As some of senescence markers were reversed by down-regulation of PKCa and PKC inhibition, effects of PKCa down-regulation on cell proliferation were evaluated. Mid-old and Mid-old HDF cells were treated with GFP or PKCa siRNA. As shown in Fig. 15, PKCa down-regulated mid-old and old HDF cells proliferated faster than GFP siRNA treated cell.
However, down-regulation of PKCa did not affect the proliferation ability in young cells.
Fig. 14. Senescence phenotype could be reversed by PKCa down-regulation. (A) PKCa was down-regulated by PKCa siRNA. Mid-old cells were treated with 20 nM PKCa siRNA or 20 nM GFP siRNA for 2 days. PKCa was down-regulated by PKCa siRNA treatment in mid-old HDF cells, but not by GFP siRNA. (B) Marked difference in ROS level between young and mid-old HDF cells, measured by FACS analysis (left panel). Young and mid-old HDF cells were seeded. After 24h, the cells were treated with H2DCF-DA 10 min prior to harvest. Changes of fluorescence by ROS generated were measured by FACS analysis. ROS level of PKCa siRNA treated mid-old cells was significantly decreased (right panel). Mid-old cells were pretreated with PKCa siRNA or GFP siRNA for 2 days and treated with H2DCF-DA 10 min prior to harvest. (C) Mid-old cells were treated with PKCa siRNA or GFP siRNA. As revealed in these figures, morphological change was induced by PKCa siRNA treatment for 2 days. (D) To evaluate change in molecular markers of senescence, immunoblot analysis of p21WAF1, p53, and SA-p-Erk1/2 were performed. p21WAF1, p53, and SA-p-Erk1/2 levels were increased in mid-old cells (left panel). However, the molecular markers of senescence of PKCa siRNA treated mid-old cell were lower than those of GFP
siRNA treated cells. (E) Non-specific PKC inhibitor, GF109203X, showed effects similar to PKCa siRNA treatment in mid-old cells. Mid-old cells were treated with vehicle (DMSO) or GF109203X (4 mM) for 3 days. Morphology of GF109203X treated cells clearly changed to young cell-like cells and the molecular markers of senescence were also decreased.
Fig. 15. PKCa down-regulated old cells regained proliferation ability. Young, mid-old, old HDF cells (4 X 103) were seeded into 12-well plates for 16 h and were treated with GFP siRNA (20 nM) or PKCa siRNA (20 nM) for indicated time. In mid-old and old HDF cells, PKCa siRNA treated cells proliferated faster than GFP siRNA treated ones (middle and right panel). On the other hand, PKCa down-regulation had no effect on proliferation ability in young cells (left panel).
Ⅳ. DISCUSSION
Replicative senescence of human fibroblasts has frequently beenused as an aging model in vitro (Cristofalo and Pignolo, 1993). In the present study,we have shown that a large amount of p-Erk1/2 proteins, denotedas SA-p-Erk1/2, were found in the cytoplasm of senescent HDF cells (Fig. 1), and that the SA-p-Erk1/2 were competent substrates for PP1/2A, PTP, and MKP-rVHR in vitro as well asin vivo (Fig. 2). During the process of cellular senescence,PP1/2A (Fig. 4B) activities were significantlyreduced; however, their protein expression levels were not affected(Figs. 4D). The decrease seemed to coincide temporarilywith the aging process, because the phosphatase activities werealready reduced in the mid-old HDF cells
The phosphorylation state of any given protein in vivo dependson the balance between the activities of phosphatases and kinases,and there is increasing evidence that relatively few Ser- orThr-specific protein phosphatases with pleiotropic action participatein cellular regulation. Therefore, the finding that a significantdecrease of PP1 activities in both the cytoplasm and nuclearfractions of the senescent cells without any change in theirprotein expression (Fig. 4D) resulted in the induction of p-Erk1/2levels that might possibly be explained by the existence andinduction of phosphatase inhibitors such as inhibitor-1 or an unidentified inhibitor during cellular senescence. Therefore,in order to verify the presence or increase of a putative inhibitorin the old cells, we carried out two approaches as follows:
detection of the known inhibitor 1 expression in the young andold cells by immunoblot
analysis, and measurement of PP1/2A activity after mixing the young and old cell lysates.
