Download Expression of insulin like growth factor binding protein-5 in

Indian J Med Res 125, January 2007, pp 43-48
Expression of insulin like growth factor binding protein-5 in drug
induced human gingival overgrowth
Tamilselvan Subramani, Anbazhagi Sakkarai*, Kamatchiammal Senthilkumar*, Soundararajan Periasamy**
Georgie Abraham** & Suresh Rao
Department of Periodontics, Sri Ramachandra Dental College & Hospital, *NEERI, Council of
Scientific & Industrial Research, & **Department of Nephrology, Sri Ramachandra Medical
College & Research Institute, Deemed University, Chennai, India
Received February 14, 2006
Background & objectives: Insulin like growth factor binding proteins (IGFBPs) control the
distribution, function and activity of insulin like growth factors (IGFs) in various cells, tissues and
body fluids, thereby modulating their metabolic and mitogenic effects. IGFBP-5, the most conserved
IGFBP, can function through IGF or directly play a role in fibrosis. Cyclosporine A (CsA) widely
used in organ transplant patients, often causes various side effects including gingival fibrotic
overgrowth. This study was carried out to assess the mRNA expression of IGFBP-5 in healthy
human gingival, chronic periodontitis and CsA induced gingival overgrowth tissues.
Methods: Total RNA was isolated from gingival tissues collected from eight patients with chronic
periodontitis, eight patients with CsA induced gingival outgrowth and an equal number of healthy
individuals, and subjected to reverse transcription (RT)-PCR for IGFBP-5 gene expression.
Results: CsA induced gingival overgrowth tissues expressed increased IGFBP-5 mRNA compared
to control and chronic periodontitis.
Interpretation & conclusion: Increased mRNA expression of IGFBP-5 in CsA induced gingival
outgrowth tissues may be associated with increased collagen synthesis, thereby promoting
fibrogenesis.
Key words - Cyclosporine - IGFBP-5 - overgrowth - periodontitis
Insulin like growth factor binding proteins
(IGFBPs) are a family of six circulating proteins
which bind insulin like growth factors I and II (IGFI and II) with high affinity and thus control their
distribution, function and activity in various cells,
tissues and body fluids, thereby modulating their
metabolic and mitogenic effects1-3. The liver is the
major source of circulatory IGFs and IGFBPs. There
are changes in IGFBP expression and concentration
in different tissues in many pathological states.
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INDIAN J MED RES, JANUARY 2007
IGFBP-5 is the most conserved of the six IGFBPs.
IGFBP-5, 29 kD glycoprotein, potentiates the action
of IGF-I in smooth muscle cells, fibroblasts and
osteoblasts4. IGFBP-5 also binds with high affinity
to extracellular matrix components (ECM) which
protects it from proteolysis5. Drug-induced gingival
overgrowth (DIGO) is known to be caused as side
effect of certain drugs such as immunosuppressive
agents (cyclosporine A, CsA), antiepileptic drugs
(phenytoin) and calcium channel blockers
(nifidipine)6,7. The pathogenic mechanisms of DIGO
involve changes in the metabolism of connective
tissue, causing an increased proliferation or
decreased apoptosis, increased ECM production8,
etc., leading to fibrotic overgrowth. In pulmonary
fibrosis, IGFBP-5 was found to be increased9. Since
the gingival overgrowth is similar to pulmonary
fibrosis, the present study was carried out to examine
the expression of IGFBP-5 mRNA in CsA induced
human gingival overgrowth and compared with
chronic periodontitis and healthy gingival tissues.
