ARTICLE |
Correspondence to: Dina Lewinson, Dept. of Anatomy and Cell Biology, The Bruce Rappaport Faculty of Medicine, TechnionIsrael Institute of Technology, POB 9649, Haifa 31096, Israel. E-mail: dinal@tx.technion.ac.il
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bone cells respond to mechanical stimulation by gene expression. The molecular events involved in the translation of mechanical stimulation into cell proliferation and bone formation are not yet well understood. We looked for the expression of early-response genes of the AP-1 transcription factor complex in an in vivo bone regeneration system subjected to mechanical forces because these genes were found to be related to mechanotransduction and important for bone development. Sheep maxillary bone was distracted daily for 15 days. c-Jun and c-Fos were evaluated by Northern blotting analysis and immunohistochemistry in biopsy specimens removed at 8 and 15 days and were compared with post-osteotomy but not distracted repair tissue. Elevated levels of c-Jun and c-Fos mRNA were found after 8 days of distraction. Likewise, mesenchyme-like and fibroblast-like cells composing the 8-day distracted regeneration tissue showed increases in the intensity of immunostaining compared to cells in the corresponding non-distracted fracture repair tissue. After 15 days of distraction, when bone trabeculae start to form distally and proximally in the distracted regeneration tissue, mostly preosteoblasts and osteoblasts retained c-Fos and c-Jun immunoreactivity, similar to bone-associated cells in control non-distracted fracture repair tissue. We propose that the elevated expression of c-Jun and c-Fos is related to mechanical stimulation in this in vivo bone regeneration system.
(J Histochem Cytochem 51:11611168, 2003)
Key Words: AP-1 proteins, c-Fos, c-Jun, distraction osteogenesis, bone regeneration, mechanotransduction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
OVEREXPRESSION or targeted gene ablation studies have implicated c-fos and other Fos- and Jun-related proteins in the regulation of bone tissue formation (
The nuclear protooncogenes c-fos, c-jun, fra-2, and jun-D, the expression of which has been followed in the present study, are members of a family of transcription factors that contain a leucine-rich region and a basic region, which are necessary for dimer formation and DNA binding, respectively. Dimerization is a prerequisite for DNA binding to a consensus response element site (5'-TGAG/CTCA-3') designated activator protein-1 (AP-1) (
In the present study we took advantage of an in vivo membranous bone regeneration system that is under continuous mechanical stimulation, to gain more insight into the translation of mechanical force into gene expression in developing bone (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Seven young adult sheep aged 1 year were operated on in this study. The sheep head was chosen because the sheep is a large mammal and can provide successive biopsies. Surgery was performed under general anesthesia. All the sheep underwent complete maxillary osteotomy from the right side to the left side as described earlier by us (
Biopsies were removed under general anesthesia 30 min after the ninth and sixteenth distraction manipulations following 8 or 15 days of distraction, respectively, and in parallel from the non-distracted sheep on the corresponding days. The biopsies bridged the whole distracted or the non-distracted regeneration tissue from the first to the second osteotomy line. These two temporal points were chosen because bone trabeculae start to form proximally and distally from the cut edges of the osteotomized bone usually later than 8 days of distraction. No osteogenic process is yet under way after 8 days of distraction and the same is true for the corresponding controls. By 15 days of distraction the osteogenic process is well under way and trabeculae can be observed to grow from both bone edges in the direction of the center of the regeneration tissue. Each temporally different biopsy was removed from one side of the face and the next one from the other side as described in detail earlier by us (
RNA Extraction and Northern Blotting Analysis
Total RNA was isolated using Tri-Reagent (Molecular Research Center; Cincinnati, OH) according to the manufacturer's protocol. Each sample of 25 µg of total RNA was separated by electrophoresis in 2.2 mol/liter formaldehyde with 1.0% agarose gel containing 10 mg/ml ethidium bromide. After electrophoresis, the gel was examined with UV light to visualize ribosomal RNA bands to ensure quality and equal loading of the samples. RNA was transferred to a nylon membrane (Amersham; Arlington Heights, IL). Membrane was incubated for 3 hr with hybridization buffer [1 M NaH2PO4, 1 M Na2HPO4, 7% sodium dodecyl sulfate (SDS), 0.5 M EDTA, 1% bovine serum albumin (BSA), pH 8.0] and then incubated at 65C in the same buffer containing the [-32p]-d-ATP labeled specific complementary DNA (cDNA) for human c-jun and c-fos (Oncogene Research Products; Cambridge, MA) that was labeled with [
-32p]-ATP. Membrane was washed for the first time with 2 x saline sodium citrate (SSC) (1 x = 1.5 mM sodium citrate, 15 mM NaCl, pH 7) and 0.1% SDS at RT, followed by another wash with 2 x SSC and 0.1% SDS at 65C for 30 min and a third wash with 2 x SSC and 0.1% SDS for 30 min at RT. After a last wash with 2 x SSC for 5 min at RT, the membrane was exposed to autoradiographic film (Kodak; Rochester, NY) for 5 days at -70C.
