I would dissolve your inhibitor in DMSO to make a 10mM stock. The typical inhibitors used are hydroxamates from Calbiochem or Tocris. Then I would add directly to cell culture media to 1-10uM if the IC50 is 1-10nM. Typically one uses 100 x the Ki of the in vitro enzymatic assay. The final DMSO concentration should not exceed 0.1%. The MMP inhibitors are not very soluble in water/buffers, so if you need to dilute the inhibitor, you can make a 10% DMSO stock at 100-300uM first. You would do this, if you want to make the final concentration 1uM and the piper ting volume is too low if you pipettes directly from the 10mM stock.  Absolutely do not make a 200uM-10mM stock in buffer. It won't be soluble.

I am not an expert in this particular MMP and you don't provide specific details on the inhibitor (name?) to answer your question, but I always liked fluorescent protease substrates to assay protease activity. They are basically small peptides containing a fluorescent label and a fluorescence quencher. Digestion with you favorite protease cuts the peptide and separates the label from the quencher to produce fluorescence. You can easily find out conditions that lead to a complete inhibition of MMP9 by assaying your cells after treatment with MMP9 fluorescent protease substrates:

https://www.google.com/search?q=MMP9+inhibitor&sourceid=ie7&rls=com.microsoft:en-US:IE-ContextMenu&ie=&oe=&gws_rd=ssl#q=MMP9+fluorescent+substrate

Dear Jordan,

The following cover the answer to your question. Important paragraphs are marked with bold font.

1- Protease Inhibitor Treatments Reveal Specific Involvement of Matrix Metalloproteinase-9 in Human Adipocyte Differentiation

Virginie Bourlier,

Alexia Zakaroff-Girard,

Sandra De Barros,

Christophe Pizzacalla,

Véronique Durand de Saint Front,

Max Lafontan,

Anne Bouloumié and

Jean Galitzky

http://jpet.aspetjournals.org/content/312/3/1272.full

Unité de Recherche sur les Obésités, Institut National de la Santé et de la Recherche Médicale, Institut Louis Bugnard, Hôpital Rangueil, Université Paul Sabatier, Toulouse, France (V.B., A.Z.-G., S.D.B., C.P., V.D.d.S.F., M.L., J.G.); and Institut für Kardiovaskuläre Physiologie, Frankfurt/Main, Germany (A.B.)

Address correspondence to:

Dr. Virginie Bourlier, Institut National de la Santé et de la Recherche Médicale U586, 37 Allées Jules Guesde, 31073 Toulouse, France. E-mail:[email protected]

Next Section

Abstract

We previously showed that human and murine 3T3-F442A preadipocytes produced and released matrix metalloproteinases (MMPs) 2 and 9 and that a treatment by MMP inhibitors resulted in the blockade of murine fat cell adipose conversion. In parallel, investigators reported that other protease inhibitors, the human immunodeficiency virus (HIV) protease inhibitors (PIs) involved in lipodystrophy in humans, also reduced the adipocyte differentiation process of several murine cell lines. The present work was performed to define the effects of MMP inhibitors and HIV-PIs on the human adipocyte differentiation process, to clarify the involvement of MMPs in the control of human adipogenesis, and to determine whether HIV-PIs interact with MMPs in the control of this process. The effect of two MMP inhibitor and four HIV-PI treatments on the differentiation of primary culture human preadipocytes, as well as the putative relationships between HIV-PIs and MMP-2 and -9 expression, release, or activity were investigated. We showed that MMP inhibitors and HIV-PIs reduced the human adipocyte differentiation process as assessed by the decrease of cell protein and/or triglyceride contents and expression of fatty acid binding protein and hormone-sensitive lipase, two adipocyte markers. Unlike MMP inhibitors, HIV-PIs were devoid of any effect per se on recombinant MMP-2 and 9 activities but reduced the expression and release of MMP-9 by human preadipocytes. Thus, the present study indicates that the modulation of the extracellular matrix components through the production and/or activity of MMPs, and, more precisely, MMP-9 might be a key factor in the regulation of human adipose tissue development.

