Annals of Clinical & Laboratory Science 38:25-30 (2008)
© 2008 Association of Clinical Scientists
The Expression of Neurofibromin in Human Osteoblasts and Chondrocytes
Hui Chen1,*,
Yong Qiu1,
Leilei Chen2,
Lei Li2,
Junhao Chen2,
Chaoying Zhang3,
Bin Wang1,
Yang Yu1,
Zezhang Zhu1,
Feng Zhu1,
Bangping Qian1 and
Weiwei Ma1
1 Spine Surgery Department and 2 Central Laboratory, Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, and 3 Department of Biology, Nanjing Normal University, Nanjing, China
Address correspondence to Yong Qiu, M.D., Spine Surgery, Affiliated Drum Tower Hospital, 321 Zhongshan Road, Nanjing 210008, China; tel 86 25 8310 5121; fax 86-25-83105121; e-mail scoliosis2002{at}sina.com.
 |
Abstract
|
|---|
The goal of this study was to determine if neurofibromin is expressed in cultured human osteoblasts and chondrocytes that were isolated from ilia and ilial growth plate. Purity of the osteoblast and chondrocyte cultures was confirmed by alkaline phosphatase and toluidine blue staining, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect neurofibromin mRNA. Indirect immunofluorescence and Western blot studies were done to delineate the cellular distribution and expression levels of neurofibromin. These experiments show that neurofibromin is expressed at low levels in human osteoblasts and chondrocytes and is located mainly in the cytoplasm. Only the type II isoform of neurofibromin is detected in these cells. These findings suggest that the type II isoform of neurofibromin plays a physiological role in human osteoblasts and chondrocytes. Whether functional deficiency of neurofibromin is responsible for skeletal abnormalities remains to be established.
Keywords: neurofibromin, osteoblast, chondrocyte, neurofibromatosis-1, von Recklinghausen disease
|
Introduction
|
|---|
Type 1 neurofibromatosis (NF1), also termed peripheral neurofibromatosis or von Recklinghausen disease, is an autosomal dominant disease with high mutation rate. Its main clinical characteristics include neurofibromas, café-au-lait spots, freckles, Lisch nodules, bone deformities, learning disabilities, and predisposition to neoplasia. Skeletal abnormalities occur in nearly half of NF1 patients; scoliosis constitutes the major skeletal manifestation, with an incidence of about 20% [1,2]. Generally combined with kyphosis and dystrophic changes, scoliosis in NF1 patients occurs earlier and progresses more rapidly than its idiopathic counterparts. Dystrophic changes and low bone mineral density make the deformity difficult to correct, resulting in frequent pseudarthrosis and lack of remediation [3–5].
Neurofibromin, encoded by the NF1 gene, is a guanosine triphosphatase (GTPase)-activating protein (GAP). The various GAP proteins accelerate the hydrolysis of Ras-GTP to Ras-guanosine diphosphate (GDP), converting it from an active form to the inactive form, and thereby negatively regulating the Ras signal [6]. Ras is a small G-protein involved in transmitting signals from growth factor receptors to a large number of downstream signaling molecules that eventually alter gene expression in the nucleus. Studies indicate that besides its tumor suppressor role, neurofibromin regulates cellular proliferation, differentiation, development, and homeostasis in many tissues [7–17]. Neurofibromin functions in neuron differentiation [8], cardiac development [9], repair of the vascular endothelium [10], lymphocyte genesis and function [11], and wound healing mediated by fibroblasts [12]. Functional deficiency of neurofibromin is now regarded as the basis of various phenotypes [13].
Kuorilehto et al [14] recently demonstrated the expression of neurofibromin in murine bone. Kolanczyk et al [15] reported that neurofibromin-deficient mice show bowing of the tibia and diminished growth, with functional deficits in osteoblasts and chondrocytes. Yu et al [16] reported that neurofibromin is necessary for osteoprogenitor cells to differentiate into osteoblasts in mice. Since the research on neurofibtomin in bone has been limited to mice, knowledge about its role in the skeletal changes of NF1 patients is deficient. The expression of neurofibromin in human musculoskeletal cells has not hitherto been reported. In the present study, we tested the expression of neurofibromin in human osteoblasts and chondrocytes using RT-PCR, immunofluorescent, and Western blot techniques.
|
Materials and Methods
|
|---|
Source of samples.