Material & Methods
Materials: Gingival tissues were collected at the
time of resective surgery from marginal papillary
and attached gingiva of eight chronic periodontitis
patients (aged 35-55 yr; 4 male and 4 female). CsA
induced gingival overgrowth tissue samples ware
obtained from lower anterior and upper posterior
of eight patients (3 male and 5 female) aged 4060 yr. Eight control samples (4 male and 4 female)
were also obtained from upper and lower premolar
region of healthy individuals, aged 13-17 yr, who
have undergone orthodontic treatment. Drug
induced gingival overgrowth tissue samples were
obtained from cyclosporine-A medicated organ
transplant recipients during gingivectomies required
to treat drug induced gingival overgrowth. Patients,
who were under CsA medication for minimum
period of one year, and were taking the average dose
of 3.6 mg/kg of body weight per day and developed
clinically significant gingival overgrowth within 6
months of intake of the drug were included in the
study. All the DIGO samples represented moderate
to severe degree of gingival overgrowth. All the
chronic periodontitis samples expressed clinical
signs of inflammation and were taken from sites
with a probing depth greater than 6 mm with
evidence of bleeding on probing. The control
subjects were periodontally healthy without
gingival inflammation and showed no evidence of
bleeding on probing with a probing depth less than
4 mm. Gingival tissue samples which would
normally be discarded at the time of periodontal
surgery were retained if they meet the inclusion
criteria. All the patients and healthy subjects were
nonsmokers. Subjects, who were medically
compromised, or had antibiotics within the
preceding 6 months and who had undergone any
periodontal therapy within 6 months were excluded
from the study. The number of samples was
restricted to 8 based on the availability of samples.
The study was carried out in Sri Ramachandra
Medical College and National Environmental
Engineering Research Institute, Chennai, India. The
duration of the study was one year from June 2003
to May 2004. All the procedures were performed
with appropriate informed consent from all subjects
and the study protocol was approved by the
Institutional Ethical Committee and Review Board
of Sri Ramachandra Medical College and Research
Institute, Deemed University.
RNA isolation and RT-PCR: The total RNA was isolated
from the gingival tissues by single step, acid guanidium
thiocyanate-phenol-chloroform extraction method 10.
Briefly, gingival tissue was homogenized with 1 ml of
solution containing 4 M guanidium thiocyanate; 25 mM
sodium citrate, pH 7.0; 0.5 per cent sarcosyl; 0.1 M 2mercaptoethanol. 100 µl of 2M sodium acetate, pH 4;
1 ml of H2O-saturated phenol; 200 µl of a 49:1 mixture
of CHCl3: isoamyl alcohol (Merck, India) were added
to the homogenized tissues and mixed thoroughly by
inversion after addition of each reagent. The final
mixture was shaken vigorously on vortex for 10 sec
and transferred to ice for 15 min then centrifuged at
10,000 xg for 20 min at 4°C. The RNA-containing
aqueous phase was transferred to a fresh tube, and the
DNA and proteins which should be present in the phenol
SUBRAMANI et al: IGFBP-5 IN DRUG INDUCED GINGIVAL OVERGROWTH
and at the interface was discarded. One volume of
isopropanol was added to the aqueous phase and
precipitated at -20°C for at least 1 h and centrifuged at
10,000 xg for 20 min at 4°C. The pellet was transferred
to a 1.5 ml microcentrifuge tube and precipitated the
RNA again in 1 volume of isopropanol (or two volumes
of ethanol) at -20°C for at least 1 h. After an hour, it
was centrifuged at high speed (13,000 xg in a
microcentrifuge) for 10 min at 4°C. The RNA pellet
was washed in 75 per cent ethanol, centrifuged again
and vacuum desiccated. The total RNA was transcribed
to cDNA using First Strand cDNA synthesis Kit
(Qbiogene, USA) for RT-PCR according to
manufacturer instructions. The IGFBP-5 was designed
from the known sequence, as IGFBP-5: 5’GGCTCCGAATCTAAGTGCTG- 3’ (sense) and 5’GCAGCCCTGTCTCACTAACC-3’ (antisense). The
primers (Biocorporals, India) were predicted to amplify
457 bp base pairs 11 . As a positive control
b-actin
primer
designed
as:
5’AAGGATTCCTATGTGGGC-3’ (sense) and 5’CATCTCTTGCTCGAAGTC-3’ (antisense), which
were predicted to amplify a 300 base pair DNA
fragment. The amplification profile was as follows:
initial denaturing at 94 0 for 5 min, followed by
denaturing at 940C for 90 sec; annealing at for 60 sec;
extension at 720C for 60 sec. The cDNA was amplified
for 25 cycles for IGFBP-5 and 36 cycles for b-actin;
followed by a step of 7 min at 720C to extend the
partially amplified products. The PCR products were
electrophoresed on 1.5 per cent agarose gel and
visualized by ethidium bromide staining. The gels were
photographed and their image data were analyzed for
band quantification using 1D image analysis software
(Applied Biosystems, USA). The relative amount of
each IGFBP-5 gene expression was calculated as the
ratio of the individual IGFBP-5 to the intensity of bactin gene products as the control. The relative
expression of each IGFBP-5 gene from control, diseased
and drug induced gingival overgrowth tissues were
compared.
