Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes

BACKGROUND Matrix metalloproteinases(MMPs),including MMP-9,are an integral part of the immune response and are upregulated in response to a variety of stimuli.New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion.There is significant evidence for regulation of inflammation by dimethyl sulfoxide(DMSO)and 3',5'-cyclic adenosine monophosphate(cAMP),thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factorα(TNFα)secretion by human monocytes was of high interest.The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFαsecretion by distinct mechanisms.AIM To investigate the regulation of lipopolysaccharide(LPS)-stimulated MMP-9 and tumor necrosis factorαsecretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.METHODS The paper describes a basic research study using THP-1 human monocyte cells.All experiments were conducted at the University of Missouri-St.Louis in the Department of Chemistry and Biochemistry.Human monocyte cells were grown,cultured,and prepared for experiments in the University of Missouri-St.Louis Cell Culture Facility as per accepted guidelines.Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFαproduction.Inhibitors including DMSO,cAMP regulators,and anti-TNFαantibody were added to the cells prior to LPS treatment.MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software.TNFαsecretion was analyzed by enzyme-linked immuno sorbent assay.All data is presented as the average and standard error for at least 3 trials.Statistical analysis was done using a two-tailed paired Student t-test.P values less than 0.05 were considered significant and designated as such in the Figures.LPS and cAMP regulators were from Sigma-Aldrich,MMP-9 standard and antibody and TNFαantibodies were from R&D Systems,and amyloid-βpeptide was from rPeptide.RESULTS In our investigation of MMP-9 secretion from THP-1 human monocytes,we made the following findings.Inclusion of DMSO in the cell treatment inhibited LPSinduced MMP-9,but not TNFα,secretion.Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dosedependent fashion.A cell-permeable cAMP analog,dibutyryl cAMP,inhibited both LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFαin a dose-dependent fashion.Pre-treatment of monocytes with an anti-TNFαantibody blocked LPSinduced MMP-9 and TNFαsecretion.Amyloid-βpeptide induced MMP-9 secretion,which occurred much later than TNFαsecretion.The latter two findings strongly suggested an upstream role for TNFαin mediating LPS-stimulate MMP-9 secretion.CONCLUSION The cumulative data indicated that MMP-9 secretion was a distinct process from TNFαsecretion and occurred downstream.First,DMSO inhibited MMP-9,but not TNFα,suggesting that the MMP-9 secretion process was selectively altered.Second,cAMP inhibited both MMP-9 and TNFαwith a similar potency,but at different monocyte cell exposure time points.The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFαand that TNFαmay a key component of the pathway leading to MMP-9 secretion.This temporal relationship fit a model whereby early TNFαsecretion directly led to later MMP-9 secretion.Lastly,antibody-blocking of TNFαdiminished MMP-9 secretion,suggesting a direct link between TNFαsecretion and MMP-9 secretion.     

2-Aminobenzamide, an inhibitor of ADP-ribosylation, antagonizes induced DNA hypomethylation during differentiation of murine Friend erythroleukemia cells by N''-methylnicotinamide

The ADP-ribose synthesis inhibitor 2-aminobenzamide was found to reduce the induction of haemoglobin synthesis in murine Friend erythroleukemia cells, cultured continuously for 96 h with 5 mM N'-methylnicotinamide, with over 50% inhibition at equimolar doses. 2-Aminobenzamide was optimally effective as an inhibitor of differentiation if present only during the time period 24-48 h after initial N'-methylnicotinamide exposure. A transient, genome-wide DNA hypomethylation accompanies induction by N'-methylnicotinamide. Although 2-aminobenzamide does not seem to affect normal DNA methylation in cultured cells, the observed DNA hypomethylation in cells cultured for 24 h with 5 mM N'-methylnicotinamide was antagonized over 50% by equimolar 2-aminobenzamide. We suggest that ADP-ribosylation has a positive modulatory role in erythroid differentiation, and possibly in the process(es) leading to DNA hypomethylation following inducer exposure.