Parathyroid Hormone-Smad3 Axis Exerts Anti-apoptotic Action and Augments Anabolic Action of Transforming Growth Factor β in Osteoblasts*

Although several studies indicated that parathyroid hormone (PTH) exerted anabolic action on bone, its precise mechanisms have been unknown. On the other hand, transforming growth factor β (TGF-β), abundantly stored in bone matrix, stimulates bone formation with a local injection in rodents. Although our previous study suggested that Smad3 is an important molecule for the stimulation of bone formation, no reports have been available about the effects of PTH on Smad3. In this present study, we examined the effects of PTH on Smad3 and the physiological significance in mouse osteoblastic cells. PTH promoted the expression of Smad3 mRNA within 10 min and the protein level in a dose-dependent manner in MC3T3-E1 and rat osteoblastic UMR-106 cells. Protein kinase A (PKA) activator as well as protein kinase C (PKC) activators increased Smad3 protein level, and both PKA and PKC inhibitors antagonized PTH-induced Smad3, indicating that PTH promotes the production of Smad3 through both PKA and PKC pathways. Next, we examined anti-apoptotic effects of PTH and Smad3 in these cells, employing trypan blue, transferase-mediated nick end labeling, and Hoechst staining. Pretreatment with PTH or overexpression of Smad3 decreased the number of apoptotic cells induced by dexamethasone and etoposide. Moreover, a dominant negative mutant, Smad3ΔC, abrogated PTH-induced anti-apoptotic effects. On the other hand, PTH augmented TGF-β-induced transcriptional activity. Furthermore, PTH enhanced TGF-β-induced production of type I collagen, whereas it did not affect TGF-β-reduced proliferation in MC3T3-E1 cells. These observations indicated that PTH amplified the anabolic effects of TGF-β by accelerating the transcriptional activity of Smad3. In conclusion, we first demonstrated that PTH-Smad3 axis exerts anti-apoptotic effects in osteoblasts and reinforces the anabolic action by TGF-β in osteoblasts. Hence, PTH-Smad3 axis might be involved in the bone anabolic action of PTH.

The bone is a highly specialized and dynamic organ with continuous regeneration, called remodeling. Bone remodeling consists of two different events, resorption by osteoclasts and formation by osteoblasts. For an increase in bone mineral density, a positive balance, in which the formation is prior to the resorption, should be gained. The ability to gain the positive balance may be a therapeutic strategy for osteoporosis.
Although several agents are capable of decreasing bone resorption and halting further bone loss in osteopenic states, the ideal drug would be an anabolic agent that increases bone mass by stimulating bone formation. It has been well established that daily injections of low dose of parathyroid hormone (PTH), 1 a main regulatory hormone in calcium and bone metabolism, increase bone mass in animals and humans (1)(2)(3)(4)(5)(6)(7)(8). However, the mechanisms by which PTH possesses bone anabolic action in vivo are not fully known.
On the other hand, transforming growth factor ␤ (TGF-␤) is most abundant in bone matrix, compared with other tissues (9). TGF-␤ is stored in an inactive form, released from the bone matrix, and activated in the bone microenvironment (10). It is produced by osteoblasts and appears to regulate bone metabolism in various ways, including skeletal development and bone remodeling (11). TGF-␤ modulates the proliferation, differentiation, and production of bone matrix proteins of osteoblasts (10). Several reports demonstrated that TGF-␤ induced bone formation when it was locally administered into bone tissues in rat (12)(13)(14)(15). The Smad family proteins are critical components of the TGF-␤ signaling pathways (16,17), and TGF-␤ regulates the transcriptional response of the target genes through the two receptor-regulated Smads, Smad2 and Smad3 (16,17). Receptor-mediated phosphorylation of Smad2 or Smad3 induces their association with the common partner Smad4, followed by translocation into the nucleus where these complexes activate transcription of specific genes (16,17). We recently reported that Smad3 promotes the production of type I collagen, alkaline phosphatase activity, and mineralization in mouse osteoblastic MC3T3-E1 cells (18,19). Moreover, the mice with the target disruption of Smad3 exhibited the osteopenia caused by the decreased bone formation (20). Based on these data, we have proposed that Smad3 is a molecule of promoting bone formation.
