help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, H.
Right arrow Articles by Waters, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, H.
Right arrow Articles by Waters, M. J.
Endocrinology Vol. 139, No. 9 3855-3862
Copyright © 1998 by The Endocrine Society

ARTICLES

Growth Hormone and Insulin-Like Growth Factor I Induce Bone Morphogenetic Proteins 2 and 4: A Mediator Role in Bone and Tooth Formation?1

H. Li, P. M. Bartold, C. Z. Zhang, R. W. Clarkson, W. G. Young and M. J. Waters

Departments of Dentistry (H.L., P.M.B., C.Z.Z., W.G.Y.) and Physiology and Pharmacology (R.W.C., M.J.W.), University of Queensland, St. Lucia, Queensland 4072, Australia

Address all correspondence and requests for reprints to: Prof. M. J. Waters, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4072, Australia. E-mail m.waters{at}mailbox.uq.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH is known to increase the formation of bone and hard tissues of the tooth (dentine, cementum, and enamel), as do bone morphogenetic proteins. GH receptors are expressed in these tissues and could mediate local growth responses. Here we report that both GH and insulin-like growth factor I (IGF-I) are able to increase expression of bone morphogenetic protein-2 and -4 messenger RNAs 4- to 5-fold in human dental pulp fibroblasts in vitro. Induction was seen at physiological concentrations of hormone (25–100 ng/ml GH; 50–200 ng/ml IGF-I) and reached a maximum at 4–8 h. Immunoblot analysis demonstrated that the increase in messenger RNAs resulted in an increase in expressed protein. Anti-IGF-I inhibition experiments indicate that GH is able to induce the response without a requirement for local IGF-I production. These results raise the possibility that bone morphogenetic proteins mediate the local osteogenic actions of GH and IGF-I, and lend support to the view that GH can act through the mediation of factors other than IGF-I. These factors may combine with IGF-I in different tissues to enhance GH action and specificity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A NUMBER of studies have shown that GH is able to increase the formation of hard tissues in vivo [e.g. bone (1, 2, 3), dentine (4, 5, 6), enamel (6, 7), and cementum (8)]. Several in vitro studies have shown GH has the ability to stimulate the proliferation of osteoblasts, including bone marrow stromal osteoblasts (9), and to induce markers of bone formation such as Gla protein and {alpha}1-procollagen (3). Likewise, GH is able to induce proliferation of epithelial stem cells in molar tooth buds, along with preameloblast differentiation and dentine matrix formation (10). It is believed that these actions on bone-forming cells are mediated by local or endocrine insulin-like growth factor I (IGF-I) (11, 12), according to the somatomedin hypothesis. However, evidence is accumulating that GH may also use other mediators to sustain its actions, e.g. hepatocyte growth factor (HGF) in liver (13), fibroblast growth factor in chondrocytes (14), epidermal growth factor (EGF) in kidney and liver (15, 16), and estrogen in uterus (17), through induction of either the growth factor or its receptor. Because of the potent morphogenetic effects of bone morphogenetic proteins (BMPs) in bone, tooth, and cartilage formation, we have sought to test the possibility that GH induces these proteins, providing an additional avenue for its actions.

BMPs have been shown to be involved in a number of processes, such as induction of mesoderm (18), skeletal patterning (19), tooth development (20), and limb morphogenesis (21). They regulate diverse processes, including cell proliferation, determination, differentiation, morphogenesis, and apoptosis (22). Indeed, one of the actions of BMPs is to promote the entry of uncommitted multipotent stem cells into the chondrogenic or osteogenic, rather than myogenic, lineages (22). BMPs also mediate the epithelial-mesenchymal interactions needed for formation of many mixed lineage tissues such as tooth (20) and kidney (22). In particular, BMP-4 is involved in earlier stages of tooth formation, and BMP-2 is involved in later stages (20), where BMP-2 mediates epithelial-mesenchymal interactions through the epithelial enamel knot. In later stages of tooth formation, BMPs have been shown to be expressed in ameloblasts, odontoblasts, and dentine matrix and can induce established dental pulp mesenchymal cells to proliferate and then differentiate into odontoblast cells (20, 23). Consequently, BMPs are able to induce osteodentine and tubular dentine formation in vivo. As we have reported the ability of GH to induce dentine, enamel, and cementum formation (24), and the concurrent expression of GH receptor and GH itself in different stages of tooth formation (25), it was appropriate to determine whether GH is able to induce BMPs in dental pulp fibroblasts. We report here the use of a sensitive and specific competitive PCR assay to establish that GH is able to induce BMP-2 and -4 mRNA expression by these cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human GH (hGH) was a gift from Bresagen (Adelaide, Australia), and recombinant hIGF-I was purchased from Gropep (Adelaide, Australia). Recombinant BMP-2 and -4 were obtained from the Genetics Institute (Cambridge, MA). Goat antihuman BMP-2/4 polyclonal antibody (sc-6267) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-hIGF-I monoclonal antibody (Sm 1.2) was purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture
Human third molars extracted for orthodontic or prosthetic reasons were used to obtain explant cultures of fibroblasts from dental pulp of these teeth (26). For the experiments described here, fifth passage cells were cultured in DMEM (ICN Flow, Sydney, Australia) supplemented with 10% FCS (CSL, Victoria, Australia). Upon reaching confluence (4 x 105 cells/well, six-well plate), cells were treated with varying concentrations of recombinant hGH (25, 50, 100, and 200 ng/ml) or recombinant hIGF-I (50, 100, 200, and 300 ng/ml) in 10% FCS, normally for 48 h. To study the response over time, cells were treated with hGH (100 ng/ml) or hIGF-I (100 ng/ml) for 2, 4, 8, 24, and 48 h. Antihuman IGF-I monoclonal antibody (40 µg/ml) was added to the cultured cells with hGH (100 ng/ml) or IGF-I (100 ng/ml) where appropriate.