Immunohistochemistry
The antibodies used to identify cells that express the early-response genes of the AP-1 family were anti-c-fos, anti-c-jun, anti-fra-2, and anti-jun-D (Santa Cruz Biotechnology; Santa Cruz, CA). All were affinity-purified rabbit polyclonal antibodies. According to the manufacturer, anti-c-fos does not crossreact with fos-B, fra-1, or fra-2. Anti c-jun does not crossreact with jun-B or jun-D. Anti-fra-2 does not crossreact with c-fos, fra-1, or fos-B.
The immunohistochemical staining was performed either on paraffin sections or on frozen sections. Ablation of endogenous peroxidase activity by 3% H2O2 in absolute methanol (not performed on frozen sections) was followed by blocking of unspecific binding by 1% BSA in PBS containing 0.01% Tween-20. The sections were then immunostained by the primary antibodies for 11.5 hr at RT. This was followed by the biotinylated goat anti-rabbit second antibody, employing the streptavidinbiotinperoxidase method and AEC as substrate according to the manufacturer's directions (Histostain plus kit; Zymed Laboratories, San Francisco, CA). Replacing the primary antibody by pre-immune rabbit serum served as negative control.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Northern Blotting Analysis
RNA was extracted from biopsies that contained the whole callus that was last distracted 30 min earlier. The biopsies were removed from experimental sheep that were distracted for 8 and 15 days after a prior latency period of 5 days and from the corresponding fractured, but not distracted sheep maxillae that served as controls. Because the yield of mRNA was very poor, we had to pool the extract from the callus of the two experimental animals and from the two control sheep. The Northern blotting analysis for c-fos mRNA showed that the mRNA levels were upregulated in regeneration tissue removed from animals that were distracted for 8 days (receiving the last stimulus of distraction 30 min before removal of the tissue) compared with the mRNA levels in tissues from the corresponding control animals. At this stage of regeneration the distracted tissues consist of mesenchyme and fibroblast-like cells. No overt bone elements are yet present. A representative gel separation of c-fos is shown in Fig 1A. c-fos mRNA level has increased about ninefold over control as calculated from densitometry measurements (Fig 1C). mRNA levels of c-jun were likewise elevated after daily distractions for 8 days in the experimental samples relative to the control samples about fivefold, (Fig 1B and Fig 1C). Northern blotting analysis for c-jun mRNA from 15-day distracted callus did not show any difference between control and experimental samples (not shown). c-fos mRNA levels in 15-day tissues was not analyzed because of the low yields of tissue from the control non-distracted sheep. Control for loading is shown by ethidium bromide staining of 18S and 28S RNAs. We could not use GAPDH for normalization because there is no specific probe for sheep tissues.
