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Departments of Research (R.C.P., E.C.) and Medicine (E.C.) , Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105; University of Connecticut School of Medicine (E.C.), Farmington, Connecticut 06030; and Regeneron Pharmaceuticals, Inc. (A.N.E.), Tarrytown, New York 10591
Address all correspondence and requests for reprints to: Ernesto Canalis, M.D., Department of Research, Saint Francis Hospital and Medical Center, 114 Woodland Street, Hartford, Connecticut 06105-1299. E-mail: ecanalis{at}stfranciscare.org
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Introduction |
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BMPs, like other growth factors, are regulated at the level of expression and activity, and the effects of BMPs can be modulated by a group of polypeptides that antagonize BMP action (5, 10). BMP antagonists prevent BMP signaling by binding BMPs with high affinity, therefore precluding their binding to specific cell surface receptors (11, 12, 13, 14, 15). Polypeptides antagonizing BMP action include noggin, chordin, and the Dan/cerberus family of genes, which is comprised of the head inducer cerberus, the tumor suppresser Dan, gremlin and its rat homologue drm, the protein related to Dan and cerberus, and Dte (11, 12, 14, 15, 16, 17, 18, 19, 20). Noggin, but not chordin, was recently shown to be expressed by skeletal cells, and studies in noggin null mice demonstrate that it is essential for proper skeletal development (8, 21, 22). Noggin null mice have excessive cartilage and fail to initiate joint formation, probably due to excessive BMP action. Noggin blocks the effect of BMPs on osteoblastogenesis and osteoblastic function, and although unstimulated osteoblasts express modest levels of noggin, they express noggin messenger RNA (mRNA) and protein after BMP exposure (8, 21). This would suggest that induction of BMP antagonists may be a protective mechanism to prevent excessive exposure of skeletal cells to BMPs. However, except for noggin and chordin, there is no information about the expression and function of BMP antagonists in osteoblasts.
Gremlin was cloned from a Xenopus ovary library as a novel gene with axial patterning activities. The product of the gene is a secreted polypeptide with a Mr of 28 kDa, and, like noggin, it prevents BMP receptor binding and activities in a variety of assays, but not the activity of other members of the TGFß family (14). There is no information about the expression or activity of gremlin in osteoblasts. In the present study we examined the expression of gremlin in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells) and determined its regulation by BMPs and other growth regulators. We also examined whether gremlin modified the actions of BMPs in cultured osteoblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50,000 cells/cm2). For
the nuclear run-on experiment, first passage cultures were used. Ob
cells were cultured in DMEM (Life Technologies, Inc.,
Grand Island, NY), supplemented with nonessential amino acids and 10%
FBS (Summit Biotechnologies, Fort Collins, CO). Immortalized
osteoblastic mouse MC3T3-E1 cells were plated at a density of 14,000
cells/cm2 in 
MEM (Life Technologies, Inc.) containing 20 mM HEPES and 10% FBS and were
grown under the same conditions as Ob cells to confluence (100,000
cells/cm2) (24). Cells were exposed
to serum-free medium for 20–24 h and then exposed to test or control
medium in the absence of serum for 2–24 h. Recombinant human BMP-2 (a
gift from Genetics Institute, Cambridge, MA), TGFß1 (a
gift from Genentech, Inc., South San Francisco, CA),
fibroblast growth factor-2 (FGF-2), platelet-derived growth factor BB
(PDGF-BB), insulin-like growth factor I (IGF-I; Austral, San Ramon,
CA), and gremlin (Regeneron Pharmaceuticals, Inc.,
Tarrytown, NY) were added directly to the medium. Recombinant human
BMP-4 and BMP-6 were dissolved in 0.1% trifluoroacetic acid and
diluted 1:8,000 and 1:4,000 in culture medium. PTH-(1–34)
(Bachem, Torrance, CA) was dissolved in 0.05 N
HCl containing 4 mg/ml BSA and diluted 1:10,000 or greater in culture
medium. Porcine insulin (Sigma, St. Louis, MO) was
dissolved in 0.001 N HCl and diluted 1:1,000 in DMEM,
recombinant human GH (a gift from P. A. Kelly, Paris, France) was
dissolved in distilled water, and T3
(Sigma) was added directly to the culture medium.
1,25-Dihydroxyvitamin D3 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), cortisol, and
cycloheximide (both from Sigma) were dissolved in ethanol
and diluted 1:1,000 in DMEM. Control cultures contained equal amounts
of solvent. For RNA analysis, the cell layer was extracted with
guanidine thiocyanate at the end of the incubation and stored at -70
C. For the nuclear run-on assay, nuclei were isolated by Dounce
homogenization (Kontes Co., Vineland, NJ). For gremlin protein
analysis, the medium was collected in the presence of 0.1%
polyoxyethylene sorbitan monolaurate (Tween-20, Pierce Chemical Co., Rockford, IL). For DNA and protein synthesis and
determination of alkaline phosphatase activity (APA), the cell layer
was extracted and stored at -70 C.
Northern blot analysis
Total cellular RNA was isolated using the RNeasy kit according
to the manufacturer’s instructions (QIAGEN, Chatsworth,
CA). The RNA recovered was quantitated by spectrometry, and equal
amounts of RNA from control or test samples were loaded on a
formaldehyde agarose gel after denaturation. The gel was stained with
ethidium bromide to visualize RNA standards and ribosomal RNA,
confirming equal RNA loading of the various experimental samples. The
RNA was blotted onto GeneScreen Plus charged nylon
(DuPont/NEN, Wilmington, DE), and the uniformity of
transfer was confirmed by revisualization of ethidium bromide-stained
ribosomal RNA. A 0.6-kb XhoI/NotI human gremlin
complementary DNA (cDNA; Regeneron Pharmaceuticals, Inc.)
was purified by agarose gel electrophoresis and labeled with
[-32P]deoxy-CTP and
[-32P]deoxy-ATP (50 µCi each at a specific
activity of 3000 Ci/mmol; DuPont/NEN) using the random
hexanucleotide primed second strand synthesis method (25).
Hybridizations were carried out at 42 C for 16–72 h, followed by two
posthybridization washes at room temperature for 15 min in 1 x
saline sodium citrate (SSC) and a third wash performed at 65 C for 30
min in 1 x SSC. The blots were stripped and rehybridized with an
-32P-labeled 752-bp
BamHI/SphI restriction fragment of the murine 18S
ribosomal RNA cDNA (American Type Culture Collection,
Manassas, VA) under conditions previously described (8).
The bound radioactive material was visualized by autoradiography on
Kodak X-AR5 film (Eastman Kodak Co.,
Rochester, NY), employing Cronex Lightning Plus
(DuPont/NEN) or Biomax MS (Eastman Kodak Co.)
intensifying screens. Relative hybridization levels were determined by
densitometry, and levels of gremlin expression were normalized to the
levels of 18S hybridization. The Northern analyses shown are
representative of three or more cultures.
Nuclear run-on assay
To examine changes in the rate of transcription, nuclei were
isolated by Dounce homogenization in a Tris buffer containing 0.5%
IGEPAL-650 (Sigma) (26). Nascent transcripts
were labeled by incubation of nuclei in a reaction buffer containing
500 µM each of ATP, CTP, and GTP; 150 U RNasin
(Promega Corp., Madison, WI); and 250 µCi
[-32P]UTP (800 Ci/mM;
DuPont/NEN) (26). RNA was isolated by
treatment with DNase I and proteinase K, followed by phenol-chloroform
extraction and ethanol precipitation. Linearized gremlin cDNA was
immobilized onto GeneScreen Plus by slot blotting according to the
manufacturer’s directions (DuPont). The plasmid vector pGL2-Basic
(Promega Corp.) was used as a control for nonspecific
hybridization, and 18S ribosomal RNA cDNA was used to estimate
uniformity of counts applied to the membrane. Equal counts per min of
[-32P]RNA from each sample were hybridized
to cDNAs at 42 C for 72 h and washed in 1 x SSC at 65 C for
60 min. Hybridized cDNAs were visualized by autoradiography.
Western blot analysis
To examine changes in gremlin protein levels, aliquots of the
culture medium representing one culture well were precipitated with 5%
trichloroacetic acid and mixed with Laemmli sample buffer to give a
final concentration of 2% SDS. To test for the presence of
oligosaccharides, the precipitated samples were resuspended in sodium
phosphate buffer and exposed to a mixture of deglycosylating enzymes
before electrophoresis, in accordance with the manufacturer’s
instructions (glycoprotein deglycosylation kit,
Calbiochem, La Jolla, CA). Supernatant from COS-7 cells
overexpressing human gremlin was used as a standard. Samples were
fractionated by PAGE on a 15% denaturing gel in the presence of
reducing agents (27). Proteins were transferred to
Immobilon P membranes (Millipore Corp., Bedford, MA),
blocked with 2% BSA, and exposed to 20 ng/ml of a rat monoclonal
antibody raised against human gremlin (Regeneron Pharmaceuticals, Inc.) in 1% BSA overnight. Blots were exposed to a goat antirat
IgG antiserum conjugated to horseradish peroxidase and developed with a
horseradish peroxidase chemiluminescent detection reagent. Western
blots are representative of three or more cultures.
DNA, collagen, and noncollagen protein synthesis
DNA synthesis was studied by measuring effects on the
incorporation of
[methyl-3H]thymidine (5
µCi/ml; SA, 78 Ci/mmol; DuPont/NEN) into acid-insoluble
extracts during the last 2 h of culture, as previously described
(28). Data are expressed as disintegrations per
min/0.32-cm2 culture well. Collagen and
noncollagen protein synthesis were determined by measuring the
incorporation of [2,3-3H]proline (12.5
µCi/ml; SA, 40 Ci/mmol; DuPont/NEN) into
collagenase-digestible protein and noncollagen protein according to the
method of Peterkofsky and Diegelmann (28, 29). Data are
expressed as disintegrations per min/2-cm2
culture well.
APA
APA was determined in 0.5% Triton X-100 cell extracts by
hydrolysis of p-nitrophenyl phosphate to
p-nitrophenol and was measured by spectroscopy at 410 nm
after 30 min of incubation according to the manufacturer’s
instructions (Sigma). Data are expressed as picomoles of
p-nitrophenol released per min/µg protein. Total protein
content in the extracts was determined by the Coomassie brilliant blue
G-250 dye-binding assay of Bradford in accordance with the
manufacturer’s instructions (Bio-Rad Laboratories, Inc.,
Richmond, CA).
Statistical analysis
Data are expressed as the mean ± SEM.
Statistical differences were determined by ANOVA and
post-hoc examination by Ryan-Einot-Gabriel-Welch F test,
using a Crunch Statistical Package (Crunch Software Corp., Oakland, CA)
(30).
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Results |
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). The effect was more pronounced after
24 h and varied somewhat from experiment to experiment, so that
BMP-2 at 3.3 nM increased gremlin mRNA by about 4- to
8-fold after 6 h and by about 15-fold after 24 h. BMP-4
caused a similar stimulation of gremlin transcripts, whereas BMP-6 had
a more modest effect (not shown). The stimulatory effect of BMP-2 was
dose-dependent, and treatment of Ob cells with BMP-2 at 0.3–3.3
nM (10–100 ng/ml) for 6 h increased gremlin mRNA
levels (Fig. 2). Western blot analysis of
conditioned medium from Ob cells revealed that BMP-2 at 3.3
nM for 24 h, but not for 2 or 6 h, induced a
protein reacting with the gremlin antibody and migrating with a
Mr of approximately 28 kDa, the known
Mr of gremlin (Fig. 3) (15). The protein
comigrated with one of the products of COS-7 cells overexpressing human
gremlin; however, the conditioned medium of COS-7 and Ob cells
contained additional proteins that reacted with the gremlin antibody
and could not be characterized. To determine whether some of the
proteins in Ob cell-conditioned medium represented different degrees of
gremlin glycosylation, medium samples were precipitated and exposed to
a mixture of deglycosylating enzymes. The predominant immunoreactive
band induced by BMP-2 migrated with a Mr of 28
kDa before and 26 kDa after deglycosylation, confirming prior
observations that glycosylation plays a role in the
Mr heterogeneity of gremlin (31).
BMP-2 increased gremlin polypeptide at 1 and 3.3 nM, and at
lower doses it had an inconsistent effect (not shown).
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). BMP-2 increased
gremlin transcripts by (mean ± SEM; n = 3)
7.7 ± 0.5-fold in the absence of cycloheximide and by 10.1
± 4.1-fold in the presence of cycloheximide. To determine whether
BMP-2 modified gremlin gene transcription, nuclear run-on assays were
performed twice on nuclei from Ob cells. The assay demonstrated that
BMP-2 for 6 h induced the rate of gremlin transcription by about
2-fold (Fig. 5).
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). The degree of the stimulatory effect
in the experiment shown in Fig. 6 was difficult to determine accurately
because gremlin mRNA levels were below the level of detection in
control cultures. After 6 h, FGF-2 and PDGF BB increased gremlin
mRNA, after correction for 18S hybridization, by 3- to 4-fold. In
contrast, TGFß1 at 1.2 nM and IGF-I at 100 nM
had no effect (not shown). Therefore, the induction of gremlin appeared
to be selective to BMPs, FGF-2, and PDGF-BB. Treatment with hormones,
such as GH at 50 µM, insulin at 100 nM,
T3 at 10 nM, cortisol at 1
µM, PTH at 10 nM, and 1,25-dihydroxyvitamin
D3 at 100 nM, for 2, 6, and 24 h
did not modify gremlin expression in Ob cells (not shown). Similar to
its effects in Ob cells, BMP-2 also increased gremlin mRNA levels in
the mouse MC3T3 osteoblastic cell line (Fig. 7). The effect was noted after 2 h,
was maximal after 6 h, and was sustained for 24 h.
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).
Gremlin at 30 nM decreased basal DNA synthesis and
prevented the effect of BMP-2 at 3.3 nM on DNA, collagen
and noncollagen protein synthesis, and APA in Ob cells.
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Discussion |
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FGF-2 and PDGF-BB induced gremlin, but other hormones and growth factors known to regulate bone cell function failed to up-regulate gremlin expression. Although PDGF-BB and FGF-2 have important mitogenic activity for skeletal cells, they do not acutely modify the differentiated function of osteoblasts like BMPs (33, 34). It is possible that during periods of mitogenesis, there is a need to prevent the effects of BMPs on cell differentiation, and gremlin induction could play such a role. It is important to note that in contrast to their actions on gremlin induction, neither FGF-2 nor PDGF-BB induces noggin transcripts in osteoblasts, whereas BMPs induce both noggin and gremlin (8).
In the present studies we confirm that BMP-2 has modest mitogenic properties for Ob cells, stimulates collagen and noncollagen protein synthesis, and increases APA in osteoblasts (8). Gremlin, like noggin, decreased the biological effect of BMP-2 on osteoblastic function, indicating that it is capable of attenuating the effects of BMPs in osteoblasts, as previously shown in stromal cells (14). Our studies also indicate that gremlin, like noggin, can antagonize selected actions of endogenous BMPs, such as DNA labeling and collagenase expression (35). These observations in conjunction with an induction of gremlin and noggin after exposure to BMPs indicate a possible role for these two factors in limiting the actions of BMPs in cells of the osteoblastic lineage. It is intriguing that the expression of noggin and gremlin in unstimulated cells is limited. This would suggest that they act as BMP-inducible binding proteins to prevent overexposure of osteoblasts to the morphogenetic proteins. There have been no reports of direct cellular actions of gremlin or noggin or of the existence of specific receptors for either peptide, suggesting that their primary function is to act as BMP-binding proteins. As such, they could prevent the effects of BMP, prolong their half-lives, and store or transport BMPs to other target cells in the bone microenvironment. Our studies also confirm recent observations demonstrating posttranslational modifications of gremlin in the form of glycosylation (31).
The induction of noggin and gremlin by BMPs should not be surprising, as they may regulate the amount of effective or biologically available BMPs in a manner analogous as that described for IGFs and IGF-binding proteins (IGFBP) (36). Frequently, agents that induce IGF I expression also enhance the synthesis of selected IGFBPs by the osteoblast. This would suggest not only a coordinated synthesis among binding proteins and IGF-I, but also a possible role for the binding proteins in modifying the activity of IGF I in bone (37, 38). In addition, IGF I itself induces the transcription of IGFBP-5 in osteoblasts, so that the induction of a binding protein by a growth factor is not unique to BMPs (39).
Binding proteins for other members of the TGFß family of peptides also have been reported. Follistatin binds and regulates the activity of activin in skeletal and nonskeletal cells, although there are no reports of follistatin induction by activin in osteoblasts (40, 41). The induction of gremlin and noggin by BMPs may be a necessary protective mechanism to avoid excessive exposure of skeletal cells to BMPs in the bone healing and remodeling processes. In fact, inappropriate expression of BMP-4 is associated with fibrodysplasia ossificans progressiva, and transgenic mice overexpressing TGFß2 in the bone microenvironment develop osteopenia, suggesting that excessive cellular exposure to growth factors may be detrimental (42, 43). Recently, we demonstrated the suppression of BMP-4 by BMPs, suggesting additional negative feedback loops to regulate BMP levels and action in the skeletal system (5). These observations, in conjunction with the induction of gremlin after exposure to BMPs, indicate a complex interaction between BMPs and their antagonists to control BMP activity in the bone microenvironment.
In conclusion, the present studies demonstrate that BMPs cause an induction of gremlin mRNA and protein levels in skeletal cells by transcriptional mechanisms, and gremlin prevents the effects of BMPs in osteoblasts. The induction of gremlin by BMPs could be an additional mechanism to limit the effects of BMP in bone.
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Acknowledgments |
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Footnotes |
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Received April 26, 2000.
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