RNA isolation and complementary DNA (cDNA) synthesis
Total cellular RNA was isolated from cultured cells by guanidine hydrochloride extraction (27). The RNA concentration of all samples was determined by spectrometer at the same time, and RNA quality was checked by gel electrophoresis. Extracted RNA was reverse transcribed under the following conditions: 1 µg RNA with 12.5 ng/µl oligo(deoxythymidine)15 (Promega, Madison, WI) made to 12 µl with distilled water were heated at 70 C for 10 min, then at 37 C for 60 min with reverse transcription buffer (20 mM Tris-HCl, pH 8.3; 375 mM KCl; and 15 mM MgCl), 50 mM dithiothreitol, 1 U/µl RNasin (Promega), 500 mM mixed deoxy (d)-NTPs (dATP, dGTP, dCTP, and dTTP), and 5 U/µl Moloney’s murine leukemia virus reverse transcriptase (Promega) (28).

Construction of BMP-2 and BMP-4 competitor cDNA
Competitive PCR requires an internal reference DNA to amplify along with target cDNA, using the same primers (29, 30). We constructed homologous reference DNAs of a different size from target cDNA to allow for visualization and quantitation of target and reference products on the PCR gel (Fig. 1Go) (31). To construct the BMP-2 competitor, BMP-2 PCR sequence-1 (886 bp) was synthesized using the primers A-1 (5'-CTA CAT TCT AGA CCT GTA TCG C) and B-2 (5'-TGC TGT ACT ATC GAT ACC CAC; see below for conditions). Primer A contains an XbaI site, and primer B contains a ClaI site (underlined). The BMP-2 PCR sequence-1 was then subcloned into the XbaI and ClaI sites of pBluescript II KS- (Stratagene, La Jolla, CA) to create pBII-BMP2. Unique NcoI and AflII sites within the subcloned BMP-2 PCR sequence were used to excise a 200-bp fragment from pBII-BMP2 (Fig. 1Go). The resulting truncated construct (pBII-BMP2COM) was recircularized by end-filling with Klenow polymerase (New England Biolabs, Beverly, MA) and blunt end ligated with T4 DNA ligase (Boehringer Mannheim, Mannheim, Germany).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. A schematic diagram showing the construction of a BMP-2 competitor DNA. Primers A and B are forward and reverse primers, common to both human BMP-2 cDNA and competitive DNAs.

 
To construct BMP-4 competitor DNA, two oligonucleotide primers, A (5'-ACT TCG AGG CGA CAC TTA TGC) and B (5'-GCT GAA GTC CAC ATA GAG CGA GTG), were used to PCR the BMP-4-coding sequence (788 bp; see below for conditions). BMP-4 competitive DNA (495 bp) was created by digestion with ApaI to remove a 293-bp fragment and religation with T4 DNA ligase.

The sizes of BMP-2 and BMP-4 PCR products and their respective competitor DNA are shown in Fig. 2Go. Dideoxy sequencing of the BMP-2 and BMP-4 primer products yielded a sequence identical to the predicted sequence.



View larger version (64K):
[in this window]
[in a new window]
  		Figure 2. PCR products synthesized from BMP-2 and BMP-4 cDNA in hormone-treated human cultured dental pulp fibroblasts and their competitive DNAs. The upper left band is BMP-2 PCR product (818 bp); the lower left band is BMP-2 competitor DNA PCR product (618 bp). The upper right band is BMP-4 PCR product (788 bp); the lower right band is BMP-4 competitor DNA PCR product (495 bp). Upper bands were amplified from same amount of target cDNA. Lower bands were amplified from a dilution series of competitor DNA. For BMP-2, competitor DNA concentrations were, from the left, 2.4, 1.2, 0.6, 0.3, 0.15, 0.075, 0.038, and 0.019 pg. For BMP-4, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.02 pg of competitor DNA were used.

