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1 Reports An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13 Seniz Inanc, Didem Keles, and Gulgun Oktay Dokuz Eylul University, School of Medicine, Department of Medical Biochemistry, Izmir, Turkey BioTechniques 63: (October 2017) doi / Keywords: collagen zymography; MMP-1; MMP-8; MMP-13; type I collagen Supplementary material for this article is available at /article/ Collagen zymography is an SDS-PAGE based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/ml type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3 1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3 1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions. It has been well-documented that matrix metalloproteinases (MMPs), which are zinc-dependent endopeptidases, participate in proteolytic turnover and cleavage of several extracellular matrix (ECM) components and non-ecm molecules (1). MMP-1, MMP-8, and MMP-13 are major secreted collagenases capable of destroying type I, II, III, V, and IX collagen as well as native fibrillary collagen (2). These proteases play a significant role in tissue regeneration and organ development during ECM remodeling. It has been demonstrated that dysregulation of collagenases can lead to a wide range of pathological conditions, including rheumatoid arthritis (3), osteoarthritis (4), chronic ulcer (5), and tumor progression and metastasis (6). Therefore, detection of the activities of these enzymes could provide significant diagnostic and prognostic information, leading to optimal selection of treatment options. Substrate zymography is one of the most widely used techniques for detecting proteolytic activity (7). In this method, proteins are separated in an SDS-PAGE gel copolymerized with a specific substrate (e.g., gelatin, casein, or collagen) under non-reducing conditions and without heating. SDS induces non-proteolytic activation of the proenzyme, which triggers degradation of the substrate by the active enzyme. This phenomenon makes it possible to analyze both the proenzyme and the active form (8). When the gels are stained, the digested areas appear as clear bands against a dark background. Collagen zymography is the preferred method for evaluating collagenase 174 (MMP-1, MMP-8, and MMP-13) activities for the following reasons: (i) it is easy and inexpensive compared with techniques such as western blot, ELISA, and activity assays, and (ii) it allows for analyses of not only the proenzyme and active forms but also glycosylated isoforms. However, there are technical problems that need to be considered, including selection of the appropriate collagen type and concentration, determination of collagenase sensitivity, and identification of collagenases separately despite their similar molecular weights. Collagen zymography was developed by Gogly et. al. (9). Although the authors optimized their method to analyze MMP-1 activity, they did not investigate MMP-8 and MMP-13 activities. Here, we improve on the collagen zymography method for quantitative analysis of collagenases (MMP-1, MMP-8, REPRINT WITH PERMISSION ONLY METHOD SUMMARY Collagen zymography was optimized by using type I collagen extracted from rat tail tendon in order to evaluate glycosylated, latent, and active forms of human collagenases. In addition, standard curves were generated to quantify the collagenase activity of unknown samples.

2 REPORTS and MMP-13) by creating standard curves for each collagenase. Materials and methods Extraction and purification of type I collagen from rat tail tendon Rat tails were obtained from 6 7-month-old Wistar rats (Dokuz Eylul University, Experimental Animals Laboratory) and stored at -20 C until the extraction procedure. The protocol was approved by the Ethics Committee at Dokuz Eylul University. Rat tails were incubated in 70% ethanol for 20 min at room temperature. After removing the skin, tendons were dissected out and minced with a scalpel under aseptic conditions. The tendons were transferred to 17 mm acetic acid (50 ml per tail) and stirred for 48 h at 4 C. The insoluble materials were centrifuged at 30,000 x g for 60 min at 4 C. The supernatant, which contained collagen, was neutralized to ph 7.0 with 0.1 M NaOH and then stirred for 90 min at 4 C. The precipitated collagen was centrifuged at 10,000 x g for 20 min at 4 C, and the pellet was dissolved in 17 mm acetic acid and stirred for 48 h at 4 C (10). The total collagen concentrations were measured by the BCA (bicinchoninic acid) assay (Thermo Fisher Scientific, Waltham, MA). SDS-PAGE The purity of the extracted type