Six patients with congenital scoliosis (3 males and 3 females) were included in our study. The average age was 11 yr (7~13 yr). The diagnosis was confirmed by X-ray visualization of semivertebra or unsegmented vertebrae. Pelvic X-rays showed a Risser sign of 0 to +++, but no abnormality was detected in the ilia. Samples of cancellous iliac bone and lateral half of the iliac growth plate were harvested at surgery, with the approval of the ethics committee of the hospital and the consent of the patients and their families.
Cultures of osteoblasts and chondrocytes.
The samples of cancellous bone and growth plate weighed about 700 and 500 mg, respectively. An explant culture system was used to isolate osteoblasts. Chondrocytes were isolated by sequential digestion with hyaluronidase, trypsin, and type II collagenase (Sigma, St. Louis, MO). The culture medium was DMEM/F12 1:1 (Hyclone, Logan, UT) with 10% fetal bovine serum (Hyclone), penicillin, 100 U/ml, streptomycin, 100 µg/ml, and ascorbic acid, 50 µg/ml (all from Sigma). The cells were split with 0.25% trypsin/EDTA (Hyclone) at a 1:3 ratio when their growth was near confluence. Staining of alkaline phosphatase (modified Gomori’s method) and toluidine blue were done to confirm the purity of the cultured osteoblasts and chondrocyes, respectively. The cells of second passage (P2) were assayed.
RT-PCR Procedure.
Normal human brain tissue was selected as the positive control (with the permission of the ethics committee). The total RNA was extracted from brain tissue, osteoblasts, and chondrocytes with Trizol (Invitrogen, Carlsbad, CA), and quantitated with a spectrophotometer. The A260/280 ratio was between 1.8 and 2.1. A Titanium One-Step RT-PCR Kit (Clontech, Mountain View, CA) was used. Beta-actin was selected as the internal control. The 50 µl reaction system included 20 pmol primer, 10 U M-MuLV reverse transcriptase, 2 U Taq DNA polymerase, 10 nmol dNTP, 75 nmol/L MgCl2, and buffer. The primers were: beta-actin (500 bp):
sense 5'-ccaaggccaaccgcgagaagatgac-3',
antisense 5'-agggtacatggtggtggtgccagac-3';
neurofibromin: sense 5'-cagaattcccccctcaacttcgaagt-3',
antisense 5'-tgcgtgctgcatcaaagttgcttttcac-3'.
Both type I and type II neurofibromin mRNA can be amplified with these primers, 303 bp and 366 bp, respectively [17]. The mRNA template was transcribed at 50°C for 60 min and then at 94°C for 10 min. Afterwards 30 cycles of polymerase chain reaction were run with 45 sec denaturation at 94°C, 60 sec annealing at 64°C, 60 sec extension at 72°C, and finally 10 min at 72°C. The reaction products were resolved by electrophoresis with 2% agar gel and stained with ethidium bromide (Sigma). A FR-200 gel imaging system was used to analyze the results. Reaction products were sequenced by the Shanghai Sangon Biological Engineering & Technology Service Co. (Shanghai, China).
Immunofluorescence.
Cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 10 min. The slides were incubated in blocking solution for 30 min. Primary antibody for osteocalcin (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) or type II collagen (1:100, Santa Cruz) was added and incubated for 30 min. Fluorescein isothiocyanate (FITC)-labeled secondary antibody was then added and incubated for another 30 min (1:100, Zhongshan Golden Bridge Biotechnology Co., Beijing, China). For neurofibromin detection, primary antibody for neurofibromin was incubated for 30 min (1:100, Novus, Littleton, CO), followed by the same incubation period for rhodamine-labeled secondary antibody (Beijing Zhongshan Golden Bridge Biotechnology Co., 1:100). Finally Hochest 33258 (Sigma) was added for 5 min. Slides were rinsed 3~5 times with 0.01 M PBS after each incubation. Slides were mounted with neutral resin after air-drying; the slides were observed and photographed by confocal microscopy.
Immunoprecipitation and Western blot studies.