Fluorometric Activity Assay on Recombinant MMP-2 and -9. In vitro assays were performed on 96-well plates. Human recombinant activated MMP-2 (2 nM) or MMP-9 (4 nM) (Calbiochem) were preincubated in the presence or absence of increasing concentrations of batimastat (from 0.01 nM to 0.01 μM) or HIV-PIs (from 1 nM to 100 μM) for 30 min at 37°C. Twenty micromolar of the fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dnp-Ala-Arg (Bachem, Bubendorf, Switzerland) were then added to each well, and fluorescence (excitation, 325 nm; emission, 390 nm) was recorded every 2 min for 60 min. MMP activity was evaluated by the maximum rate per minute on five separate measurements in each well.

2- Am J Pathol. 2010 Dec; 177(6): 2870–2885.

doi:  10.2353/ajpath.2010.090050

PMCID: PMC2993298

Matrix Metalloproteinase-9 and Cell Division in Neuroblastoma Cells and Bone Marrow Macrophages

M. Gloria Sans-Fons, Sonia Sole, Coral Sanfeliu, and Anna M. Planas

Author information ► Article notes ► Copyright and License information ►

See "This Month in AJP" in volume 177 on page 2681.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993298/

Go to:

Abstract

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions in cell development, cancer, injury, and regeneration. In addition to its well recognized extracellular action, functional intracellular MMP activity under certain conditions is supported by increasing evidence. In this study, we observed higher gelatinase activity by in situ zymography and increased MMP-9 immunoreactivity in human neuroblastoma cells and in bone marrow macrophages undergoing mitosis compared with resting cells. We studied the pattern of immunoreactivity at the different stages of cell division by confocal microscopy. Immunostaining with different monoclonal antibodies against MMP-9 revealed a precise, dynamic, and well orchestrated localization of MMP-9 at the different stages of cell division. The cellular distribution of MMP-9 staining was studied in relation to that of microtubules. The spatial pattern of MMP-9 immunoreactivity suggested some participation in both the reorganization of the nuclear content and the process of chromatid segmentation. We then used several MMP-9 inhibitors to find out whether MMP-9 might be involved in the cell cycle. These drugs impaired the entry of cells into mitosis, as revealed by flow cytometry, and reduced cell culture growth. In addition, the silencing of MMP-9 expression with small interfering RNA also reduced cell growth. Taken together, these results suggest that intracellular MMP-9 is involved in the process of cell division in neuroblastoma cells and in primary cultures of macrophages.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade several components of the extracellular matrix. MMPs are normally found as latent zymogens that become active through proteolytic cleavage1 by mechanisms ensuring sensitive and complex regulation of MMP activity in vivo.2 The matrix undergoes intense remodeling during development, neoplasia, inflammation, and tissue injury and recovery, which often involves enhanced release of certain proteases. MMPs are thought to have key functions in various pathological conditions, such as diseases of the central nervous system3 and cancer.4 Indeed, invasiveness requires that tumoral cells degrade the basement membrane, and this is associated with release of MMPs.4 In accordance, MMP inhibitors attenuate tumor cell growth and/or invasive capacity.5,6 For instance, MMP inhibitors with a broad inhibitory spectrum suppress cervical lymph node metastasis.7Furthermore, increased MMP-9 expression is associated with enhanced proliferative capacity in mouse aortic smooth muscle cells,8 and mice injected with lymphoma cells constitutively expressing MMP-9 develop thymic lymphoma more rapidly than mice injected with control lymphoma cells.9

Growth factors up-regulate certain MMPs, such as gelatinases MMP-2 and MMP-9, in gliomas.10 MMPs are downstream targets of the epidermal growth factor receptor (EGFR) signaling pathway.11 Indeed, exposure of breast or carcinoma cells to EGFR ligands increases cell proliferation and induction of MMP-9,12,13,14which is mediated by mitogen-activated protein kinases.15 In addition, the EGFR signaling pathway is associated with the proteolytic and invasive phenotype in head and neck squamous cell carcinomas.16Transforming growth factor-α (TGF-α) is a ligand for EGFR with mitogenic activity for epithelial and mesenchymal cells.17,18 TGF-α enhances MMP-9 in cervical-carcinoma SKG-IIIb cells.19

Although MMPs exert multiple functions in the extracellular space, several lines of evidence support the possibility that MMPs could also play roles in functions inside the cells.20,21 In this study we examined the intracellular expression and activation of MMP-9 in neuroblastoma cells and in primary cultures of rat macrophages, the effects of MMP-9 inhibitors or of silencing MMP-9 expression in cell cycle and/or cell growth, and the response to growth stimulation with TGF-α. The results suggest that MMP-9 is involved in certain steps of cell division in neuroblastoma cells and in macrophages.