Although several studies indicated that PTH increases TGF-␤ expression and secretion in osteoblasts (21,22), there have been no papers that reported the effects of PTH on Smad3 in osteoblasts. Hence, in our present study, we examined the effects of PTH on the expression and the transcriptional activity of Smad3, and also its physiological significance in osteoblasts.
Construct and Transient Transfection-Myc-tagged Smad2 and Smad3 and flag-tagged Smad4 were prepared, as previously described (23). Smad3 DNA was derived from rat. A mutant form of Myc-tagged Smad3 (Smad3⌬C), in which the MH2 domain corresponding to amino acid residues 278 -425 was removed, was kindly provided by Dr. Y. Chen. Myc-Smad3, Myc-Smad3⌬C, and empty vector (pcDNA3.1ϩ) (each 3 g) were transfected to MC3T3-E1 and UMR-106 cells with LipofectAMINE (Invitrogen). Six h later, the cells were fed with fresh medium containing 10% FBS. Forty-eight h later, the transfected cells were used for the experiments. To rule out the possibility of clonal variation, we characterized at least three independent clones for each transfection. Empty vector-transfected cells were used as the control.
Luciferase Assay-Cells were seeded at a density of 2 ϫ 10 5 cells/6well plate. Twenty-four h later, cells were transfected with 3 g of the reporter plasmid (p3TP-Lux) and the pCH110 plasmid expressing ␤-galactosidase (1 g), using LipofectAMINE (Invitrogen). Fifteen h later, the medium was changed to a fresh one containing 4% FBS, and the cells were incubated for an additional 9 h. Thereafter, cells were cultured for 24 h in the presence or absence of 5.0 ng/ml TGF-␤ in medium containing 0.2% FBS. Cells were lysed, and the luciferase activity was measured and normalized to the relative ␤-galactosidase activity as described (18).
Protein Extraction and Western Analysis-Cells were lysed with radioimmunoprecipitation buffer containing 0.5 mM PMSF, complete protease inhibitor mixture, 1% Triton X-100, and 1 mM sodium orthovanadate. Cell lysates were centrifuged at 12,000 ϫ g for 20 min at 4°C, and the supernatants were stored at Ϫ80°C. Protein quantitation was performed with BCA protein assay reagent (Pierce). Twenty g of proteins were denatured in SDS sample buffer and separated on 10% polyacrylamide-SDS gels. Proteins were transferred in 25 mM Tris, 192 mM glycine, and 20% methanol to polyvinylidene difluoride. Blots were blocked with Tris-buffered saline (20 mM Tris-HCl (pH 7.5) and 137 mM NaCl) plus 0.1% Tween 20 containing 3% dried milk powder. The antigen-antibody complexes were visualized using the appropriate secondary antibodies (Sigma), and the enhanced chemiluminescence detection system, as recommended by the manufacturer (Amersham Biosciences, Buckinghamshire, United Kingdom).
For co-immunoprecipitation experiments, cells were lysed with a buffer containing 1% Triton X-100, 1% deoxycholate, 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1.5 mM MgCl 2 , 2 mM EGTA, plus a protease inhibitor mixture for 30 min at 4°C, and insoluble materials were separated by centrifugation at 4°C for 30 min at 14,000 ϫ g. The supernatant containing 1 mg of protein was clarified and incubated with anti-flag antibody (Sigma) on a rocking platform at 4°C overnight. The immune complexes were collected with Protein G Plus/Protein A-agarose beads (Calbiochem-Novabiochem Corp., San Diego, CA) for 30 min at 4°C. The beads were washed three times with the lysis buffer, resuspended in 2ϫ sample buffer, and boiled for 5 min. Immunoprecipitated proteins were then analyzed by SDS-PAGE and subjected to Western blot analysis employing anti-Myc antibody (Sigma) as described above.
