect under any of these conditions. Thus, whereas cellular sulfated GAGs inhibit BMP-4-induced SaOS-2 cell differentiation, sulfated GAGs shed into the medium by SaOS-2 cells do not have any effect, even when cellular GAGs are depleted of sulfation.
Effect of Accumulation of Abnormal GAGs on BMP-4 Signaling
Finally, we examined the effect on BMP-4 signaling of accumulation of cellular GAGs in a human disease. Using microarrays, we first assessed whether GAGs accumulated in cells in Hurler syndrome altered expression of genes in the BMP signaling pathway. As shown in Table 2, MAPC obtained and cultured from the bone marrow of normal donors and patients with Hurler syndrome expressed transcripts for various BMPs, their receptors, the extracellular antagonist chordin, the intermediate signaling Smads, and target proteins; e.g., inhibitors of DNA binding (Id proteins). Whereas there were differences in the levels of expression of different members of each class of transcripts, there were no statistically significant differences in expression of any of these transcripts between normal and Hurler MAPC.
To examine the efficiency of BMP-4 signaling in Hurler syndrome, we next measured BMP-4-induced phosphorylation of nuclear Smad-1 in normal and Hurler MAPC. As with SaOS-2 cells, the effect of BMP-4 (25 ng/ml) was maximal at 30 min (data not shown). As shown in Fig. 5, BMP-4 did induce concentration-dependent phosphorylation of nuclear Smad-1 at 30 min in Hurler MAPC. However, BMP-4 signaling was significantly enhanced (P = 0.0004) after removal of accumulated GAGs by treatment of Hurler MAPC with recombinant IDUA enzyme. The inhibition of BMP-4 signaling by accumulated GAGs was most evident at low-intermediate concentrations of BMP-4 but could be overcome by increasing BMP-4 concentration to ≥10 ng/ml (Fig. 5C). The ED50 of BMP-4 for Smad-1 phosphorylation in untreated Hurler MAPC was almost threefold higher than that in IDUA-treated cells (2.2 and 0.8 ng/ml, respectively). High concentrations of BMP-4 (≥10 ng/ml) were able to activate essentially 100% of both untreated and IDUA-treated cells and normal cells. These data indicate that the abnormally accumulated GAGs impair BMP-4 signaling in Hurler syndrome, which can be restored by enzymatic removal of these GAGs.
Expression and activity of the TGF-β superfamily, which includes the BMPs, plays an important role in tumorigenesis, progression, and metastasis and on host responses (angiogenesis, ECM production, and suppression of immune surveillance) (2, 3). We show that both endogenous and exogenous (soluble) GAGs directly modulate the biological activity of BMP-4 and that this effect is dependent on specific sulfate residues of the GAGs.
The net effects of GAGs on the biological activity of BMPs are extremely variable since they depend on multiple factors, including 1) the specific BMP examined (11, 12, 28, 29, 34, 37); 2) the type, sulfation pattern, concentration, and source of GAGs (11, 37); 3) the types and levels of BMP receptors and proteoglycans expressed by the cell type tested (11, 12); 4) the presence or absence of serum in the medium; 5) the availability or absence/modification (by enzymatic cleavage or chlorate-induced inhibition of sulfation) of cell surface GAGs (11, 12, 28, 29); 6) the presence or absence of endogenous and/or exogenously provided heparin-binding BMP antagonists such as chordin (12) or Noggin (28); and 7) the assay system used; e.g., monolayers of cultured cells (11, 34, 37) vs. in vivo systems where diffusion and gradients of BMPs vs. antagonists may be critical determinants of tissue differentiation (12, 27–29). For these reasons, GAGs have been found to have opposite effects in different in vitro and in vivo systems, enhancing BMP activity in certain systems, and inhibiting it in other systems. All our studies were performed in serum-free media and thus represent the specific effects of BMP-4 in the absence of confounding factors such as other cytokines or exogenous BMP antagonists.
