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 Blanquaert, F.
Right arrow Articles by Canalis, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blanquaert, F.
Right arrow Articles by Canalis, E.
Endocrinology Vol. 140, No. 3 1069-1074
Copyright © 1999 by The Endocrine Society

ARTICLES

Fibroblast Growth Factor-2 Induces Hepatocyte Growth Factor/Scatter Factor Expression in Osteoblasts1

Frederic Blanquaert, Anne M. Delany and Ernesto Canalis

Departments of Research and Medicine (F.B., A.M.D., E.C.), Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105; and The University of Connecticut School of Medicine (A.M.D., E.C.), Farmington, Connecticut 06030

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor with a major role in tissue morphogenesis and repair. It stimulates the proliferation of cells of the osteoblast and osteoclast lineages. Mitogenic factors playing a role in fracture repair may act by regulating HGF/SF expression or activity in bone-forming cells. We investigated the effect of fibroblast growth factor-2 (FGF-2) on the expression of HGF/SF and its receptor, encoded by c-met, in the MC3T3-E1 osteoblastic cell line. MC3T3-E1 cells expressed low levels of HGF/SF messenger RNA (mRNA), which were markedly increased by FGF-2 in a dose- and time-dependent manner. FGF-2 also induced HGF/SF polypeptide synthesis. The stimulation of HGF/SF mRNA expression by FGF-2 was blocked by cycloheximide, a protein synthesis inhibitor, but not by DNA or prostaglandin synthesis inhibitors. FGF-2 increased the rate of HGF/SF gene transcription by approximately 2-fold, as determined by nuclear run-on assays, and did not modify the decay of HGF/SF mRNA in transcriptionally arrested cells. FGF-2 also caused a dose- and time-dependent stimulation of c-met mRNA. In conclusion, FGF-2 induces HGF/SF expression in osteoblasts and may promote HGF/SF activity by increasing the expression of its receptor. Through these mechanisms, HGF/SF could mediate FGF actions on bone repair.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HEPATOCYTE GROWTH FACTOR/SCATTER factor (HGF/SF) is a pleiotropic factor produced by cells of mesodermal origin. It exerts mitogenic, motogenic, and morphogenic effects on a wide variety of target cells (1, 2, 3, 4). HGF/SF is secreted as an inactive single chain precursor, which is processed to an active heterodimer by limited proteolysis. Four proteases are known to activate HGF/SF in vitro, including the serine protease HGF activator, blood coagulation factor XIIa, urokinase, and tissue-type plasminogen activator (5, 6, 7, 8). In addition, a HGF/SF activator inhibitor was recently reported (9). The active form of HGF/SF is a disulfide-linked heterodimeric protein composed of a 69-kDa {alpha}-chain, with four kringle domains, and a 34-kDa ß-chain, with a serum protease-like sequence (1). HGF/SF binds its target cells through a tyrosine kinase receptor, a 190-kDa disulfide-linked heterodimer, encoded by the proto-oncogene c-met (10, 11).

HGF/SF was initially identified as a potent mitogen for hepatocytes and subsequently found to stimulate the proliferation of epithelial and endothelial cells (1, 3, 4, 12, 13). As a motogenic factor, HGF/SF promotes the dissociation or "scattering" of epithelial cell colonies, increasing the motility and invasiveness of individual cells by enhancing the synthesis of enzymes involved in matrix proteolysis (3, 4, 13, 14, 15). HGF/SF induces the formation of branching tubules or capillary-like tubes by epithelial and endothelial cells in vitro, and the formation of blood vessels in vivo (2, 12).

HGF/SF is a critical factor for tissue repair and morphogenesis. HGF/SF is essential for the development of skeletal muscle and participates in liver and kidney repair (3, 4, 16, 17, 18). Transgenic mice lacking a functional HGF/SF gene display embryonic lethality and impaired development of the liver and placenta (19, 20). Liver regeneration is faster in transgenic mice overexpressing HGF/SF than in wild-type mice, or in normal mice treated with exogenous HGF/SF (16, 17). The effects of HGF/SF on tissue regeneration are likely due to its potent mitogenic, motogenic, and angiogenic activities.

HGF/SF is considered a regulator of skeletogenesis and bone remodeling (18, 19). It stimulates the proliferation of chondrocytes, osteoclasts, osteoblasts, and pluripotent hematopoietic progenitors, all of which express c-met (21, 22, 23, 24, 25, 26). Constitutive synthesis of HGF/SF has been reported in osteoclasts and stromal cells but not in osteoblasts (21, 24, 27). HGF/SF modulates bone resorption by increasing osteoclast number, motility, and spreading and stimulates alkaline phosphatase activity in osteoblasts (21, 23, 28).

HGF/SF is known as a key factor for tissue repair. It stimulates bone cell mitogenesis and neoangiogenesis, which are essential in fracture healing, suggesting that HGF/SF may also be implicated in bone repair (3, 4, 12, 16). We therefore postulated that growth factors playing a role in bone healing, and displaying similar biological effects to those of HGF/SF, could regulate HGF/SF expression and modulate its activity in bone cells. Among these factors, fibroblast growth factors (FGFs) are especially important (29, 30, 31, 32). These pleiotropic growth factors are known for their mitogenic and angiogenic properties and for their capacity to stimulate hematopoiesis and tissue repair (29, 30). Their effects on the skeleton are well characterized (33). FGFs are produced by bone cells, increase the proliferation of osteoblastic cells, and stimulate bone formation and fracture healing (34, 35, 36, 37, 38). FGFs are expressed during the early phases of fracture repair and are detected in the hematoma and granulation tissue (31, 32). Their stimulatory effect on bone repair is thought to be related to their ability to stimulate the proliferation of osteoblastic precursors, subsequently contributing to new bone formation, and to their capacity to stimulate neoangiogenesis.

