The gene expression and protein profiles of ADAMTS and TIMP in human chondrosarcoma cell lines induced by insulin: The potential mechanisms for skeletal and articular abnormalities in diabetes
Electronic Journal of General Medicine
The delay in wound healing, decrease in the long bones resilience to fracture, and delay in fracture healing are among common complications diabetes mellitus (DM) patients, and they still remain as challenging issues to be solved. The mechanism has not been fully understood yet, but high sugar and/or insulin deficiency or unresponsiveness to insulin in blood are potential causes to blame. Extracellular matrix degradation/remodeling is one of the important mechanisms whereby cell
... ell differentiation, bone remodeling and wound repair can be regulated. A disintegrin and metalloproteinase with a thrombospondin type 1 motif (ADAMTS) proteins play important roles in cartilage/bone metabolism. This study aimed to determine whether ADAMTS/Tissue inhibitors of metalloproteinases (TIMP) proteins were affected by insulin application in OUMS-27 (chondrosarcoma) cells. Material and Methods: OUMS-27 cells were induced by 10μg/mL insulin for 1, 3, 7, and 11 days. Cells were harvested, mRNA and protein extractions were performed. Total mRNA and cDNA levels were measured by qRT-PCR and protein levels were detected by WB. Results: ADAMTS1,5, and 7 levels were significantly decreased, while TIMP-3 levels were detected increased (mRNA/protein concentrations). Conclusion: Pathologies and disturbances of cartilage/bone metabolism, delayed fracture healing in particular, in patients with DM may result from insulin deficiency. ADAMTS genes that play a role in healing process are increased during insulin deficiency, which consequently interrupts healing process by causing cartilage extracellular matrix (ECM) degradation. Akyol et al. / ADAMTS and TIMP in human chondrosarcoma 2 / 17 http://www.ejgm.co.uk Recent studies have demonstrated that insulin stimulates bone cells to produce an endocrine hormone that increases the efficiency of glucose metabolism (3). Maxillary sinus graft application is a commonly used dental process to increase bone volume before the insertion of dental implants (4-6). This technique is claimed to be contradictory when used for tooth implants in patients with uncontrolled DM (7). In animal model studies, it is found that DM decreases the bone formation, results in osteopenia and delayed fracture healing (8). In studies examining the impact of DM on bone formation, it is seen that DM delays the fracture healing in experimental fractures of the tibia or femur and that insulin replacement reversed this effect (9). In an experiment conducted on rabbits, whose pancreatic β-cells were damaged by Alloxan which finally causes type-1 DM, the researchers found decreased levels of newly formed bone mass with blood vessels, as well as the number of osteoblasts, collagen and serum-osteocalcin (10). The treatment of insulin has increased the osteogenesis in the extended maxillary spaces of diabetic rabbits. Thus, decreased bone formation in diabetes models in animals can be prevented via insulin therapy. Fractures are a significant health issue for patients with both type-1 and -2 DM (11). During the healing process of fractures, when hematoma occurs due to the rupture of blood vessels, progenitor cells come to the damaged area and proliferate to differentiate into osteoblasts and chondrocytes. Chondrocytes produce a cartilage, which forms a soft cartilaginous callus, then turns into calcified form, and finally takes the damaged area under protection. While osteoclasts, turned into apoptosis, are taken away from the environment of mineralized cartilage by chondrocytes, osteoblasts start the endochondral bone formation. Another major problem in type-1 DM is the decrease in bone mineral density and bone formation, which results in osteopenia (12,13). Case-controls and clinical studies of patients with DM also showed prolonged healing of wounds and fractures compared to healthy cases (14,15). Although most of the studies are focused on the bone, the deterioration of cartilage production, which is one of the stages of fracture healing in diabetics, is one of the subjects that need to be investigated. Chondrocyte differentiation and proliferation, which normally occur during fracture healing, have considerably declined in patients with DM (16, 17) . Matrix metalloproteinases are capable of digesting extracellular matrix (ECM) macromolecules and non-ECM molecules including receptors, cytokines, chemokines, growth factors, all of which are determinants of the tissue microenvironment. They are present in both acute and chronic wounds. Together with their inhibitors, they play a pivotal role in regulating ECM degradation and deposition (18). It has been proven that a disintegrin and metalloproteinase with a thrombospondin type 1 motif (ADAMTS) proteins, which are members of the metzincins superfamily of zinc-based proteinases, play important roles during growth-development, progression of joint diseases and the metabolism of the cartilage ECM (19). Although the roles of ADAMTS of the chondrocyte growth, differentiation and maturation on the epiphyseal plate are known, their roles in endochondral ossification are not fully proven (20). Researchers have shown the ADAMTS9 mRNA expression during the chondrogenesis of insulin-induced ATDC5 cells. ADAMTS9 is expressed in these chondrocytes and together with the proliferation of chondrocytes and matrix degradation; they help to contribute to its hypertrophy and endochondral ossification process (21). After in vitro induction of chondrogenesis by insulin in ATDC5 cells, the ADAMTS9 expression increased by having/reaching two peaks within 8th-14th days and later within 28th-35th days. Recent studies of joint diseases in mice lacking metalloproteinase have shown that these proteases have important roles in bone biology and pathology (22). It is not yet understood from which molecular mechanisms these observed phenotypes are disclosed/revealed. As each metalloproteinase cuts its own substrate at a certain point, multiple biological processes arise. Also if we take into account the argument that metalloproteinases serve as