Tumors may vary in their endogenous expression of BMPs, as exemplified by the methylation-induced silencing of BMP-2 in gastric carcinomas (40). Furthermore, the expression level and activity of specific receptor subtypes in different benign and malignant cells may have markedly different effects on the biological activity of individual BMPs, inducing augmentation of the activity of some BMPs and simultaneous reduction in the activity of other BMPs. Thus deletion of BMP receptor 2 (BMPR2) reduces BMP2 signaling but paradoxically enhances BMP-6 and -7 signaling (43) due to activation of ActRIIa and utilization of type I receptors distinct from those used by BMP2. Loss of function mutations in BMP receptors occur in malignant cells; e.g., BMPR2 in renal cell cancer and prostate cancer (18, 19, 24, 38) and in Smads (2). Using qRT-PCR, we found comparatively high levels of expression of BMP-2, -4, and -6 and BMP-R1A, BMP-R2, ACVR2B, Smad-1 and -5 expression in malignant SaOS-2 cells, consistent with previous nonquantitative data on other osteosarcoma cells (8). In Hurler syndrome MAPC, we found that accumulated GAGs do not change the expression of various BMPs, their receptors, chordin, Smads, and Ids by progenitor cells, even though expression of many other proteins is altered (unpublished observations).
Time- and concentration-dependent activation of BMP-4 signaling varies with cell type, different BMPs and Smads, and experimental conditions (7, 10, 11, 13). In SaOS-2 cells, we found that the BMP-4 signaling pathway is functionally expressed, as evidenced by phosphorylation of nuclear Smad-1, activation of the Id-1 promoter, and acquisition of alkaline phosphatase, a marker of osteogenic differentiation. The optimal concentration of BMP-4 was 25 ng/ml, and the maximal activation of Smad-1 occurred in 30–60 min. Soluble heparin inhibited BMP-4 activity as measured by all the above assays. Zhao et al. (44) found that heparin (5 μg/ml) prolongs the duration of phosphorylation of Smad-1/5/8 induced by BMP-2 (100 ng/ml) in C2C12 myoblasts. In those conditions, heparin prolonged the half-life of BMP-2 in the culture medium, possibly by inhibition of degradation. The mechanism underlying the latter effect remains to be determined. Somewhat different from its effect on the activity of BMP-2 on C2C12 myoblasts (44), we found that heparin inhibits peak induction of phosphorylation of nuclear Smad-1 stimulated by BMP-4 in SaOS-2 cells.
We demonstrated that in the presence of unaltered cell surface + ECM GAGs in the microenvironment of SaOS-2 cells, soluble heparin in the culture medium modulates BMP-4 signaling. Heparin by itself inhibited BMP-4-mediated phosphorylation of nuclear Smad-1 at 30 min and also augmented the inhibitory effect of 500 ng/ml chordin. These effects were partially dependent on both N- and O-sulfation of the GAG, as shown by the experiments using NDSNAc. Since heparin contains both O- and N-sulfate residues but NDSNAc contains O- but not N-sulfation, conclusions about the requirement for N-sulfation are based on the observed differences between heparin and NDSNAc in our studies. Furthermore, our own preliminary studies as well as data from other investigators (37) show that CDSNAc (chemically modified heparin depleted of both O- and N-sulfates) does not have any effect on BMP binding/signaling. Therefore, conclusions regarding the effect of O-sulfates are based on the observed effect of NDSNAc in the current studies.
In rat osteosarcoma cells, heparin appears to similarly inhibit BMP-2/4 as well as BMP-7 signaling (11). In these cells, BMP-7 binds to cell surface HS, and BMP-7-induced signaling is inhibited by removal/desulfation of cell surface GAGs. Taken together, these results show that N-sulfation is essential for inhibition of BMP-7 signaling but not to the same extent for inhibition of BMP-4 signaling. Since both BMP-4 and chordin bind heparin, it is possible that the increase in the inhibitory activity of chordin by heparin-NDSNAc may be at least partly due to heparin-induced facilitation of the interaction of BMP-4 with chordin, as has been shown for other proteins such as the interaction between FGF-2 and FGF receptors (1, 22). However, determination of the exact mechanism of action of heparin requires further investigation. Furthermore, in addition to the major Smad pathway, some of the effects of BMPs may be mediated via the MAP kinase and STAT pathways (5, 20, 23, 32). We have not examined the latter pathways in the current study.