In the present study, we have investigated the effect of FGF-2 on the expression of HGF/SF and its receptor, c-met, in the osteoblastic cell line MC3T3-E1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Early passages of MC3T3-E1 cells, a mouse osteoblastic cell line derived from fetal mouse calvaria, were used and cultured in {alpha}MEM (Gibco BRL, Grand Island, NY) containing 20 mM HEPES and 10% FBS (Summit Biotechnology, Fort Collins, CO) in a humidified 5% CO2 incubator at 37 C until reaching confluence (39). At confluence, the medium was replaced with serum-free medium containing 0.1% BSA (Fluka Chemical Co., Ronkokoma, NY) and 50 µg/ml ascorbic acid for 24 h. Cells were then exposed to test or control medium in the absence of serum for 2–48 h. In 48 h treated cultures, the medium was replaced after 24 h with fresh control or test solutions. FGF-2 (Austral, San Ramon, CA) and hydroxyurea (Sigma Chemical Co., St. Louis, MO) were dissolved in {alpha}MEM. Cycloheximide, indomethacin, and 5, 6-dichlorobenzimidazole riboside (DRB) (all from Sigma Chemical Co.) were dissolved in absolute ethanol, and diluted in {alpha}MEM. At a dilution of less than 1:10,000, an equal amount of ethanol was added to control cultures.

Northern blot analysis
For RNA analysis, the cell layer was extracted with the lysis buffer provided with the Qiagen RNeasy kit (Qiagen, Chatsworth, CA) and stored at -70 C. Total cellular RNA was isolated according to manufacturer’s instructions (Qiagen), quantitated by spectrophotometry, and equal amounts of RNA from control and test samples were loaded on a formaldehyde-agarose gel following denaturation. The gel was stained with ethidium bromide to visualize ribosomal RNA under UV light, before and after transfer, documenting uniformity of RNA loading and transfer of the various experimental samples. The RNA was then blotted onto Gene Screen Plus charged nylon (DuPont, Wilmington, DE). Restriction fragments containing a 1.4-kb rat HGF/SF complementary DNA (cDNA) (kindly provided by Dr. T. Nakamura, Osaka, Japan), a 4.0-kb mouse c-met cDNA (kindly provided by Dr. C. C. Lee, Bethesda, MD) and a 0.75-kb murine 18S ribosomal RNA cDNA (American Type Culture Collection, Rockville, MD) were labeled with [{alpha}-32P] deoxycytidine triphosphate and [{alpha}-32P]-deoxyadenosine triphosphate (specific activity 3,000 Ci/mmol; DuPont) using the random hexanucleotide primed second strand synthesis method (1, 40, 41). Hybridizations were carried out at 42 C for 16–72 h. Post hybridization washes were performed in 0.5 x saline-sodium citrate (SSC) at 65 C for HGF/SF and c-met, and in 0.1 x SSC at 65 C for 18S ribosomal RNA. The bound radioactive material was visualized by autoradiography on Kodak Biomax or X-AR5 films (Eastman Kodak Co., Rochester, NY) employing Biomax (Kodak) or Cronex Lightning Plus (DuPont) intensifying screens. Relative hybridization levels were determined by densitometry. Northern analyses shown are representative of three or more cultures.

Measurement of immunoreactive HGF/SF
At the end of the culture period, conditioned medium was aspirated and stored at -70 C after the addition of Tween 20 (Sigma Chemical Co.) to a final concentration of 0.1%. An enzyme-immunoassay (EIA) detection kit (Institute of Immunology Co. Ltd., Tokyo, Japan) was used to measure immunoreactive rodent HGF/SF (42). Briefly, medium samples were thawed and cleared by centrifugation, and a 50-µl aliquot of the supernatant was dispensed in duplicate into a 96-well plate precoated with antirat HGF/SF mouse monoclonal antibody. HGF/SF standard solutions were provided by the manufacturer and used for a standard curve. After an overnight incubation at room temperature, the plate was washed and incubated with antirat HGF/SF rabbit polyclonal antibody. Peroxidase-labeled goat antirabbit immunoglobulin was added, and the presence of HGF/SF was detected by colorimetry after an enzymatic reaction using a peroxidase substrate (o-phenylenediamine) and measurement of the product with a microplate spectrophotometer at 490 nm. The sensitivity of the assay is 0.4 ng/ml. Data are expressed in ng/ml or ng/mg protein, determined using a Bio-Rad DC protein assay kit according to manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA).

