Concentration-dependent switch-on and -off mechanism of cell-growth promoting activity of TIMP-1

已经报道了TIMP-1在其细胞生长促进活性上显示出典型的钟形剂量曲线，表明TIMP-1的细胞生长促进活性存在一些浓度依赖性调节（Hayakawa et al.，J. Cell Sci. 107，2373-2379，1994; Yamashita等人，FEBS Lett. 396，103-107，1996）。为了证实这一点，首先制备人TIMP-1基因转染的[CGEMT 1]和空载体转染的[CGEMEV] HT 1080细胞，并比较它们在不含胎牛血清的Dulbecco改良的Eagle最小必需培养基（DMEM）中的增殖和TIMP-1表达/分泌。CGEMT 1细胞分泌TIMP-1，并在2天的滞后期后增殖。然而，CGEMEV细胞既不分泌TIMP-1也不增殖。加入微量的抗TIMP-1单克隆抗体后，CGEMT 1细胞的增殖完全被抑制，表明细胞的增殖完全依赖于细胞产生的TIMP-1。 ...更多信息 自身，即TIMP-1的细胞生长促进活性的自分泌机制。此外，细胞增殖在培养第4天左右趋于平稳，TIMP-1分泌停止并达到平台。培养液中TIMP-1浓度约为100 ng/ml。然后，我们将CGEMT 1细胞保存在补充有100 ng/ml人重组TIMP-1的D-MEM中。细胞既不增殖也不分泌TIMP-1，但从仅用DMEM替换培养基后1天开始分泌TIMP-1并增殖。在HT 1080细胞的细胞内信号转导方面，TIMP-1在低浓度（~ 25 ng/ml）时对MEK和p238 MAPK的激活作用最强，而在高浓度（>100 ng/ml）时则无激活作用。然而，ERK 1/2在所有检测浓度（25 ~ 150 ng/ml）下均被TIMP-1持续激活。这些结果有力地支持了我们的假设，即，用于控制TIMP-1的细胞生长促进活性的浓度依赖性开关机制的存在。少

It has been already reported that TIMP-1 shows a typical bell-shaped dose curve on its cell-growth promoting activity, suggesting the presence of some concentration-dependent regulation of the cell-growth promoting activity of TIMP-1(Hayakawa et al., J. Cell Sci. 107, 2373-2379, 1994; Yamashita et al., FEBS Lett. 396, 103-107, 1996). To confirm this point, human TIMP-1 gene-transfected [CGEMT1] and empty vector-transfected [CGEMEV] HTlO8O cells were first prepared, and compared their proliferation and TIMP-1 expression/secretion in fetal calf serum-free Dulbecco's modified Eagle's minimal essential medium(DMEM) were examined. CGEMT1 cells secreted TIMP-1 and proliferated after 2 days lag period. CGEMEV cells, however, neither secreted TIMP-1 nor proliferated at all. Proliferation of CGEMT1 cells were completely suppressed by the addition of a minute amount of anti-TIMP-t monoclonal antibody, clearly indicating that the proliferation is totally depend on the TIMP-1 produced by cells the … More mselves, that is, an autocrine mechanism of the cell-growth promoting activity of TIMP-1. Furthermore, cell proliferation leveled off at around 4 culture day, and TIMP-1 secretion ceased and got a plateau. TIMP-1 concentration in the culture medium was around 100ng/ml. Then, we kept CGEMT1 cells in D-MEM supplemented with 〜160 ng/ml human recombinant TIMP-1. The cells neither proliferated nor secreted TIMP-1, but started to secrete TIMP-1 and proliferate from 1 day after replacing the culture medium with DMEM alone. Concerning to the intracellular signal transduction in HT1080 cells, TIMP-1 activated both MEK and p238 MAPK maximally at the low concentration (〜25 ng/ml), but not activated at the higher concentration (>100 ng/ml). ERK1/2, however, were activated constantly by TIMP-1 at all the concentrations examined (25〜150 ng/ml). These results strongly support our hypothesis, i.e., the presence of a concentration-dependent switch-on and -off mechanism for the control of cell-growth promoting activity of TIMP-1. Less