The linear regression curve for IGFBP-5 in DIGO
was performed against the control tissue and the
differences in the levels of expression of IGFBP-5
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in different groups of patients and healthy controls
were analyzed using ANOVA for the significance.
Results & Discussion
IGFBP-5 was expressed in all examined tissue
samples collected from DIGO, chronic periodontitis
and healthy subjects. The level of the IGFBP-5
mRNA expression from chronic periodontitis and
DIGO tissue samples was found to be more than that
in the uninflamed tissues (Fig. 1). On the other hand,
DIGO tissue samples showed significantly increased
expression of IGFBP-5 mRNA compared to chronic
periodontitis and control (Fig. 2). The linear
regression curve of IGFBP-5 in DIGO against healthy
control tissues (Fig. 3) showed the increased
expression of IGFBP-5 in DIGO tissue samples.
Binding proteins are produced locally by different
tissues and could therefore act in local regulation.
IGFBPs modulate the half-life and activity of IGFs12.
IGFBP-5 is unique, in that it is the only binding
protein that has been shown to consistently stimulate
cell proliferation in vitro 13. IGFBP-5 is produced
locally in all tissues and plays a major role in
decreasing apoptosis and increasing proliferation14.
IGFBP-5 enhanced the mitogenic actions of IGF-I on
cultured fibroblasts and smooth muscle cells by
associating with extracellular matrix (ECM) near the
target cell3. However, IGFBP-5, also has an important
role in controlling cell survival, differentiation and
apoptosis15.
ECM associated IGFBP-5 may act by
accumulating IGF-I near its receptor by modulating
the interaction of IGF-I3. The expression of IGFBP5 gene is cell type specific. High levels of IGFBP-5
mRNA have been found in fibroblasts, giloblastoma
cells, skeletal muscle cells, osteoblasts, and
chondrocytes 16 . In our study we found IGFBP-5
mRNA in healthy human gingival tissues. McCarthy
et al 17 have shown that IGFBP-5 mRNA was
increased 2.4 fold in inflamed rat colon compared
with control. Similar to earlier studies, our results
showed that the expression of IGFBP-5 mRNA was
4-fold increased in chronic periodontitis tissues
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INDIAN J MED RES, JANUARY 2007
Fig. 1. IGFBP-5 mRNA expression in human gingival tissue samples obtained from patients with cyclosporine-A induced gingival
overgrowth (DIGO), chronic periodontitis (CP), and normal healthy subjects (Control). Total RNA was isolated and RT-PCR was
performed using gene specific primers. b-actin was used as the internal control for PCR.
