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Endocrinology Vol. 138, No. 5 1972-1978
Copyright © 1997 by The Endocrine Society

Articles

Expression of Indian Hedgehog in Osteoblasts and Its Posttranscriptional Regulation by Transforming Growth Factor-ß1

Shunichi Murakami, Akira Nifuji and Masaki Noda

Department of Molecular Pharmacology, Division of Functional Disorder Research, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan

Address all correspondence and requests for reprints to: Dr. Masaki Noda, Department of Molecular Pharmacology, Division of Functional Disorder Research, Medical Research Institute, Tokyo Medical and Dental University, 3–10 Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo 101, Japan. E-mail: noda.mph{at}mri.tmd.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Indian hedgehog (Ihh) was recently reported to be expressed in chondrocytes and to regulate chondrocyte differentiation. This report examined the expression of Ihh in osteoblastic cells and its regulation by calcitropic cytokines. We found that Ihh messenger RNA (mRNA) was expressed as a single 2.5-kilobase band at a modest level in rat osteoblastic osteosarcoma ROS17/2.8 cells. In sharp contrast to the previous observation of dpp regulation of hedgehog expression in Drosophila embryos, bone morphogenetic protein-2 did not affect Ihh expression in these cells. On the other hand, treatment with 2 ng/ml transforming growth factor-ß1 (TGFß1) increased the steady state level of Ihh mRNA 2- to 4-fold. Western blot analysis of the cell lysates using antisera also showed enhancement of the Ihh protein level by TGFß1 treatment. The effect of TGFß1 on Ihh mRNA abundance started within 3 h, peaked at 24 h and lasted at least 48 h after the initiation of the treatment. The effect of TGFß1 on the increase in Ihh mRNA was dose dependent, starting at 0.2 ng/ml and saturating at 2 ng/ml. Neither actinomycin D nor cycloheximide blocked this effect. Experiments using 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole showed an enhancement of Ihh mRNA stability by TGFß1, indicating the presence of posttranscriptional regulation. We then examined the effects of TGFß1 on Ihh mRNA in osteoblast-enriched cells isolated from neonatal rat calvariae. TGFß1 also enhanced Ihh mRNA expression in these cells. Our data indicate for the first time that Ihh is one of the members of the cytokines produced by osteoblastic cells and that the expression of Ihh is regulated posttranscriptionally by TGFß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOBLASTS play a central role in bone formation and maintenance of the balance of bone metabolism. The function of osteoblasts is regulated by systemic factors, such as hormones and local factors. Osteoblasts secrete such local factors, and they also possess receptors for such factors, indicating the auto/paracrine nature of these local mediators. These include many growth factors and cytokines. Among those, TGFß superfamily members such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) have been regarded as some of the most potent regulators of osteoblastic function, whereas these molecules also play critical roles in morphogenesis and pattern formation, suggesting a close link between the repertoires of local regulators acting on osteoblasts and those acting in embryonic development of limb and axial structures.

Recent analyses have revealed a remarkable degree of conservation in the mechanisms involved in pattern formation between vertebrates and invertebrates. In Drosophila, dpp regulates the dorsoventral pattern of the embryos (1) and the differentiation and patterning of cells in imaginal discs (2, 3, 4, 5). Its vertebrate homolog, BMP-2, on the other hand, is involved in the development of limb buds as well as the induction of osteoblastic cell differentiation. Hedgehog (hh) has been identified as a member of segment polarity genes that play multiple inductive roles during fly development. hh encodes a secreted protein that regulates embryonic segmentation (6) and patterning of adult appendages (7, 8). Recently, several vertebrate homologs of hh have been identified (9, 10, 11, 12). Sonic hedgehog (Shh) mediates the activity of the zone of polarizing activity (ZPA); ectopic expression of Shh in the anterior part of the limb bud causes respecification of anterior cells and results in mirror-image skeletal duplications, similar to those caused by a ZPA graft (11, 13). Shh has also been implicated in floor plate induction (12) and induction of sclerotome from presomitic mesoderm (14, 15).