RNA Extraction and Northern Analysis-Total RNA was prepared from cells using the acid guanidinium-thiocyanate-phenol-chloroform extraction method. Twenty g of total RNA was denatured, run on a 1% agarose gel containing 2% formaldehyde, transferred to a nitrocellulose membrane, and fixed with ultraviolet light (Funa-UV-Linker, Funakoshi, Tokyo, Japan). The membrane was hybridized to a DNA probe labeled with 32 P (Amersham Biosciences) overnight at 42°C. The hybridization probe was the 2.8-kb fragment of the ␣1 gene of type I procollagen (a gift from Dr. T. Kimura, Osaka University, Osaka, Japan). After hybridization, the filter was washed twice with 2ϫ standard saline citrate (SSC) containing 0.5% SDS and subsequently washed twice with 0.1ϫ SSC containing 0.5% SDS at 58°C for 1 h. The filter was exposed to x-ray film, using intensifying screen at Ϫ80°C. All values were normalized for RNA loading by probing blots with human ␤-actin cDNA (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-Reverse transcription of 5 g of cultured cell total RNA was carried out for 50 min at 42°C and then 15 min at 70°C, using Super Script TM First-Strand Synthesis System for RT-PCR (Invitrogen), which contained RT buffer, oligo(dT) [12][13][14][15][16][17][18] , 5ϫ First-Strand Solution, 10 mM dNTP, 0.1 M DTT, SuperScript II (RT-enzyme), and RNase H (RNase inhibitor). PCR using primers to unique sequences in each cDNA was carried out in a volume of 10 l of reaction mixture for PCR (as supplied by TaKaRa, Otsu, Japan), supplemented with 2.5 units of TaKaRa Taq TM , 1.5 mM amount of each dNTP (TaKaRa), and 10ϫ PCR buffer, which contained 100 mM Tris-HCl (pH 8.3), 500 mM KCl, and 15 mM MgCl 2 . Twenty-five ng of each primer and 1 l of template (from a 50-l RT reaction) were used. Thermal cycling conditions and primer sequences are described below: 1) initial denaturation at 96°C for 2 min; 2) cycling for cDNA-specific number of cycles; 96°C for 1 min, cDNA-specific annealing temperature for 2 min, and 72°C for 2 min; and 3) final extension at 72°C for 5 min. Primer sequences, annealing temperature, and cycle numbers were as follows. Smad3 sequences were 5Ј-GAGTAGAGACGCCAGTTCTACC-3Ј and 5Ј-GGTTTGGAGAA-CCTGCGTCCAT-3Ј (62°C; 25 cycles) (25), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) sequences were 5Ј-ATCCCATCA-CCATCTTCCAGGAG-3Ј and 5Ј-CCTGCTTCACCACCTTCTTGATG-3Ј (58°C; 22 cycles). For semiquantitative RT-PCR, the number of cycles was chosen so that amplification remained well within the linear range, as assessed by densitometry (NIH Image J, version 1.08i, public domain program). An equal volume from each PCR was analyzed by 6% nondenaturing polyacrylamide gel electrophoresis, and ethidium bromidestained PCR products were evaluated. Marker gene expression was normalized to GAPDH expression in each sample.
Determination of Osteoblast Apoptosis-Trypan blue staining (Invitrogen; 0.1% final concentration) was used for routine quantification of apoptosis (26). In brief, a drop of the cell suspension was mixed with a drop of the trypan blue solution. The ratio of each volume was 1:1. The total numbers of viable and nonviable cells were calculated under the light microscopy (27). Apoptosis cleaves cellular DNA into histoneassociated fragments. As the more specific staining to detect apoptosis, transferase-mediated dUTP nick end labeling (TUNEL) staining and Hoechst staining were employed. In brief, cells were cultured on round cover glasses (Fisher Scientific, Pittsburgh, PA) set in 6-well plates and the glasses attached by cells were rinsed with ice-cold PBS two times, followed by fixation with 4% neutral formaldehyde. Then, these fixed cell layers on the glasses were stained. TUNEL reaction was performed using an Apoptosis in Situ Detection kit (Wako), following the standard protocol, as described (28). Hoechst staining was performed to view the pyknotic fragmented nuclei typical of apoptotic cells, using Hoechst 33258 (Sigma). In each experiment, the percentages of numbers of apoptotic cells per total ones in randomly selected fields were calculated using hemocytometer. Each experiment was performed at least three times.