 
Semiquantitative competitive PCR
For competitive PCR, the target template is coamplified together with a dilution series of the competitor DNA of known concentration using the same specific oligonucleotide primers (Table 1Go) (29, 30). PCR reactions were carried out simultaneously in a total volume of 20 µl, comprising target cDNA (4 µl, from 0.2 µg RNA), competitive DNA (4 µl), 1x reaction buffer, dNTPs (0.4 mM), MgCl2 (1.5 mM), 0.5 U Taq DNA polymerase (Advanced Biotechnologies, Surrey, UK), and 0.2 mM of each primer. Amplification was carried out in a PTC-100TM thermal cycler (Bresatec, Adelaide, Australia). Cycle conditions for both BMP-2 and BMP-4 were as follows: denaturation at 93 C for 3 min, followed by 35 cycles of denaturation at 93 C for 1 min, annealing at 60 C for 30 sec, and extension at 72 C for 1.5 min, followed by further extension at 72 C for 10 min. For each treatment, target cDNAs were amplified from three separate cell cultures.


View this table:
[in this window]
[in a new window]
  		Table 1. PCR primer sequences for both target cDNA and competitive DNA

 
Analysis of competitive PCR products
After competitive PCR, samples were electrophoresed in 1.0% agarose Tris-borate EDTA gels and photographed. The amounts of target DNA and competitive DNA present in each band were determined by densitometry using the NIH Image program (see Fig. 3Go). The band intensity data were converted to ratios of target DNA to competitive DNA and plotted logarithmically against the known concentrations of competitive DNA. The unknown target DNA concentration was determined from the equation of the linear regression line. The point of equivalence (1:1 ratio when log y = 0) is where competitive DNA equals target DNA and represents the concentration of target cDNA in the unknown.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Standard curve for BMP-2 and BMP-4 mRNA determination in cultured human pulp fibroblasts. Ratios of target BMP-2 and BMP-4 cDNA to their competitor DNA (cDNA) were plotted logarithmically against the known concentrations of competitor DNA. Unknown target DNA (tDNA) was determined from the equation of the linear regression line shown under the graph.

 
Immunoblot analysis
Cells were scraped from 10-cm dishes, pelleted in cold PBS, and then resuspended in 300 µl/well lysis buffer (10 mM Tris-HCl, pH 8.0; 5 mM MgCl2; 25 mM KCl; 0.5% Nonidet P-40; 2 mM phenylmethylsulfonylfluoride; 2 µg/ml leupeptin; 2 µg/ml aprotinin; and 1 mM EDTA). The lysate was passed 15 times through a 27-gauge needle and incubated in ice for 10 min, and NaCl was then added to 0.25 M. Cell supernatants were obtained by centrifugation at 15,000 rpm for 1 h in a microfuge at 4 C.

Expressed BMP-2/4 was immunoprecipitated with 10 µg/ml goat antihuman BMP-2/4 polyclonal antibody for 1 h at room temperature, followed by incubation with 20 µl protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) at a 1:1 suspension in PBS for 3 h at 4 C. The bound antibody complexes were collected by centrifugation at 10,000 x g for 30 sec and then washed twice with immunoprecipitation buffer (as for lysis buffer, but without Nonidet P-40) by resuspension, followed by centrifugation.

For immunoblot analysis, 20 µl Laemmli sample buffer (2% SDS, 10% glycerol, 100 mM dithiothreitol, and 60 mM Tris-HCl pH 6.8) were added to protein G pellets, and they were heated at 80 C for 5 min. All of the liquid was then loaded onto 12.5% (for BMP-2) or 10% (for BMP-4) SDS-polyacrylamide gels. Proteins were transferred onto nitrocellulose membrane (Hybond C, Amersham, Aylesbury, UK) using semidry transfer. Membranes were then blocked for 45 min with 5% low fat milk-TBST (20 mM Tris base, 137 mM NaCl, and 0.1% Tween-20, pH 7.6). To detect the BMP-2/4 proteins, blocked membranes were incubated with 2 µg/ml goat antihuman BMP-2/4 polyclonal antibody in 5% low fat milk-TBST for 3 h at room temperature, then washed three times for 10 min each time in TBST. Membranes were subsequently incubated with antigoat horseradish peroxidase-conjugated antibody (NA 9310, Amersham) for 20 min at room temperature, then washed with TBST three times for 10 min each time. Horseradish peroxidase activity was detected using enhanced chemiluminescence (ECL+plus, Amersham), followed by exposure to x-ray film.

Statistics
In all cases comparisons between experimental groups were undertaken with pooled data using ANOVA and Tukey’s post-hoc test for multiple comparisons. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of GH and IGF-I on BMP expression in human dental pulp fibroblasts
As shown in Fig. 4Go, both hGH and IGF-I are able to induce BMP-2 messenger RNA (mRNA) and BMP-4 mRNA. GH is able to stimulate mRNA expression up to 5-fold for BMP-2 and 4-fold for BMP-4, whereas IGF-I gives a maximum 5-fold stimulation with BMP-2 message and a 3-fold stimulation for BMP-4 mRNA. These stimulations are seen well within the range of circulating hormone concentrations. The dose-response curve for GH declines at higher GH concentration for reasons that are unclear, but the decline is not a result of toxicity, as this hGH preparation supported maximal proliferation of a Baf/B03 line expressing the hGH receptor up to 500 ng/ml (M. J. Waters, unpublished observation). Although receptor site 1 occupancy by GH at high concentrations can produce a bell-shaped curve, the decline is normally seen only above 1000 ng/ml (32).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 4. Expression of BMP-2 and -4 RNA in response to varying concentration of hGH and IGF-I. Relative to control cultures in 10% FCS, significant differences for each treatment are shown as P < 0.05 (*) and P < 0.01 (**) by ANOVA with Tukey’s multiple range test. The error bars represent the SD. This experiment was performed three times, each with three separate wells.