Nuclear run-on assay
To determine changes in the rate of transcription, nuclei were isolated by Dounce homogenization in Tris buffer containing 0.5% Nonidet P-40 (Sigma Chemical Co.). Nascent transcripts were labeled by incubation of nuclei in a reaction buffer containing 500 µM each of adenosine, cytidine, and guanosine triphosphates, 150 U RNasin (Promega Corp., Madison, WI), and 250 µCi [{alpha}-32P]-uridine triphosphate (800 Ci/mM, DuPont) (43). RNA was isolated by treatment with DNase I and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. Linearized plasmid DNA containing 1 µg of cDNA was immobilized onto GeneScreen Plus by slot blotting according to manufacturer’s directions (DuPont). The plasmid vector pGL3-Basic (Promega Corp.) was used as a control for nonspecific hybridization, and murine 18S cDNA was used to estimate uniformity of the radioactive counts applied to the membrane. Equal cpm of [{alpha}-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 30 min. Hybridized cDNAs were visualized by autoradiography.

Statistical methods
Significant differences of the slopes of messenger RNA (mRNA) decay were analyzed using the method of Sokal and Rohlf (44).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Northern blot analysis of total RNA extracted from MC3T3-E1 cells revealed predominant HGF/SF transcripts of 6.3, 3.7, and 3.1 kb (Fig. 1Go). Continuous treatment of MC3T3-E1 cells with FGF-2 caused a time-dependent increase in HGF/SF steady-state transcripts. FGF-2 at 30 ng/ml increased HGF/SF mRNA levels by approximately 2-fold to over 100-fold after 2–48 h, as determined by densitometry (Fig. 1Go). The effect of FGF-2 was dose dependent, and continuous treatment of MC3T3-E1 cells with FGF-2 for 24 h at 10–30 ng/ml increased HGF/SF transcripts by approximately 20- to 100-fold (Fig. 2Go). Immunoreactive HGF/SF protein was undetectable (<0.4 ng/ml) in conditioned medium of control cultures, and only cultures exposed to FGF-2 contained detectable levels of HGF/SF. The concentration of HGF/SF in cultures exposed to FGF-2 at 30 ng/ml for 24 h was (mean ± SEM, n = 3) 1.6 ± 0.2 ng/ml. A 48-h exposure to FGF-2 resulted in an increase from undetectable levels in control cultures to 3.3 ± 0.2 ng/ml or 9.4 ± 0.7 ng/mg protein (n = 4).



View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Effect of FGF-2 at 30 ng/ml on HGF/SF mRNA expression in cultures of MC3T3-E1 cells treated for 2, 6, 24, or 48 h. Total RNA from control (-) or FGF (+) treated cultures was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled rat HGF/SF cDNA. The blot was stripped and rehybridized with labeled murine 18S cDNA. HGF/SF mRNA was visualized by autoradiography and is shown in the upper panel, and 18S ribosomal RNA is shown below.

 


View larger version (63K):
[in this window]
[in a new window]
  		Figure 2. Effect of FGF-2 at 0.1 to 30 ng/ml on HGF/SF mRNA expression in cultures of MC3T3-E1 cells treated for 24 h. Total RNA from control (0) or FGF treated cultures was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled rat HGF/SF cDNA. The blots were stripped and rehybridized with labeled murine 18S cDNA. HGF/SF mRNA was visualized by autoradiography and is shown in the upper panel, and 18S ribosomal RNA is shown below.

 
To determine whether the effect of FGF-2 on HGF/SF mRNA levels was dependent on protein, DNA or prostaglandin synthesis, MC3T3-E1 cells were treated with FGF-2 at 30 ng/ml for 24 h in the presence or absence of cycloheximide at 3.6 µM, hydroxyurea at 1 mM, or indomethacin at 10 µM. Previous work demonstrated that these doses of inhibitors block protein, DNA, and prostaglandin synthesis in osteoblasts (45, 46, 47). Treatment with cycloheximide for 24 h superinduced HGF/SF transcripts and prevented the stimulatory effect of FGF-2 (Fig. 3Go). In contrast, hydroxyurea and indomethacin did not modify HGF/SF mRNA expression in control or FGF-2-treated cultures (Fig. 3Go).



View larger version (57K):
[in this window]
[in a new window]
  		Figure 3. Effect of FGF-2 at 30 ng/ml, in the presence or absence of cycloheximide (Cx) at 3.6 µM, hydroxyurea (HU) at 1 mM, or indomethacin (Indo) at 10 µM, on HGF/SF mRNA expression in cultures of MC3T3-E1 cells treated for 24 h. Total RNA from control (C) or FGF-2 treated cultures was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled rat HGF/SF cDNA. The blot was stripped and rehybridized with labeled murine 18S cDNA. HGF/SF mRNA was visualized by autoradiography and is shown in the upper panel, and 18S ribosomal RNA is shown below.

 
To determine whether the effect of FGF-2 on HGF/SF mRNA levels was due to changes in transcript stability, cultures of MC3T3-E1 cells were exposed to {alpha}MEM in the presence or absence of FGF-2 at 30 ng/ml for 2 h and then treated with the RNA polymerase II inhibitor DRB for 1.5–8 h (45, 48). The half-life of HGF/SF mRNA was approximately 7.5 h in transcriptionally arrested osteoblasts, and it was not affected by FGF-2 (Fig. 4Go). To determine the effect of FGF-2 on the transcription of the HGF/SF gene, nuclear run-on assays were performed. FGF-2 at 30 ng/ml for 2, 6, or 24 h increased the rate of HGF/SF transcription by approximately 2-fold (Fig. 5Go).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Effect of FGF-2 at 30 ng/ml on HGF/SF mRNA decay in transcriptionally arrested MC3T3-E1 cells. Cultures were exposed to control or FGF-containing medium 2 h before the addition of DRB at 75 µM. Total RNA, obtained 1.5–8 h after the addition of DRB alone or in the presence of FGF, was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled rat HGF/SF cDNA, visualized by autoradiography, and quantitated by densitometry. Values are expressed as means ± SEM for three cultures and presented as percentage of HGF/SF mRNA levels relative to the time of DRB addition. Slopes were analyzed by the method of Sokal and Rohlf and not found to be statistically different.