[ 3 H]Thymidine Incorporation Assay-MC3T3-E1 cells were seeded at 2 ϫ 10 4 cells/well in 24-well plates. These cells were maintained in ␣-MEM with 10% FBS. After 48 h of culture, cells were labeled with 0.5 Ci/ml [ 3 H]thymidine (Amersham Biosciences) for 4 h. The incubation was terminated by removal of the medium, washed with PBS twice, and followed by the addition of 5% trichloroacetic acid on ice in 10 min. After removal of the trichloroacetic acid, the residue was dissolved in 20 mM NaOH at 37°C, and scintillation mixture was added. Each sample was counted in a liquid scintillation counter.
Statistics-Data were expressed as means Ϯ S.E. Statistical analysis was performed using an unpaired t test or analysis of variance.

RESULTS
PTH Stimulated the Expression of Smad3 mRNA and Protein-First, we examined the effects of PTH on the expressions of Smad3 mRNA and protein. In MC3T3-E1 cells, PTH-(1-34) stimulated the expression of Smad3 mRNA in a dose-dependent manner (Fig. 1A). PTH-(1-34) also stimulated the expression of Smad3 protein in both MC3T3-E1 and UMR-106 cells (Fig. 1, B and C). Next, we performed the time-course experiments. As shown in Fig. 2A, PTH-(1-34) promoted the expression of Smad3 mRNA within 10 min in MC3T3-E1 cells. The expression of Smad3 protein was enhanced by PTH-(1-34) within 1 h in MC3T3-E1 and UMR-106 cells (Fig. 2, B and C). These effects of PTH-(1-34) were sustained at least for 24 h. Moreover, PTH-(1-34) increased the level of phosphorylated Smad3 within 6 h (Fig. 2D). Because the up-regulation of Smad3 by PTH-(1-34) was initiated in early times, PTH might stimulate the expression of Smad3 without protein synthesis de novo. We therefore employed cycloheximide, a protein synthesis inhibitor, and actinomycin D, a transcription inhibitor. Although 10 M cycloheximide did not affect PTH-induced expression of Smad3 mRNA, PTH-induced Smad3 protein was reduced with 10 M actinomycin D (Fig. 3). These findings suggested that PTH promoted the expression of Smad3 at the transcriptional level independently of protein synthesis de novo in osteoblasts.
PTH Promoted Smad3 Expression via PKA and PKC Pathways-PTH binds to PTH/PTH-related protein receptor, which is a G-protein-coupled seventh transmembrane-type receptor. The PTH signals are known to be transduced through PKA and PKC pathways (1). We therefore investigated whether PTH would up-regulate the Smad3 expression through PKA and/or PKC pathway(s). As shown in Fig. 4, activators for PKA pathways, db-cAMP and (S p )-cAMPS as well as forskolin, and an activator for PKC pathway, phorbol 12-myristate 13-acetate, promoted the expression of Smad3 mRNA and protein in MC3T3-E1 cells (Fig. 4, A and B) and UMR-106 cells (Fig. 4C). In addition, a specific inhibitor of PKA pathway, H89, as well as the inhibitors of PKC pathways, staurosporine and H7, antagonized PTH-induced expression of Smad3 mRNA and protein in MC3T3-E1 cells (Fig. 5, A and B) and UMR-106 cells (Fig. 5C). These findings indicated that PTH stimulates the expression of Smad3 mRNA and protein through both PKA and PKC pathways in osteoblasts.
