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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Members of the transforming growth factor-β (TGF-β) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-β signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-β superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-β1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-β1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-β/activin-restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-β superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Members of the transforming growth factor-β (TGF-β) family transduce signals through type I and type II serine/threonine kinase receptors.(1–3) The intracellular signal transduction through serine/threonine kinase receptors has been well characterized for TGF-β. Both type I and type II TGF-β receptors (TβR) are required for signal transduction. TβR-I is a substrate for TβR-II and propagates the signal downstream upon activation.(4) The mechanisms by which bone morphogenetic proteins (BMPs) and related members of the TGF-β superfamily transduce signals are likely to be analogous to TGF-β1.(5)

The recently identified Smad protein family mediates the downstream intracellular signaling by TGF-β family receptors. Smad proteins are 50–70 kDa intracellular proteins which transduce signals from the serine/threonine kinase receptors for the TGF-β family proteins to the nucleus.(6–8) Certain Smads act in a lineage-specific manner. Smad2 and Smad3 are activated by TβR-I or the activin type IB receptor (ActR-IB),(9–12) whereas Smad1 and Smad5 are activated by the type expression receptors for bone morphogenetic proteins (ActR-1/ALK-2, BMP receptor [BMPR]-IA/ALK-3, and BMPR-IB/ALK-6).(13–17) These pathway-restricted Smads transiently interact with and become phosphorylated by the activated type I receptor, and thereafter form heteromeric complexes with common-mediator Smad4 (also termed DPC-4).(18–20) The heteromeric complexes translocate into the nucleus and regulate the transcription of specific genes. Smad6 and Smad7 have more recently been identified in mammals and are distantly related to other Smads. Interestingly, Smad6 and Smad7 act as inhibitors in TGF-β family or BMP family protein signals.(21–24)

There is growing evidence that TGF-β superfamily proteins, in particular BMPs, play a morphogenetic role in regulation of the mesenchymal cell migration, proliferation, and differentiation that lead to new bone formation.(25) Previous studies have shown that BMPs and other corresponding receptors are upregulated during fracture healing.(26) In the present study, we examined the spatial and temporal distribution of Smad family members during endochondral ossification in the epiphyseal growth plate of growing rat and compared them with the presence of TGF-β1, BMP-2/4, and their corresponding receptors, TβRs, and BMPRs, respectively.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Materials

Five Wistar rats aged 6 weeks were used for our study. They were kept in cages without external fixation. The experimental procedures were performed in compliance with the guiding principles in the Care and Use of Animals described in the American Journal of Physiology and the guidelines established by the Institute of Laboratory Animal Science of Medicine of Kagoshima University.

The proximal part of the tibia was resected en bloc after sacrifice and fixed in 10% neutral-buffered formalin for 24 h at 4°C. After washing with phosphate-buffered saline (PBS) overnight, the tissues were decalcified with 0.36 M EDTA (pH 7.0–7.2) and embedded in paraffin. Sections were cut at 3–5 μm thickness and subjected to hematoxylin and eosin staining and immunostaining for Smad1 through Smad7, TGF-β1, BMP-2/4, TβR-I, TβR-II, BMPR-IA, and BMPR-II.

Preparation of antibodies

Anti-ligand antibodies

Anti-human TGF-β1 latency-associated peptide neutalizing antibody (R&D Systems, Inc., Minneapolis, MN, U.S.A.) was used for TGF-β1 immunostaining. This antibody is specific for the LAP derived from the TGF-β1 precursor and was used for the detection of latent TGF-β1.(27)

A previously described polyclonal antiserum that detects BMP-2 and BMP-4 was used.(28) The antibody was raised in rabbits against the bacterially expressed mature domain of human BMP-2.(29) The BMP-2/4-specific immunoglobulin G (IgG) fraction was enriched on a Protein A Sepharose (KabiPharmacia, Stockholm, Sweden) column. The specificity of antibody was determined by Western blotting using recombinant BMP-2, BMP-4, BMP-3 and BMP-7/OP-1, as previously reported.(28)

Anti-receptor antibodies

TβR-I, TβR-II, BMPR-IA, BMPR-II were used as reported previously.(30–32)