Recently, another member of the hedgehog gene family, Indian hedgehog (Ihh) has been shown to be expressed in prehypertrophic chondrocytes (16, 17), and it has been implicated in the regulation of terminal differentiation of chondrocytes. In this report, we examined the expression of Ihh and its regulation in osteoblastic osteosarcoma ROS17/2.8 cells. This cell line has been shown to possess many characteristics associated with mature osteoblasts, including functional vitamin D3 (18) and PTH receptors (19); production of type I collagen (20), alkaline phosphatase (19), osteopontin (21), and osteocalcin (22); and the ability to form bone in vivo (23). We also examined nontransformed primary bone cell cultures obtained from newborn rat calvariae. We found that Ihh is expressed in these cells, and its transcript stabilization is regulated by TGFß1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant human TGFß1 was purchased from King Brewing Co. (Kakogawa, Japan). Recombinant basic FGF was obtained from R&D Systems (Minneapolis, MN). Recombinant human BMP-2 (24) was kindly provided by Dr. J. Wozney (Genetics Institute, Cambridge, MA). 1,25-Dihydroxyvitamin D3 and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Indian hedgehog cDNA and antibody against N-terminal of Shh/Ihh were kindly provided by Dr. McMahon. Chicken Shh-cDNA was a generous gift from Dr. Tabin.

Cell cultures
Rat osteoblastic osteosarcoma ROS17/2.8 cells (19) were kindly provided by Dr. G. Rodan (Merck Research Laboratories, West Point, PA). They were maintained in modified F-12 medium (Life Technologies, Grand Island, NY) supplemented with 5% FBS (Life Technologies). The cells were grown in 56-cm2 Costar (Cambridge, MA) or 145-cm2 Nunclon (Nunc, Roskilde, Denmark) tissue culture dishes at 37 C in a humidified atmosphere with 5% CO2. For the experiments, the cells were grown to confluence and were treated for the indicated periods of time with TGFß1 or other agents.

Rat osteoblast-enriched cells, fraction 3, were obtained from newborn rat (Sprague-Dawley) calvariae by collecting the cells released between 30–50 min of enzymatic digestion (25, 26) in 0.2% collagenase (Wako Pure Chemical Industries, Osaka, Japan) and 0.1% hyaluronidase. These cells were grown in modified Ham’s F-12 medium supplemented with 5% FBS and 0.1 mg/ml kanamycin.

RNA preparation and Northern analysis
Cells grown to confluence in 56-cm2 culture dishes were lysed with 4 M guanidium thiocyanate, and total cellular RNA was extracted according to the single step guanidium thiocyanate-phenol-chloroform method (27). Aliquots of 20 µg of the total RNA/lane were fractionated by electrophoresis on 1% agarose gels containing 0.22 M formaldehyde, transferred onto nylon filters (Hybond-N, Amersham Corp., Arlington Heights, IL) by electroblotting (28), and cross-linked to the filters by exposure to UV light. The filters were prehybridized overnight at room temperature in hybridization buffer containing 50% formamide, 5 x SSC (1 x SSC consists of 0.15 M NaCl and 10 mM sodium citrate), 5 x Denhardt’s solution, 0.1% SDS, and 100 µg/ml sheared and denatured herring sperm DNA. The 1.8-kilobase mouse Indian hedgehog complementary DNA (cDNA) insert was excised with EcoRI, gel-purified, and then radiolabeled by the random primer method using Klenow fragment (Pharmacia, Piscataway, NJ) and [{alpha}-32P]deoxy-CTP (New England Nuclear-DuPont, Boston, MA), as described by Feinberg and Vogelstein (29), to a specific activity greater than 108 cpm/µg DNA. This cDNA probe has been shown to be specific for Ihh among other hedgehog gene families in Northern hybridization analysis (9). The chick sonic hedgehog cDNA probe was also used to rule out cross-hybridization. Hybridization was performed in fresh hybridization solution supplemented with 1 x 106 cpm/ml denatured 32P-labeled probe for 16–24 h at 37 C. Filters were washed in 1 x SSC-0.1% SDS three times at room temperature for 5 min each time, followed by 20 min washing in 0.5 x SSC-0.1% SDS at 60 C. The filters were subjected to autoradiography using x-ray films (Fuji Photo Film Co., Minamiashigara, Japan) and intensifying screens (New England Nuclear-DuPont) at -80 C for several days. Equal loading of the RNA in each lane was checked by either ethidium bromide staining or hybridization to a ß-actin probe. For quantification, the ethidium bromide staining or the hybridization signals were measured by scanning densitometry (ZERO-Dscan, Scanalytics, Billerica, MA).