Smad3 Is an Essential Molecule for PTH-induced Anti-apoptotic Effects in Osteoblasts-PTH exerts anti-apoptotic signals in osteoblasts (29). Recent study revealed that mice with the targeted disruption of Smad3 exhibited osteopenia caused by decreased bone formation, including the promoted apoptosis of osteoblasts and osteocytes (20). These evidence suggested that Smad3, as well as PTH, possesses anti-apoptotic effects in osteoblasts. We therefore examined the effects of PTH and Smad3 on dexamethasone-or etoposide-induced apoptosis in MC3T3-E1 and UMR-106 cells. We employed trypan blue staining, TUNEL staining, and Hoechst staining methods to detect the apoptotic cells. The trypan blue stain method is generally used to distinguish the viable cells from nonviable ones. As shown in Fig. 6, dexamethasone and etoposide induced the number of nonviable cells in MC3T3-E1 (A and B) and UMR-106 cells (C and D). Treatment with PTH-(1-34) as well as Smad3 overexpression antagonized dexamethasone-and etoposide-induced cell death. The truncated Smad3 mutant, Smad3⌬C, lacks its MH2 region in C terminus and possesses dominant negative effects on endogenous Smad3 activity, as previously described (18). Smad3 inactivation with Smad3⌬C antagonized the PTH-induced anti-apoptotic effects on the cells treated with dexamethasone and etoposide, although Smad3⌬C expression itself did not affect them (Fig. 6, A and C). These findings suggested that PTH and the overexpression of Smad3 antagonized dexamethasone-and etoposide-induced apoptosis, and that Smad3 was indispensable in the PTH-induced anti-apoptotic signals in osteoblasts. Cell death detected by trypan blue assay may include death by mechanisms other than apoptosis. TUNEL stain method is more specific for the detection of apoptotic cells, because cells with TUNEL-positive nucleus mean ones with DNA fragmentations. In both empty vector-and Smad3⌬C-transfected UMR-106 cells, dexametha-sone increased the numbers of TUNEL-positive cells (Fig. 7, A  and C). PTH-(1-34) did not affect dexamethasone-induced TUNEL-positive cell number in Smad3⌬C-transfected cells, although PTH antagonized it in empty vector-transfected cells (Fig. 7, A and C). Hoechst staining method is generally recommended to detect apoptotic nucleus morphologically. As shown in Fig. 7B, the nucleus of the empty vector-transfected cells treated with dexamethasone exhibited the dot-spot appearance, indicating that the cells fell in apoptosis. As seen in Fig.  7D, dexamethasone increased the number of apoptotic cells transfected with empty vector and Smad3⌬C. However, PTH did not affect the number of apoptotic cells transfected with Smad3⌬C, although PTH antagonized it in empty vector-transfected cells (Fig. 7D). These findings indicated that Smad3 is an essential molecule for PTH-induced anti-apoptotic action in osteoblasts.
PTH Augmented Smad3-induced Transcriptional Activity-Our previous study revealed that Smad3 promoted the expression of type I procollagen, resulting in acceleration of mineralization in MC3T3-E1 cells (19). We therefore examined the effects of PTH on Smad3-induced transcriptional activity with luciferase assay using 3TP-Lux containing the promoter of plasminogen inhibitor 1 with a Smad3-specific responsive element. TGF-␤ increased the transcriptional activity in the absence of PTH-(1-34) in MC3T3-E1 cells (Fig. 8A). PTH-(1-34) significantly augmented TGF-␤-induced transcriptional activity, although PTH-(1-34) alone did not affect them (Fig. 8A). These results suggested that PTH-(1-34) augmented TGF-␤induced transcriptional activity of Smad3. Although PTH increased the expression of TGF-␤ in MC3T3-E1 and UMR-106 cells (Fig. 8B), PTH alone did not affect the transcriptional activity in MC3T3-E1 cells, and TGF-␤ pretreatment as well as PTH pretreatment alone did not affect the transcriptional activity (data not shown). Moreover, PTH pretreatment did not affect TGF-␤-induced phosphorylation of Smad3 (data not shown). These findings suggest that PTH augmented TGF-␤induced transcriptional activity in a manner independent of PTH-induced expression of TGF-␤ and Smad3. In TGF-␤ signaling, receptor-mediated phosphorylation of Smad2 or Smad3 induces their association with the common partner Smad4, followed by translocation into the nucleus where these complexes activate transcription of specific genes (16,17). We examined the effects of PTH on the association of Smad2 and Smad3 with Smad4. As shown in Fig. 8 (D and E), when Myc-Smad2 or Myc-Smad3 was co-transfected with flag-Smad4 in MC3T3-E1 and UMR-106 cells, Smad2 or Smad3 was coimmunoprecipitated with Smad4 with TGF-␤ treatment. However, PTH-(1-34) did not affect the association of Smad2 or Smad3 with Smad4 in TGF-␤ signaling (Fig. 8, D (for Smad2) and E (for Smad3)). Moreover, PTH did not affect the expression of Smad2 and Smad4 in MC3T3-E1 and UMR-106 cells (Fig. 8C). These findings suggest that PTH specifically increased the expression of Smad3, and that PTH-augmented transcriptional activity by TGF-␤ are independent of Smad2 and Smad4. Next, we examined the nuclear translocation of Smad3 by using Smad3-transfected MC3T3-E1 cells. As shown in Fig. 8F, PTH-(1-34) did not affect TGF-␤-induced translocation of Smad3 into nucleus. These results suggested that PTH stimulated the transcriptional activity of Smad3, after Smad3 was translocated into nucleus.