Materials and Methods

Neuroblastoma Cell Cultures and Drug Treatments

Human neuroblastoma SH-SY5Y cells (European Collection of Cell Cultures, Salisbury, Wiltshire, UK) were grown in Ham’s F12 and Eagle’s minimal essential medium (1:1) containing 15% fetal calf serum, 1% nonessential amino acids, 2 mmol/L glutamine, and 20 μg/ml gentamicin at 37°C and 5% CO2 in a humidified atmosphere. N-Myc amplified human neuroblastoma SK-N-BE-2C cells were kindly provided by Dr. Jaume Mora (Hospital de Sant Joan de Deu, Barcelona, Spain). All culture media and reagents were from Invitrogen (GIBCO, Invitrogen SA, Barcelona, Spain). Cells were seeded at the density of 3 to 4 × 103cells/cm2, and drug treatment was initiated on the next day.

The MMP-9 inhibitors, inhibitor A (MMP-2/MMP-9 Inhibitor II 444249) and inhibitor B (MMP-9/MMP-13 Inhibitor I 444252), and the broad MMP inhibitor GM6001 were obtained from Calbiochem (Darmstadt, Germany). Stock solutions were prepared in dimethyl sulfoxide (0.5% in the final concentration), and concentration-response curves were studied. In each experiment, control cells (0 μmol/L inhibitor) were exposed to the vehicle for the same time. Human recombinant TGF-α (Calbiochem) was dissolved in 10 mmol/L acetic acid and then PBS to yield a neutral pH. The day after seeding cells were exposed to TGF-α (10 ng/ml), and studies were performed at different time points ranging from 1 to 6 days. In some experiments the tyrosine kinase activity of EGFR was inhibited with 10 μmol/L 4,5-dianilinophthalimide (RBI, Köln, Germany).

3- Article

Experimental & Molecular Medicine (2009) 41, 259-268; doi:10.3858/emm.2009.41.4.029

Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media

Yun-Song Lee1, Huong Thi Lan Tran1 and Quang Van Ta1

1Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746, Korea.

Correspondence: Yun-Song Lee, Tel, 82-31-299-6190; Fax, 82-31-299-6209; E-mail, [email protected]

Accepted 3 December 2008

http://www.nature.com/emm/journal/v41/n4/full/emm200931a.html

Topof pageAbstract

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-α, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-γ. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-γ. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.

Keywords:

anthra(1,9-cd)pyrazol-6(2H)-one; autocrine communication; JNK mitogen-activated protein kinases; macrophages; matrix metalloproteinase 9; serum

Methods

Reagents

DMEM, BSA, and FBS were purchased from Gibco (Gaithersburg, MD). Control, JNK1, or JNK2 siRNA was purchased from Dharmacon (Chicago, IL). SP600125 was purchased from Alexis (San Diego, CA) and the JAK inhibitor pyridone 6 (P6) was from Calbiochem (San Diego, CA). Antibodies to p-JNK, p-ERK, p-Akt, Akt, p-c-Jun, p-p38 MAPK, and p-p65 were from Cell Signaling Technology (Boston, MA). Antibodies to ERK, α-tubulin, and p38 MAPK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to p65 was from Upstate Biotechnology (Lake Placid, NY). LPS (O26:B6), IFN-γ, gelatin, and other chemicals were acquired from Sigma-Aldrich (St. Louis, MO).

Cell culture

Raw 264.7 cells from Korean Cell Line Bank (Seoul, Korea) were grown in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37℃ in a 5% CO2 humidified incubator. Before experiments, the cells were incubated in DMEM containing 0.02% BSA overnight, and the media was replaced again 4 h prior to each experiment. All the data are representative of triplicate experiments.

