The airway epithelium provides a protective barrier against inhaled environmental toxins and microorganisms and epithelial injury initiates a number of processes to restore its barrier integrity including activation of matrix metalloproteinases such as MMP-9 (92-kD gelatinase B). or MMP-9 activation is definitely unknown. Using main or immortalized human being bronchial epithelial cells we demonstrate that low concentrations of NO promote epithelial cell migration and wound restoration in an wound assay which was associated with improved localized manifestation and activation of MMP-9. In addition in HBE1 cells that were stably transfected with inducible NOS (NOS2) to mimic constitutive epithelial NOS2 manifestation wound model using an exogenous NO donor (DETA NONOate) and stable transfection with NOS2 in an attempt to mimic continuous NO production by airway epithelial cells (27). Cells were grown and managed as previously explained (33) and experiments were performed in 6- 12 or 24-well plates (Corning Corning NY) or in 8-well chamber slides (Nunc Rochester NY). After changing the medium or after cell injury (next section) cells were treated with the slow-releasing NO-donor diethylenetriamine NONOate (DETA NONOate 10-500 μM; Cayman Chemical Ann Arbor MI; t1/2 = 20 h at 37°C) human being TNF-α (100 ng/ml; Sigma St Louis MO) or 8-bromoguanosine 3′5′-cyclic monophosphate (8-Br-cGMP 10 μM; Sigma) for up to 24 h. Cells were pretreated where indicated for 30 min with the soluble guanylyl cylase inhibitor 1H-[1 2 4 3 (ODQ 10 μM; Sigma) endogenous NOS activity was clogged using NG-monomethyl-L-arginine (l-NMMA 1 mM; Alexis San Diego CA) or the specific NOS2 inhibitor 1400 (100 μM; Alexis). Where indicated the PKG inhibitors KT5823 (10 μM; Sigma) and DT-2 (10 μM) (28) or the MMP inhibitor (MMP Inhibitor II 1 μM; Calbiochem La Jolla CA) were added 30 min before experimentation. None of them of the providers used significantly affected cell morphology or viability under these conditions. Wound Restoration Assay To investigate the effects of NO on Rabbit polyclonal to ACSM5. epithelial cell migration we used a common wound assay in which confluent cell monolayers are mechanically wounded by developing a linear scuff. Although wound restoration in such injury models is definitely a complex process including both cell migration and proliferation the initial phase of wound restoration involves primarily cell migration (29 30 After intro of a linear wound of ～ 0.5 mm width using a sterile P-200 pipette tip cell monolayers were washed with media to remove cell debris fresh media was added to each well and appropriate reagents were given. Closure of linear wounds was adopted for 24 h using an IX70 inverted microscope (Olympus Center Valley PA) using UltraView v4 software (Perkin Elmer Existence Sciences Wellesley MA). Wound closure was indicated as a percentage of the initial wound area quantitated using NIH ImageJ software. Cell KOS953 Migration For more quantitative analysis of epithelial cell migration cells were seeded at 5.0 × 104 cells/well in 8.0 μm polycarbonate membranes (Nunc) and the ability of KOS953 cells to migrate into these membranes was followed. Twenty-four hours after cell treatments nonmigrated cells were removed having a cotton swab and cells that migrated through the membrane pores were fixed with 4% paraformaldehyde (PFA) stained with 0.5% crystal violet in 20% methanol and extracted in 0.2 M sodium acetate (pH 4.5) for quantitation by absorbance at 562 nm using a Biotek Synergy HT microplate reader (Winooski VT). Analysis KOS953 of MMP-9 by Gelatin Zymography Secretion of MMP-9 in the tradition media was analyzed by gelatin zymography as explained previously (26). Gelatinolytic bands were scanned and digitized for quantitation of band intensity using NIH ImageJ software. RT-PCR Analysis of MMP-9 Manifestation Total RNA was extracted using KOS953 TRIzol (Invitrogen Carlsbad CA) according to the manufucturer’s protocol. Reverse transcription (RT) was performed using 2-5 μg of total RNA and PCR reactions were performed using a GeneAmp PCR System 9 700 (Applied Biosystems Foster City CA) as detailed previously (26). PCR products were resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The manifestation of MMP-9 and uPA was normalized to that of GAPDH by band densitometry analysis using NIH ImageJ software. MMP-9 Reporter Assay To determine the transcriptional activation of MMP-9 by NO cells were transiently transfected with the pGL3/MMP-9/Luc-670 create (generously provided by Dr. D. Boyd University or college of Texas Houston TX) (31). Briefly 1 μg of construct was incubated with the transfection reagent (Lipofectamine and Plus Reagent; Invitrogen) at room temperature and.