Anti-Smad antibodies

Smad1, Smad2, Smad3, and Smad4 antisera have been described by Nakao et al.(20,33) Specific antisera were raised against synthetic peptides corresponding to amino acid sequences of the variable proline-rich linker regions of Smad1 (peptide: TFPDSFQQPNSHPFHSP), Smad2 (peptide: DQQLNQSMDTGSPAELSPTTL), Smad3 (peptide: DHQMNHSMDAGSPNLSPNPM), Smad4 (peptide: HPPSNRASTETYSTPALLA), Smad5 (peptide: SSNMIPQTMPSISSRDVQP), Smad6 (peptide: ESPPPPYSRLSPRDEYKPLD), and Smad7 (peptide: KERQLELLLQAVESRGGTRTA). the peptides were coupled to keyhole limpet haemocyanin (Calbiochem-Behring, La Jolla, CA, U.S.A.) with glutaraldehyde, mixed with Freund adjuvant, and used to immunize rabbits. The antisera were tested for specificity by immunoprecipitation on metabolically labeled COS cells transfected with different Smads. Details of characterization will be published elsewhere.

Immunohistochemistry

Immunohistochemistry was performed by the avidin–biotin peroxidase complex method.(34) An Elite ABC GOAT IgG KIT (Vector Laboratories, Burlingame, CA, U.S.A.) was used for immunostaining by antibodies against TGF-β1. An Elite ABC RABBIT IgG KIT was used for immunostaining by antibodies against BMP-2/4, TβR-I, TβR-II, BMPR-IA, BMPR-II, and Smad1–Smad7. After deparaffinization and hydration, endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide. Specimens were incubated with PBS containing 5% normal goat serum and 1% bovine serum albumin for 30 minutes at room temperature to eliminate nonspecific binding, and then with the appropriate concentrations of primary antibodies at 4°C overnight in a humidified chamber. Washing with PBS three times for 5 minutes or overnight was followed by incubation with biotinylated secondary antibody and avidin–biotin peroxidase complex in a humidified chamber for 30 minutes at room temperature. Color was developed using 3,3′-diaminobenzidine tetrachloride (Dojindo Chemical Laboratories, Kumamoto, Japan). No counterstaining was performed in the immunostaining. For negative controls, PBS, normal rabbit IgG, or normal goat IgG was used instead of the primary antibodies.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Because the tissue specimens of epiphyseal plate obtained from individual rats showed a comparable histology and immunostaining pattern, we summarized our results as below.

Histology

The epiphyseal plate of growing rats aged 6 weeks exhibited a cellular morphology characteristic of the epipyyseal plate cartilage with four successively layered zones of chondrocytes with distinct appearances. From the epiphysis to the diaphysis, these were: the zone of resting cartilage, the zone of proliferating cartilage, the zone of maturing cartilage, and the zone of calcifying cartilage.

In the zone of resting cartilage, which lies adjacent to the epiphyseal bone, chondrocytes of moderate size were scattered irregularly throughout intracellular substance. Proliferating chondrocytes in the second zone were thin and wedge-shaped, and piled up on top of one another like stacks of coins, forming columns perpendicular to the plate. The mitotic figures were comparable among these cells. The third zone contained large chondrocytes in various stages of maturation. Those near the proliferating chondrocytes were the least mature, and those nearest the diaphysis were the most mature. The calcifying zone was very thin, directly abutted the diaphysis and contained chondrocytes that are undergoing hypertrophy.

Immunostaining

TGF-β1 and TGF-β receptors

TGF-β1 immunostaining was observed weak in the zone of resting cartilage. TGF-β1 immunostaining was equally strong in the zones of proliferating cartilage and maturing cartilage. The hypertrophied chondrocytes showed a decreased TGF-β1 immunostaining. The extracellular matrix in the cartilage showed a weak expression of TGF-β1. TGF-β1 staining was also noted in osteoblast-like cells of the trabecular bone of epiphysis and diaphysis adjoining the epiphyseal plate (Fig. 1).