Western blot analysis
ROS17/2.8 cells grown to confluence in 145-cm2 dishes were cultured in modified F-12 medium with or without TGFß1 at 4 ng/ml for 22 h. The cells were then lysed in a buffer containing 20 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM phenylmethanesulfonylfluoride. The cell lysates were concentrated 7-fold by a Centricon-10 (Amicon, Beverly, MA) at 4 C according to the manufacturer’s instructions. The protein concentration was determined by the Bradford method (30), and 1.5 mg of each sample were separated by 15% SDS-PAGE (31). The proteins were electrophoretically transferred to nitrocellulose filters (Hybond-C, Amersham, Little Chalfont, UK) as described previously (32). The filters were then blocked for 3 h in 3% nonfat dry milk in Tris-buffered saline followed by overnight incubation with anti-Shh/Ihh serum (Ab80) (33) at a dilution of 1:300. To visualize the signals, the filters were incubated with biotinylated antirabbit IgG (Vector Laboratories, Burlingame, CA) for 1.5 h, followed by incubation for 1.5 h with avidin-biotin-peroxidase complex (ABC Vecta Stain Kit, Vector Laboratories). Color was developed with 0.04% 3,3'-diaminobenzidine tetrahydrochloride in 0.1 M Tris-HCl, pH 7.5, containing 0.03% H2O2. To confirm the specificity of the visualized signals, the same samples were either analyzed with antiserum that had been preincubated with recombinant sonic hedgehog N-terminus peptide, or the incubation step with antiserum was simply omitted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first examined whether Ihh is expressed in osteoblastic osteosarcoma ROS17/2.8 cells. Northern blot analysis indicated constitutive expression of a single 2.5-kilobase Ihh messenger RNA (mRNA) band in these cells (Fig. 1AGo). No signals were detected when the filters were hybridized with Shh cDNA probe (data not shown). To examine the regulation of the Ihh mRNA expression, we screened calcitropic hormones and cytokines. Although dpp has been known to regulate hedgehog expression in Drosophila embryos (34), BMP-2 did not affect Ihh expression in ROS17/2.8 cells (data not shown; partly in Fig. 6Go). On the other hand, TGFß was the only cytokine that regulated Ihh expression in these cells. TGFß1 treatment enhanced the steady state Ihh mRNA level 2- to 4-fold in a dose-dependent manner, starting at 0.2 ng/ml and saturating at 2 ng/ml (Fig. 1Go, A and B). The abundance of ß-actin mRNA was not affected by TGFß1, indicating the specificity of the TGFß1 effect on Ihh mRNA expression.



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  		Figure 1. TGFß1 enhancement of the steady state levels of IHH mRNA. Confluent ROS17/2.8 cells were treated with the indicated doses of TGFß1 for 24 h. Total RNA was isolated as described in Materials and Methods and was subjected to Northern blot analysis. The positions of IHH, ß-actin, and 18S and 28S ribosomal RNA are indicated. A, Dose dependence of TGFß1 effects on IHH mRNA level. B, Quantification of IHH mRNA bands in A. A and B represent one of two experiments with similar results.

 


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  		Figure 6. Specificity of the TGFß1 effect on the enhancement of IHH mRNA levels. Confluent ROS17/2.8 cells were treated for 24 h with combinations of TGFß1 and other agents as indicated. - and + indicate the absence and the presence of TGFß1 (2 ng/ml), respectively. Total RNA was isolated as described in Materials and Methods and was subjected to Northern blot analysis. The band intensity relative to the control value (after normalization to actin level) was 0.96 for BMP-2 treatment alone, 0.98 for basic FGF alone, and 0.96 for 1,25-dihydroxyvitamin D3 alone. Moreover, no agent altered TGFß1 enhancement of Ihh expression by more than 20%; the ratio of TGFß1 treatment in combination with each agent over TGFß1 treatment alone was 1.19 for BMP-2, 1.08 for basic FGF, and 0.85 for 1,25-dihydroxyvitamin D3. The positions of IHH, ß-actin, and 18S and 28S ribosomal RNA are indicated. The figure represents one of two experiments with similar results.

 
The time course of this effect is shown in Fig. 2Go, A and B. We first examined the time course of the effect of TGFß1 on Ihh mRNA expression over a 72-h period. There was a time-dependent increase in Ihh mRNA expression, which peaked at 24 h (3.9- and 4.5-fold in two independent experiments) and lasted at least up to 48 h of the initiation of treatment. We then further analyzed the early time course of this effect and found that the TGFß1 enhancement of Ihh mRNA started as early as 3 h.