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 5. Effect of FGF-2 at 30 ng/ml on HGF/SF transcription rates in cultures of MC3T3-E1 cells treated for 2, 6, and 24 h. Nascent transcripts from control (C) or FGF-treated cultures were labeled in vitro with [{alpha}-32P]-uridine triphosphate, and the labeled RNA was hybridized to immobilized cDNA for HGF/SF. Murine 18S cDNA was used to demonstrate loading, and pGL3-Basic vector DNA was used as a control for nonspecific hybridization.

 
FGF-2 also caused a time- and dose-dependent increase in c-met mRNA, detected as an 8.6-kb transcript (Fig. 6Go). Either a shadow from the main transcript or a possible secondary transcript of smaller size was noted in overexposed Northern blots, a pattern similar to that observed in certain blots detecting c-met mRNA in other cells (11, 49, 50). FGF-2 at 10 ng/ml (not shown) and 30 ng/ml for 6, 24, or 48 h (Fig. 6Go) increased c-met transcripts by (mean ± SEM; n = 3) 2.2 ± 0.2-fold, 2.4 ± 0.5-fold, and 2.6 ± 0.4-fold respectively, following normalization to 18S. Cycloheximide superinduced c-met without modifying the stimulatory effect of FGF-2, whereas hydroxyurea and indomethacin did not modify the expression of c-met mRNA in control or FGF-2 treated cells (Fig. 7Go).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 6. Effect of FGF-2 at 30 ng/ml on c-met mRNA expression in cultures of MC3T3-E1 cells treated for 6, 24, or 48 h. Total RNA from control (-) or FGF (+) treated cultures was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled mouse c-met cDNA. The blot was stripped and rehybridized with labeled murine 18S cDNA. c-met mRNA was visualized by autoradiography and is shown in the upper panel, and 18S ribosomal RNA is shown below.

 


View larger version (58K):
[in this window]
[in a new window]
  		Figure 7. Effect of FGF-2 at 30 ng/ml, in the presence or absence of cycloheximide (Cx) at 3.6 µM, hydroxyurea (HU) at 1 mM, or indomethacin (Indo) at 10 µM, on c-met mRNA expression in cultures of MC3T3-E1 cells treated for 24 h. Total RNA from control (C) or FGF-treated cultures was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled mouse c-met cDNA. The blot was stripped and rehybridized with labeled murine 18S cDNA. c-met mRNA was visualized by autoradiography and is shown in the upper panel, and 18S ribosomal RNA is shown below.

 
To determine whether the effects of FGF-2 on c-met mRNA levels were related to changes in transcript stability, cell cultures were exposed to {alpha}MEM in the presence or absence of FGF-2 at 30 ng/ml for 24 h and then treated with DRB for 1.5–8 h. The half-life of c-met mRNA was approximately 6.5 h, and FGF-2 did not modify the decay of c-met mRNA (Fig. 8Go). To confirm that FGF-2 modified the transcription of the c-met gene, nuclear run-on assays were performed on nuclei from MC3T3-E1 cells treated for 6 and 24 h with FGF-2 at 30 ng/ml. These assays showed that FGF-2 increased the rate of c-met transcription by about 2-fold (Fig. 9Go).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 8. Effect of FGF-2 at 30 ng/ml on c-met mRNA decay in transcriptionally arrested MC3T3-E1 cells. Cultures were treated with control or FGF-containing medium for 24 h before the addition of DRB at 75 µM. Total RNA, obtained 1.5–8 h after the addition of DRB alone or in the presence of FGF, was subjected to Northern blot analysis and hybridized with [{alpha}-32P]-labeled mouse c-met cDNA, visualized by autoradiography and quantitated by densitometry. Values are expressed as means ± SEM for three cultures and presented as percentage of c-met mRNA levels relative to the time of DRB addition. Slopes were analyzed by the method of Sokal and Rohlf and not found to be statistically different.

 