|
AP-1 Protein Immunohistochemistry
Compatible with the mRNA findings, c-Fos and c-Jun proteins, as exposed by the IHC staining, were revealed in paraffin sections of 8-day distracted tissues in more than 50% of the cells. In comparison, the control non-distracted tissues did not reveal positively stained cells (Fig 2A2E, Table 1). The staining was mainly confined to the cytoplasmic compartment of the polymorphic mesenchyme-like cells, which are the main components of the distracted callus at this stage. These cells might be regarded as preosteogenic cells because they already express Cbfa-1 (Lewinson et al. unpublished data). Fra-2 protein immunostaining was quite impressive in both 8-day distracted tissues and in the respective non-distracted control regeneration tissue (Fig 2G and Fig 2H). In the 8-day distracted samples, Jun-D protein was expressed in lining cells of the capillaries and in some mesenchyme-like cells (Fig 2F). Control tissues were not checked for Jun-D. Tissues in which the primary antigen was substituted with non-immune rabbit serum were negative (Fig 2I).
|
|
After 15 days of distraction, the distraction gap consists mainly of a fibrous zone, the cells of which are fibroblast-like secreting a collagen-rich matrix (
Prominent immunostaining for c-Fos, Jun-D, and Fra-2 proteins was demonstrated in the longer (15-day) distracted calluses in many of the fibroblast-like cells (Fig 3A, Fig 3C, and Fig 3D), and less intense expression of c-Jun and in fewer cells (Fig 3B; Table 1), but most intense in the new bone trabeculae that begin to form at the edges of the biopsies. All proteins were intensely revealed in preosteoblasts, osteoblasts, and osteocytes (Fig 3E3L). In parallel, in the control fractured but non-distracted repair tissues (of a total regeneration time of 20 days), similar strong immunostaining was revealed in all the bone-associated cells and the mesenchyme-like cells that are still recruited to the newly formed trabeculae but, interestingly, no staining could be observed in the fibrous zone that separates the edges of the bone trabeculae (Fig 4A and Fig 4B). When rabbit non-immune serum was substituted for the primary antibody, no positive staining could be observed (Fig 4C). A semiquantitative evaluation of the results of the immunostaining of all the proteins tested in this study in distracted tissues is summarized in Table 1.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bone formation by osteoblasts is essential not only for skeletal growth and bone remodeling but also for bone healing and repair. Several hormones and growth factors that are implicated in the regulation of bone physiology are now known to upregulate the expression of proteins of the AP-1 complex (
In the sheep model of the present study, a continuous mechanical strain/stress is exerted on the regenerating callus beginning 5 days after an osteotomy and external fixation of the maxilla (which is a component of the craniofacial complex). It is assumed that the distraction force, which distracts both edges of the fractured bone one from the other for 1 mm daily, maintains the strain force as a result of the external fixation until the next distraction on the following day. Several studies as well as our own have confirmed the very early observations of Ilizarov that the distraction force enhances and maintains proliferation of the cells that constitute the callus replacing the initial hematome (
We show an increase in the expression of c-fos and c-jun both on the mRNA level and, by IHC, on the protein level. The latter technique offers the advantage of identifying the specific cells that synthesize the proteins and their intracellular localization. Although AP-1 family proteins are usually regarded to be nuclear, we found that most of the immunostaining was cytoplasmic (
In conclusion, we have demonstrated that mechanical stimulation of regenerating bone by daily distraction and under normal physiological conditions stimulates the expression of early-response genes of the AP-1 family of transcription factors.
![]() |
Acknowledgments |
---|
Supported in part by the Lazaroff Fund for Research in Maxillofacial Surgery and by a grant from the Ministry of Health, grant number 3785, Israel.