 
Induction of cellular BMP-2 and BMP-4 proteins was demonstrated by immunoblot analysis after concentration of cellular BMP by immunoprecipitation (Fig. 5Go). In accord with the absolute levels of mRNA, induction of BMP-2 protein was clearly seen after 6 h of hormone treatment, with a low level of induction seen in the 10% FCS medium control. Induction of BMP-4 protein was more difficult to visualize because the protein runs just above the light chain of the immunoprecipitating antibody. Again in agreement with the absolute levels of BMP-4 mRNA, induction of BMP-4 protein was seen after an overnight (16-h) incubation with GH and IGF-I.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 5. Immunoblot showing induction of BMP-2 and -4 protein by GH and IGF-I. Human pulpal fibroblasts were treated with hormones in 10% FCS or 10% FCS alone (6 h for BMP-2, 16 h for BMP-4) and solubilized, then BMPs were immunoprecipitated with goat anti-BMP-2/4 antibody, immunoblotted with the same antibody, and then visualized by ECL as described in Materials and Methods. A: Lane 1, Recombinant BMP-2, without immunoprecipitation; lane 2, immunoprecipitation without cells; lane 3, 10% FCS medium control; lane 4, hGH (100 ng/ml); lane 5, IGF-I (100 ng/ml). B: Lane 1, Recombinant BMP-4; lane 2, immunoprecipitation without cells; lane 3, 10% FCS medium control; lane 4, hGH (100 ng/ml); lane 5, IGF-I (100 ng/ml). As can be seen from the upper constitutive band, in this experiment immunoprecipitation recovery from the GH treatment was low, and that from the IGF-I treatment was high, but in both cases an induced band was present that is not seen in the control lanes.

 
Time course of induction
Induction of BMP-2 and -4 mRNA at optimum hormone concentrations is shown in Fig. 6Go, A–D, along with the 10% FCS medium control. Absolute levels of BMP-2 mRNA increased rapidly to a maximum at 4 h, declined, and then increased slowly over 48 h (Fig. 6AGo). When expressed as fold induction over the 10% FCS medium control, however, there was a sustained relative induction of BMP-2 mRNA (Fig. 6CGo). Absolute levels of BMP-4 mRNA increased more slowly (Fig. 6BGo), but the induction relative to 10% FCS was quite rapid, reaching a maximum by 4 h and being sustained thereafter (Fig. 6DGo).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. Time course of induction by GH (100 ng/ml) and IGF-I (100 ng/ml) of BMP-2 mRNA and BMP-4 mRNA in cultured human dental pulp fibroblasts. A and B, Absolute levels of mRNA, showing values for 10% FCS medium control for comparison. C and D, Values expressed as fold induction at each time relative to the 10% FCS medium control. The error bars represent the SD. Significant differences from 10% FCS alone were seen at all time points. The experiment was performed three times, in each case in triplicate.

 
Effect of IGF-I antibody neutralization
To determine whether the actions of GH are mediated through the induction of IGF-I, anti-hIGF-I was coincubated with the dental pulp cells during hormone induction. As is apparent from Fig. 7Go, although the antibody was effective in abrogating the IGF-I stimulation, it was without effect on the GH stimulation of either BMP-2 or BMP-4 mRNA.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 7. Antibodies against human IGF-I (IGF-IAb) in the culture medium neutralize the ability of IGF-I, but not GH, to induce BMP mRNA: GH (100 ng/ml) or IGF-I (100 ng/ml) in 10% FCS or 10% FCS alone (medium control) was added to human dental pulp fibroblasts and incubated for 48 h. The third column in each set shows the effect of simultaneous incubation with anti-hIGF-I (40 µg/ml). Pooled data from three experiments are shown, each with three wells per point. Values are the mean ± SD. Significant increases relative to 10% FCS were seen only with IGF-I alone, but with GH, the antibody incubations were also significantly different from the 10% FCS control value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates for the first time that both GH and IGF-I are able to induce two important morphogens, BMP-2 and BMP-4. A 4- to 5-fold local induction of these proteins could be expected to result in many of the inductive effects of BMP seen in the tooth, such as odontoblast differentiation, dentine formation, and enamel formation. Epithelial-mesenchymal interactions play a central role in the regulation of these events. When tissues are dissected from early tooth germs before the bud stage, only the dental epithelium instructs tooth development if cultured with nondental neural crest-derived mesenchyme. After the bud stage, only dental mesenchyme instructs tooth development when cultured with nondental epithelium (33, 34). Along with other molecules in the transforming growth factor-ß superfamily, BMPs are thought to be involved in tooth development partly because of their expression in developing tooth germs and partly because of their inductive ability in vivo (20). BMP-4 mRNA is present in the thickened presumptive dental epithelium before the tooth bud stage, and expression subsequently shifts to the condensing dental mesenchyme. BMP-2 mRNA is expressed in dental epithelium from the early bud stage until the cap stage, then shifts to mesenchyme (20). Such localization data suggest that BMPs are associated with the shift of developmental potential from epithelium to mesenchyme. It is of considerable relevance, therefore, that GH, GH receptor, and binding protein are detected at these tissue and developmental stages in rat tooth germs (25). Furthermore, application of BMPs to exposed dental pulp mesenchyme (corresponding to the fibroblasts studied here) resulted in stimulation of rapid fibrodentine (osteodentine) matrix formation and polarized odontoblast-like cells, which are an indication of cell differentiation, and are only found in association with this primitive osteotypic response (23, 35). Likewise, in the same model of the developing tooth germ, we have demonstrated that both GH and IGF-I exert inductive effects on differentiation of dental epithelium and mesenchymal cells (the odontoblasts) in vitro (10, 24).