View larger version (34K):
[in this window]
[in a new window]
  		Figure 9. Effect of FGF-2 at 30 ng/ml on c-met transcription rates in cultures of MC3T3-E1 cells treated for 6 and 24 h. Nascent transcripts from control (C) or FGF-treated cultures were labeled in vitro with [{alpha}-32P]-uridine triphosphate, and the labeled RNA was hybridized to immobilized cDNA for c-met. Murine 18S cDNA was used to demonstrate loading, and pGL3-Basic vector DNA was used as a control for nonspecific hybridization.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HGF/SF modulates bone cell proliferation and function and could be a coupling agent in bone remodeling (21). In the present study, we demonstrated that HGF/SF mRNA transcripts were expressed at low levels in control cultures of osteoblastic MC3T3-E1 cells and were markedly increased by FGF-2 in a dose- and time-dependent manner. The effect was dependent on de novo protein synthesis and was independent of the growth factor mitogenic properties (34, 35). FGF-2 induces prostaglandin synthesis in osteoblasts, and prostaglandins induce HGF/SF in nonskeletal cells, but the effect of FGF-2 on HGF/SF expression was not due to increased prostaglandin synthesis (49, 51, 52). Furthermore, prostaglandin E2 does not increase HGF/SF mRNA levels in MC3T3-E1 cells, indicating that the mechanisms regulating the expression of HGF/SF in osteoblasts and nonskeletal cells differ (Blanquaert, F., and E. Canalis, unpublished observations). Although FGF-2 increased HGF/SF transcripts by 10- to 100-fold, it increased HGF/SF gene transcription rate by only about 2-fold and did not modify the stability of HGF/SF mRNA in transcriptionally arrested MC3T3-E1 cells. Cytokines and growth factors that induce HGF/SF in fibroblast cultures also have a modest effect on transcription (53, 54). For instance, the effect of IL-1 on HGF/SF levels in lung fibroblasts is of a comparable magnitude as that of FGF-2 in osteoblasts, and IL-1, like FGF-2, increases HGF/SF transcription rate by about 2-fold (53). It is possible that FGF-2 increases HGF/SF mRNA stability, but the effect was not detectable under conditions of transcriptional arrest, which may have suppressed the expression of genes coding for proteins required to regulate HGF/SF transcript stability.

The levels of immunoreactive HGF/SF in control untreated MC3T3 cells were below the limit of detection of the assay (< 0.4 ng/ml), suggesting that, under basal conditions, osteoblasts do not synthesize significant amounts of HGF/SF. These results are in agreement with previous observations in human osteoblasts (12). In contrast, stromal cells synthesize HGF/SF constitutively, and our studies demonstrate that MC3T3 cells can be induced to express HGF/SF mRNA and protein (24, 27). In addition, the basal level of HGF/SF mRNA increased modestly in MC3T3 cells induced to form nodules in vitro, an effect that may be related to an accumulation of endogenous bone morphogenetic proteins, which also cause a modest increase in HGF/SF mRNA in osteoblastic cells (Blanquaert, F., A. M. Delany, and E. Canalis, unpublished observations). In the present studies, we did not determine whether the protein secreted is biologically active. Skeletal cells have the capacity to activate HGF/SF, suggesting that they secrete at least one of the proteases known to activate the growth factor (21). Therefore, even if HGF/SF secreted by osteoblasts is inactive, it is likely to be activated in the bone microenvironment. HGF/SF is known to bind to the extracellular matrix, due to its ability to bind to heparan sulfate glycosaminoglycans, and it may accumulate in the bone matrix, although this has not been reported (55).

We showed that MC3T3-E1 cells express c-met mRNA, and FGF-2 increased its expression in a dose- and time-dependent manner. Through this mechanism, FGF-2 might promote the biological activity of HGF/SF on osteoblastic cells. The induction of c-met expression by HGF/SF has been reported in epithelial cells and described as a mechanism of auto-amplification (56). Nevertheless, the stimulation of c-met by FGF-2 in MC3T3-E1 cells was probably not mediated by HGF/SF because it was independent of protein synthesis. The stimulation of c-met transcripts by FGF-2 also was independent of DNA and prostaglandin synthesis. FGF-2 did not modify the stability of c-met mRNA in transcriptionally arrested MC3T3-E1 cells and increased c-met gene transcription and transcript levels to a similar extent, indicating that transcriptional mechanisms are responsible for the effect observed.

The effects of HGF/SF and its expression during skeletogenesis suggest that this factor could play a key role in bone metabolism or development (21, 22). The production of HGF/SF by osteoblasts indicates that this factor might participate in the regulation of bone remodeling through autocrine and paracrine regulatory mechanisms. In addition, HGF/SF may be essential for bone repair and could mediate selected effects of growth factors, such as FGFs, in this process. FGFs induce bone formation, participate in fracture repair, and stimulate the formation of a soft fracture callus (31, 32, 36, 37, 38). The stimulatory effect of FGFs on bone healing is considered to be related to their mitogenic and angiogenic properties, allowing the proliferation of osteoblast precursors and the formation of a capillary network essential for tissue healing (57, 58). Because HGF/SF stimulates bone cell proliferation and blood vessel formation, it could contribute to bone repair and mediate selected FGF effects in this process (13, 57).

The role of HGF/SF in skeletal tissue repair is supported by the recent discovery that HGF/SF stimulates the repair of full thickness articular cartilage defects in rabbits (59). The healing of these osteochondral defects requires the participation of undifferentiated mesenchymal cells originating from bone marrow spaces of subchondral bone (60). The defect is colonized by these cells, which differentiate and produce new subchondral bone and fibrocartilage. It is possible that the stimulatory effect of HGF/SF on this process is related to its ability to stimulate the migration and proliferation of undifferentiated mesenchymal cells in the defect, subsequently promoting the formation of bone and cartilage and restoring the articular cartilage function.

In conclusion, the present study demonstrates that FGF-2 induces the expression of HGF/SF in osteoblasts and increases the expression of its receptor, potentially promoting its biological activity. Through these effects, HGF/SF could mediate FGF actions in bone metabolism and fracture repair.