About 5 x 106 P2 osteoblasts or chondrocytes were rinsed with cold PBS and lysed in 200 µl RIPA buffer (containing 5 µl protease inhibitor cocktail). Protein concentration was determined with a modified Lowry protein assay reagent kit (Pierce, Rockford, IL). The cleared cell lysate was pretreated with 50% (v/v) protein A-Sepharose. Two µg of neurofibromin polyclonal antibody (Novus) was added to each sample and incubated overnight at 4°C with gentle mixing on a rotator; 50 µl protein A-Sepharose was then added and further incubated overnight at 4°C. The rinsed immunoprecipitates were dissolved in SDS loading buffer and boiled. Proteins were resolved on 7% SDS-PAGE gel. The gel was wet transferred overnight at 4°C to a PVDF membrane. The membrane was blocked with 5% milk, and incubated with monoclonal antibody against neurofibromin (Novus, 1:1000) overnight at 4°C; secondary antibody was then added and incubated for 3 hr at 25°C. The protein was finally detected with the ECL chemoluminescence system (Pierce).
|
Results
|
|---|
Osteoblast and chondrocyte culture.
The growth to confluence of primary cultured osteoblasts took 4~5 wk and 2~3 x 106 cells were obtained from 700 mg of cancellous bone. Trypan blue staining showed a cell survival rate of about 80%. The osteoblasts appeared shuttle-like, cuboid, and irregularly polygonal upon examination with an inverted microscope. The cells on coverslips were stained positively for alkaline phosphatase. About 106 chondrocytes were isolated through enzymatic digestion from 500 mg samples of growth plate. Trypan blue staining showed a survival rate of about 75%. Chondrocytes appeared cuboid and irregularly polygonal, and a typical paving-stone pattern was seen after confluence. The chondrocytes were stained positively with toluidine blue.
Expression of neurofibromin mRNA in osteoblasts and chondrocytes.
The results of RT-PCR studies showed that neurofibromin mRNA was expressed in the osteoblasts and chondrocytes from all 6 cases (Fig. 1A, 1B
). The expression levels of neurofibromin in the osteoblasts and chondrocytes was lower than in the brain tissue. Type I and type II neurofibromin mRNA were both expressed in the brain tissue, with type I mRNA being dominant. In contrast, in osteoblasts and chondrocytes, only type II neurofibromin mRNA was detected (Fig. 1C, 1D). The sequence of the RT-PCR product was analyzed and found to be identical to the Genebank sequence of neurofibromin.
View larger version (80K):
[in this window]
[in a new window]
|
Fig. 1. Expression of neurofibromin mRNA in human osteoblasts and chondrocytes. (A) Expression of neurofibromin mRNA in osteoblasts of 6 cases (lanes 1 to 6 represent cases 1 to 6, respectively). (B) Expression of neurofibromin mRNA in chondrocytes of 6 cases (lanes 1 to 6 represent cases 1 to 6, respectively). (C) Type I and type II neurofibromin (303 bp and 366 bp, respectively) are both expressed in brain tissue (lanes 1 and 2; lane 1 is β-actin) with type I being dominant. Only type II neurofibromin is expressed in osteoblasts (lanes 3 and 4, lane 3 is β-actin). (D) Compared to brain tissue (lanes 1 and 2; lane 1 is β-actin), only type II neurofibromin is expressed in chondrocytes (lanes 3 and 4; lane 3 is β-actin).
|
|
Expression of neurofibromin protein in osteoblasts and chondrocytes.
Osteocalcin and type II collagen are specific proteins of osteoblasts and chondrocytes, respectively. Positive immunofluorescent staining of these 2 proteins confirmed the cell identities as osteoblasts and chondrocytes (FITC-labeled, green fluorescence, Fig. 2A, 2D). Immunofluorescent staining of neurofibromin was positive in both osteoblasts and chondrocytes (rhodamine-labeled, red fluorescence, Fig. 2B, 2E), but the fluorescence intensity was low. In addition, the fluorescence was seen mainly in the cytoplasm. These indirect immunofluorescent staining results demonstrate that neurofibromin is expressed in human osteoblasts and chondrocytes at low levels and is mainly localized in cytoplasm (Fig. 2C, 2F).