communicator molecules between certain bone cells, it shows the necessity to research the subject further to reveal different aspects. Although it is known that ADAMTS 4 and 5 within the matrix metalloprotease are major aggrecanases in the human cartilage, it is not clearly known how these zymogen (inactive) enzyme forms become active and how these activities are managed in vivo. As DM affects the cartilage formation and resorption, it has a negative effect on fracture healing. Although the issue is clinically known, the molecular mechanism is not yet fully understood. How insulin affects the cartilage formation and resorption, which is one of the important stages of fracture healing in in vitro and in vivo conditions, remains as an original subject to study for years to come. The aim of this study is to investigate the quantity and behavioral changes of ADAMTS proteins, which are thought to be related to production and degradation of cartilage synthesized by chondrocytes, during an insulin implementation to the OUMS-27 chondrosarcoma cells. For this purpose, the ECM formation, cartilage production and ADAMTS1, 4, 5, 7, 15 and 18, which have been thought to play an important role in bone formation, and Tissue inhibitors of metalloproteinases (TIMP)-3 and 4, which are inhibitors of the functions of these enzyme, were investigated in terms of mRNA and protein product levels. ). OUMS-27 cells were cultured in DMEM containing 10% BSA and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2. The cells were sub-cultured using trypsin-EDTA at every 7-10 days. The medium was changed every-other-day with either control media or control media supplemented with 10 μg/mL insulin for 11 days. Four groups of cells (two of each group, one for qRT-PCR and one for Western blot (WB) analysis) were subjected to insulin: For day-1 (D1) experiment, 2×10 5 cells, for day-3 (D3) experiment 1×10 5 cells, for day-7 (D7) experiment 5×10 4 cells, and for day-11 (D11) experiment 3×10 4 cells were plated in 20-mm dishes and exposed to the same concentration of insulin on the days indicated. Insulin powder was dissolved within 0.01N HCl. The stock solution had 2 mg/mL insulin in 0.01N HCl, and working solution had 10 μg/mL in medium. Cells were incubated with insulin in medium for one, three, seven, and eleven days. Cells were plated in five dishes for each condition. After the experiment, cells were harvested, and total RNA/protein isolations were performed. RNA isolation and qRT-PCR: Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Two-micrograms of RNA were reverse transcribed with RevertAid M-MuLV Reverse Transcriptase and random hexamers (Thermo Scientific, Waltham, MA) with random primers (Table 1) . Human GAPDH was amplified as a control for the PCR. Samples lacking reverse transcriptase were amplified as a control for genomic DNA contamination. RNase-free water (Qiagen GmbH, Germany) was used to elute total RNA. Ultraviolet-visible spectrophotometry was used to quantify and determine the purity of samples (required a 260/280 ratio of 2.0 and a 260/230 ratio of 1.7 for adequate purity). Quantitative real-time (qRT)-PCR was performed on cDNA samples (Qiagen-Rotor-Gene Q-RT-PCR) as described previously (23) . Total RNA RT-PCR section uses intercalating dye SYBR® green (Thermo Scientific Maxima/SYBR ®Green/ROX) in presence of primer pairs. PCR mixture consisted of SYBR® Green PCR Master Mix including 10 pmol forward and reverse primers, and cDNA of samples in total 20 μL. GAPDH was amplified for normalizing the efficiency of cDNA synthesis and the amount of RNA applied. PCR was performed with initial denaturation at 95 °C for 5-min, followed by amplification for 40 cycles, each cycle consisting of denaturation at 95 °C for 30s, annealing at 57 °C for 30s, polymerization at 72 °C for 60s and, polymerization at 72 °C for 5 min. The results pertaining to ADAMTS1, 4, 5, 7, 15, 18, were represented as graphics. The bars and error bars represent means and standard deviations (SD) of amplicon concentrations. Gradient/conventional PCRs: Although primers purchased were supplied with theoretical Tm, we calculated the annealing temperature of the thermal cycling reaction for our assays. As the starting point, a gradient was set up so that it spans the calculated annealing temperature of the primers approximately ±10 °C (51.0, 52.7, 55.3, 58.5, 61.2, and 62.9 °C). PCR was performed in the same conditions as stated above by using BioRad T-100 Thermal Cycler. Once the Akyol et al. / ADAMTS and TIMP in human chondrosarcoma 6 / 17 http://www.ejgm.co.uk The antibody used for ADAMTS4 protein detection gave two bands on 68 kDa/55 kDa. At the D1 of insulin application, ADAMTS4 protein level decreased sharply. The decreases of protein amount for first band continued up to D3 and then started to increase from D7. Regarding the second band, the decrease observed on day-1 was permanent/constant up to D11 (Figure 2B) . Quantitative nucleic acid concentrations of ADAMTS5 in D1, D3, D7, and D11 groups were decreased compared to control, but only D1 and D7 values were significant (P= 0.009 and P= 0.016, respectively) ( Figure 3A) . It generally means that the application of insulin led to a crucial decrease in mRNA amount of ADAMTS5 in at least two study groups. It could not be observed for D3 and D11 groups. Furthermore, there were statistically significant differences of amplicon concentration between D1-D11 and D7-D11 (P= 0.009 and P= 0.036, respectively) ( Figure 3A ). Figure 2: (A) qRT-PCR test results of A disintegrin and metalloproteinases with thrombospondin type 1 motif 4 (ADAMTS4) in OUMS-27 chondrosarcoma cells. Values were standardized and normalized by proportioned with GAPDH values. Bars and error bars represent mean values and standard deviations, respectively. There were no statistically significant differences between the groups. (B) The electrophoretic image of ADAMTS4 protein of OUMS-27 cells by Western blotting technique and their graphics (two different bands) obtained by the calculation of band densities by CCD camera and ImageJ program. The x-axis shows insulin application time intervals (in days) and y-axis shows band densities of ADAMTS4. The densities for ADAMTS4 were normalized with GAPDH densities.