PTH Augmented Anabolic but Not Catabolic Effects of TGF-␤ in Osteoblasts-TGF-␤ stimulates type I collagen expression
and inhibits proliferation of MC3T3-E1 cells, as previously described (30). As shown in Fig. 9 (A and B), PTH-(1-34) accelerated TGF-␤-induced expression of type I procollagen mRNA and type I collagen protein in MC3T3-E1 cells. These  (Fig. 9C). These results suggested that PTH-(1-34) augmented anabolic but not catabolic effects of TGF-␤ in osteoblasts. DISCUSSION The present study indicated three important new points in osteoblasts. First, PTH promoted the expression of Smad3, a crucial mediator in TGF-␤ signaling. Second, Smad3 was in-volved in the anti-apoptotic effects of PTH. Finally, PTH augmented bone anabolic actions of TGF-␤.
In this study, PTH promoted the expression of Smad3 in osteoblasts. Smad3 is a critical component of the TGF-␤ signaling pathways, and receptor-mediated phosphorylation of Smad2 or Smad3 induces their association with the common partner Smad4, followed by translocation into the nucleus where these complexes activate transcription of specific genes (16,17). Thus, Smad3 is considered to play as a signal transduction molecule and a transcriptional regulator in cytoplasm and nucleus, respectively. Previous studies indicated that PTH stimulates TGF-␤ expression in osteoblasts (22). In the present study, PTH stimulated Smad3 expression within only 1 h and its effects were independent of the protein synthesis de novo. These findings indicated that Smad3 expression induced by PTH was not through TGF-␤. Smad3 might be a transcriptional regulator for bone formation partly in a manner independent of TGF-␤ in osteoblasts, as previously reported (18,19). It has been reported that PTH affects the transcriptional factors through activator protein-1 (AP-1), cAMP-responsive element-binding protein (CREB), and Runt-related transcriptional factor 2 (Runx2) (31). As for the AP-1 family, c-Fos, Fra-1, and ⌬FosB play important roles in bone formation in the previous studies (32)(33)(34), and PTH stimulated the expression of c-Fos, c-Jun, Fra-1, Fra-2, and FosB (35)(36)(37)(38)(39). Moreover, the phosphorylation of CREB in response to PTH treatment was required for PTH-stimulated expression of c-Fos (35,39). Runx2 has been characterized as an osteoblast-specific tran-scriptional activator (40 -42), and it induced promoter activity in the osteocalcin, osteopontin, type I collagen, and collagenase-3 promoters (40,(43)(44)(45)(46)(47)(48). In UMR-106 -01 cells, a rat osteoblastic cell line, PTH led to activation of Runx2 (49) and the physical interaction between AP-1 and Runx2 was required for the PTH-stimulated transcriptional activity of the collagenase-3 promoter in MC3T3-E1 cells (50). These data suggest that those transcriptional regulators are important in PTH actions in bone. Our previous study suggested that Smad3 is a crucial molecule in bone formation (18,19). The present findings, therefore, suggested that Smad3 functions, as a transcriptional regulator downstream of PTH signaling.
It was reported that several nuclear receptors of the steroid hormones interacted with Smad3; Smad3 potentiated ligandinduced transactivation of vitamin D receptor as a coactivator of this receptor (51), and glucocorticoid receptor inhibited transcriptional activation of Smad3 (52). Smad3 was an androgen receptor co-regulator in prostate cancer cells, whereas the androgen receptor repressed TGF-␤ signaling through interaction with Smad3 (53, 54). Estrogen receptor-mediated transcriptional activation enhanced by TGF-␤ signaling was through Smad3 (55). Although these findings suggested that Smad3 is related to the action of steroid hormones, no reports have been available about the significance of these interactions in osteoblasts. Moreover, whether the peptide hormone would regulate the expression and the transcriptional activity of Smad3 was not known. Our present report was the first that calciotropic hormone affected the expression and transcriptional activity of Smad3.