View larger version (64K):
[in this window]
[in a new window]
|
Fig. 2. Detection of neurofibromin in human osteoblasts and chondrocytes by indirect immunofluorescence. (A) Abundant expression of osteocalcin (FITC-labeled) in osteoblasts. (B) Low expression of neurofibromin (rhodamine-labeled) in osteoblasts. (C) Nuclear staining with Hochest 33258 shows that neurofibromin is mainly located in osteoblast cytoplasm. (D) Abundant expression of type II collagen (FITC-labeled) in chondrocytes. (E) Low expression of neurofibromin (rhodamine-labeled) in chondrocytes. (F) Neurofibromin is mainly located in the cytoplasm of chondrocytes.
|
|
By means of a simple Western blot technique, it was not feasible to detect neurofibromin in the cultured osteoblasts or chondrocytes (data not shown). However, after enrichment by immunoprecipitation, low levels of neurofibromin were detected in human osteoblasts and chondrocytes by Western blot analysis (Fig 3A, 3B); the molecular weight of neurofibromin was about 220 kDa.
View larger version (52K):
[in this window]
[in a new window]
|
Fig. 3. Neurofibromin detection in human osteoblasts and chondrocytes by Western blot analysis (lanes 1 to 6 represent cases 1 to 6, respectively). (A) Neurofibromin expression in osteoblasts of the 6 cases. (B) Neurofibromin expression in chondrocytes of the 6 cases.
|
|
|
Discussion
|
|---|
Clinical manifestations of NF1 include a variety of skeletal changes (eg, scoliosis, vertebral dysplasia, short stature, sphenoid wing dysplasia, and pseudarthrosis or bowing of the tibia [18]. Other less common abnormalities of the skeleton include local overgrowth, abnormalities of the rib cage, genu varum/valgum, lytic bone lesions, osteosclerosis, rib fusion, absence of the patella, syndactyly, and sundry congenital bone malformations [19]. These clinical findings in patients with NF1 suggest a role for neurofibromin in skeletal development.
Several recent studies in mice support this concept. Neurofibromin is a cytoplasmic protein that is predominantly expressed in neurons, Schwann cells, oligodendrocytes, astrocytes, and leukocytes. Kuorilehto et al [14] reported the expression of neurofibromin in growth plate, periosteum, and tracebular bone of mice, and the expression in growth plate was mainly located in chondrocytes of the hypertrophic layer. Yu et al [16] reported that upon the activation of Ras signals, Nf1+/– murine osteoprogenitor cells show increased proliferation and premature apoptosis; the osteoprogenitor cells also exhibit a lower rate of differentiation to osteoblasts [16]. Yu et al [16] considered neurofibromin and its role as Ras signal regulator to be necessary for osteoblast function. Kolanczyk et al [15] found that osteoblasts from Nf1Prx1(Nf1+/–) mice show increased proliferation and decreased abilities to differentiate and mineralize, whereas chondrocytes demonstrate a lower proliferation rate and defective differentiation.
These previous studies were all limited to mice. Until now, no one has tested whether neurofibromin is expressed in human musculoskeletal cells. Based on RT-PCR, immunofluorescence, and Western blot experiments, we now report in vitro expression of neurofibromin in human osteoblasts and chondrocytes. Our study shows that neurofibromin is weakly expressed in these cultured human cells and is mainly located in cytoplasm; the molecular weight of neurofibromin is about 220 kDa.
Stevenson et al [20] recently investigated the affected skeletal tissue from patients with tibial pseudarthrosis and found additional mutations of the second NF1 allele, which indicates that the homozygous loss of NF1 function can be detrimental for normal bone development. The report of Stevenson et al [20] and our findings provide an interesting comparison, with positive neurofibromin expression in normal human musculoskeletal cells and negative expression in pseudarthrosis. These results support a role of neurofibromin in normal bone development and remodeling in humans.