I collagen was evaluated by SDS-PAGE (11). Briefly, samples were mixed with 2 sample buffer (125 mm Tris, 4% SDS, 10% b-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, ph 6.8) and heated at 95 C for 5 min. Extracted collagens were separated by 5% resolving gels (110 V for 60 min) and stained with 0.5% Coomassie Blue R-250 (Sigma- Aldrich, Taufkirchen, Germany) (w/v) (40% methanol, 10% acetic acid). Protein molecular weight markers (4 250 kda) (SeeBlue Plus 2 Prestained Standard, Cat no. LC5925; Invitrogen Corporation, Carlsbad, CA) were used to calculate the relative molecular weights of purified type I collagen specific bands by LabWorks 4.6 Image Acquisition and Analysis Software (UVP Inc, Upland, CA). Preparation of commercial type I and II collagen Type I collagen from rat tail tendon and type II collagen from chicken sternal cartilage (Sigma-Aldrich) were reconstituted to 5 mg/ml in 0.01 M and M acetic acid, respectively. The solutions were stirred at 4 C for 48 h until the collagen dissolved. Collagen zymography Collagen zymography was standardized to analyze the proenzyme and active forms of the collagenases (MMP-1, MMP-8, and MMP-13) according to the method of Heussen and Dowdle (12). SDS-PAGE gels (10% polyacrylamide) were copolymerized with mg/ml of purified type I collagen, commercial type I collagen, or type II collagen. Recombinant human pro-collagenases (1 100 ng) (rh-prommp-1, rh-prommp-8, and rh-prommp-13) (R&D Systems, Minneapolis, MN) were mixed with non-reducing 2 sample buffer (0.625 M Tris, 20% glycerol, 4% SDS, 0.004% bromophenol blue, ph 6.8) and then electrophoresed at a constant voltage of 110 V for 2 h at 4 C. Gels were washed twice in 2.5% Triton X-100 (v/v) for 15 min to remove SDS and then incubated in developing buffer (50 mm Tris, 10 mm CaCl 2, 50 mm NaCl, 0.05% Brij 35 (Sigma-Aldrich), ph 7.6) at 37 C for 48 h. After incubation, gels were stained with 0.5% Coomassie Blue R-250 (w/v) and then destained in 40% methanol and 10% acetic acid solution. Collagenolytic activities were detected as clear bands against a Coomassie Blue stained gel background. Semiquantification of digested bands was performed with LabWorks 4.6 Image Acquisition and Analysis Software. Cell culture The SV40-transfected and immortalized normal human primary thyroid follicular epithelial cell line Nthy-ori 3 1 and the human undifferentiated anaplastic thyroid cancer cell line 8505C were purchased from the DSMZ - German Collection of Microorganisms and Cell Culture (DMSZ, Braunschweig, Germany). Nthy-ori 3 1 and 8505C cells were cultured in RPMI-1640 (Lonza, Basel, Switzerland) supplemented with 10% FBS, 2 mm L-glutamine, and 100 U/mL penicillin 100 µg/ml streptomycin. All cells were grown in a humidified incubator (5% CO 2 at 37 C). For zymographic analyses, 1x10 6 of the Nthy-ori 3 1 and 8505C cells were seeded into 25 cm 2 cell culture flasks and allowed to adhere overnight. 175 Figure 1. SDS-PAGE of type I collagen extracted from five different rat tail tendon samples. Lane 1: molecular weight marker (MW); Lanes 2 6: extracted type I collagen. Molecular weights for the collagen a 1, a 2, and b chains were calculated based on the retention factor (Rf) values for the corresponding gel bands. Subsequently, the cells were incubated in serum-free medium for 24 h. The conditioned medium was collected and concentrated with a 3-kDa Amicon Ultra Centrifugal Filter (EMD Millipore, Bedford, MA) according to manufacturer s instructions. Twenty-five micrograms of the conditioned medium and 10 ng of rh-prommp-1 were loaded on collagen zymogram gels containing 0.3 mg/ml extracted type I collagen, and collagen zymography was performed as previously described. Western blot One-hundred micrograms of conditioned medium and 10 ng of rh-prommp-1 were loaded onto the SDS-PAGE gel and electrophoresed at 4 C under denaturing conditions. Following electrophoresis, gels were transferred to the PVDF membranes and incubated at 4 C overnight. The membranes were blocked with 5% skim milk powder in TBS-T (w/v) for 1 h at room temperature and then treated with primary antibodies anti-mmp-1 (dilution 1:200) (Cat no. AB6002; EMD Millipore; RRID: AB_ ) and antib-actin (dilution 1:3000) (Cat no. 4967S; Cell Signaling Technology, Beverly, MA; RRID: AB_330288) overnight at 4 C. After washing, membranes were incubated with diluted HRP-conjugated secondary antibody (Cell Signaling Technology; AB_ ) for 1 h. The protein bands were detected by ECL reagent (Thermo Fisher Scientific), and images were captured using the KODAK 4000MM Imaging System (Carestream, Rochester, NY).

