Assessing Gelatinase Activity in Normal and Disease Uterine Tissue and Cells Via Gelatin Zymography

Matrix metalloproteinases (MMPs) are critical for the maintenance and remodeling of the extracellular matrix (ECM) under normal physiological conditions such as pregnancy and wound healing. However, an increase of MMPs in uterine diseases, such as adenomyosis, endometrial cancer, endometriosis, and uterine fibroids, has been observed and suspected to contribute to important pathophysiology phenotypes like invasion and migration. Of note, MMP-2 (also referred to as gelatinase A) and MMP-9 (also referred to as gelatinase B) are common gelatinases that have demonstrated increased activity in uterine diseases and in several cancer types, such as breast cancer, to promote cancerous phenotypes like increased invasion and migration. In-gel zymography is a useful technique for the detection of MMP activity via degradation of gelatin in gelatin-based gels. Using zymography, it is possible to assess the activity levels of MMP-2 and MMP-9 via gelatin degradation during the zymography process. Here, we will describe the process of zymography and assessment of MMP activity levels (MMP-2 and MMP-9) for both uterine tissues and cancerous cell lines. Access this chapter Tax calculation will be finalised at checkout Purchases are for personal use only Similar content being viewed by others

Granelli-Piperno A, Reich E (1978) A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med 148:223–234 Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102:196–202 Nikolov A, Popovski N (2021) Role of gelatinases MMP-2 and MMP-9 in healthy and complicated pregnancy and their future potential as preeclampsia biomarkers. Diagnostics (Basel) 11 Nagase H, Visse R, Murphy G (2006) Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 69:562–573 Cabral-Pacheco GA, Garza-Veloz I, Castruita-De la Rosa C, Ramirez-Acuña JM, Perez-Romero BA (2020) The roles of matrix metalloproteinases and their inhibitors in human diseases. Int J Mol Sci 21 Brew K, Nagase H (2010) The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta 1803:55–71 Murphy G (2011) Tissue inhibitors of metalloproteinases. Genome Biol 12:233 Lopata A et al (2003) Detection of endometrial cancer by determination of matrix metalloproteinases in the uterine cavity. Gynecol Oncol 90:318–324 Li T, Li YG, Pu DM (2006) Matrix metalloproteinase-2 and -9 expression correlated with angiogenesis in human adenomyosis. Gynecol Obstet Investig 62:229–235 Itoh Y, Nagase H (2002) Matrix metalloproteinases in cancer. Essays Biochem 38:21–36 Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2:161–174 Scheau C, Badarau IA, Costache R, Caruntu C, Mihai GL, Didilescu AC et al (2019) The role of matrix metalloproteinases in the epithelial-mesenchymal transition of hepatocellular carcinoma. Anal Cell Pathol (Amst) 2019:9423907 Hwang KE, Kim HJ, Song IS, Park C, Jung JW, Park DS et al (2021) Salinomycin suppresses TGF-β1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer. Int J Med Sci 18:715–726 Ries C (2014) Cytokine functions of TIMP-1. Cell Mol Life Sci 71:659–672 Ogata Y, Itoh Y, Nagase H (1995) Steps involved in activation of the pro-matrix metalloproteinase 9 (progelatinase B)-tissue inhibitor of metalloproteinases-1 complex by 4-aminophenylmercuric acetate and proteinases. J Biol Chem 270:18506–18511 Itoh Y, Ito A, Iwata K, Tanzawa K, Mori Y, Nagase H (1998) Plasma membrane-bound tissue inhibitor of metalloproteinases (TIMP)-2 specifically inhibits matrix metalloproteinase 2 (gelatinase a) activated on the cell surface. J Biol Chem 273:24360–24367 DeClerck YA, Yean TD, Lu HS, Ting J, Langley KE (1991) Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2. J Biol Chem 266:3893–3899 Peeney D, Liy Y, Lazaroff C, Gurung S, Stetler-Stevenson WG (2022) Unravelling the distinct biological functions and potential therapeutic applications of TIMP2 in cancer. Carcinogenesis 43:405–418 Costanzo L, Soto B, Meier R, Geraghty P (2022) The biology and function of tissue inhibitor of metalloproteinase 2 in the lungs. Pulm Med 2022:3632764 Arpino V, Brock M, Gill SE (2015) The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix Biol 44-46:247–254 Bernardo MM, Fridman R (2003) TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP. Biochem J 37:739–745 Fan D, Kassiri Z (2020) Biology of tissue inhibitor of metalloproteinase 3 (TIMP3), and its therapeutic implications in cardiovascular pathology. Front Physiol 11:661 Harper E, Bloch KJ, Gross J (1971) The zymogen of tadpole collagenase. Biochemistry 10:3035–3041 Li QL, Wang HM, Lin HY, Liu DL, Zhang X, Liu GY et al (2002) Expression of gelatinases and their tissue inhibitors in rat corpus luteum during pregnancy and postpartum. Mol Reprod Dev 63:273–281 Nothnick WB (2001) Disruption of the tissue inhibitor of metalloproteinase-1 gene in reproductive-age female mice is associated with Estrous cycle stage-specific increases in stromelysin messenger RNA expression and activity1. Biol Reprod 65:1780–1788 Hu J, Zhang X, Nothnick WB, Spencer TE (2004) Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus. Biol Reprod 71:1598–1604 Gonçalves AN, Meschiari CA, Stetler-Stevenson WG, Nonato MC, Alves CP, Espreafico EM et al (2012) Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli. J Biotechnol 157:20–24 Yabluchanskiy A, Ma Y, Iyer RP, Hall ME, Lindsey ML (2013) Matrix metalloproteinase-9: many shades of function in cardiovascular disease. Physiology (Bethesda) 28:391–403 Wechselberger C, Doppler C, Bernhard D (2019) An inexpensive staining alternative for gelatin zymography gels. Methods Protoc 2 Author information Authors and Affiliations Corresponding author Editor information Editors and Affiliations Rights and permissions Copyright information © 2025 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature About this protocol Cite this protocol Peterson, R., Nothnick, W.B. (2025). Assessing Gelatinase Activity in Normal and Disease Uterine Tissue and Cells Via Gelatin Zymography. In: Khalil, R.A. (eds) Zymography. Methods in Molecular Biology, vol 2918. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-4482-9_19 Download citation DOI: https://doi.org/10.1007/978-1-0716-4482-9_19 Published: Publisher Name: Humana, New York, NY Print ISBN: 978-1-0716-4481-2 Online ISBN: 978-1-0716-4482-9 eBook Packages: Springer Protocols