Denner Darcy R, Udan-Johns Maria Ld, Nichols Michael R
Department of Chemistry and Biochemistry, University of Missouri-St. Louis, St Louis, MO 63121, United States.
World J Biol Chem. 2021 Jan 27;12(1):1-14. doi: 10.4331/wjbc.v12.i1.1.
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 factor α (TNFα) secretion by human monocytes was of high interest. The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFα secretion by distinct mechanisms.
To investigate the regulation of lipopolysaccharide (LPS)-stimulated MMP-9 and tumor necrosis factor α secretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.
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 TNFα production. Inhibitors including DMSO, cAMP regulators, and anti-TNFα antibody were added to the cells prior to LPS treatment. MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software. TNFα secretion 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 -test. 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 TNFα antibodies were from R&D Systems, and amyloid-β peptide was from rPeptide.
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 LPS-induced MMP-9, but not TNFα, secretion. Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dose-dependent fashion. A cell-permeable cAMP analog, dibutyryl cAMP, inhibited both LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFα in a dose-dependent fashion. Pre-treatment of monocytes with an anti-TNFα antibody blocked LPS-induced MMP-9 and TNFα secretion. Amyloid-β peptide induced MMP-9 secretion, which occurred much later than TNFα secretion. The latter two findings strongly suggested an upstream role for TNFα in mediating LPS-stimulate MMP-9 secretion.
The cumulative data indicated that MMP-9 secretion was a distinct process from TNFα secretion 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 TNFα with 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 TNFα and that TNFα may a key component of the pathway leading to MMP-9 secretion. This temporal relationship fit a model whereby early TNFα secretion directly led to later MMP-9 secretion. Lastly, antibody-blocking of TNFα diminished MMP-9 secretion, suggesting a direct link between TNFα secretion and MMP-9 secretion.
基质金属蛋白酶（MMPs），包括MMP - 9，是免疫反应的一个组成部分，并且在对多种刺激的反应中上调。关于介导MMP - 9分泌的机制和调节途径的新细节不断涌现。有大量证据表明二甲基亚砜（DMSO）和3'，5'-环磷酸腺苷（cAMP）可调节炎症，因此研究这两种分子如何调节人单核细胞分泌MMP - 9和肿瘤坏死因子α（TNFα）备受关注。本研究检验的假设是DMSO和cAMP通过不同机制调节MMP - 9和TNFα的分泌。
研究二甲基亚砜和cAMP对脂多糖（LPS）刺激的THP - 1人单核细胞中MMP - 9和肿瘤坏死因子α分泌的调节作用。
本文描述了一项使用THP - 1人单核细胞的基础研究。所有实验均在密苏里大学圣路易斯分校的化学与生物化学系进行。按照公认的指南，人单核细胞在密苏里大学圣路易斯分校细胞培养设施中生长、培养并准备用于实验。细胞用LPS处理选定的暴露时间，收集条件培养基用于分析MMP - 9和TNFα的产生。在LPS处理之前，将包括DMSO、cAMP调节剂和抗TNFα抗体在内的抑制剂添加到细胞中。通过凝胶电泳/蛋白质免疫印迹分析MMP - 9分泌，并通过ImageJ软件进行定量。通过酶联免疫吸附测定分析TNFα分泌。所有数据均表示为至少3次试验的平均值和标准误差。使用双尾配对学生t检验进行统计分析。P值小于0.05被认为具有统计学意义，并在图中如此标注。LPS和cAMP调节剂购自西格玛奥德里奇公司，MMP - 9标准品、抗体和TNFα抗体购自R&D系统公司，淀粉样β肽购自rPeptide公司。
在我们对THP - 1人单核细胞MMP - 9分泌的研究中，我们有以下发现。细胞处理中加入DMSO可抑制LPS诱导的MMP - 9分泌，但不抑制TNFα分泌。在细胞处理中加入不同浓度的DMSO以剂量依赖性方式抑制LPS诱导的MMP - 9分泌。一种细胞可渗透的cAMP类似物，二丁酰cAMP，可抑制LPS诱导的MMP - 9和TNFα分泌。用腺苷酸环化酶激活剂福斯高林预处理细胞可抑制LPS诱导的MMP - 9和TNFα分泌。用通用的cAMP磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX）预处理细胞以剂量依赖性方式降低LPS诱导的MMP - 9和TNFα分泌。用抗TNFα抗体预处理单核细胞可阻断LPS诱导的MMP - 9和TNFα分泌。淀粉样β肽诱导MMP - 9分泌，其发生时间比TNFα分泌晚得多。后两个发现强烈表明TNFα在介导LPS刺激的MMP - 9分泌中起上游作用。
累积数据表明MMP - 9分泌是一个与TNFα分泌不同的过程，且发生在下游。首先，DMSO抑制MMP - 9，但不抑制TNFα，表明MMP - 9分泌过程被选择性改变。其次，cAMP以相似的效力抑制MMP - 9和TNFα，但在不同的单核细胞暴露时间点。cAMP对这两种分子的抑制模式表明MMP - 9分泌位于TNFα下游，并且TNFα可能是导致MMP - 9分泌途径的关键组成部分。这种时间关系符合一个模型，即早期TNFα分泌直接导致后期MMP - 9分泌。最后，TNFα的抗体阻断减少了MMP - 9分泌，表明TNFα分泌与MMP - 9分泌之间存在直接联系。