IGFBP-5/beta-actin ratio
2
1.5
1
0.5
0
Control
CP
DIGO
Gingival tissue samples
Fig. 2. mRNA expression of IGFBP-5 in human gingival samples obtained from patients with cyclosporin-A induced gingival
overgrowth (DIGO), chronic periodontitis (CP), normal healthy subjects (Control). Patients with CsA-induced gingival overgrowth
had higher IGFPB-5 mRNA in gingival tissues than chronic periodontitis and healthy subjects. **P<0.001 vs control (n=8 in each
case).
compared to control. Zimmermann et al 18
demonstrated the expression of IGFBP-5 mRNA in
rat intestinal smooth muscle and increased IGFBP-5
mRNA in chronically inflamed intestine. The role
of gingival fibroblasts proliferation in the setting of
inflammation is poorly understood but is likely
caused by several inflammatory mediators including
IGF-I19. Kelly et al20 suggested that IGFBP-5 may
play an important role in the enhancement of the
fibrogenic actions of IGF-I.
IGFBP-5 induced production and deposition of
collagen and fibronectin in primary adult lung
fibroblasts. Unlike most of the other binding proteins,
which act as competitive inhibitors of IGF-I receptor,
IGFBP-5 acts to enhance IGF-I actions. IGF-I
increases the synthesis of both IGFBP-5 and
collagen21. Moreover, IGF-I’s biological activity on
fibroblasts includes stimulation of collagen production
and downregulation of collagenase production,
suggesting that IGF-I may be an important mediator
in the development of gingival fibrosis through
IGFBP-511. Feghali et al22 have demonstrated a 20fold increase in IGFBP-5 expression in fibroblast from
the fibrotic skin of patients than control skin. The
interaction of IGFBP-5 to collagen type I and
fibronectin in ECM may form the basis for the
initiation of signal and its perpetuation since
stimulation of collagen and fibronectin production
provides additional IGFBP binding partners in the
ECM, thus generating an uncontrolled loop of
increased ECM components and increased binding
SUBRAMANI et al: IGFBP-5 IN DRUG INDUCED GINGIVAL OVERGROWTH
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IGFBP-5 mRNA in control (ug/mg tissue)
230
220
210
200
190
180
170
160
150
140
130
900
1000
1100
1200
1300
1400
1500
1600
IGFBP-5 mRNA in DIGO (ug/mg tissue)
Fig. 3. Linear regression curve showing the expression of IGFBP-5 in drug induced gingival overgrowth using control as a references
sample set. The dotted lines denote the confidence band. SE=7.9, R-Sq = 94.1% and R-Sq(adj) = 93.1%.
sites for IGFBPs. IGFBPs contribute to the
propagation of the fibrotic phenotype, but our
observation of increased expression of IGFBP-5
implicates IGFBPs in the initiation phase of fibrosis,
where the inflammation factor was uncontrolled. This
is further supported by other findings showing
increased IGFBP-5 mRNA levels in fibrotic skin of
patients with systemic sclerosis23,24.
Further support comes from micro array analysis
that revealed increased IGFBP-5 mRNA levels in
human idiopathic pulmonary fibrosis lung tissues and
increased IGFBP-5 in lung tissues of bleomycintreated mice25,26. The greater expression of IGFBP-5
in periodontal ligament fibroblast (PDLF) together
with IGF-I induced reduction of apoptosis in PDLF,
suggests a potential role of IGFBP-5 in the
upregulation of IGF-I pathway 10. Taken together,
these observations suggest that IGFBPs may be
involved early in the cascade of fibrosis.
In this pilot study, we kept the methodology to
the minimum and the limitation of the study was
not carrying out the protein estimation. In
conclusion, drug induced gingival tissues showed
increased mRNA expression of IGFBP-5 compared
to chronic periodontitis and healthy gingival tissues
which may be associated with increased collagen
synthesis, thereby promoting fibrogenesis. Further
studies need to be done to understand the
mechanism.
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Reprint requests: Dr R. Suresh, Professor & Head, Department of Periodontics, Sri Ramachandra Dental College & Hospital
Sri Ramachandra Medical College & Research Institute, Deemed University, Porur, Chennai 600116, India
e-mail: [email protected]