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Figure FIG. 1.. Immunostaining of TGF-β1 and BMP-2/4 in the epiphyseal plate (original magnification ×35). (A) TGF-β1. (B) BMP-2/4. EB, epiphyseal bone; R, zone of resting cartilage; P, zone of proliferating cartilage; M, zone of maturing chondrocytes; CC, zone of calcifying cartilage; MB, metaphyseal bone.

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In accordance with localization of ligand, the expression of TβR-I was strong in both proliferating and maturing chondrocytes and less intense in hypertrophied chondrocytes. TβR-II colocalized with TβR-I: both receptors showed a similar expression pattern in the cartilage. They were variously expressed in the zone of resting cartilage and the trabecular bone of epiphysis and metaphysis (Fig. 2).

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Figure FIG. 2.. Immunostaining of TβR-I and TβR-II in the epiphyseal plate (original magnification ×35). (A) TβR-I. (B) TβR-II. EB, epiphyseal bone; R, zone of resting cartilage; P, zone of proliferating cartilage; M, zone of maturing chondrocytes; CC, zone of calcifying cartilage; MB, metaphyseal bone.

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Expression of BMP-2/4 and BMP receptor

BMP-2/4 immunostaining was weakly observed in the zone of resting cartilage. Strong staining was present in proliferating chondrocytes and maturing chondrocytes near the zone of proliferating cartilage. Hypertrophied chondrocytes showed a moderate staining. It appears that the expression of BMP-2/4 was more prominent in chondrocytes that are undergoing maturation, BMP-2/4 was also expressed in osteoblast-like cells in the trabecular bone of epiphysis and metaphysis. The extracellular matrix was strongly or moderately stained in both the zone of proliferating cartilage and the zone of maturing cartilage (Fig. 1).

BMPR-IA and BMPR-II exhibited a comparable expression pattern with their ligand. BMPR-IA was actively expressed in proliferating chondrocytes and maturing chondrocytes near the zone of the proliferating cartilage. Decreased expression was observed in the hypertrophied chondrocytes. In addition to its localization in proliferating and least mature chondrocytes, the BMPR-II expression, however, stayed more active in the deeper zone of matured chondrocytes. The expression of both BMPR-IA and BMPR-II was found weakly or moderately in resting chondrocytes, and variously in the trabecular bone of the epiphysis and metaphysis (Fig. 3).

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Figure FIG. 3.. Immunostaining of BMPR-IA and BMPR-II in the epiphyseal plate (original magnification ×35). (A) BMPR-IA. (B) BMPR-II. EB, epiphyseal bone; R, zone of resting cartilage; P, zone of proliferating cartilage; M, zone of maturing chondrocytes; CC, zone of calcifying cartilage; MB, metaphyseal bone.

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The decreased expression of TβRs and BMPRs in hypertrophied chondrocytes may simply be a result of the receptors spreading over a larger area of the membrane of hypertrophied chondrocytes, which are much bigger than the least mature chondrocytes or advanced proliferating chondrocytes.

Expression of Smads

The TGF-β/activin-restricted Smads, i.e., Smad2 and Smad3, were expressed in a nearly complimentary fashion. Smad2 was expressed strongly in proliferating chondrocytes and weakly in hypertrophied chondrocytes. In contrast, Smad3 was expressed strongly in maturing chondrocytes, especially in the least mature near the zone of proliferating cartilage. Smad2 and Smad3 were also variably expressed in the zone of resting cartilage and the trabecular bone near the calcifying cartilage (Fig. 4).

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Figure FIG. 4.. Immunostaining of Smad2 and Smad3 in the epiphyseal plate (original magnification ×35). EB, epiphyseal bone; R, zone of resting cartilage; P, zone of proliferating cartilage; M, zone of maturing chondrocytes; CC, zone of calcifying cartilage; MB, metaphyseal bone.

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BMP-restricted Smads, i.e., Smad1 and Smad5, showed virtually a similar localization, showing a high expression in proliferating chondrocytes near the zone of maturing cartilage and maturing chondrocytes near the zone of proliferating cartilage. The Smad1 and Smad5 expression was weak in proliferating chondrocytes near the zone of resting cartilage and hypertrophied chondrocytes. Some selected chondrocytes in the zone of the resting cartilage appeared positive for Smad1 and Smad5 staining. In accordance with the high BMP immunostaining, the localization of Smad1 protein was observed in the osteoblast-like cells of the developing trabeculae near the calcifying cartilage. The expression of Smad5 was, however, weak (Fig. 5).