3 REPORTS A B C D E F Figure 2. Optimization of collagen zymography. Serial dilutions of rh-prommp-1, rh-prommp-8, and rh-prommp-13 were loaded into collagen zymogram gels containing 0.3 mg/ml extracted type I collagen. (A) Zymogram gel image of MMP-1. (B) Logarithmic standard curve for prommp-1 (n = 5). (C) Zymogram gel image of MMP-8. (D) Logarithmic standard curve for prommp-8 (n = 6). (E) Zymogram gel image of MMP-13. (F) Logarithmic standard curve for prommp-13 (n = 4). Results were represented as mean ± SEM. Determination of molecular weights and densitometric analysis of collagen zymograms The relative molecular weights of recombinant human pro-collagenases were determined by plotting a calibration curve of the molecular weight markers (4 250 kda) (SeeBlue Plus 2 Prestained Standard) and calculating retention factors (Rf). Densitometric analysis was carried out to quantify each collagenase proenzyme, and standard curves were plotted as optical density (OD) versus the amount of rh-prommp-1, rh-prommp-8, and rh-prommp-13 in nanograms). All analyses were done using LabWorks 4.6 Image Acquisition and Analysis Software. The graphs were created using GraphPad Prism 7.0 software (GraphPad Software, Inc, San Diego, CA). At least four independent collagen zymography experiments were performed, and the data were represented as mean ± SEM. Results and discussion Identification of Type I collagen by SDS-PAGE Type I collagen was extracted from rat tail tendon for use as a substrate in collagen zymography. SDS-PAGE was performed to confirm the identity and purity of the extracted collagen. Type I collagen is composed of b (215 kda), a 1 (130 kda), and a 2 (115 kda) chains (10). SDS-PAGE revealed the typical pattern for type I collagen, with bands at apparent molecular weights of 217 kda (b), 131 kda (a1), and 120 kda (a2), which were calculated using their Rf values (Figure 1). Optimization of collagen zymography To optimize collagen zymography, we first determined the most appropriate substrate for detecting all forms of the collagenases. We tested three different types of collagen as the substrate: (i) extracted type I collagen from rat tail tendon, (ii) commercial type I collagen from rat tail tendon, and (iii) commercial 176 type II collagen from chicken cartilage. As shown in Supplementary Figure S1 (Panels A C), we detected collagenase activity using both commercial and extracted type I collagens; however, we could not detect significant collagenase activity when using commercial type II collagen. Therefore, we performed the subsequent experiments with extracted type I collagen for three major reasons: (i) Collagenases cleave type I collagen at specific sites in the a-chain, leading to the formation of 1/4 C-terminal and 3/4 N-terminal fragments (13); (ii) type I collagen can be extracted with high purity and a significant yield (~ µg/ ml per rat tail); and (iii) the extraction procedure is easy to perform and more cost-effective than commercial alternatives. The use of three different concentrations of extracted type I collagen in SDS-PAGE gels was also examined (Supplementary Figure S1, D F). Recombinant collagenases were loaded into zymogram gels that were copoly-

4 REPORTS A B Figure 3. MMP-1 activity and expression levels in human thyroid cell lines. Conditioned media from the human thyroid cancer cell line 8505C and the human normal thyroid cell line Nthy-ori 3 1 were subjected to (A) collagen zymography and (B) western blotting. MW: molecular weight marker; rh-prommp-1: recombinant human prommp-1. merized with 0.15, 0.3, and 0.6 mg/ml collagen. Gels containing 0.15 mg/ml and 0.6 mg/ml collagen showed more weakly digested collagen bands compared with gels containing 0.3 mg/ml collagen. At the lowest collagen concentration tested (0.15 mg/ml), although all the forms of human collagenase can be visualized, the low staining capacity may lead to difficulty in the analysis of weak enzyme activity. At the highest collagen concentration tested (0.6 mg/ml), collagenases may not degrade all of the substrate during the incubation, and since the remaining substrate is stained, the proteolytic activity is barely discernible (14). Consequently, we carried out all further studies with 