   Acknowledgments
 
The authors thank Dr. T. Nakamura for the rat HGF/SF cDNA, Dr. C. C. Lee for the mouse c-met cDNA, Ms. Sheila Rydziel for advice, Ms. Cathy Boucher, Deena Durant, and Susan O’Lone for technical assistance, and Ms. Charlene Gobeli for secretarial support.


   Footnotes
 
1 Supported by Grant AR-21707 from the National Institute of Arthritis Musculoskeletal and Skin Diseases. Back

Received June 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S 1989 Molecular cloning and expression of human hepatocyte growth factor. Nature 342:440–443[CrossRef][Medline]
  2. Montesano R, Matsumoto K, Nakamura T, Orci L 1991 Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67:901–908[CrossRef][Medline]
  3. Strain AJ 1993 Hepatocyte growth factor: another ubiquitous cytokine. J Endocrinol 137:1–5[Medline]
  4. Matsumoto K, Nakamura T 1996 Emerging multipotent aspects of hepatocyte growth factor. J Biochem 119:591–600[Abstract/Free Full Text]
  5. Miyazawa K, Shimomura T, Kitamura A, Kondo J, Morimoto Y, Kitamura N 1993 Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. Structural similarity of the protease precursor to blood coagulation factor XII. J Biol Chem 268:10024–10028[Abstract/Free Full Text]
  6. Shimomura T, Miyazawa K, Komiyama Y, Hiraoka H, Naka D, Morimoto Y, Kitamura N 1995 Activation of hepatocyte growth factor by two homologous proteases, blood-coagulation factor XIIa and hepatocyte growth factor activator. Eur J Biochem 229:257–261[Medline]
  7. Naldini L, Tamagnone L, Vigna E, Sachs M, Hartmann G, Birchmeier W, Daikuhara Y, Tsubouchi H, Blasi F, Comoglio PM 1992 Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. EMBO J 11:4825–4833[Medline]
  8. Mars W, Zarnegar R Michalopoulos G 1993 Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am J Pathol 143:949–958[Abstract]
  9. Shimomura T, Denda K, Kitamura A, Kawaguchi T, Kito M, Kondo J, Kagaya S, Qin L, Takata H, Miyazawa K, Kitamura N 1997 Hepatocyte growth factor activator inhibitor, a novel kunitz-type serine protease inhibitor. J Biol Chem 272:6370–6376[Abstract/Free Full Text]
  10. Bottaro DP, Rubin JS, Faletto DL, Chan AML, Kmiecick TE, Vande Woude GF, Aaronson SA 1991 Identification of the hepatocyte growth factor receptor as the c-met protooncogene. Science 251:802–804[Abstract/Free Full Text]
  11. Naldini L, Vigna E, Narsiham R, Gaudino G, Zarnegar R, Michalopoulos G, Comoglio PM 1991 Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-met. Oncogene 6:501–504[Medline]
  12. Grant DS, Kleinman HK, Goldberg ID, Bhargava MM, Nickoloff BJ, Kinsella JL, Polverini P, Rosen EM 1993 Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci USA 90:1937–1941[Abstract/Free Full Text]
  13. Matsumoto K, Hashimoto K, Yoshikawa K, Nakamura T 1991 Marked stimulation of growth and mobility of human keratinocytes by hepatocyte growth factor. Exp Cell Res 196:114–120[CrossRef][Medline]
  14. Stoker M 1989 Effect of scatter factor on motility of epithelial cells and fibroblasts. J Cell Physiol 139:565–569[CrossRef][Medline]
  15. Weidner KM, Behrens J, Vandekerkhove J, Birchmeier W 1990 Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J Cell Biol 111:2097–2108[Abstract/Free Full Text]
  16. Roos F, Ryan AM, Chamow SM, Bennett GL, Schwall RH 1995 Induction of liver growth in normal mice by infusion of hepatocyte growth factor/scatter factor. Am J Physiol 268:G380–G386
  17. Okano JI, Shiota G, Kawasaki H 1997 Protective action of hepatocytes growth factor for acute liver injury caused by D-galactosamine in transgenic mice. Hepatology 26:1241–1249[Medline]
  18. Takayama H, LaRochelle WJ, Anver M, Bockman DA, Merlino G 1996 Scatter factor/hepatocyte growth factor as a regulator of muscle and neural crest development. Proc Natl Acad Sci USA 93:5866–5871[Abstract/Free Full Text]
  19. Schmidt C, Bladt F, Goedecke S, Brinkmann V, Zschiesche W, Sharpe M, Gheraradi E, Birchmeier C 1995 Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373:699–702[CrossRef][Medline]
  20. Uehara Y, Minowa O, Mori C, Shiota K, Kuno J, Noda T, Kitamura N 1995 Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter. Nature 373:702–705[CrossRef][Medline]
  21. Grano M, Galimi F, Zambonin G, Colucci S, Cottone E, Zambonin Zallone A, Comoglio PM 1996 Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro. Proc Natl Acad Sci USA 93:7644–7648[Abstract/Free Full Text]
  22. Takebayashi T, Iwamoto M, Jikko A, Matsumura T, Enomoto-Iwamoto M, Myoukai F, Koyama E, Yamaai T, Matsumoto K, Nakamura T, Kurisu K, Noji S 1995 Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes. J Cell Biol 129:1411–1419[Abstract/Free Full Text]
  23. Inaba M, Koyama H, Hino M, Okuno S, Terada M, Nishizawa Y, Nishino T, Morii H 1993 Regulation of release of hepatocyte growth factor from human promyelocytic leukemia cells, HL-60, by 1,25 dihydroxyvitamin D3, 12-o-tetredecanoylphorbol 13-acetate, and dibutyryl cyclic adenosine monophosphate. Blood 82:53–59[Abstract/Free Full Text]
  24. Sato T, Hakeda Y, Yamaguchi Y, Mano H, Tezuka KI, Matsumoto K, Nakamura T, Mori Y, Yoshizawa K, Sumitani K, Kodama H, Kumegawa M 1995 Hepatocyte growth factor is involved in formation of osteoclast-like cells mediated by clonal stromal cells (MC3T3–G2PA6). J Cell Physiol 164:197–204[CrossRef][Medline]
  25. Nishino T, Hisha H, Nishino N, Adachi M, Ikehara S 1995 Hepatocyte growth factor as a hematopoietic regulator. Blood 85:3093–3100[Abstract/Free Full Text]
  26. Galimi F, Bagnara GP, Bonsi L, Cottone E, Follenzi A, Simeone A, Comoglio PM 1996 Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoeitic progenitors. J Cell Biol 127:1743–1754[Abstract/Free Full Text]
  27. Takai K, Hara J, Matsumoto K, Hosoi G, Osugi Y, Tawa A, Okada S, Nakamura T 1997 Hepatocyte growth factor is constitutively produced by human bone marrow stromal cells and indirectly promotes hematopoiesis. Blood 89:1560–1565[Abstract/Free Full Text]