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  		Figure 2. Time course of TGFß1 effects on the steady state levels of IHH mRNA. Confluent ROS17/2.8 cells were cultured for the indicated periods of time (hours) in the absence (-) or presence (+) of 2 ng/ml TGFß1. Total RNA was isolated as described in Materials and Methods and was subjected to Northern blot analysis. The same filters were hybridized with 32P-labeled ß-actin probe later. The positions of IHH, ß-actin, and 18S and 28S ribosomal RNA are indicated. A, Time course of the effect of TGFß1 on the IHH mRNA level over a 72-h period. B, Early time course of the effect of TGFß1 on the IHH mRNA level. A and B represent one of two experiments with similar results.

 
To determine whether the effect of TGFß1 on Ihh mRNA is translated into its protein level, ROS17/2.8 cells were treated with TGFß1 at 4 ng/ml for 22 h, and Western blot analysis was conducted. The antiserum used in this analysis was originally raised against the N-terminal peptide of Shh and is cross-reactive with Ihh. We found that the TGFß1 treatment enhanced the level of Ihh protein, which migrated as a 25-kDa band [Fig. 3AGo, compare TGFß (-) vs. (+)]. As a positive control, the recombinant N-terminal fragment of Shh(SHH-N) was analyzed in the same Western analysis. The size of SHH-N band was approximately 27 kDa on this gel (Fig. 3AGo, rightmost lane). The reactivity of these bands was abolished when the antiserum was preincubated with the recombinant Shh protein before Western blotting, showing the specificity of this reaction (Fig. 3BGo). Similarly, the band was not detected when the reaction step with the antiserum was simply omitted (data not shown).



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  		Figure 3. TGFß1 enhancement of IHH production in ROS17/2.8 cells. Confluent cells in 145-cm2 dishes were cultured for 22 h in the absence (-) or presence (+) of 4 ng/ml TGFß1. Cell lysates were concentrated by Centricon-10, and aliquots were examined by Western blot analysis as described in Materials and Methods. A, Effect of TGFß1 on IHH protein level. Molecular masses in kilodaltons and the position of IHH are indicated. Recombinant mouse Sonic hedgehog (Shh-N) was run in parallel as a positive control. B, Complete inhibition of antibody binding by sonic hedgehog protein. The same samples were analyzed with antiserum that had been preincubated with recombinant sonic hedgehog protein to check specificity. A and B represent one of two experiments with similar results.

 
To investigate the mode of TGFß1 action in the regulation of Ihh gene expression, the cells were treated for 24 h with TGFß1 in the presence or absence of inhibitors for transcription or protein synthesis. As shown in Fig. 4Go, TGFß1 enhancement of the Ihh mRNA level was still observed in the presence of actinomycin D or cycloheximide. We then analyzed the effects of TGFß1 on Ihh mRNA stability using 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) to inhibit mRNA synthesis. The half-life of Ihh mRNA, estimated based on the quantitation by scanning densitometry, was about 4 h in the control cells and 8 h in the TGFß1-treated cells, whereas TGFß1 did not affect the mRNA stability of ß-actin (half-life, 9 h; Fig. 5Go). The stabilization of Ihh mRNA in the TGFß1-treated cells compared with that in the control cells indicates the posttranscriptional regulation of Ihh gene expression by TGFß1. On the other hand, nuclear run-on analysis did not show alteration in the transcription rate of Ihh by the TGFß1 treatment (data not shown).



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  		Figure 4. Effects of actinomycin D and cycloheximide on TGFß1-induced accumulation of IHH mRNA. Confluent ROS17/2.8 cells were treated with vehicle (-) or 2 ng/ml TGFß1 (+) for 24 h in the presence or absence of 0.2 µg/ml actinomycin D (AD) or 2 µg/ml cycloheximide (CH). Total RNA was isolated as described in Materials and Methods and was subjected to Northern blot analysis. The positions of IHH, ß-actin, and 18S and 28S ribosomal RNA are indicated. The figure represents one of two experiments with similar results.

 


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  		Figure 5. Effect of TGFß1 on the stability of IHH mRNA in ROS17/2.8 cells. DRB (25 µg/ml) was added to culture medium 15 min before the addition of vehicle (-) or 2 ng/ml TGFß1 (+). Total RNA was isolated 0, 6, 12, and 24 h after the addition of vehicle or TGFß1 as described in Materials and Methods and was subjected to Northern blot analysis. The positions of IHH, ß-actin, and 18S and 28S ribosomal RNA are indicated. The figure represents one of two experiments with similar results.