Historically, protein phosphatase inhibitor-1 (inhibitor-1, or I-1) was the first such endogenous molecule found to regulateprotein phosphatase activity in vivo (Huang and Glinsmann, 1976). This 19-kDa protein has a highly conserved primary sequence in vertebrates rangingfrom fish to mammals (Elbrecht et al., 1990). Phosphorylationat Thr35 by cAMP-dependent protein kinase (PKA) converts theinactive protein into a selective and highly potent inhibitorof the catalytic subunit of PP1 (IC50 : 1 nM) (Huang and Glinsmann, 1976; Foulkes et al., 1983). When analyzed by immunoblot analysis, the protein expression levels among the two were basically the same (data not shown).Also, when increasing amounts of the old cell lysates were added to a fixed amount of young cells and the PP1/2A activity wasmeasured, the sums of the activity measured individually werethe same as those in the mixture, indicating the absence orno increase of an inhibitor in the old cell lysates (data notshown). The above two experiments verified the fact that thedecreased PP1/2A activity in the old cells was not due to increaseof a putative inhibitor in the old cells.To investigate further the mechanistic evidence of the reducedPP1/2A activity during cellular senescence, thiol-specific reducingagents were added to the cell lysates whose cells had been pretreatedwith H2O2. As seen in Fig. 8, the reducing agents could significantlyrestore the H2O2-inhibited PP1/2A activities in the young HDF cells and increase the PP1/2A activity in mid-old cells. Thesedata strongly indicated that the inhibition of phosphatase activitymight have been due to the oxidation of reactive site cysteineresidue(s) by ROS during cellular senescence. It should be notedthat the catalytic domains of PP1, PP2A, and calcineurin arehighly homologous, i.e. 49% identity between PP1 and PP2A and40%
identity between PP2A and calcineurin. However, their substrate specificities and interactions with regulatory molecules are very different. The crystal structure of their catalytic domains revealed similarly folded catalytic cores, each of which containstwo catalytic metals (Goldberg et al., 1995; Kissinger et al., 1995). There is very little informationavailable about the effects of oxidants on the activity of PP1/2A.However, Sommer et al. (Sommer et al., 2002) recently revealed that the activitiesof PP1/2A in SK-N-SH cell lysates were reversibly inhibitedby H2O2. ROS-inhibited PP1 activity could be reversed by treatingSK-N-SH cells either with thiol-reducing agent DTT or metal-reductant ascorbate. Our results together with the above observation led us to suggest that the mechanism of inhibition of PP1/2A inHDF cells was due to direct attack of H2O2 on cysteine residue(s)of PP1/2A but not due to a regulating factor activity.As shown in Fig. 2 and Fig. 3, SA-p-Erk1/2 served as a substratefor PP1/2A. Because the inhibition constants (Ki) of PP1 andPP2A by okadaic acid are 150 nM and 32 pM, respectively (Takai et al., 1995),induction of p-Erk1/2 in the young and old HDF cells 4 h afterthe treatment with 40 nM okadaic acid (Fig. 4E, lanes 2–4 and 6–8) strongly indicated that the preferential inactivationof PP2A during cellular senescence could also be significantlyresponsible for SA-p-Erk1/2 proteins. The reason of why a robust induction of p-Erk1/2 by a single treatmentwith 10 mM H2O2 was greatly attenuated in 40 min might be foundin the rapid induction of MAPK through EGF receptor (Morey et al., 2001; Nishinaka and Yabe-Nishimura, 2001),and the significant but transient reduction of PP1 and PP2Ain HDF cells, which were significantly inhibited in 10 min butrecovered by 40 min and reached the control level in 8 h (Fig.5C). It is important to note that, when the cells were treatedwith
H2O2 every 8 h for more than 72 h, activities of PP1/2Awere persistently reduced, and the level of p-Erk1/2 was constitutivelyhigh (Fig. 5D), resulting in SA-p-Erk1/2. These findings implicatethat cells in vivo might be continuously exposed to the higherlevel of ROS during the senescence process, thus resulting in the inhibition of PP1/2A and the constitutive increase of p-Erk1/2.This was supported by the 20 times more ROS measured in themid-old cells than the young cells (Fig. 5A). When FACS analysiswas employed to measure ROS generation, data acquisition fromthe old cells was impossible to obtain population-gated, becausethe size was too large and the fragility of the oldcells, thus necessitating the use of the mid-old cells for theexperiment. Even though the cells were stimulated with 1 mMH2O2 and markedly induced p-Erk1/2 levels, the huge inductioncould completely be prevented by NAC pretreatment (Fig. 5B).Furthermore, treatment of the young cells only once with a lowerconcentration of H2O2 (200 µM) significantly inhibitedphosphatase activity along with the concomitant increase ofp-Erk1/2 proteins 5 min after the treatment. However, when treatedrepeatedly with H2O2 at every 8 h, the PP1/2A activities remained significantly inhibited even after 72 h (Fig. 5D). The levels of p-Erk1/2 in the young cells were reciprocally regulated withthe PP1/2A activities by the same treatment.