The anabolic mechanism in vivo may be partially explained by the growth factor genes expressed in activated osteoblasts following exposure to PTH. PTH is known to stimulate the expression of IGF-I, IGF-II, IGF-binding proteins, and TGF-␤ in osteoblasts (21,70,71). IGF-I is known to stimulate bone cell replication and matrix synthesis (72,73), and mice with a bone-specific mutation in IGF-I receptor exhibited a striking decrease in bone volume (74). Moreover, a neutralizing antibody against IGF-I blocked the stimulatory effect on alkaline phosphatase activity and the expression of osteocalcin induced by the intermittent exposure to PTH in osteoblast cells from rat calvariae (75). These lines of evidence suggested that the anabolic action of PTH in bone is partly mediated by IGF-I (21,70,76,77). Furthermore, IGF-I-binding proteins also might be related to anabolic action of PTH-IGF-I signals (78). On the other hand, the anabolic action of PTH has been partly ex- FIG. 8. PTH stimulates the transcriptional activity of Smad3. MC3T3-E1 cells were transfected with 3 g of the reporter plasmid (p3TP-Lux), the pCH110 plasmid expressing ␤-galactosidase (1 g) per well in 6-well plate. Forty-eight h later, cells were treated with 10 Ϫ8 M PTH for 12 h, and fed with fresh ␣-MEM with or without 5 ng/ml TGF-␤. Then, 24 h later, cells were harvested and relative luciferase activity was measured. Values of relative luciferase activity represent the mean Ϯ S.E. *, p Ͻ 0.01 from TGF-␤-untreated group. **, p Ͻ 0.01 from PTH-untreated group. B and C, after confluent MC3T3-E1 cells were cultured in serum-free medium for 12 h, cells were treated with 10 Ϫ8 M PTH-(1-34) for the indicated times. Then, protein extraction and Western blot analysis were performed, as described under "Experimental Procedures." D and E, Myc-tagged Smad2 (D) or Smad3 (E) was co-transfected into MC3T3-E1 cells with flag-tagged Smad4. Cells were treated with 5 ng/ml TGF-␤ for 1 h after pretreatment with 10 Ϫ8 PTH-(1-34) for 12 h. Cell extracts were immunoprecipitated (IP) with anti-flag, followed by immunoblotting (IB) with anti-Myc, as described under "Experimental Procedures." F, Smad3-transfected MC3T3-E1 cells were treated with 10 Ϫ8 M PTH for 12 h, and fed with fresh ␣-MEM with or without 5 ng/ml TGF-␤. One h later, subcellular fractionations and Western blot analysis were performed, as described under "Experimental Procedures." plained by evidence that PTH exerts anti-apoptotic effects on osteoblasts (29). The mice with targeted disruption of Smad3 exhibited osteopenia caused by decreased bone formation, including the promoted apoptosis of osteoblasts and osteocytes (20). These evidence raised the possibility that Smad3 possesses anti-apoptotic effects in osteoblasts. In the present study, Smad3 inhibited apoptosis and inactivation of Smad3 by dominant negative mutant attenuated PTH-induced anti-apoptotic effects in mouse and rat osteoblastic cells. These findings indicated that Smad3 was required for PTH-induced anti-apoptotic effects on osteoblasts. Apoptosis plays a critical role during embryonic limb development, skeletal maturation, bone turnover, fracture healing, and bone degeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time (79). Previous studies revealed that glucocorticoid as well as estrogen withdrawal promoted apoptosis in osteoblasts (80 -82). It was also suggested that the anti-apoptotic effects of PTH are mediated by signals transduced through the G s pathway (83). However, it is still unclear what signal transduction pathways are used in apoptosis of osteoblasts and what steps in apoptosis are targeted by PTH in its rescue effects.