Received for publication November 11, 2002; accepted April 2, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aronson L, Shen XC, Gao GG, Miller G, Quattlebaum T, Skinner RA, Badger TM et al. (1995) Sustained proliferation accompanies distraction osteogenesis in the rat. J Orthop Res 15:563-569
Candeliere GA, Prud'homme J, St-Arnaud R (1991) Differential stimulation of fos and jun family members by calcitriol in osteoblastic cells. Mol Endocrinol 5:1780-1788[Abstract]
Dony C, Gruss P (1987) Proto-oncogene c-fos expression in growth regions of fetal bone and mesodermal web tissue. Nature 28:711-714
Evans DB, Hipskind RA, Bilbe G (1996) Analysis of signaling used by parathyroid hormone to activate the c-fos gene in human SaOS2 osteoblast-like cells. J Bone Miner Res 11:1066-1074[Medline]
Fitzgerald J, HughesFulford M (1999) Mechanically induced c-fos expression is mediated by c-AMP in MC3T3E1 osteoblasts. FASEB J 13:553-557
Granet C, Guigandon A, Vico L, Alexandre C, LafageProust M-H (2002) MAP and src kinases control the induction of AP-1 members in response to changes in mechanical environment in osteoblastic cells. Cell Signal 14:679-688[Medline]
Grigoriadis AE, Wang Z, Wagner EF (1993) c-fos oncogene expression in cartilage and bone tissues of transgenic and chimeric mice. In Noda M, ed. Cellular and Molecular Biology of the Bone. New York, Academic Press, 497-537
Hollangel A, Schroder D, Gross G (1996) Domain-specific gene activation by parathyroid hormone in osteoblastic ROS17/2.8 cells. J Biol Chem 271:21870-21877
Ilizarov GA (1988) The principles of Ilizarov method. Bull Hosp J Dis Orthop Inst 48:1-11
Inaoka T, Lean JM, Bessho T, Chow JWN, Mackay A, Kokubo T, Chambers TJ (1994) Sequential analysis of gene expression after an osteogenic stimulus: c-Fos expression is induced in osteocytes. Biochem Biophys Res Commun 217:264-270
Johnson RS, Spiegelman EM, Pappaioannou V (1992) Pleiotropic effects of a null mutation in the c-fos proto-oncogene. Cell 71:577-586[Medline]
Katai H, Stephenson JD, Simkevich CP, Thompson JP, Raghow R (1992) An AP-1-like motif in the first intron of human pro alpha1 (I) collagen gene is a critical determinant of its transcriptional activity. Mol Cell Biochem 118:119-129[Medline]
Kletsas D, Basdra EK, Papavassiliou AG (2002) Effect of protein kinase inhibitors on the stretch-elicited c-Fos and c-Jun up-regulation in human PDL osteoblast-like cells. J Cell Physiol 190:313-321[Medline]
Kouzardief T, Ziff E (1988) The role of the leucine zipper in the Fos-Jun interaction. Nature 336:646-651[Medline]
Lean JM, Mackay AG, Chow JW, Chambers TJ (1996) Osteocytic expression of mRNA for c-fos and IGF-I: an immediate early gene response to an osteogenic stimulus. Am J Physiol 270:E937-945[Medline]
Lee K, Deeds JD, Chiba S, UnNo M, Bond AT, Segre GV (1994) Parathyroid hormone induces sequential c-fos expression in bone cell in vivo: in situ localization of its receptor and c-fos messenger ribonucleic acids. Endocrinology 134:441-450[Abstract]
Lewinson D, Maor G, Rozen N, Rabinowich I, Stahl S, Rachmiel A (2001) Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system. Histochem Cell Biol 116:381-388[Medline]
Li G, Simpson AHRW, Kenwright J, Triffit JT (1997) Assessment of cell proliferation in regenerating bone during distraction osteogenesis at different distraction rates. J Orthop Res 15:765-772[Medline]
Lian JB, Stein GS, Stein JL, Wijnen AJ (1998) Osteocalcin gene promoter: unlocking the secrets for regulation of osteoblast growth and differentiation. J Cell Biochem Suppl 30(31):62-72