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Figure FIG. 5.. Immunostaining of Smad1 and Smad5 in the epiphyseal plate (original magnification ×35). EB, epiphyseal bone; R, zone of resting cartilage; P, zone of proliferating cartilage; M, zone of maturing chondrocytes; CC, zone of calcifying cartilage; MB, metaphyseal bone.

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The common-mediator Smad4 was broadly localized in chondrocytes in all zones of the epiphyseal growth plate. Inhibitory Smad6 expression overlapped locally with inhibitory Smad7 expression in maturing chondrocytes near the zone of proliferating cartilage, showing a strong expression (Fig. 6).

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Figure FIG. 6.. Immunostaining of Smad4, Smad6 and Smad7 in the epiphyseal plate (original magnification ×35). EB, epiphyseal bone; R, zone of resting cartilage; P, zone of proliferating cartilage; M, zone of maturing chondrocytes; CC, zone of calcifying cartilage; MB, metaphyseal bone.

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Nuclear accumulation of the Smad proteins, Smad1 through Smad7, was clearly observed in proliferating chondrocytes and maturing chondrocytes where their intracellular immunostaining was positively recognized (Fig. 7). An increase in nuclear staining often coincided with the increased cytoplasmic staining for specific Smad proteins which was further corroborated with the ligand and receptors staining pattern.

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Figure FIG. 7.. Immunostaining showing nuclear accumulation of Smad2 in chondrocytes of the epiphyseal plate (original magnification ×150). One arrow indicates the proliferating chondrocyte and two arrows point to the maturing chondrocyte.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The longitudinal growth of long bones occurs in the epiphyseal plate during postnatal development, where typical features of endochondral bone formation are observed. Bone growth is regulated by systemic hormones as well as by locally produced peptide growth factors. Among these peptide growth factors, TGF-βs and BMPs have been found at high levels in bone and cartilage implicating that they may play a morphogenic role in chondrocyte proliferation, maturation, and calcification that are involved during endochondral bone formation.(35–38) The temporal and spatial expressions of BMPs and TGF-βs in various organ systems during embryogenesis indicate that in addition to skeletal development they may play an important role in overall tissue growth and development in adult.(39,40) The specific and interactive roles for TGF-βs and BMPs during endochondral bone formation may vary with developmental potentials of responding cells and their microenvironment. While BMPs are potent bone-inducing agents in a rat subcutaneous assay, TGF-βs and activin are not, although periosteal injection of TGF-β and activin induces bone formation.(41–43) However, a recent study showed that TGF-β1 induces endochondral bone formation in extraskeletal sites of adult baboons and synergizes with BMP-7 bone-inducing activity.(44)

We have shown previously that BMP-2/4 and BMPRs are expressed during endochondral ossification that occurs in adult fracture repair and ossification of the longitudinal ligament.(45) The present findings show a similar expression pattern during endochondral ossification of epiphyseal growth plate. TGF-β1 and BMP-2/4 were expressed in the epiphyseal cartilage together with their corresponding type I receptors and type II receptors, indicating that they may regulate the proliferation and differentiation of chondrocytes during various phases of endochondral ossification by their relative distribution and expression level.

Specific Smad proteins mediate the intracellular signals of TGF-βs and BMPs. Smad1, Smad2, Smad3, and Smad5 act in a ligand-specific manner. The ligand-specific Smads are rapidly and specifically phosphorylated by the type I receptors upon ligand-induced heteromeric complex formation with type II receptors. Then after heteromeric complex formation with Smad4, Smad complexes move into the nucleus and induce transcriptional activity.(10–11,15,20)

Closely related Smad2 and Smad3 showed somewhat different localization in cartilage, which suggests that the distinct effect of TGF-β may be mediated through these two closely structurally related Smad molecules during endochondral ossification. Recent results suggest that they activate different subsets of target genes.(46) Smad2 may act strongly for the proliferation of chondrocytes at the early stage of endochondral ossification for TGF-β signal transduction, whereas Smad3 may function strongly for both proliferation and differentiation of chondrocytes at the later stage.