It may be that GH acts on mesenchymal fibroblasts elsewhere during later fetal development and postnatally to promote the formation of tissues where BMP is thought to play such a role, e.g. bone, cartilage, kidney, lung, gut, skin, and hair, and the reproductive organs (22). GH receptors are present in all of these tissues, in some cases at a higher level perinatally than in the adult (36). For example, hypertrophic chondrocytes forming the secondary ossification center in the tibia express GH receptors in a location correlating with BMP action in endochondral ossification (37). The putative involvement of BMPs in organs undergoing branching through epithelio-mesenchymal interactions suggest that the ability of GH to induce duct formation in the mammary gland, for example (38), is a result of its ability to induce BMPs. As GH and functional GH receptors are expressed first at the very earliest stages of preimplantation development (39), the possibility arises that some of the important actions of BMP in induction of limb patterning and tissue morphogenesis could be a result of local GH action. The normalcy of these processes in GH-insensitive Laron dwarfs may be a result of the ability of IGF-I to substitute in such a role.

The question of mediation of the BMP response by IGF-I was addressed here in Fig. 7Go. The results indicate that either GH or IGF-I can induce these BMPs, and that GH can still induce BMPs when IGF-I is inhibited. This result can be explained by parallel signaling pathways that trans-activate the BMP promoters. These certainly exist; both GH and IGF-I, for example, activate insulin receptor substrate-1 and -2, phosphatidylinositol 3- kinase, and mitogen-activated protein kinase pathways (40, 41). Such parallel pathways would be advantageous in maintaining signal input; initially, the GH pulse would initiate an induction of BMP along with IGF-I, followed by a second induction as a result of IGF-I action, which would sustain the signal. This would be expected to occur in vivo; with our in vitro system it appears that local production of IGF-I is not contributing significantly to GH induction of BMPs over the 8-h maximum. Confluent adult fibroblasts such as those used here do synthesize less IGF-I in response to mitogens (42), and the IGF-I response to GH is less than that to other mitogens (43).

This study raises the issue of the primacy of IGF-I mediation in GH action. As indicated in the introduction, GH induces a number of other growth factors or their receptors that could carry out roles in addition to those of IGF-I. Examples include HGF in the liver (13) and probably in the tooth, where HGF is also involved in tooth morphogenesis (44), and fibroblast growth factor in chondrocytes, for which FGF mRNA is induced much more effectively by GH than is IGF-I mRNA (14). FGF is a powerful mitogen for chondrocytes (14). Other examples include induction of TGFß in hepatocytes (45), induction of EGF receptors in liver and granulosa cells (16, 46), induction of EGF itself in kidney (15), induction of interleukin-6 in osteoblast-like cells (47), and induction of thymulin in thymus (48). It seems likely that GH acts selectively on target tissues by inducing growth factor combinations as mediators. These may be induced either by pituitary endocrine GH or by locally synthesized GH, as in the forming tooth germ (25) or mammary tissue (49).

In conclusion, it is necessary to modify our thinking regarding the actions of GH on bone and tooth in light of the finding reported here that GH acts to induce formation of BMPs important in bone and cartilage formation. The fact that both BMP-4 and IGF-I stimulate 35SO4 incorporation into cartilage (50), and that both are locally induced by GH should alert us to the likelihood of that cartilage and bone formation involves more than just the intermediacy of IGF-I. Thus, a general multi-effector hypothesis appears more appropriate to explain the many of the actions of GH than the more traditional dual effector hypothesis (51).


   Acknowledgments
 
We thank Xiaoyi Zhu and Tang-sheng Zeng for advice.