The concept that neurofibromin deficiency underlies varied kinds of phenotypes is gradually being accepted. Abdel-Wanis and Kawahara [21] hypothesized that a defect of vertebral osteogenesis induced by neurofibromin deficiency may underlie the scoliosis in NF1 patients. The Ras-MAPK pathway is a key signal transduction pathway in osteoblasts and chondrocytes that mediates the proliferation and differentiation effects of extracellular signals such as growth factors [22,23]. Abdel-Wanis and Kawahara [24] envisioned that deficiency of neurofibromin may lead to activation of the MAPK pathway in osteoblasts; the increased mitosis signals might interact with specific transcription factors like Cbfa1, and finally lead to reduced production of type I collagen. Kuorilehto et al [14] suggested that neurofibromin plays a role as Ras-GAP in musculoskeletal cells, and that neurofibromin deficiency influences osteogenesis and finally leads to skeletal abnormalities like bone dystrophy [14]. Wu et al [25] reported that Nf1+/– mesenchymal stem/progenitor cells (MSPC) have increased proliferation and decreased differentiation. The results suggest that neurofibromin has a crucial role in modulating MSPC differentiation into osteoblasts and that the defect in osteoblast differentiation may contribute at least in part to the osseous abnormalities in NF1 patients.
As the expression of neurofibromin in brain tissue is relatively high [8], brain tissue was selected as a positive control in our RT-PCR studies. Our RT-PCR results show that the type I isoform is dominant in brain tissue, whereas the neurofibromin expressed in osteoblasts and chondrocytes is mainly type II. The difference between the 2 isoforms is an insertion of 21 amino acids (exon 23a) in type II neurofibromin. The inserted element destroys the 5c helix and leaves the functional GAP-related domain partly exposed. The enhanced electric charge of the exposed area interferes with Ras combination and thereby greatly attenuates its GAP activity [26]. Culture conditions may conceivably lead to changes in neurofibromin expression levels and isoforms. Therefore, whether low expression levels and dominancy of the type II isoform of neurofibromin also exist in vivo in human osteoblasts and chondrocytes, whether neurofibromin plays a limited role as Ras-GAP in these cells, and whether neurofibromin deficiency contributes to the skeletal abnormalities in NF1 patients all need to be investigated.
|
Footnotes
|
|---|
* Present address: Nanjing BenQ Hospital, Nanjing, China, (e-mail chenhui0802{at}126.com). 
|
References
|
|---|
- Akbarnia BA, Gabriel KR, Beckman E, Chalk D. Prevalence of scoliosis in neurofibromatosis. Spine 1992; 17:S244–248.[Medline]
- North K. Neurofibromatosis type 1: review of the first 200 patients in an Australian clinic. J Child Neurol 1993;8:395–402.[Abstract/Free Full Text]
- Kim HW, Weinstein SL. Spine updates: the management of scoliosis in neurofibromatosis. Spine 1997;22:2770–2776.[Medline]
- Zhu F, Qiu Y, Wang B, Yu Y, Zhu ZZ. Neurofibromatosis dystrophic scoliosis: imaging feature and its clinical significance. J Spine Surg 2003;2:68–71.
- Lammert M, Kappler M, Mautner VF, Lammert K, Störkel S, Friedman JM, Atkins D. Decreased bone mineral density in patients with neurofibromatosis 1. Osteoporos Int 2005;16:1161–1166.[Medline]
- Bollag G, McCormick F. Ras regulation. NF is enough of GAP. Nature 1992;356:663–664.[Medline]
- Basu TN, Gutmann DH, Fletcher JA, Glover TW, Collins FS, Downward J. Aberrant regulation of ras proteins in malignant tumor cells from type 1 neurofibromatosis patients. Nature 1992;356:713–715.[Medline]
- Yunoue S, Tokuo H, Fukunaga K, Feng L, Ozawa T, Nishi T, Kikuchi A, Hattori S, Kuratsu J, Saya H, Araki N. Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras. J Biol Chem 2003;278:26958–26969.[Abstract/Free Full Text]