RT-PCR of MMP-9 mRNA

RT-PCR was used to determine mRNA expression of MMP-9 and GAPDH. Total RNAs were obtained using a commercial kit (Intron, Sungnam, Korea) according to the manufacturer's instructions. To obtain cDNA, 0.2 µg total RNAs were added to a 20 µl reaction mixture containing 15 U/µl SSII ribonuclease H-reverse transcriptase (Superscript II; Invitrogen), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 0.5 mM dNTPs, 2.5 µM random hexamers, and 2.5 U/µl RNase inhibitor. PCR primers for mouse MMP-9 and GAPDH were as follows: MMP-9, 5'-CAAACCCTGCGTATTTCC-3' (sense) and 5'-AGAGTACTGCTTGCCCAGGA-3' (antisense); GAPDH, 5'-CTCATGACCACAGTCCATGC-3' (sense) and 5'-TTCATCGGGATGACCTT-3' (antisense). PCR was performed with 2 µl of cDNA and 0.2 mM primers using a touchdown PCR; 2 cycles of PCR at 94℃ for 1 min, 64℃ for 1 min and 72℃ for 1 min, followed by 25 cycles (MMP-9) or 20 cycles (GAPDH) of PCR at 94 ℃ for 1 min, 59℃ for 1 min and 72℃ for 1 min. PCR products (MMP-9, 223 bps; GAPDH, 155 bps) were resolved in 1.5% agarose gels containing ethidium bromide. Images were taken by a Gel-Doc apparatus (Kodak, Rochester, NY), and analyzed with Image Gauge software (Fuji, Japan).

Assay of MMP-9 activity by gelatin zymography

The gelatinolytic activity of MMP-9 secreted into the culture medium was determined by gelatin zymography (Chung et al., 2004). Briefly, conditioned media freed of cell debris by centrifugation were mixed with Laemmli buffer lacking reducing agents. After electrophoresis in an 8% SDS-PAGE gel containing 1 mg/ml gelatin, the gel was incubated in a developing buffer (20 mM Tris/HCl, pH 7.8, 1% Triton X-100, 10 mM CaCl2, 5 µM ZnCl2) for 24 h at 37℃. Thereafter, the gel was stained with 1% Coomassie Brilliant Blue R-250. Images of gelatinolytic activities were taken and analysis done as described above.

Hoping this will be helpful,

Rafik

I suppose, you can find IC50 or other measures of the effectiveness of your inhibitor in bioactivity databases like ChEMBL https://www.ebi.ac.uk/chembl/ or PubChem https://pubchem.ncbi.nlm.nih.gov/

It may become a good starting point.

Thank you all for your help, and Rafik, special thanks to you for putting so much time into your response. I appreciate it

I would dissolve your inhibitor in DMSO to make a 10mM stock. The typical inhibitors used are hydroxamates from Calbiochem or Tocris. Then I would add directly to cell culture media to 1-10uM if the IC50 is 1-10nM. Typically one uses 100 x the Ki of the in vitro enzymatic assay. The final DMSO concentration should not exceed 0.1%. The MMP inhibitors are not very soluble in water/buffers, so if you need to dilute the inhibitor, you can make a 10% DMSO stock at 100-300uM first. You would do this, if you want to make the final concentration 1uM and the piper ting volume is too low if you pipettes directly from the 10mM stock.  Absolutely do not make a 200uM-10mM stock in buffer. It won't be soluble.

I would suggest to talk to Roeland Hanemaaijer and Natascha van Lent from TNO (Leiden, The Netherlands). Roeland Hanemaaijer (https://www.linkedin.com/in/roeland-hanemaaijer-39a32920) works with MMP9 years and years. Together with Natascha van Lent (https://www.linkedin.com/in/natascha-van-der-meijden-van-lent-21963387) they make a kit for MMP9 activity assay (detection of total and active MMP9; https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314716762536/litdocs1370_20110830174054.pdf). Time ago they had only human version of this assay, but 3-4 years ago we also adapted that assay for mouse and rat samples (antibodies, time of incubation, etc.). I don't remembe details now, but Natascha and Roeland should know these things very well. Success!