   Footnotes
 
1 This work was supported by a National Health and Medical Research Council (Australia) grant. Back

Received April 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Aspenberg P, Hansson LI, Thorngren KG 1985 Modification of bone formation rate by growth hormone, melanocyte stimulating hormone and cortisone in the normal rat. Acta Anat 121:84–88[Medline]
  2. Ehrenberg A, Brosjo O, Laftman P, Nilsson O, Stromberg L 1993 Enhancement of bone formation in rabbits by recombinant human growth hormone. Acta Orthop Scand 64:562–566[Medline]
  3. Rosen HN, Chen V, Cittadini A, Greenspan SL, Douglas PS, Moses AC, Beamer WG 1995 Treatment with growth hormone and IGF-1 in growing rats increases bone mineral content but not bone mineral density. J Bone Miner Res 10:1352–1358[Medline]
  4. Hansson LI, Stenstrom A, Thorngren KG 1978 Effect of pituitary hormones on dentin production in maxillary incisors in the rat. Scand J Dent Res 86:80–86[Medline]
  5. Young WG, Zhang CZ, Li H, Lobie PE, Waters MJ 1993 A bromodeoxyuridine immunocytochemical and morphometric study of the influence of GH on cell proliferation in odontogenic mesenchyme of the Lewis dwarf rat. Arch Oral Biol 38:207–214[CrossRef][Medline]
  6. Zhang CZ, Li H, Bartold PM, Young WG, Waters MJ 1995 Effect of GH on the distribution of decorin and biglycan during odontogenesis in the rat incisor. J Dental Res 74:1636–1643[Abstract/Free Full Text]
  7. Young WG, Zhang CZ, Li H, Osborne P, Waters MJ 1992 The influence of GH on cell proliferation in odontogenic epithelia by bromodeoxyuridine immunocytochemistry and morphometry: a study in the Lewis dwarf rat. J Dental Res 71:1807–1811[Abstract/Free Full Text]
  8. Clayden AM, Young WG, Zhang CZ, Harbrow D, Romaniuk K, Waters MJ 1994 Ultrastructure of cementogenesis as affectedby GH in the molar periodontium of the hypophysectomized rat. J Periodontal Res 29:266–275[CrossRef][Medline]
  9. Kassem M, Mosekilde L, Eriksen EF 1994 Growth hormone stimulates proliferation of normal human bone marrow stromal osteoblast precursor cells in vitro. Growth Regul 4:131–135[Medline]
  10. Young WG, Ruch JV, Stevens MR, Begue-Kirn C, Zhang CZ, Lesot H, Waters MJ 1995 Comparison of the effects of GH, IGF-1 and fetal calf serum on mouse molar odontogenesis in vitro. Arch Oral Biol 40:789–799[CrossRef][Medline]
  11. Ernst M, Froesch ER 1988 Growth hormone dependent stimulation of osteoblast like cells in serum free cultures via local synthesis of insulin like growth factor-1. Biochem Biophys Res Commun 151:142–147[CrossRef][Medline]
  12. Slootweg MC 1993 Growth hormone and bone. Horm Met Res 25:335–343[Medline]
  13. Ekberg S, Luther M, Nakamura T, Jansson JO 1992 Growth hormone promotes early initiation of hepatocyte growth factor gene expression in the liver of hypophysectomized rats after partial hepatectomy. J Endocrinol 135:59–67[Abstract]
  14. Izumi T, Shida J-I, Jingushi S, Hotokebuchi T, Sugioka Y 1995 Administration of GH modulates the gene expression of basic fibroblast growth factor in rat costal cartilage, both in vivo and in vitro. Mol Cell Endocrinol 112:95–99[CrossRef][Medline]
  15. Rogers SA, Rasmussen J, Miller SB, Hammerman MR 1994 Effects of growth hormone on rat renal epidermal growth factor expression. Am J Physiol 267:F208–F214
  16. Ekberg S, Carlsson L, Carlsson B, Billig H, Jansson JO 1989 Plasma growth hormone pattern regulates epidermal growth factor (EGF) receptor messenger ribonucleic acid levels and EGF binding in the rat liver. Endocrinology 125:2158–2166[Abstract]
  17. Bezecny I, Bartova J, Skarda J 1992 GH treatment increases oestrogen receptor concentration in the guinea pig uterus. J Endocrinol 134:5–9[Abstract]
  18. Dale L, Howes G, Price BM, Smith JC 1992 Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development. Development 115:573–585[Abstract]
  19. Kingsley DM, Bland AE, Gruber JM, Marker PC, Rusell LB, Copeland NG, Jakins NA 1992 The mouse short ear skeletal morphogenesis locus is associated with defects in a BMP member of the TGFß superfamily. Cell 71:399–410[CrossRef][Medline]
  20. Vainio S, Karavanova I, Jowett A, Thesleff I 1993 Identification of BMP-4 as a signal mediating secondary induction beteen epithelial and mesenchymal tissues during early tooth development. Cell 75:45–58[CrossRef][Medline]
  21. Francis PH, Richardson MK, Brickell PM, Tickle C 1994 BMPs and a signalling pathway that controls patterning in the developing chick limb. Development 120:209–218[Abstract]
  22. Hogan BL 1996 Bone morphogenic proteins: multifunctional regulators of vertebrate development. Genes Dev 10:1580–1594[Free Full Text]
  23. Lianjia Y, Yuhao G, White F H 1993 Bovine bone morphogenetic protein-induced dentinogenesis. Clin Orthop 295:305–312
  24. Young WG 1995 Growth hormone and insulin-like growth factor-1 in odontogenesis. Int J Dev Biol 39:263–272[Medline]