0.3 mg/ml of extracted type I collagen. Under these optimized conditions, even though we used recombinant human pro-collagenases, we observed additional bands, which might belong to either active or glycosylated forms of the enzyme (Figure 2). MMP-1 is secreted as 2 different glycosylated proenzymes (57 and 52 kda) from human skin fibroblasts, and cleavage of these peptides forms 2 active enzymes (47 and 42 kda) (15). Consistently, we calculated the molecular weights of the MMP-1 bands as glycosylated prommp-1 (60 and 56 kda) and as glycosylated active MMP-1 (51 and 49 kda), respectively (Figure 2A). MMP-8 is produced as two different types. The PMN (polymorphonuclear neutrophil)-type MMP-8 is highly glycosylated and secreted as preprommp-8 (80 kda), prommp-8 (75 kda), and active MMP-8 (65 kda). Fibroblast-type MMP-8 has two less-glycosylated forms: prommp-8 (55 kda) and active MMP-8 (45 kda) (16 18). In collagen zymogram gels, MMP-8 bands were observed at 83, 73, and 57 kda, which could potentially be the PMN-type MMP-8 (Figure 2C). We detected 2 distinct MMP-13 bands of 59 and 46 kda (Figure 2E), which correspond to prommp-13 (60 kda) and active MMP-13 (48 kda) (19). Previously, we reported that despite using rh-prommp-2 and rh-prommp-7 for gelatin and casein zymography, active forms of the recombinant proenzymes could be also observed (20). These results indicate that the commercial rh-prommps probably contain low amounts of other forms of MMPs that could be detected by highly sensitive zymographic techniques. Determining sensitivity and plotting standard curves Serial dilutions of recombinant pro-collagenases were evaluated under the given experimental conditions, and collagenase activity as low as 1 ng could be quantified (Figure 2). Accordingly, a range of ng of recombinant pro-collagenases was analyzed to generate standard curves. When the results were plotted as band density versus collagenase concentration, the curves for prommp-1 (Figure 2B), prommp-8 (Figure 2D), and prommp-13 Figure 4. Scheme of the optimized collagen zymography method. 178 (Figure 2F) were logarithmic. When the collagenase activities of the samples are out of the analyzable range, samples should be diluted or concentrated to obtain more accurate quantification. MMP-1 activity in 8505C and Nthy-ori 3-1 cells We investigated collagenolytic activity in the conditioned media of a human anaplastic thyroid cancer cell line (8505C) and a normal thyroid follicular epithelial cell line (Nthy-ori 3 1). We observed that both pro- and active MMP-1 levels were elevated in 8505C cells, whereas no collagenolytic activity was detected in Nthy-ori 3 1 control cells (Figure 3A). The specific activity of prommp-1 was found to be ~9.59 ng prommp-1/µg total protein in 8505C cells using the prommp-1 standard curve. We also performed western blotting to determine whether the collagenolytic activity was due to MMP-1. Consistent with the activity results, increased prommp-1 protein levels were found in 8505C cells compared with Nthy-ori 3 1 cells (Figure 3B). These results clearly indicate that MMP-1 in 8505C cells is secreted and activated in the extracellular space. Experimental studies have shown that MMP-1 is overexpressed at both the mrna and protein levels in thyroid cancer cell lines (21) and follicular thyroid carcinomas (22,23). However, researchers could not detect MMP-1 activity levels unambiguously in thyroid carcinoma. To our knowledge, this is the first report that shows increased MMP-1 activity levels in 8505C cells. Here, we demonstrated, for the first time, an optimized collagen zymography technique that enables the evaluation of all