  28. Fuller K, Owens J, Chambers TJ 1995 The effect of hepatocyte growth factor on the behavior of osteoclasts. Biochem Biophys Res Commun 212:334–340[CrossRef][Medline]
  29. Klagsbrun M 1989 The fibroblast growth factor family: structural and biological properties. Prog Growth Factor Res 1:207–235[CrossRef][Medline]
  30. Bikfalvi A, Klein S, Pintucci G, Rifkin DB 1997 Biological roles of fibroblast growth factor-2. Endocr Rev 18:26–45[Abstract/Free Full Text]
  31. Bourque WT, Gross M, Hall BK 1993 Expression of four growth factors during fracture repair. Int J Dev Biol 37:573–579[Medline]
  32. Joyce ME, Jingushi S, Scully SP, Bolander ME 1991 Role of growth factors in fracture healing. Clinical and Experimental Approaches to Dermal and Epidermal Repair: Normal and Chronic Wounds. Wiley-Liss, Inc., 391–416
  33. Canalis E, Centrella M, McCarthy T 1988 Effects of fibroblast growth factor on bone formation in vitro. J Clin Invest 81:1572–1577
  34. McCarthy TL, Centrella M, Canalis E 1989 Effects of fibroblast growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells. Endocrinology 125:2118–2126[Abstract]
  35. Globus RK, Patterson-Buckendahl P, Gospodarowicz D 1988 Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor ß. Endocrinology 123:98–105[Abstract]
  36. Jingushi S, Heydemann A, Kana SK, Macey LR, Bolander ME 1990 Acidic fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing. J Orthop Res 8:364–371[CrossRef][Medline]
  37. Kawaguchi H, Kurokawa T, Hanada K, Hiyama Y, Tamura M, Ogata E, Matsumoto T 1994 Stimulation of fracture repair by recombinant human by basic fibroblast growth factor in normal and streptozotocin-diabetic rats. Endocrinology 135:774–781[Abstract]
  38. Mayahara H, Ito T, Nagai H, Miyajima H, Tsukuda R, Taketomi S, Mizoguchi J, Kato K 1993 In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats. Growth Factors 9:73–80[Medline]
  39. Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S 1983 In vitro differentiation and calcification in a new cell line derived from newborn mouse calvaria. J Cell Biol 96:191–198[Abstract/Free Full Text]
  40. Lee CC, Amata KM 1994 Identification of a novel type of alternative splicing of a tyrosine kinase receptor. Juxtamembrane deletion of the c-met protein kinase C serine phosphorylation regulatory site. J Biol Chem 269:19457–19461[Abstract/Free Full Text]
  41. Feinberg AP, Vogelstein B 1984 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266–267[CrossRef][Medline]
  42. Yamada A, Matsumoto K, Iwanari H, Sekiguchi K, Kawata S, Matsuzawa Y, Nakamura T 1995 Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues. Biomed Res 16:105–114
  43. Ausubel FM, Brent R, Kingsten RE, Moore DD, Seidman JG, Smith JA, Struhl K 1992 Current Protocols in Molecular Biology. Greene and Wiley Interscience, New York
  44. Sokal RR, Rohlf FJ 1981 Biometry (ed. 2). Freeman, San Francisco
  45. Delany AM, Canalis E 1998 Basic fibroblast growth factor destabilizes osteonectin mRNA in osteoblasts. Am J Physiol 274:C734–C740
  46. Centrella M, McCarthy TL, Canalis E 1991 Glucocorticoid regulation of transforming growth factor ß1 activity and binding in osteoblast-enriched cultures from fetal rat bone. Mol Cell Biol 11:4490–4496[Abstract/Free Full Text]
  47. Centrella M, McCarthy TL, Kusmik WF, Canalis E 1991 Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone. J Cell Physiol 147:420–426[CrossRef][Medline]
  48. Zandomeni R, Bunick D, Ackerman S, Mittleman B, Weinmann R 1983 Mechanism of action of DRB. Effect on specific in vitro initiation of transcription. J Mol Biol 167:561–574[CrossRef][Medline]
  49. Takahashi M, Ota, S, Mikami, Y, Azuma, N, Nakamura, T, Terano, A, Omata M 1996 Hepatocyte growth factor as a key to modulate anti-ulcer action of prostaglandins in stomach. J Clin Invest 98:2604–2611[Medline]
  50. Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S 1997 Enhanced expression of hepatocyte growth factor/c-met by myocardial ischemia and reperfusion in a rat model. Circulation 95:2552–2558[Abstract/Free Full Text]
  51. Matsumoto K, Okazaki H, Nakamura T 1995 Novel function of prostaglandins as inducers of gene expression of HGF and putative mediators of tissue regeneration. J Biochem 117:458–464[Abstract/Free Full Text]
  52. Kawaguchi H, Pilbeam CC, Gronowicz G, Abreu C, Fletcher BS, Herschman HR, Raisz LG, Hurley MM 1995 Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor. J Clin Invest 96:923–930
  53. Tamura M, Arakaki N, Tsubouchi H, Takada H, Daikuhara Y 1993 Enhancement of human hepatocyte growth factor production by interleukin-1 {alpha} and -1ß and tumor necrosis factor-{alpha} by fibroblasts in culture. J Biol Chem 268:8140–8145[Abstract/Free Full Text]
  54. Jiang J-G, Zarnegar R 1997 A novel transcriptional regulatory region within the core promoter of the hepatocyte growth factor gene is responsible for its inducibility by cytokines via the C/EBP family of transcription factors. Mol Cell Biol 17:5758–5770[Abstract]
  55. Sakata H, Stahl S, Taylor W, Rosenberg J, Sakaguchi K, Wingfield P, Rubin J Heparin binding, and oligomerization of hepatocyte growth factor/scatter factor isoforms. J Biol Chem 272:9457–9463
  56. Boccacio C, Gaudino G, Gambarotta G, Galimi F, Comoglio PM 1994 Hepatocyte growth factor (HGF) receptor expression is inducible and is part of a delayed-early response to HGF. J Biol Chem 269:12846–12851[Abstract/Free Full Text]
  57. Brighton CT, Hunt RM 1991 Early histological and ultrastructural changes in medullary fracture callus. J Bone J Surg 73:A832–A847
  58. Hulth AH 1989 Current concepts of fracture healing. Clin Orthop Relat Res 249:265–284
  59. Wakitani S, Imoto K, Kimura T, Ochi T, Matsumoto K, Nakamura T 1997 Hepatocyte growth factor facilitates cartilage repair. Full thickness articular cartilage defect studied in rabbit knees. Acta Orthop Scand 68:474–480[Medline]
  60. Buckwalter JA, Mankin HJ 1997 Articular cartilage. Part II: Degeneration and osteoarthritis, repair, regeneration and transplantation. J Bone Joint Surg 79:A612–A632