 
Hedgehog is known to interact with other growth factors; therefore, we examined the effects of TGFß1 on Ihh gene regulation in the presence of other agents known to modulate phenotypic expression of osteoblasts. As shown in Fig. 6Go, BMP-2, basic FGF, and 1,25-dihydroxyvitamin D3 did not largely affect the steady state Ihh mRNA levels in ROS17/2.8 cells in either the presence or absence of TGFß1, indicating the certain specificity of the effect of TGFß1 on Ihh expression, at least in ROS cells.

To test the generality of Ihh mRNA expression and its regulation by TGFß1 in osteoblast-like cells, we examined osteoblast-enriched cells isolated from neonatal rat calvariae. Similar to ROS17/2.8 cells, the Ihh mRNA level in these cells was increased by TGFß1 treatment about 2-fold (Fig. 7Go).



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  		Figure 7. Effects of TGFß1 on the steady state levels of IHH mRNA in neonatal rat calvaria cells. Cells were prepared and cultured as described in Materials and Methods, and then were treated with vehicle (-) or 2 ng/ml TGFß1 (+) for 24 h. Total RNA was isolated as described in Materials and Methods and was subjected to Northern blot analysis. The positions of IHH and 18S ribosomal RNA are indicated. The bottom panel indicates the ethidium bromide-stained gels shown as the control. The figure represents one of two experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper reported for the first time the expression of Ihh in osteoblast-like cells and osteoblast-enriched cells. Ihh is one of the recently identified vertebrate homologs of a Drosophila segment polarity gene, hedgehog (9). Shh, one of the isoforms of Ihh, has been shown to mediate the activity of the ZPA (11, 13), and Ihh has been implicated in the regulation of terminal differentiation in chondrocytes (16, 17). These findings indicate that hedgehog proteins play an important role in the differentiation and proliferation of mesenchymal cells. Osteoblastic differentiation and regulation of their functions have been known to be under the control of a set of cytokines, including BMPs, FGFs, and TGFßs. Shh has been known to interact with BMPs and FGFs during the embryonic morphogenesis of limbs as well as axial structures in the early embryos (35, 36, 37, 38). However, nothing was known to date regarding hedgehog expression in osteoblasts. Our data indicate that Ihh is a new member of cytokines that are produced by osteoblasts.

Interestingly, although dpp has been reported to regulate hedgehog expression in Drosophila embryos (34), BMP did not affect Ihh expression in osteoblastic cells. Our observation clearly indicates that TGFß, another member of the TGFß superfamily, regulates Ihh mRNA expression in osteoblasts. We also demonstrated the production in ROS17/2.8 cells of the Ihh protein, which migrated as a 25-kDa band in our Western blot analysis. The band intensity was enhanced by TGFß1, indicating that the effect of TGFß1 on Ihh mRNA is translated into the Ihh protein level. Although the size of the recombinant Shh N-terminus protein has been reported to be approximately 19 kDa, it migrated as 27 kDa in our gel. Therefore, the single 25-kDa band observed in our Western analysis corresponds to the N-terminus fragment of Ihh, as judged by its reactivity to the antiserum raised against the N-terminal peptide. Intramolecular proteolytic cleavage has been shown to occur in Drosophila Hh and vertebrate Shh (39), and similar autoproteolysis has been suggested in other members of the hedgehog gene family based on the well conserved sequence in the proteolytic cleavage site and the catalytic domain in the carboxyl-terminus (40, 41). Further analysis of posttranslational event may clarify the precise nature of this protein.

The effects of TGFß1 on Ihh gene expression are specific to this cytokine, as other potent modulators of osteoblastic functions, such as BMP-2, basic FGF, or 1,25-dihydroxyvitamin D3, did not largely affect Ihh expression, nor did they alter the TGFß1-induced increase in Ihh mRNA levels. TGFß1 is considered to promote bone formation based on the in vivo injection studies (42, 43) as well as numerous in vitro data suggesting its anabolic actions on osteoblastic cells (21, 44). A recent study of TGFß2 misexpression leading to osteoporosis suggested that continuous overdose of TGFß may be catabolic rather than anabolic (45). In addition to the enhancing effects of TGFß on the accumulation of extracellular matrices via stimulation of the expression of the genes encoding extracellular matrix proteins and attachment-related proteins and enzymes, TGFß promotes its own expression as one of its alternative pathways of action (46, 47, 48). TGFß1-specific enhancement of Ihh expression in the cell of osteoblastic lineage revealed a novel pathway of the action of this pluripotent growth factor in bone metabolism.