To release cytoplasmic retention of SA-p-Erk1/2, young and old HDF cells were treated with EGF or TPA. Not EGF, but TPA, induced nuclear translocation of p-Erk1/2 (Fig. 7) accompaniedby the induction of DNA synthesis (Fig. 8B), implying stronglythat the G1 arrest during cellular senescence may be causedby PKC pathway. Involvement of PKC was additionally confirmed by the use of Go6976, a PKC inhibitor, which inhibitedTPA-induced DNA synthesis in the old cells (Fig. 8C), indicating a significant role of PKC in the reversal
of senescence phenotypes. Failure of PKC activation by EGFmay be explained by recent reports of up-regulation of caveolinand down-regulation of amphiphysin-1 attenuate EGF signalingin the senescent HDF cells through the receptor-mediated endocytosis(Park et al., 2000; Park et al., 2001; Park et al., 2002). These findings are strengthened additionallyby the recent observation that reduction of caveolin expressionreverses the senescent phenotype in HDF cells (Cho et al., 2003). The earlier report that phosphorylation of RB protein occurred10–20 h after serum stimulation, accompanied by inductionof DNA synthesis (Futreal and Barrett, 1991), is in good accord with our present observationthat TPA reversed senescent cell morphology to youngcell-like in 8 hr, and that the reversal became evidentin 20 h (data not shown). TPA treatment also reversed thewell-known biochemical markers of cellular senescence in 20 h, including RB protein phosphorylation, down-regulations of p21WAF1 and p-Erk1/2, caveolin-1, and caveolin-2 expressions(Fig. 9).
MAPK pathways control cell proliferation and cell differentiation mainly through the regulation of transcription factors in the nucleus. Thus, to transmit extracellular signals to the nucleus, the terminal component of the MAPK pathways—that is, MAPK—must translocate from the cytoplasm to the nucleus. Widely accepted mechanism of nuclear translocation of activated Erk1/2 (p-Erk1/2) is that activated p-Erk1/2 is released from cytoplasmic MEK1/2 (Fukuda et al., 1997). Dissociated from MEK1/2, Erk1/2 then translocates to the nucleus by three mechanisms: passive diffusion of a monomer, active transport of a dimer, and direct interaction with the nuclear pore complex (Khokhlatchev et al., 1998; Adachi et al., 1999; Matsubayashi et al., 2001; Whitehurst et al., 2002; Kondoh et al., 2005). In this study, another possible mechanism of Erk1/2 nuclear translocation might
be proposed. ‘PKC a - Erk1/2 interaction’ by TPA treatment was increased and translocated into nucleus (Fig. 10C and Fig. 11).
However, DNA synthesis and morphological change occurred in 20 hr of TPA treatment.
At that time, PKCa was already down-regulated (Fig. 12). Therefore, PKC down-regulation may have some role in reverse of senescence phenotypes. Therefore, PKC isozymes were evaluated to elucidate which isozyme was activated by TPA treatment. PKCa and bI were activated by TPA treatment in HDF cells (Fig. 10A). As PKCa has been proposed to be implicated in control of the G1/S transition (Sasaguri et al., 1993; Frey et al., 1997; Besson and Yong, 2000; Frey et al., 2000), PKCa was focused in this study. Protein level of PKCa was not changed during the senescence process, however, its activity of old cells was greater than that of young cells. It seemed that the change was controlled by cellular ROS level (Fig.
13C). ROS, such as H2O2 and oxidative stress, are already known to activate PKC activity (Konishi et al., 1997; Min et al., 1998; Wang et al., 2004). Treatment of young cells with H2O2 induced PKCa activation. In addition, PKC a activity was decreased in NAC, ROS scavenger, treated old cells. These observation strongly suggests that increased ROS level of old cells constitutively activates PKCa. However, H2O2 failed to change intracellular localization of PKCa (Fig. 13D and E). These observations were evidenced the increase of PKCa activity, but no translocation of PKCa in old cells.
