Since TPA is known not only as a PKC activator and a down-regulator of PKC (Lindner et al., 1991; Li et al., 1998; Lu et al., 1998) as well as reversal of senescence phenotype in 20 hr after TPA treatment, we measured PKC a expression level (Fig. 12). PKCa in the old cells was regulated from 8 hr after TPA treatment. p-Erk1/2 level was also down-regulated from 12 hr after TPA treatment. From these results, we suggested that the expression and activity of PKCa could be related with maintenance of senescence phenotype. To investigate whether there is any change in PKCa expression level or activity, young, mid-old, and old HDF cells were used for PKCa immunoblot analyses (Fig. 13A).
Protein expression level was not significantly changed between young and old cells, however, activity of PKCa activity was significantly higher in the mid-old cells than young
cells (Fig. 13B). ROS such as H2O2 is able to activate PKCa (Konishi et al., 1997; Min et al., 1998; Wang et al., 2004) and ROS level is increased in old cells. To determine whether ROS is a major factor in PKCa activation, we assessed the PKCa activity after H2O2 or NAC treatment. H2O2 induced activity of PKCa in young HDF, conversely, NAC, as a ROS scavenger, reduced PKCa activity (Fig. 13C). To evaluate intracellular localization of activated PKCa with ROS, subcellular fractionation of HDF cells and immunocytochemistry were performed. H2O2 treatment failed to induce translocations of PKCa and p-Erk1/2 from cytoplasm to nuclear fraction (Fig 13D right panel), and the result is well accordant with the previous report (Rybin et al., 2004). In addition, H2O2 treatment neither translocated PKCa from soluble to particulate fractions (Fig 13D left panel). As shown in Fig 13E, H2O2 did not induce intracellular localization of PKCa. Together, these results indicate that ROS induced activity of PKCa but not its translocation.
I. Senescence phenotype is reversed by PKCa down-regulation.
To determine the correlation between PKCa and senescence phenotype, PKCa was down-regulated by using PKCa siRNA. Fig. 14A shows that PKCa was down-down-regulated by PKCa siRNA treatment, but not by GFP siRNA, in mid-old HDF cells. Increased ROS content is one of the characteristics in senescence process. ROS level of mid-old HDF was greater than that of young HDF (Fig. 14B left panel). In mid-old HDF, PKCa siRNA treatment decreased ROS level (Fig. 14B right panel). Another characteristic feature of senescent cell is wide and flat morphology. Fig. 14C shows that PKCa siRNA treatment also changed morphology of
HDF cells from flat and large to slim and small. It was the same as TPA treatment. To determine the change of molecular markers of senescence, p53, p21WAF1, and SA-p-Erk1/2 were evaluated. As shown in Fig. 14D, they were up-regulated in mid-old cells. However, these markers were significantly decreased by treatment with PKCa siRNAs. These phenomena were also repeated by NAC treatment. The result was is in good accordance with Fig. 13C. NAC treatment inhibited PKCa activation by ROS, therefore, the molecular markers of senescence observed in mid-old HDF were reversed. To investigate whether PKC inhibitor reproduces the same effects as those of PKCa siRNA treatment, mid-old HDF cells were treated with GF109203X, PKC inhibitor. Indeed, the inhibitor could induce morphological change and decrease of the molecular markers of senescence (Fig. 14E).
These data suggest that PKCa has an important role in maintenance of senescence phenotypes.
Fig. 11. Activated PKCa interacts with p-Erk1/2. (A) To investigate whether the PKCa was bound to p-Erk1/2 or not and also to compare the bound p-Erk1/2 between the young and old cells, 500 mg of each cell lysates were immunoprecipitated with anti-PKCa antibody.
Activated PKCa by TPA treatment interacted with p-Erk1/2 15 min after TPA treatment.
However, their interaction was abrogated after 4 hr. (B) Interaction between p-Erk1/2 and PKCa was increased in young HDF cells by TPA treatment.
Fig. 12. Persistent treatment of TPA down-regulated PKCa. Mid-old HDF cells were treated with TPA (50 ng/ml) and harvested at indicated time for immunoblot analysis.
Protein level of PKCa was decreased from 8 hr after TPA treatment. p-Ekr1/2 level was also decreased from 12 hr after treatment.
Fig. 13. Increased ROS level activated PKCa, but did not induce intracellular translocation. (A) In young, mid-old, and old HDF cells, there was no marked difference in
PKCa protein expression level. (B) To investigate the change in PKCa activity during the senescence process, young and mid-old HDF cell lysates were immunoprecipitated with PKCa antibody, and the The PKCa immunoprecipitates were washed twice with immunoprecipitation buffer, then twice with kinase buffer and resuspended in 20 ml of kinase buffer. The kinase assay was initiated by adding 40 ml of kinase buffer containing 5 mg of MBP substrate and 5 mCi of [g-32P]ATP. The reactions were performed at 30oC for 30 min. The reactions were terminated by adding SDS sample buffer and boiled for 5 min. The reaction products were analyzed by SDS-PAGE and autoradiography. In mid-old cells, the activity of PKCa was greater than that of young cells. (C) ROS activated PKCa activity.
Young HDF cells were treated with 1mM H2O2 or 50 ng/ml TPA for 15min, IP-kinase assay was preformed. In H2O2 and TPA treated young cells, PKCa was activated. However, NAC (10 mM) treatment reduced PKC a activity in mid-old cells. (D) To evaluate intracellular localization of PKCa with ROS treatment, subcellular fractionation of young cells was performed. H2O2 treatment did not induce translocation of PKCa from soluble fraction(S) to particulate fraction(P) (right panel). In addition, H2O2 did not translocate of PKCa from cytoplasm(C) to nucleus(N) (left panel). (E) Young and old cells were treated with 1 mM H2O2 for 15 min. And intracellular localization was determined by immunocytochemistry. In accordance with subcellular fractionation, PKCa did not translocate by H2O2 treatment.