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
N. Chattopadhyay, R. J. MacLeod, J. Tfelt-Hansen, and E. M. Brown
1alpha ,25(OH)2-vitamin D3 inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells
Am J Physiol Endocrinol Metab, January 1, 2003; 284(1): E219 - E227.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Onimaru, Y. Yonemitsu, M. Tanii, K. Nakagawa, I. Masaki, S. Okano, H. Ishibashi, K. Shirasuna, M. Hasegawa, and K. Sueishi
Fibroblast Growth Factor-2 Gene Transfer Can Stimulate Hepatocyte Growth Factor Expression Irrespective of Hypoxia-Mediated Downregulation in Ischemic Limbs
Circ. Res., November 15, 2002; 91(10): 923 - 930.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
M. R. Rubin and J. P. Bilezikian
The Role of Parathyroid Hormone in the Pathogenesis of Glucocorticoid-Induced Osteoporosis: A Re-Examination of the Evidence
J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4033 - 4041.
[Full Text] [PDF]

Home page
 Genes Dev.Home page
D. M. Ornitz and P. J. Marie
FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease
Genes & Dev., June 15, 2002; 16(12): 1446 - 1465.
[Full Text] [PDF]

Home page
 IOVSHome page
H. Shibuki, N. Katai, S. Kuroiwa, T. Kurokawa, J. Arai, K. Matsumoto, T. Nakamura, and N. Yoshimura
Expression and Neuroprotective Effect of Hepatocyte Growth Factor in Retinal Ischemia-Reperfusion Injury
Invest. Ophthalmol. Vis. Sci., February 1, 2002; 43(2): 528 - 536.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
T. Iwata, L. Chen, C.-l. Li, D. A. Ovchinnikov, R. R. Behringer, C. A. Francomano, and C.-X. Deng
A neonatal lethal mutation in FGFR3 uncouples proliferation and differentiation of growth plate chondrocytes in embryos
Hum. Mol. Genet., July 1, 2000; 9(11): 1603 - 1613.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
F. Blanquaert, R. C. Pereira, and E. Canalis
Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts
Am J Physiol Endocrinol Metab, March 1, 2000; 278(3): E509 - E515.
[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 Blanquaert, F.
Right arrow Articles by Canalis, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blanquaert, F.
Right arrow Articles by Canalis, E.

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