| Aging involves a progressive loss of physiological capacities at both cellular and tissue levels and is an ultima and congenerous behavior of cells excitated by diversely physiological stimulations. A mass of experiments demonstrated that signal pathway was in charge of initiation and persistence of aging. In this paper, by using human replicative senescent diploid fibroblast WI-38 cells, we try to find out the changs of Stat3 avtivity, which is an important member of JAK/STAT signal pathway, and the changes of MMPs/TMPs system in replicative senescence, as well as the role of angiotensin n in aging.By Western blotting we found that senescent cells expressed a higher level of total StatS protein than young cells. Angiotensin II stimulation did not modulate the expression of total StatS in these cells. However, angiotensin II treatment induced StatS phosphorylation in both young and senescent cells. Examination of nuclear extracts revealed that young cells had a higher increase in the amount of p-Stat3 in the nuclei than in that of senescent cells, suggesting a failure to translocate phosphorylated StatS to the nucleus in senescent cells. Indirect immunofluorescence staining supported the above results. In untreated young and senescent cells, the immunoreactivity was distributed weakly and diffusely in both cytoplasm and nucleus. A 60 minexposure to angiotensin II resulted in a rapid increase of immunofluorescence activity in the nucleus of young cells. In contrast, the staining signals were not concentrated in the nuclei but diffusely distributed within the cells in senescent cells. Therefore, this experiment provided further evidence that nuclear translocation of phosphorylated activated Stat3 was decreased in the senescent cells. Similar experiments were performed to detect Stall, whereas the levels of p-Statl in both cell lysates and nuclear extracts were very weak with or without angiotensin II stimulation in both young and senescent cells, suggesting that angiotensin II induce primarily Stat3 phosphorylation in human fetal lung diploid fibroblast. EMSAs showed that the DNA-binding activity of Stat3 was significantly decreased in senescent cells. Angiotensin II induced the DNA-binding activity of Stat3 in both time- and dose-dependent manners in senescent cells via ATI receptor. The maximal SIF formation was at 60min after angiotensin II stimulation. Supershift analysis demonstrated that the predominant complexs formed was SEF-A and SIF-B. These results prove that in senescent cells, the constitutive or growth stimulated activity of Stat3 was reduced when compared to that in young cells and that angiotensin II could induce Stat3 activity through ATI receptor in time- and dose-dependent manners.Furthermore, gelatin zymography showed that WI-38 cells primarily produced MMP-2. MMP-2 activity was decreased in senescent cells when compared with young cells. The activity of MMP-9 was very weak and comparable in both senescent and young cells. Moreover, angiotensin n slightly increased the levels of MMP-2 but not MMP-9 in senescent or young cells. Interestingly, senescent cells appeared to express more TIMP-1 protein than young cells. Exposure of senescent cells to angiotensin II resulted in marked up-regulation of TIMP-1 protein expression. As TEVIP-1 promoter contains the SIF binding site, we further investigated whether the up-regulation of TIMP-1 expression by angiotensin II stimulation require theactivation of Stat3 signal protein in senescent cells. Senescent cells were transiently transfected with Stat3 antisense plasmid U6-Stat3-AS targeting the translation start site of Stat3 messenger RNA and dominant negative Stat3 mutant constructs (3F, where tyrosine 705 in Stat3 is replaced by phenylalanine), then treated with angiotensin II. The results manifested the up-regulation of TDvtP-1 protein expression following angiotensin II stimulation was significantly inhibited in senescent cells transfected with Stat3 antisense plasmid or dominant negative Stat3 mutant plasmid. In contrast, trea...
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