Liang JD, Hock JM, Sandusky GE, Santerre RF, Onyia LE (1999) Immunohistochemical localization of selected early response genes expressed in trabecular bone of young rats given hPTH 134. Calcif Tissue Int 65:369-373[Medline]
McCabe LR, Banerjee RK, Kundu R, Harrison RJ, Dobner PR, Stein JL, Lian JB et al. (1996) Developmental expression and activities of specific Fos and Jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun-D during differentiation. Endocrinology 137:4398-4408[Abstract]
McCabe LR, Kockx M, Lian JB, Stein J, Stein G (1995) Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. Exp Cell Res 218:255-262[Medline]
Moally MR, Caldwell J, Patil PV, Goldstein SA (2000) An in vivo model for investigations of mechanical signal transduction in trabecular bone. J Bone Miner Res 15:1346-1353[Medline]
Nomura S, TakanoYamamoto T (2000) Molecular events caused by mechanical stress in bone. Matrix Biol 19:91-96[Medline]
Ohta S, Yamamoto T, Lee K, Okumura T, Kasai R, Hiraki Y (1991) Fracture healing induces expression of the proto-oncogene c fos in vivo. Possible involvement of the Fos protein in osteoblastic differentiation. FEBS Lett 284:42-45[Medline]
Palcy S, Bolivar I, Goltzman D (2000) Role of activator protein I transcriptional activity in the regulation of gene expression by transforming growth factor ß1 and bone morphogenetic protein in ROS 17/2.8 osteoblast-like cells. J Bone Miner Res 15:2352-2361[Medline]
Peverali FA, Basdra EK, Papavassiliou AG (2001) Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells. Mol Med 7:68-78[Medline]
Rachmiel A, Laufer D, Jackson IT, Lewinson D (1998) Midface membranous bone lengthening: a one year histological and morphological follow-up of distraction osteogenesis. Calcif Tissue Int 62:370-376[Medline]
Rachmiel A, Rozen N, Peled M, Lewinson D (2002) Characterization of midface maxillary membranous bone formation during distraction osteogenesis. Plast Reconstruct Surg 109:1611-1620[Medline]
Rather U, Garber C, Komitowski D, Muller R, Wagner EF (1987) Deregulation of c-fos expression interferes with normal bone development in transgenic mice. Nature 325:412-416[Medline]
Sabatkos G, Sims NA, Chen J, Aoki K, Keltz MB, Amling M, Bouali Y et al. (2000) Overexpression of DeltaFosB transcription factor(s) increases bone formation and inhibits adipogenesis. Nature Med 6:985-990[Medline]
Sims NA, Sabatkos G, Chen JS, Keltz MB, Nestler EJ, Baron R (2002) Regulating DeltaFosB expression in adult tet-off-DeltaFosB transgenic mice alters bone formation and bone mass. Bone 30:32-39[Medline]
Stanislaus D, Devanarayan V, Hock JM (2000) In vivo comparison of activated protein-1 gene activation in response to human parathyroid hormone (hPTH)(134) and hPTH(184) in the distal femur metaphysis of young mice. Bone 27:819-826[Medline]
Suda M, Tanaka K, Sakuma Y, Yasoda A, Fukata J, Narumiya S, Nakao K (2000) Prostaglandin E2 (PGE2) induces c-fos and c-jun expressions via the EP1 subtype of PGE receptor in mouse osteoblastic MC3T3E1 cells. Calcif Tissue Int 66:217-223[Medline]
Varghese S, Rydziel S, Canalis E (2000) Basic fibroblast growth factor stimulates collagenase-3 promoter activity in osteoblasts through an activator protein-1-binding site. Endocrinology 141:2185-2191
Wang Z, Ovitt C, Grigoriadis AE, MohleSteinlein U, Rother U, Wagner EF (1992) Bone, haematopoietic defects in mice lacking c-fos. Nature 360:741-745[Medline]
Wozniak M, Hruska KA (2001) Differential expression of the AP-1 protein complex in mechanically strained human osteoblasts. J Bone Miner Res 16(suppl):5255