  25. Zhang CZ, Li H, Young WG, Bartold PM, Chen C, Waters MJ 1997 Evidence for a local action of growth hormone in embryonic tooth development in the rat. Growth Factors 14:131–143[Medline]
  26. Moule AJ, Li H, Bartold PM 1995 Donor variability in the proliferation of human dental pulp fibroblasts. Aust Dent J 40:110–114[Medline]
  27. Evans R, Kamdar SJ 1990 Stability of RNA isolated from macrophages depends on the removal of an RNA-degrading activity early in the extraction procedure. BioTechniques 8:357–360[Medline]
  28. Miettinen PJ, Heikinheimo K 1992 Transforming growth factor-{alpha} (TGF-{alpha}) and insulin gene expression in human fetal pancreas. Development 114:833–834[Abstract]
  29. Gilliland G, Perrin S, Bunn HF 1990 Competitive PCR for quantitation of mRNA. In: Innis MA, Gelfand DH, Sninsky JH, White TJ (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp 60–69
  30. Siebert PD, Larrick JW 1992 Competitive PCR. Nature 359:557–558[CrossRef][Medline]
  31. Zarlenga D S, Canals A, Gasbarre L 1995 Method for constructing internal standard for use in competive PCR. BioTechniques 19:324–326[Medline]
  32. Fuh G, Cunningham BC, Fukunaga R, Nagata S, Goeddel, DV, Wells JA 1992 Rational design of potent antagonists to the human GH receptor. Science 256:1677–1680[Abstract/Free Full Text]
  33. Ruch JV 1995 Tooth crown morphogenesis and cytodifferentiation: candid questions and critical comments. Connect Tissue Res 31:1–8
  34. Thesleff I, Vaahtokari A, Partanen A-M 1995 Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int J Dev Biol 39:35–50[Medline]
  35. Nakashima M 1994 Induction of dentine formation on canine amputated pulp by recombinant human bone morphogenetic proteins BMP-2 and -4. J Dent Res 73:1515–1522[Abstract/Free Full Text]
  36. Waters MJ The GH receptor. In: Kostyo JL (ed) Handbook of Physiology, vol 5, chapt 14. Oxford University Press, Oxford, in press
  37. Barnard R, Haynes KM, Werther GA, Waters MJ 1988 The ontogeny of GH receptors in the rabbit tibia. Endocrinology 122:2562–2569[Abstract]
  38. Silberstein GB, Daniels CW 1987 Investigation of mouse mammary ductal growth. Regulation using slow release plastic implants. J Dairy Sci 70:1981–1990
  39. Pantaleon M, Harvey MB, Barnard RT, Waters MJ, Kaye PL 1997 Functional GH receptors and GH mRNA are expressed by preimplantation mouse embryos: a role for GH in early embryogenesis? Proc Natl Acad Sci USA 94:5125–5130[Abstract/Free Full Text]
  40. Argetsinger LS, Carter-Su C 1996 Mechanism of signaling by GH receptor. Physiol Rev 76:1089–1107[Abstract/Free Full Text]
  41. LeRoith D, Werner H, Beitner-Johnson D, Roberts CT 1995 Molecular and cellular aspects of the IGF-1 receptor. Endocr Rev 16:143–163[CrossRef][Medline]
  42. Clemmons DR, Shaw DS 1983 Variables controlling somatomedin production by human fibroblasts. J Cell Physiol 115:137–142[CrossRef][Medline]
  43. Clemmons DR 1984 Multiple hormones stimulate the production of somatomedin by cultured human fibroblasts. J Clin Endocrinol Metab 58:850–856[Abstract]
  44. Tabata MJ, Kim K, Liu J-G, Yamashita K, Matsumara T, Kato J, Iwamoto M, Wakisaka S, Matsumoto K, Nakamura T, Kumegawa M, Kurisu K 1996 Hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars. Development 122:1243–1251[Abstract]
  45. Zvibel I, Halay E, Reid LM 1991 Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: relevance to clonal growth of tumors. Mol Cell Biol 11:108–116[Abstract/Free Full Text]
  46. Hattori MA, Shinohara Y, Yoshino E, Kanzaki M, Kojima I, Horiuchi R 1994 Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells. J Endocrinol 142:69–75[Abstract]
  47. Saggese G, Federico G, Cinquanta L 1993 In vitro effects of growth hormone and other hormones on chondrocytes and osteoblast like cells. Acta Paediatr Scand [Suppl 391] 82:54–59
  48. Timsit J, Savino W, Safieh B, Chanson P, Gagnerault MC 1992 Growth hormone and insulin like growth factor-1 stimulate hormonal function and proliferation of thymic epithelial cells. J Clin Endocrinol Metab 75:183–188[Abstract]
  49. Mol JA, Van Garderen E, Rutteman GR, Rijnberk A 1996 New insights in the molecular mechanism of progestin-induced proliferation of mammary epithelium: induction of the local biosynthesis of GH in the mammary gland of dogs, cats and humans. J Steroid Biochem 57:67–71
  50. Luyten FP, Chen P, Paralkar V, Reddi AH 1994 Recombinant BMP 4, transforming growth factor ß1 and activin A enhance the cartilage phenotype of articular chondrocytes in vitro. Exp Cell Res 210:224–232[CrossRef][Medline]
  51. Isaksson OGP, Lindahl A, Nilsson A, Isgaard J 1987 Mechanism of the stimulatory effect of GH in longitudinal bone growth. Endocr Rev 8:426–438[Medline]
  52. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA 1988 Novel regulators of bone formation: molecular clones and activities. Science 242:1528–1534