Duration of PKCa activation might be important in G1 phase arrest or senescence process. Transient activation of PKCa leads to cell proliferation, however, sustained activation of PKCa with synthetic DAG analog induces p21WAF1 induction and G1 phase cell cycle arrest (Frey et al., 1997), and constitutive expression of an active PKCa mutant in
HepG2 induces gene expression of p16INK4a and growth inhibition of HepG2 (Wen-Sheng, 2006).
To define roles of PKCa in senescence process, mid-old cells were treated with PKCa siRNA. Some senescence phenotypes were reversed in PKCa down-regulated mid-old cells.
In addition, the levels of ROS, cell cycle regulator (p53, p21WAF1), and SA-p-Erk1/2 were decreased (Fig. 14. B-D). PKC inhibitor as well as PKCa siRNA decreased expression of molecular markers and induced morphological change of senescence. Most characteristic feature of senescent cells is diminished proliferative ability. In Fig. 15, the effect of PKCa regulation on proliferation ability was evaluated. In young cells, PKCa down-regulation has no effect on cell proliferation, however, PKCa down-down-regulation in mid-old and old cells accelerated cellular proliferation. These observations strongly support that PKCa might play a major role for maintenance of senescence phenotype.
When pancreatic cancer cell (Detjen et al., 2000) and mammary epithelial cells (Slosberg et al., 1999) were treated with TPA, PKC a activation and p21WAF1-mediated G1 arrest were induced. In mammary epithelial cells, activated PKCa leads to increased transcription of p21WAF1. The increased level of p21WAF1 protein retards cell cycle progression by binding to and inhibiting the kinase activity of G1 cyclin-CDK complexes (cyclinD-CDK4, cyclineE-CDK2), thereby causing an inhibition of G1 progression.
Therefore, during the senescence process, elevated ROS level activated PKCa, and activated PKCa then induced increase of SA-p-Erk1/2, p21WAF1, ROS level, and senescence-characteristic morphology and G1 cell cycle arrest (Fig. 16).
Therefore, future work is needed to elucidate the exact role of PKCa in senescence
process or in cell proliferation and to provide important insight not only into reversal of senescence, but also into cancer cell senescence, thus facilitating the development of strategies for aging or cancer therapy.
Fig. 16. Role of PKCa in the senescence process and in the reversal of senescence. The figure represents a suggestive model depicting a role of PKC a in senescent process (upper panel). Activated PKCa induced senescence associated features. When PKCa was activated, the levels of ROS, SA-p-Erk1/2, p53, and p21WAF1 were increased. However, when PKCa was inhibited by an inhibitor or down-regulated by long-term TPA treatment or PKCa siRNA treatment, senescent phenotypes were reversed and G1→S phase of cell cycle was progressed (lower panel).
Ⅴ. CONCLUSION
In summary, SA-p-Erk1/2 proteins accumulated in the cytoplasmof old HDF cells were due to the reduced PP1/2A activities, resulting in retention ofp-Erk1/2 in the cytoplasm and prevention of their translocationto the nucleus. Failure of nuclear translocation of p-Erk1/2 might result in stoppage or slow down of mitosis in old cells.
TPA treatment induced DNA synthesis and change of senescent morphology. TPA induced PKC a activation and nuclear translocation with p-Erk1/2. However, activated PKCa by TPA was down-regulated. Down-regulation of PKCa seemed to have effects on reversal of senescence phenotype.
Protein level of PKCa was not changed during the senescence process. However, its activity in old cells was greater than that of young cells. It seemed that change of this activity was controlled by cellular ROS level. ROS such as H2O2 and oxidative stress are known to activate the PKC activity. Treatment of young cells with H2O2 induced PKCa activation. In addition, PKCa activity was decreased in NAC, ROS scavenger, treated old cells. These observation strongly suggest that increased ROS level of old cells constitutively activates PKCa.
Constitutive activation of PKCa seemed to have effects on maintenance of senescent phenotype. Active PKCa induced ROS, p53, p21WAF1, and SA-p-Erk1/2 in old cells. Induced p21 WAF1 by activated PKCa mediated G1 arrest in old cells. p21WAF1 inhibited the kinase activity of G1 cyclin-CDK complexes (cyclinD-CDK4, cyclineE-CDK2), thereby causing an
inhibition of G1 progression.
Therefore, during the senescence process, elevated ROS level activated PKCa, and activated PKCa then induced the increase of SA-p-Erk1/2, p21WAF1, and senescence-characteristic morphology.
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