The effect of TGFß on Ihh gene expression was via Ihh mRNA stabilization, which results in an increased translation of the polypeptide. TGFß1-induced Ihh mRNA accumulation was not completely blocked by actinomycin D treatment, and our nuclear run-on assay showed no TGFß1 effect on the Ihh transcription rate. Furthermore, experiments using DRB showed a marked enhancement of Ihh mRNA stability by TGFß1. TGFß has been implicated in the posttranscriptional regulation of a number of genes in various cell types. TGFß increases the stability of type I collagen and fibronectin mRNA in fibroblasts (49, 50). It also enhances its own mRNA in a human osteosarcoma cell line by a posttranscriptional mechanism (48). Although autologous stimulation of TGFß1 and TGFß1 enhancement of type I collagen and fibronectin gene expression were nullified by cycloheximide in these studies, Ihh mRNA accumulation in ROS 17/2.8 cells was resistant to the protein synthesis inhibitor, suggesting the presence of a distinct pathway that does not require new protein synthesis to stabilize the Ihh mRNA. This mRNA stabilization would be due to the activation of preexisting molecules via phosphorylation and/or dephosphorylation events initiated by serine-threonine kinase activity of type I and type II TGFß receptors. Regulation of mRNA stability has not previously been noted for any of the hedgehog genes. Our data suggest that this posttranscriptional mechanism could be an additional type of control for these important developmental factors.

Osteoblastic cells derived from rat calvariae have been shown to undergo differentiation in cell culture systems; these cells express various markers of osteoblastic differentiation in a time-dependent manner (51). Rat calvaria cells in long term cultures increased the expression of Ihh mRNA in response to TGFß1 treatment. This is consistent with the TGFß1 enhancement of Ihh expression in ROS17/2.8 cells, which express numerous features of mature osteoblasts. These findings indicate that Ihh mRNA is up-regulated by TGFß1 in normal osteoblasts.

Recent histological analyses have revealed the expression of Ihh in prehypertrophic chondrocytes in mouse embryos as well as 3-week-old chicks (16, 17). Although these studies did not describe any Ihh signal expression in osteoblasts, we observed Ihh expression in relatively mature osteoblasts. These findings suggest that the Ihh expression in the two cell types (osteoblasts and chondrocytes) may be spatially and temporally separate, and Ihh may play different roles in the two, quite different, steps or pathways of ossification. One of the major questions is the function of Ihh in osteoblasts. Our preliminary experiments did not detect any effect of Shh on the expression of PTHrP in osteoblasts, although Shh treatment has been shown to enhance PTHrP expression in periarticular perichondrium in chick and mouse embryonic bones (17). Shh treatment did not show an obvious effect on the expression of some of the osteoblastic phenotype-related genes (data not shown). It is possible, therefore, that Ihh secreted by TGFß activation may act on other types of cells located in a distance, as exemplified by prehypertrophic chondrocytes (17); however, possible direct Ihh actions on osteoblasts are not still excluded.

Bone remodeling is based on the balance of concerted regulation by multiple calcitropic factors of osteoblasts and osteoclasts. Among those, TGFß family members are implicated in the core events in bone formation. However, null mutation of TGFß1 gene did not show obvious alteration in bone structures (52, 53). One of the possible explanations is the presence of other TGFß isoforms, although this may not be as likely because the promoter regions of the TGFß isoforms are quite distinct (54, 55, 56). Alternatively, it is suggested that the presence of a cytokine network system compensates for the loss of a single component in the process of bone formation. Our data clearly indicate that Ihh is a new member of this network system. Future characterization of the role of the Ihh gene in knockout mice will clarify the role of this TGFß1-Ihh axis system in bone metabolism.


   Acknowledgments
 
We thank Andrew P. McMahon and Cliff Tabin for scientific discussions as well as the cDNAs and antibody, and Prof. Kenichi Shinomiya, Prof. Kohtaro Furuya and Dr. Haruyasu Yamamoto (Department of Orthopedic Surgery, Tokyo Medical and Dental University) for their continuous support of this research.


   Footnotes
 
1 This work was supported by grants in aid from the Ministry of Education (05404053, 07557096, 08307012, and 08044258) and Grant 0076 from the Japan Orthopedics and Traumatology Foundation, Inc. Back

Received September 23, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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