Because PTH augmented TGF-␤-induced transcriptional activity in these cells and our previous reports indicated that TGF-␤ as well as Smad3 inhibited osteoblast proliferation, it is possible that PTH induces some signaling leading to the enhancement of Smad3 signaling-mediated reduction in proliferation. Borton et al. (20) reported that TGF-␤ did not affect osteoblast proliferation in Smad3-deficient osteoblasts. Therefore, it seemed improbable that TGF-␤ inhibited osteoblast proliferation through the signal other than Smad3. Surprisingly, although PTH augmented TGF-␤-induced type I collagen expression, anabolic effects of TGF-␤ on osteoblasts, PTH did not affect the TGF-␤-reduced proliferation, catabolic effects in the present study. These data suggested that the regulation of Smad3 by PTH specifically contributed to anabolic action of TGF-␤ in osteoblasts.
Because PTH increased Smad3 expression and its phosphorylation level in the present study, it might be somewhat puzzling that PTH itself did not affect the transcriptional activity. The precise reason is unknown. However, the existence of sufficient endogenous TGF-␤ stimulation may be necessary to induce transcriptional activity of 3TP-Lux. Moreover, it seems that PTH-induced Smad3 levels are not sufficient for the activation of the reporter gene or that an additional factor or mechanism is needed for this activity. Previous studies indicated that TGF-␤ as well as Smad3 increased the expression of type I collagen in MC3T3-E1 cells (18,19,30). However, PTH itself did not increase the expression of type I collagen (data not shown), which was compatible with the previous reports (59,60), although PTH increased the expression of Smad3 in the present study. Because PTH also regulates the expression of numerous genes that inhibit the expression of type I collagen, PTH-induced proteins may inhibit the expression of type I collagen against the effects of Smad3 or TGF-␤. Alternatively, it appears that PTH-mediated induction of Smad3 alone is not sufficient for gene activation.
In the present study, although PTH stimulates the expression of TGF-␤, PTH did not affect TGF-␤-induced phosphorylation of Smad3, association of Smad2/3 and Smad4, the nuclear translocation of Smad3 in MC3T3-E1 cells, suggesting that the augmentation of PTH on transcriptional activity of Smad3 was through the events in nucleus. The previous evidence suggested that PTH affected osteoblasts through some transcriptional regulators, such as AP-1 family, CREB, and Runx2. It was also reported that Smad3 interacted with c-Jun, CREB, and Runx2 (84 -86). We therefore speculated that those transcriptional regulators induced by PTH affected the transcriptional activity of Smad3 by the interaction. Further study is necessary to clarify these issues.
It has been widely believed that both PTH and TGF-␤ possess the divergent and complex effects on osteoblasts, bone anabolic and catabolic effects. Our previous and present study suggested that Smad3 was an important molecule in antiapoptotic action of PTH and anabolic action of TGF-␤. Smad3 seemed to be an important molecule that activates the bone anabolic signals in the intracellular pathways of PTH and TGF-␤. Mammalians have got the complex network systems to maintain homeostasis under variable circumstances in development. The systems consist of systemic and local regulators, such as systemic hormones, local growth factors, and cytokines. Those factors exert the positive or negative effects in the appropriate balance on the tissues for homeostasis, and the endocrine disease is caused by the disorder of homeostasis. Bone is a dynamic connective tissue, undergoing remodeling, a continual process of resorption and renewal. In the remodeling, osteoblasts, which contribute to the renewal process, are regulated by several hormones and growth factors. These systemic and local factors cross-talk with each other, resulting in the regulation of the number and function of osteoblasts. We speculate that Smad3 is one of key molecules that arrange the signals from systemic and local factors and convert them into anabolic signals in osteoblasts, because our previous study suggested that Smad3 signal might exert anabolic actions of TGF-␤ in osteoblasts (18,19), and Smad3 leads to PTH-induced anti-apoptotic effects. Furthermore, PTH augmented anabolic effects of Smad3 on osteoblasts in the present study.
In conclusion, we first demonstrated that PTH stimulated the expression of Smad3 in osteoblasts. The pathway, the "PTH-Smad3 axis," was essential in PTH-induced anti-apoptotic effects and reinforced the anabolic effects of Smad3. Hence, PTH-Smad3 axis might be involved in the bone anabolic action of PTH, and nominated for the therapeutic strategy, targeting bone degenerated disease, including osteoporosis.