5 R REPORTS human collagenases (MMP-1, MMP-8, and MMP-13), including their latent, active, and glycosylated forms, and we created standard curves to facilitate analysis of activity levels of individual collagenases in unknown samples (Figure 4). Previously, Gogly et. al. described collagen zymography for examining both pro- and 4-aminophenylmercuric acetate (APMA) activated MMP-1 (9). They applied the zymography using gels that contained 0.5 mg/ml commercial calf skin type I collagen, whereas we extracted type I collagen from rat tail tendon with an affordable and easyto-implement procedure. In contrast to our current results, Gogly et al. could not distinguish glycosylated forms of MMP-1 under their assay conditions and did not determine the activity levels of the other collagenases, MMP-8 and MMP-13. Although Gogly et al. detected lower amounts of prommp-1 (sensitivity as low as 10 pg of prommp-1 and 0.1 pg of APMA-activated MMP-1) compared with our measurement limits, a lower detection limit can be achieved by extending incubation time. In addition, in cases where samples have low collagenase activity (<1 ng), the samples can be concentrated and/or activated with APMA to increase sensitivity. We believe that our optimized collagen zymography method will prove useful for both clinical and experimental applications. It should be noted that if a sample contains three collagenases at the same time, separation of individual collagenase bands can be a problem due to their narrow molecular weight distribution. However, this problem can be overcome using 2-D collagen zymography, where additional separation by isoelectric focusing is possible. Author contributions G.O. conceived of and designed the experiments. S.I. and D.K. designed experimental approaches, performed the experiments, and analyzed the data. S.I., D.K., and G.O. wrote the manuscript. Acknowledgments This research was supported in part by a grant (no KB.SAĞ.10) from the Dokuz Eylul University Scientific Research Project Coordination Unit. References 1. Apte, S.S. and W.C. Parks Metalloproteinases: A parade of functions in matrix biology and an outlook for the future. Matrix Biol : Ala-aho, R. and V.M. Kahari Collagenases in cancer. Biochimie 87: Sorsa, T., Y.T. Konttinen, O. Lindy, C. Ritchlin, H. Saari, K. Suomalainen, K.K. Eklund, and S. Santavirta Collagenase in synovitis of rheumatoid arthritis. Semin. Arthritis Rheum. 22: Billinghurst, R.C., L. Dahlberg, M. Ionescu, A. Reiner, R. Bourne, C. Rorabeck, P. Mitchell, J. Hambor, et al Enhanced TRANSFORMING RNA ANALYSIS WITH AUTOMATED SAMPLE QC. The Fragment Analyzer simultaneously quantifies and qualifies RNA samples, whether you re performing total RNA, mrna or small RNA analysis, or working with degraded materials like formalin fixed tissues. cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J. Clin. Invest. 99: Vaalamo, M., L. Mattila, N. Johansson, A.L. Kariniemi, M.L. Karjalainen-Lindsberg, V.M. Kahari, and U. Saarialho-Kere Distinct populations of stromal cells express collagenase-3 (MMP-13) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds. J. Invest. Dermatol. 109: Egeblad, M. and Z. Werb New functions for the matrix metalloproteinases in cancer progression. Nat. Rev. Cancer 2: Vandooren, J., N. Geurts, E. Martens, P.E. Van den Steen, and G. Opdenakker lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFP RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small R mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnr snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-dep ed RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Tot RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small RNA, mrna FFPE RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, sm RNA, mrna, JUST FFPE RNA, Total RNA, RIGHT ribo-depleted RNA, lnrna, snrna lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-depletedd RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFP RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small R mrna, FFPE RNA, Total FOR RNA, ribo-depleted QC RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnr snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-dep ed RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Tot RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small RNA, mrna FFPE RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, sm RNA, mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnrna, Total R mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-depleted RNA, lnr snrna, Micro RNA, small RNA, mrna, FFPE RNA, Total RNA, ribo-dep ed RNA, lnrna, snrna, Micro RNA, small RNA, mrna, FFPE RNA, Tot RNA, ribo-depleted RNA, lnrna, snrna, Micro RNA, small RNA, mrna Competing interests The authors declare no competing interests. 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