The effect of endogenous and exogenous GAGs on the biological activity of BMPs appears to depend considerably on the type of cell examined and the specific BMP and GAG used, but the mechanism(s) responsible for the effects of GAGs remain unclear (11, 12, 34, 37). Even though BMP-2 and -4 share structural similarity and both bind BMPR2 (2), additional factors result in differences between their activities. For example, only BMP-2 (and not BMP-4) also binds ActRIIB (2) and demonstrates biological activity on certain target cells (3). Takada et al. (37) showed that in cell lines, heparin increased osteogenic differentiation induced by BMP homo- and heterodimers. This effect was most evident in the presence of BMP-2 or its heterodimers; heparin did not induce osteogenic differentiation with BMP-6 or -7. Furthermore, BMP-2 activity was enhanced by intestine-derived but not kidney-derived HS and also not by any of the three chemically desulfated heparin derivatives. Because C2C12 myoblasts require prolonged and continuous stimulation by BMP-2 to undergo osteogenic differentiation, the authors suggested that heparin was binding and retaining BMPs in the culture medium and preventing their binding to cell surface/matrix GAGs. Nevertheless, it remains uncertain how heparin mediates these effects on BMP-2 activity, since heparin decreased BMP-2 binding to its receptor. In contrast, other studies (14) show that heparin inhibits BMP-2 signaling and activity in C2C12 myoblasts, whereas sulfated endogenous HSPG are required for internalization of BMP-2 but inhibit its osteogenic differentiating activity. Using embryonic chicken limb bud cells, Ruppert et al. (34) demonstrated that the biological activity of BMP-2 was increased both by exogenous soluble heparin and by inhibition of binding of BMP-2 to cell surface GAGs by a soluble peptide. However, since heparin induced a similar increase in the activity of a recombinant BMP-2 variant lacking the GAG-binding domain, it remained unclear how heparin was modulating the observed effects. The authors speculated that heparin might be acting via its effects on other proteins or receptors. In murine M2–10B4 stromal cells, the activity of BMP-4 is not affected by desulfation of cell surface GAGs (12). Interestingly, exogenous HS enhances BMP-2-induced chondrogenic differentiation, whereas endogenous HSPGs inhibit this effect (4).
We examined the impact of cellular and exogenously provided soluble sulfated GAGs on the proliferation and differentiation of malignant SaOS-2 cells. Our data indicate that cellular sulfated GAGs promote the growth of malignant SaOS-2 cells induced by endogenous mitogens and exogenous BMP-4, while inhibiting BMP-4-induced differentiation. That cell growth of chlorate-treated cells was not restored by soluble heparin suggests that cell proliferation specifically required cellular sulfated GAGs. An important implication of our studies is that endogenous sulfated GAGs appear to promote proliferation and impair differentiation of malignant SaOS-2 human osteosarcoma cells. These data provide a rationale for investigation of the effect on malignant cells of growth factor-binding GAGs/GAG mimetics or agents that affect endogenous GAG synthesis (42).
Finally, our results with BMP-4 signaling in Hurler MAPC points to the existence of a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses. That BMP-4 signaling was improved following clearance of accumulated GAGs and oligosaccharides suggests that activity of these morphogens may be impaired by structurally and functionally abnormal GAGs that accumulate in these diseases. These results are consistent with and extend our previous findings showing that the progressive accumulation of GAGs in Hurler MAPC interfere with FGF-2 signaling and activity and that this is likely attributable to the abnormal HS (30). However, since both HS and DS accumulate in Hurler syndrome and both are cleared by treatment with IDUA enzyme, we cannot rule out a contributory role of DS in the impaired BMP-4 signaling we observed in Hurler MAPC. These studies suggest that impairment of cytokine/morphogen signaling and activity may be a common thread that contributes to the pathophysiology of the mucopolysaccharidoses.
This work was supported by National Institutes of Health R01-NS-48606 and the Department of Veterans Affairs.
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