This article has been cited by other articles:


Home page
 Eur J EndocrinolHome page
M. Raschke, M. H. Rasmussen, S. Govender, D. Segal, M. Suntum, and J. S. Christiansen
Effects of growth hormone in patients with tibial fracture: a randomised, double-blind, placebo-controlled clinical trial
Eur. J. Endocrinol., March 1, 2007; 156(3): 341 - 351.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Yaar, C. Wu, H.-Y. Park, I. Panova, G. Schutz, and B. A. Gilchrest
Bone Morphogenetic Protein-4, a Novel Modulator of Melanogenesis
J. Biol. Chem., September 1, 2006; 281(35): 25307 - 25314.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. E. Rowland, L. M. Kerr, M. White, P. G. Noakes, and M. J. Waters
Heterozygote Effects in Mice with Partial Truncations in the Growth Hormone Receptor Cytoplasmic Domain: Assessment of Growth Parameters and Phenotype
Endocrinology, December 1, 2005; 146(12): 5278 - 5286.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Q. Xu, B. S. Emerald, E. L. K. Goh, N. Kannan, L. D. Miller, P. D. Gluckman, E. T. Liu, and P. E. Lobie
Gene Expression Profiling to Identify Oncogenic Determinants of Autocrine Human Growth Hormone in Human Mammary Carcinoma
J. Biol. Chem., June 24, 2005; 280(25): 23987 - 24003.
[Abstract] [Full Text] [PDF]

Home page
 J. Dent. Res.Home page
J.R. Smid, J.E. Rowland, W.G. Young, T.J. Daley, K.T. Coschigano, J.J. Kopchick, and M.J. Waters
Mouse Cellular Cementum is Highly Dependent on Growth Hormone Status
J. Dent. Res., January 1, 2004; 83(1): 35 - 39.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. J. Kopchick, C. Parkinson, E. C. Stevens, and P. J. Trainer
Growth Hormone Receptor Antagonists: Discovery, Development, and Use in Patients with Acromegaly
Endocr. Rev., October 1, 2002; 23(5): 623 - 646.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Graichen, D. Liu, Y. Sun, K.-O. Lee, and P. E. Lobie
Autocrine Human Growth Hormone Inhibits Placental Transforming Growth Factor-beta Gene Transcription to Prevent Apoptosis and Allow Cell Cycle Progression of Human Mammary Carcinoma Cells
J. Biol. Chem., July 12, 2002; 277(29): 26662 - 26672.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
D. Le Roith, C. Bondy, S. Yakar, J.-L. Liu, and A. Butler
The Somatomedin Hypothesis: 2001
Endocr. Rev., February 1, 2001; 22(1): 53 - 74.
[Abstract] [Full Text]

Home page
 Exp. Biol. Med.Home page
J.-L. Liu, S. Yakar, and D. LeRoith
Conditional Knockout of Mouse Insulin-Like Growth Factor-1 Gene Using the Cre/loxP System
Experimental Biology and Medicine, April 1, 2000; 223(4): 344 - 351.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J.-L. Liu and D. LeRoith
Insulin-Like Growth Factor I Is Essential for Postnatal Growth in Response to Growth Hormone
Endocrinology, November 1, 1999; 140(11): 5178 - 5184.
[Abstract] [Full Text]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Shimasaki, R. J. Zachow, D. Li, H. Kim, S.-i. Iemura, N. Ueno, K. Sampath, R. J. Chang, and G. F. Erickson
A functional bone morphogenetic protein system in the ovary
PNAS, June 22, 1999; 96(13): 7282 - 7287.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. C. Mertani, T. Zhu, E. L. K. Goh, K.-O. Lee, G. Morel, and P. E. Lobie
Autocrine Human Growth Hormone (hGH) Regulation of Human Mammary Carcinoma Cell Gene Expression. IDENTIFICATION OF CHOP AS A MEDIATOR OF hGH-STIMULATED HUMAN MAMMARY CARCINOMA CELL SURVIVAL
J. Biol. Chem., June 8, 2001; 276(24): 21464 - 21475.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, H.
Right arrow Articles by Waters, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, H.
Right arrow Articles by Waters, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals