Molecular Biology of the Cell Vol. 3, 189-196, February 1992
Bone Morphogenetic Proteins-2 and -4 are Involved in the Retinoic Acid-Induced Differentiation of Embryonal Carcinoma Cells Melissa B. Rogers,* Vicki Rosen,t John M. Wozney,t and Lorraine J. Gudas** *Pharmacology Department, Cornell University Medical College, New York, New York, 10021; and tGenetics Institute, Cambridge, Massachusetts 02140 Submitted October 1, 1991; Accepted November 19, 1991
Bone morphogenetic proteins-2 and -4 (BMPs-2 and -4) are transforming growth factor (related proteins that can induce bone formation in vivo. We observed that the level of endogenous BMP-2 mRNA increased an average of 11-fold on differentiation of F9 embryonal carcinoma cells into parietal endoderm after treatment with retinoic acid (RA) and cAMP, whereas the message for the closely related BMP-4 decreased 12-fold after this treatment. Therefore, the effects of exogenous recombinant BMP-2 protein on the RAinduced differentiation of F9 embryonal carcinoma cells were investigated. BMP-2 addition altered the growth and morphology of RA-treated but not untreated cells. Moreover, the abundance of several messages was affected by exogenous BMP-2 treatment. Notably, the BMP-2 and -4 messages themselves were reduced by the addition of exogenous BMP-2. The observations suggest that RA, which is known to affect bone morphogenesis, may regulate the osteoinductive proteins, BMP-2 and -4. Furthermore, BMP-2 and -4 may be involved in preimplantation embryogenesis. INTRODUCTION Retinoids, such as retinoic acid (RA),1 are extremely teratogenic to developing vertebrate embryos, a fact that diminishes their clinical usefulness. In particular, specific malformations of skeletal structures are often observed. For instance, RA induces craniofacial abnormalities in developing avian (Wedden et al., 1988) and mammalian embryos (Morriss-Kay, 1991). The effects of RA on the developing chick limb and the regenerating amphibian limb provide striking examples of how RA can alter long-bone pattern. RA affects both developing and regenerating limbs by reproducibly inducing limb duplications (for review, see Brockes, 1989). Local application of RA to the anterior margin of the developing chick limb causes anterior-posterior duplication, whereas exogenous RA causes proximal-distal duplication in the t Corresponding author.
l BMP, bone morphogenetic protein; CRABP I, cellular retinoic acidbinding protein I; RA, retinoic acid; RhBMP, recombinant human BMP; RACT, retinoic acid, dibutyryl cAMP, and theophylline; RAR, retinoic acid receptor; TGF, transforming growth factor.
© 1992 by The American Society for Cell Biology
regenerating amphibian limb. These effects may result from alterations in a preexisting RA gradient, the existence of which is supported by measurements of endogenous chick limb retinoids (Thaller and Eichele, 1987) or by the induction of a specialized group of cells, the zone of polarizing activity, which subsequently organizes a new pattern (Noji et al., 1991; Wanek et al., 1991). Bone morphogenetic proteins (BMPs) are molecules that can induce cartilage and bone growth in vivo (Urist, 1965; Urist et al., 1973) and, as such, are potential targets for the teratogenic effects of retinoids. Moreover, many BMPs have been cloned and sequenced and shown to be members of the transforming growth factor beta (TGF B) superfamily (BMPs-2-6) (Wozney et al., 1988; Celeste et al., 1990). This is significant because many TGF 3s are regulated by RA or act specifically on RA-treated cells (for review, see Sporn and Roberts, 1991). It is likely that BMPs play roles other than in bone induction. In situ hybridization analyses indicate that the messages for BMP-2, -4, and -6 (Vgr-1) are expressed in many nonbony tissues, including the developing heart, hair and whisker follicles, central nervous system, 189
M.B. Rogers et al.
and preimplantation embryos (Lyons et al., 1989; 1990; Jones et al., 1991). Moreover, several members of the TGF ,B family have been shown to induce mesoderm in Xenopus animal cap explants (BMP-4 [Koster et al., 1991], activin A and B [Thomsen et al., 1990], and TGF ,B2 [Rosa et al., 1988] and 3 [Roberts et al., 1990]) and axial structures in isolated chick epiblasts (PIF/activin [Mitrani et al., 1990]). Many additional TGF-/3-like factors are expressed in early embryos (e.g., Vg-1 and TGF /5 (Xenopus) and BMP-6, mouse [Lyons et al., 1989]) but have not, as yet, been demonstrated to induce mesoderm (for review, see Melton, 1991). Mitrani et al. (1990) have suggested that activin /A iS the endogenous axial mesoderm inducer, as it is expressed in the chick hypoblast, which is known to induce axial pattern in the overlying epiblast. As the mouse extraembryonic endoderm is thought to perform a similar function in the mouse embryo (Hogan et al., 1986), similar mesoderm-inducing molecules might also be identified in this tissue. Although the in vivo studies indicate quite clearly that RA can alter bone pattern, it is difficult to analyze the molecular mechanism of this effect in the models described above. Likewise, mesoderm induction is difficult to study in the small and inaccessible mouse embryo. F9 embryonal carcinoma cells can be induced to differentiate into cells that resemble extraembryonic endoderm in the early mouse embryo (Strickland et al., 1980; Hogan et al., 1981). RA alone induces primitive endoderm, and RA with agents that increase intracellular cAMP levels (e.g., dibutyryl cAMP and theophylline) induces parietal endoderm. This differentiation has been well characterized at the molecular level, and the steady-state levels of many messages increase or decrease during differentiation. Moreover, genes that are regulated by RA in F9 cells, for example, the RA receptor a (RAR /3), are also regulated by RA in embryonic chick limbs (Noji et al., 1991). Therefore, we have utilized this in vitro RA-responsive model to ask, first, whether BMPs-2 and -4 are regulated by RA and, second, whether these factors affect RA-treated cells. We report here that the BMP-2 and -4 messages are expressed in a regulated manner during the RA-induced differentiation of F9 cells. In addition, we show that recombinant human BMP-2 (rhBMP-2) affects the morphology, growth, and gene expression of F9 embryonal carcinoma cells. The data suggest that RA and BMP-2 together may affect embryonic differentiation and bone formation. MATERIALS AND METHODS Cell Culture and Differentiation Monolayer F9 embryonal carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum and 2 mM glutamine and were induced to differentiate into primitive endoderm by the addition of 0.5MuM RA and into parietal
190
endoderm by the addition of 0.5 MM RA, 250 MM cAMP, and 500 AM theophylline. RhBMP-2 at >95% purity was provided by Genetics Institute, Cambridge, MA.
RNA Isolation and Analysis Total cellular RNA was isolated by the guanidinium isothiocyanate method (Kingston, 1987), fractionated on 1% agarose/2.2M formaldehyde gels, transferred to nitrocellulose and hybridized to [32PJ-labeled probes (Feinberg and Vogelstein, 1983; LaRosa and Gudas, 1988a). Radioactive signal due to hybridization was measured directly from the nitrocellulose on a Betascope (Betagen, Waltham, MA) or indirectly by densitometry tracing of the autoradiograms using a Hewlett-Packard (Boston, MA) Scanjet Plus with Enhance 1.0.1 software.
Plasmids All cDNA clones employed were of murine origin, except those for human RAR a, #, and ,y. PAct-G (actin), PGemERA Bam 3 (Hox 1.6), pCI56 (laminin B1), pLamB2-1.5gem (laminin B2), and pCI5 (collagen IV[al]) are described in Rogers et al. (1990). P2-5 (Rex-i) is described in Hosler et al. (1989). cDNA plasmids encoding RAR a, ,B, and ,y were a gift from Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, FR), and probes were prepared as described in Hu and Gudas (1990). pCRABP gem 10 cellular RA-binding protein I [CRABP I]) (Stoner and Gudas, 1989) was constructed by Dr. C. Stoner. pMTM.2B2 (thrombomodulin) (Dittman et al., 1988) was a gift from Dr. W. Dittman (Duke University, Durham, NC). Fragments of BMP2 (BMP2-68) and BMP-4 (BMP4-40) cDNAs were subcloned into pGEM3. The regions of the BMP-2 and -4 transcripts chosen for probes are the least-conserved portions of the proregions. These probes detect distinct transcripts on Northern blots and in situ hybridized embryo sections and do not cross-hybridize to other BMPs.
RESULTS BMP-2 and -4 mRNA Expression in F9 Cells To determine if BMP-2 and -4 mRNAs were expressed in untreated F9 cells or in differentiated F9 cells, radioactive cDNA probes for BMP-2 and -4 were hybridized to Northern blots of total RNA isolated from F9 cells grown in monolayer for 1 or 3 d without drugs or with RA or RA + dibutyryl cAMP and theophylline (RACT) added to induce primitive and parietal endoderm, respectively (Figure 1A). The results indicate that BMP-2 mRNA levels are low in untreated F9 cells and are high in F9 cells induced to differentiate into parietal endoderm by RACT. In marked contrast, BMP-4 mRNA levels are high in untreated F9 cells and low in differentiated cells. Treatment of F9 cells with dibutyryl cAMP and theophylline alone or with retinol, retinyl acetate, or retinal (which do not induce differentiation) does not significantly affect the abundance of the BMP-2 and -4 mRNAs (Figure 1B). Quantitation of the signal intensities in Figures 1, A and B, and 4 indicate that, on average, after 3 d of drug treatment, the abundance of BMP-2 mRNA was increased 2-fold by RA and 11-fold by RACT, whereas the BMP-4 mRNA was decreased 6-fold by RA and 12-fold by RACT. Thus, expression of the BMP-2 and -4 genes is clearly modulated by RA in F9 cells. Molecular Biology of the Cell
Bone Morphogenetic Proteins and Differentiation
A
RA CT D
_
_
_
_
1
+
+
+
+
-
+
+
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3
1
3
3
BMP 2 *
BMP 4
A
ACTIN
&Mfi: innn i*
B
.-
4 L
(IU I-
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Uc 4
Bone Morphogenetic Proteins-2 and -4 are Involved in the Retinoic Acid-Induced Differentiation of Embryonal Carcinoma Cells Melissa B. Rogers,* Vicki Rosen,t John M. Wozney,t and Lorraine J. Gudas** *Pharmacology Department, Cornell University Medical College, New York, New York, 10021; and tGenetics Institute, Cambridge, Massachusetts 02140 Submitted October 1, 1991; Accepted November 19, 1991
Bone morphogenetic proteins-2 and -4 (BMPs-2 and -4) are transforming growth factor (related proteins that can induce bone formation in vivo. We observed that the level of endogenous BMP-2 mRNA increased an average of 11-fold on differentiation of F9 embryonal carcinoma cells into parietal endoderm after treatment with retinoic acid (RA) and cAMP, whereas the message for the closely related BMP-4 decreased 12-fold after this treatment. Therefore, the effects of exogenous recombinant BMP-2 protein on the RAinduced differentiation of F9 embryonal carcinoma cells were investigated. BMP-2 addition altered the growth and morphology of RA-treated but not untreated cells. Moreover, the abundance of several messages was affected by exogenous BMP-2 treatment. Notably, the BMP-2 and -4 messages themselves were reduced by the addition of exogenous BMP-2. The observations suggest that RA, which is known to affect bone morphogenesis, may regulate the osteoinductive proteins, BMP-2 and -4. Furthermore, BMP-2 and -4 may be involved in preimplantation embryogenesis. INTRODUCTION Retinoids, such as retinoic acid (RA),1 are extremely teratogenic to developing vertebrate embryos, a fact that diminishes their clinical usefulness. In particular, specific malformations of skeletal structures are often observed. For instance, RA induces craniofacial abnormalities in developing avian (Wedden et al., 1988) and mammalian embryos (Morriss-Kay, 1991). The effects of RA on the developing chick limb and the regenerating amphibian limb provide striking examples of how RA can alter long-bone pattern. RA affects both developing and regenerating limbs by reproducibly inducing limb duplications (for review, see Brockes, 1989). Local application of RA to the anterior margin of the developing chick limb causes anterior-posterior duplication, whereas exogenous RA causes proximal-distal duplication in the t Corresponding author.
l BMP, bone morphogenetic protein; CRABP I, cellular retinoic acidbinding protein I; RA, retinoic acid; RhBMP, recombinant human BMP; RACT, retinoic acid, dibutyryl cAMP, and theophylline; RAR, retinoic acid receptor; TGF, transforming growth factor.
© 1992 by The American Society for Cell Biology
regenerating amphibian limb. These effects may result from alterations in a preexisting RA gradient, the existence of which is supported by measurements of endogenous chick limb retinoids (Thaller and Eichele, 1987) or by the induction of a specialized group of cells, the zone of polarizing activity, which subsequently organizes a new pattern (Noji et al., 1991; Wanek et al., 1991). Bone morphogenetic proteins (BMPs) are molecules that can induce cartilage and bone growth in vivo (Urist, 1965; Urist et al., 1973) and, as such, are potential targets for the teratogenic effects of retinoids. Moreover, many BMPs have been cloned and sequenced and shown to be members of the transforming growth factor beta (TGF B) superfamily (BMPs-2-6) (Wozney et al., 1988; Celeste et al., 1990). This is significant because many TGF 3s are regulated by RA or act specifically on RA-treated cells (for review, see Sporn and Roberts, 1991). It is likely that BMPs play roles other than in bone induction. In situ hybridization analyses indicate that the messages for BMP-2, -4, and -6 (Vgr-1) are expressed in many nonbony tissues, including the developing heart, hair and whisker follicles, central nervous system, 189
M.B. Rogers et al.
and preimplantation embryos (Lyons et al., 1989; 1990; Jones et al., 1991). Moreover, several members of the TGF ,B family have been shown to induce mesoderm in Xenopus animal cap explants (BMP-4 [Koster et al., 1991], activin A and B [Thomsen et al., 1990], and TGF ,B2 [Rosa et al., 1988] and 3 [Roberts et al., 1990]) and axial structures in isolated chick epiblasts (PIF/activin [Mitrani et al., 1990]). Many additional TGF-/3-like factors are expressed in early embryos (e.g., Vg-1 and TGF /5 (Xenopus) and BMP-6, mouse [Lyons et al., 1989]) but have not, as yet, been demonstrated to induce mesoderm (for review, see Melton, 1991). Mitrani et al. (1990) have suggested that activin /A iS the endogenous axial mesoderm inducer, as it is expressed in the chick hypoblast, which is known to induce axial pattern in the overlying epiblast. As the mouse extraembryonic endoderm is thought to perform a similar function in the mouse embryo (Hogan et al., 1986), similar mesoderm-inducing molecules might also be identified in this tissue. Although the in vivo studies indicate quite clearly that RA can alter bone pattern, it is difficult to analyze the molecular mechanism of this effect in the models described above. Likewise, mesoderm induction is difficult to study in the small and inaccessible mouse embryo. F9 embryonal carcinoma cells can be induced to differentiate into cells that resemble extraembryonic endoderm in the early mouse embryo (Strickland et al., 1980; Hogan et al., 1981). RA alone induces primitive endoderm, and RA with agents that increase intracellular cAMP levels (e.g., dibutyryl cAMP and theophylline) induces parietal endoderm. This differentiation has been well characterized at the molecular level, and the steady-state levels of many messages increase or decrease during differentiation. Moreover, genes that are regulated by RA in F9 cells, for example, the RA receptor a (RAR /3), are also regulated by RA in embryonic chick limbs (Noji et al., 1991). Therefore, we have utilized this in vitro RA-responsive model to ask, first, whether BMPs-2 and -4 are regulated by RA and, second, whether these factors affect RA-treated cells. We report here that the BMP-2 and -4 messages are expressed in a regulated manner during the RA-induced differentiation of F9 cells. In addition, we show that recombinant human BMP-2 (rhBMP-2) affects the morphology, growth, and gene expression of F9 embryonal carcinoma cells. The data suggest that RA and BMP-2 together may affect embryonic differentiation and bone formation. MATERIALS AND METHODS Cell Culture and Differentiation Monolayer F9 embryonal carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum and 2 mM glutamine and were induced to differentiate into primitive endoderm by the addition of 0.5MuM RA and into parietal
190
endoderm by the addition of 0.5 MM RA, 250 MM cAMP, and 500 AM theophylline. RhBMP-2 at >95% purity was provided by Genetics Institute, Cambridge, MA.
RNA Isolation and Analysis Total cellular RNA was isolated by the guanidinium isothiocyanate method (Kingston, 1987), fractionated on 1% agarose/2.2M formaldehyde gels, transferred to nitrocellulose and hybridized to [32PJ-labeled probes (Feinberg and Vogelstein, 1983; LaRosa and Gudas, 1988a). Radioactive signal due to hybridization was measured directly from the nitrocellulose on a Betascope (Betagen, Waltham, MA) or indirectly by densitometry tracing of the autoradiograms using a Hewlett-Packard (Boston, MA) Scanjet Plus with Enhance 1.0.1 software.
Plasmids All cDNA clones employed were of murine origin, except those for human RAR a, #, and ,y. PAct-G (actin), PGemERA Bam 3 (Hox 1.6), pCI56 (laminin B1), pLamB2-1.5gem (laminin B2), and pCI5 (collagen IV[al]) are described in Rogers et al. (1990). P2-5 (Rex-i) is described in Hosler et al. (1989). cDNA plasmids encoding RAR a, ,B, and ,y were a gift from Dr. P. Chambon (Institut de Chimie Biologique, Strasbourg, FR), and probes were prepared as described in Hu and Gudas (1990). pCRABP gem 10 cellular RA-binding protein I [CRABP I]) (Stoner and Gudas, 1989) was constructed by Dr. C. Stoner. pMTM.2B2 (thrombomodulin) (Dittman et al., 1988) was a gift from Dr. W. Dittman (Duke University, Durham, NC). Fragments of BMP2 (BMP2-68) and BMP-4 (BMP4-40) cDNAs were subcloned into pGEM3. The regions of the BMP-2 and -4 transcripts chosen for probes are the least-conserved portions of the proregions. These probes detect distinct transcripts on Northern blots and in situ hybridized embryo sections and do not cross-hybridize to other BMPs.
RESULTS BMP-2 and -4 mRNA Expression in F9 Cells To determine if BMP-2 and -4 mRNAs were expressed in untreated F9 cells or in differentiated F9 cells, radioactive cDNA probes for BMP-2 and -4 were hybridized to Northern blots of total RNA isolated from F9 cells grown in monolayer for 1 or 3 d without drugs or with RA or RA + dibutyryl cAMP and theophylline (RACT) added to induce primitive and parietal endoderm, respectively (Figure 1A). The results indicate that BMP-2 mRNA levels are low in untreated F9 cells and are high in F9 cells induced to differentiate into parietal endoderm by RACT. In marked contrast, BMP-4 mRNA levels are high in untreated F9 cells and low in differentiated cells. Treatment of F9 cells with dibutyryl cAMP and theophylline alone or with retinol, retinyl acetate, or retinal (which do not induce differentiation) does not significantly affect the abundance of the BMP-2 and -4 mRNAs (Figure 1B). Quantitation of the signal intensities in Figures 1, A and B, and 4 indicate that, on average, after 3 d of drug treatment, the abundance of BMP-2 mRNA was increased 2-fold by RA and 11-fold by RACT, whereas the BMP-4 mRNA was decreased 6-fold by RA and 12-fold by RACT. Thus, expression of the BMP-2 and -4 genes is clearly modulated by RA in F9 cells. Molecular Biology of the Cell
Bone Morphogenetic Proteins and Differentiation
A
RA CT D
_
_
_
_
1
+
+
+
+
-
+
+
_
3
1
3
3
BMP 2 *
BMP 4
A
ACTIN
&Mfi: innn i*
B
.-
4 L
(IU I-
en
0CR
' 4
w
Uc 4