- Lakkis MM, Epstein J A. Neurofibromin modulation of ras activity is required for normal endocardial mesenchymal transformation in the developing heart. Development 1998;125:4359–4367.[Abstract]
- Hamilton SJ, Friedman JM. Insights into the pathogenesis of neurofibromatosis 1 vasculopathy. Clin Genet 2000;58:341–344.[Medline]
- Ingram DA, Zhang L, McCarthy J, Wenning MJ, Fisher L, Yang FC, Clapp DW, Kapur R. Lymphoproliferative defects in mice lacking the expression of neurofibromin: functional and biochemical consequences of Nf1 deficiency in T-cell development and function. Blood 2002;100:3656–3662.[Abstract/Free Full Text]
- Atit RP, Crowe MJ, Greenhalgh DG, Wenstrup RJ, Ratner N. The Nf1 tumor suppressor regulates mouse skin wound healing, fibroblast proliferation, and collagen deposited by fibroblasts. J Invest Dermatol 1999;112:835–842.[Medline]
- Kim HA, Ling B, Ratner N. Nf1-deficient mouse Schwann cells are angiogenic and invasive and can be induced to hyperproliferative: reversion of some phenotypes by an inhibitor of farnesyl protein transferase. Mol Cell Biol 1997;17:862–872.[Abstract/Free Full Text]
- Kuorilehto T, Nissinen M, Koivunen J, Benson MD, Peltonen J. NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos. J Bone Mineral Res 2004; 19:983–989.[Medline]
- Kolanczyk M, Kossler N, Kühnisch J, Lavitas L, Stricker S, Wilkening U, Manjubala I, Fratzl P, Spörle R, Herrmann BG, Parada LF, Kornak U, Mundlos S. Multiple roles for neurofibromin in skeletal development and growth. Hum Mol Genet 2007;16:874–886.[Abstract/Free Full Text]
- Yu X, Chen S, Potter OL, Murthy SM, Li J, Pulcini JM, Ohashi N, Winata T, Everett ET, Ingram D, Clapp WD, Hock JM. Neurofibromin and its inactivation of Ras are prerequisites for osteoblast functioning. Bone 2005;36:793–802.[Medline]
- Aaltonen V, Bostrom PJ, Soderstrom KO, Hirvonen O, Tuukkanen J, Nurmi M, Laato M, Peltonen J. Urinary bladder transitional cell carcinogenesis is associated with down-regulation of NF1 tumor suppressor gene in vivo and in vitro. Am J Pathol 1999;154:755–765.[Abstract/Free Full Text]
- Alwan S, Tredwell SJ, Friedman JM. Is osseous dysplasia a primary feature of neurofibromatosis 1 (NF1)? Clin Genet 2005;67:378–390.[Medline]
- Ruggieri M, Pavone V, De Luca D, Franzo A, Tine A, Pavone L. Congenital bone malformations in patients with neurofibromatosis type 1 (Nf1). J Pediatr Orthop 1999;19:301–305.[Medline]
- Stevenson DA, Zhou H, Ashrafi S, Messiaen LM, Carey JC, D’Astous JL, Santora SD, Viskochil DH. Double inactivation of NF1 in tibial pseudarthrosis. Am J Hum Genet 2006;79:143–148.[Medline]
- Abdel-Wanis ME, Kawahara N. Bone development in neurofibromatosis 1. Med Hypotheses 2003;60:459–462.[Medline]
- Hipskind RA, Bilbe G. MAP Kinase signaling and gene expression in osteoblasts. Front Biosci 1998;3:804–816.
- Stanton LA, Underhill TM, Beier F. MAP kinases in chondrocyte differentiation. Dev Biol 2003;263:165–175.[Medline]
- Abdel-Wanis ME, Kawahara N. The role of neurofibromin and melatonin in pathogenesis of pseudarthrosis after spinal fusion for neurofibromatous scoliosis. Med Hypotheses 2002;58:395–398.[Medline]
- Wu X, Estwick SA, Chen S, Yu M, Ming W, Nebesio TD, Li Y, Yuan J, Kapur R, Ingram D, Yoder MC, Yang FC. Neurofibromin plays a critical role in modulating osteoblast differentiation of mesenchymal stem/progenitor cells. Hum Mol Genet 2006;15:2837–2845.[Abstract/Free Full Text]
- Scheffzek K, Ahmadian MR, Wiesmüller L, Kabsch W, Stege P, Schmitz F, Wittinghofer A. Structural analysis of the GAP-related domain from neurofibromin and its implications. EMBO J 1998;17:4313–4327.[Medline]
Top
Abstract
Introduction
