Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. Induction and endogenous activation of collagenase, 72-kDa gelatinase, and Pump-1 is accompanied by the suppression of the tissue inhibitor of matrix metalloproteinases.
Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. Induction and endogenous activation of collagenase, 72-kDa gelatinase, and Pump-1 is accompanied by the suppression of the tissue inhibitor of matrix metalloproteinases.
复制标题
刀豆球蛋白 A 在人成纤维细胞中产生基质降解表型。
DOI：
10.1016/s0021-9258(17)45338-5
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
J. Sodek
中科院分区：
文献类型：
--
作者：
C. Overall;J. Sodek
凝集素伴刀豆球蛋白A（ConA）导致成纤维细胞获得树枝状形态并表达升高水平的胶原酶。ConA调节基质金属蛋白酶（MMPs）和基质金属蛋白酶组织抑制剂（TIMP）的时间和机制方面的特点在早期传代人成纤维细胞。胶原酶（MMP-1），在不存在TIMP的情况下通过功能测定测量，也作为免疫沉淀的[35 S]甲硫氨酸标记蛋白，在ConA（20 μ g/ml，2 × 10（-7）M）处理24-72 h后增加10-20倍，活性胶原酶约占总胶原酶活性的20%。通过比较，MMP-2（72 kDa明胶酶;分子量，72 kDa，+二硫苏糖醇; 66 kDa，-二硫苏糖醇），通过酶谱分析和亲和纯化后，增加了不到2倍的刀豆蛋白A，并完全作为一个激活的，61 kDa（+二硫苏糖醇; 59 kDa，-二硫苏糖醇）的形式。北方杂交分析表明，ConA提高了MMPs的稳态mRNA水平;胶原酶mRNA增加了约16倍，MMP-2增加了2倍，并且诱导了最近描述的MMP基因Pump-1。同时，TIMP蛋白和mRNA水平下降10倍ConA发生。相比之下，12-O-十四烷酰基佛波醇-13-乙酸酯（50 ng/ml，8 × 10（-8）M）也强烈刺激胶原酶表达（大于30倍），提高TIMP蛋白和mRNA水平（分别为2和3倍），但不影响MMP-2表达。蛋白质合成抑制剂放线菌酮阻断了ConA诱导的MMP和TIMP mRNA水平的变化，胶原酶和MMP-2 mRNA的半衰期（分别为53和46小时）不受影响，表明ConA通过需要从头蛋白质合成的途径在转录上发挥作用。通过核连续分析证实了MMP基因的转录增加; MMP-1转录增加了25倍以上，MMP-2增加了约3倍，Pump-1增加了约7倍。相比之下，Timp基因转录减少了约80%，揭示了再吸收细胞表型诱导过程中MMPs和TIMP的相互调节。条件培养基中胶原蛋白和纤连蛋白的量减少，但不减少MMP（分泌蛋白，酸性和富含半胱氨酸）的量是MMP活性的结果，因为稳态mRNA水平和相应基质蛋白基因的转录不受ConA的影响。
The lectin concanavalin A (ConA) causes fibroblasts to acquire an arborized morphology and to express elevated levels of collagenase. The temporal and mechanistic aspects of ConA regulation of matrix metalloproteinases (MMPs) and the tissue inhibitor of matrix metalloproteinases (TIMP) were characterized in early passage human fibroblasts. Collagenase (MMP-1), measured by functional assays in the absence of TIMP and also as immunoprecipitated [35S]methionine-labeled protein, was increased 10-20-fold following ConA (20 micrograms/ml, 2 x 10(-7) M) treatment for 24-72 h, with active collagenase comprising approximately 20% of the total collagenase activity. By comparison, MMP-2 (72-kDa gelatinase; molecular mass, 72 kDa, +dithiothreitol; 66 kDa, -dithiothreitol), analyzed by enzymography and following affinity purification, was increased less than 2-fold by ConA and was present entirely as an activated, 61-kDa (+dithiothreitol; 59 kDa, -dithiothreitol) form. Northern hybridization analyses revealed that ConA elevated the steady-state mRNA levels for MMPs; collagenase mRNA increased approximately 16-fold, MMP-2 increased 2-fold, and Pump-1, a recently described MMP gene, was induced. Concomitantly, a 10-fold reduction in TIMP protein and mRNA levels by ConA occurred. In comparison, 12-O-tetradecanoylphorbol-13-acetate (50 ng/ml, 8 x 10(-8) M), which also stimulates collagenase expression strongly (greater than 30-fold), elevated TIMP protein and mRNA levels (2- and 3-fold, respectively) and did not affect MMP-2 expression. The changes in MMP and TIMP mRNA levels induced by ConA were blocked by the protein synthesis inhibitor cycloheximide, and the half-lives of collagenase and MMP-2 mRNAs (53 and 46 h, respectively) were unaffected, indicating that ConA exerts its effects transcriptionally, through pathways requiring de novo protein synthesis. Increased transcription of the mmp genes was confirmed by nuclear run-on analyses; mmp-1 transcription was increased by greater than 25-fold, mmp-2 by approximately 3-fold, and Pump-1 by approximately 7-fold. In contrast, Timp gene transcription was reduced by approximately 80%, revealing reciprocal regulation of MMPs and TIMP during the induction of a resorptive cell phenotype. Decreased amounts of collagen and fibronectin, but not of SPARC (secreted protein, acidic and rich in cysteine) in the conditioned medium was the result of MMP activity since steady-state mRNA levels and transcription of the respective matrix protein genes were unaffected by ConA.