Smad2, Smad3, and Smad4 were expressed by overlapping each other in various zones of chondrocytes in this study. TβR activation-dependent interaction was shown to occur between Smad2 and Smad3 in vitro. Smad2, Smad3, and Smad4 accumulated in the nucleus upon TGF-β1 treatment and showed a synergistic effect in a transcriptional reporter assay, using the TGF-β–inducible plasminogen activator inhibitor-I promoter.(20) However, Smad2 and Smad3 were found to positively and negatively regulate the mammalian forkhead domain protein, FAST2.(47) Taken together, Smad2 and Smad3 may function alone in chondrocytes that selectively express one isoform. In the limited chondrocytes where expression of both Smads overlaps, they may act synergistically or even antagonistically. Recent knock-out studies for Smad2 and Smad3 show that one cannot substitute for another. Smad2 null mice are embryonically lethal due to a lack of methoderm induction,(48–50) while Smad3 null mice are viable with a high incidence of colon cancer.(51)

Smad1 is highly homologous to Smad5. BMP-2 and BMP-7 caused serine phosphorylation of Smad5 as well as Smad1. They induced alkaline phosphatase activity and osteoblast differentiation when transfected into C2C12.(52) Distinctive functional differences between Smad1 and Smad5 have not been apparent in BMP signal transduction during BMP induced osteoblastic differentiation.(53) Interestingly, the closely related BMP-restricted Smad1 and Smad5 showed almost the same staining pattern in the cartilage of the epiphyseal plate. They showed a strong colocalization at the late stage of proliferating chondrocytes and the early stage of maturing chondrocytes, suggesting their direct involvement in growth and differentiation of chondrocytes at various stages of endochondral ossification.

Nuclear accumulation of these Smads was clearly observed in proliferating chondrocytes and maturing chondrocytes, where the cytoplasmic expression was already shown to be high. Previous studies have shown that the transfection of pathway-restricted Smads into chondro/osteoprogenitor cells increased cellular responsiveness to the ligand.(33,52,53) Thus, the increased cytoplasmic staining of pathway-restricted Smads observed in chondrocytes may also indicate an enhanced cellular responsiveness to TGF-βs and BMPs.

The inhibitory Smads are gaining importance because they may play a role in providing balance between activation and deactivation of morphogenic signals during development. Smad7 interacts with the activated TGF-β type I receptor, thereby blocking the association, phosphorylation, and activation of Smad2.(23) However, Smad6 was reported to inhibit the phosphorylation of Smad2 and Smad1.(21) The expression of Smad6 mRNA was dramatically induced by BMP-2 or BMP-7 in various cell types, indicating a feedback circuit to regulate BMP signaling.(54,55) Interestingly, expressions of Smad6 and Smad7 were similar, showing increased staining in maturing chondrocytes. Smad6 and Smad7 may exert inhibitory effects on the TGF-β or BMP signaling and thus may modulate the proliferation and differentiation of chondrocytes. We also found that Smad6 and Smad7 were accumulated in the nucleus. Recent studies suggest that inhibitory Smads can reside in the nucleus, which upon ligand activation translocate into cytoplasm.(56) It is possible that Smad6 and Smad7 have a nuclear function in addition to interfering with the phosphorylation of lineage-specific Smads by type I receptors.

In conclusion, TGF-β1 and BMP-2/4 seem to participate strongly in the regulation of proliferation and differentiation of chondrocytes during endochondral ossification. Their receptors and ligand-specific Smad proteins play essential roles in the transduction of downstream signaling of TGF-β1 and BMP-2/4, which in turn are responsible for up- and or down-regulation of transcriptional events that occur during endochondral bone formation. The intracellular signaling Smads for TGF-β1 and BMP-2/4 exhibited an intricate expression pattern during endochondral ossification, suggesting their morphogenic role in various development phases of cell proliferation and differentiation. Additional studies are needed to elucidate further the detailed physiological function of these Smads during endochondral ossification in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank T. Imamura for valuable discussions, Y. Ishidou and A. Hanyu for preparing affinity-purified antibodies of Smad proteins, and K. Miyazono for providing TβR antibodies.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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