Chapter 4 P27kip1 Down-Regulation Was Not an End-Point in Rapamycin Inhibition of Transforming Growth Factor (TGF)-β1-Stimulated Cell Cycle Progression in C3H-10T½ Murine Embryonic Fibroblasts
Rapamycin is an antifungal macrolide that inhibited transforming growth factor-β1 (TGF-β1)-mediated DNA synthesis. TGF-β1 down-regulated the cyclin-dependent kinase inhibitory protein p27kip1 in various cellular systems including C3H-10T½ murine embryonic fibroblasts. P27kip1 was therefore a potential contributor to rapamycin inhibition of TGF-β1-mediated cell cycle entry. Immunoblot analysis of p27kip1 demonstrated that TGF-β1 down-regulated this cdk inhibitor. However, rapamycin did not accumulate p27kip1. Furthermore, rapamycin alone induced p27kip1, suggesting a rapamycin-sensitive stress-like response that may be independent from a TGF-β1-regulated pathway. TGF-β1 down-regulation of cyclin D1 was not affected by rapamycin in these fibroblasts and cyclin-dependent kinase (cdk) 4 levels were constitutive across prescribed conditions.
These results suggest that in C3H-10T½ murine embryonic fibroblasts, p27kip1 regulation contributed to TGF-β1-mediated stimulation but was not an end-point in rapamycin effects. Independent pathways from down-regulating p27kip1 were probably present in rapamycin-sensitive signaling.
Cell cycle transition is governed by interactions between accelerating and braking components comprising the cell cycle machinery. Stoichiometric behavior between stimulatory components and inhibitory components in the cell cycle enables extracellular signals to regulate phase progression by altering the availability of participating components. Changing the abundance and activity of one component would affect all interactive partners of that component. Regulating protein availability rather than phosphorylation is energetically conservative, where, under stressful conditions, the cell avoids expending energy to synthesize protein components towards post-translational modification.
Accelerating components of the cell cycle include the periodically expressed cyclins and respective kinase partners. Braking components include families of inhibitory proteins grouped according to structural homology. Cyclin D1 is an important G1 cyclin that prompts G1/S progression by hyperphosphorylating the tumor suppressor retinoblastoma protein (pRB). Conformational changes in pRB from successive hyperphosphorylation events result in the dissociation of sequestered transcription factor complex E2F/DP-1.
In addition to catalyzing cell cycle progression, cyclin D1 conferred proliferative potential towards tissue regeneration, as cyclin D1-dependent kinase activity was demonstrated to increase during G1 phase following partial hepatectomy to promote liver regeneration (Kato et al, 1998). Cdk-inhibitors brake against cyclin-cdk complex acceleration into the cell cycle. Cdk-inhibitors include members of the CIP family ("Cyclin Inhibitory Protein" p21cip1, p27kip1, and p57kip2) and the INK4 family ("cdk 4-Inhibitor Kinase gene" p15ink4b, p16ink4a, p18, and p19) categorized by structural homology. P27Kip1 was discovered as an accumulated heat labile factor during cell cycle arrest and provided a putative link between TGF-β1-mediated growth-arrest to cell cycle components (Polyak et al, 1994). In various systems, p27kip1 was induced under stress-related or inhibitory signals by anti-mitogens such as rapamycin and wortmannin, and appeared to be targeted by various anti-proliferative pathways. P27kip1 inhibits the activation of cyclin E-cdk 2 complexes and tightly binds to cyclin D1-cdk 4. The inhibition by p27kip1 of cyclin E is titratable by accumulating cyclin D1 complexes.
P27Kip1 is notably regulated at the protein level rather than at the mRNA level. P27kip1 is induced under treatment with many cell cycle inhibitors and was observed to participate in inflammation, apoptosis, cellular stress responses, and differentiation. Given the important roles of p27kip1 and cyclin D1 in cell cycle progression, and given observations that p27kip1 and cyclin D1 were often associated with growth factor-mediated responses as well as growth inhibitor-mediated cell cycle arrest, the effects of rapamycin on p27kip1 and cyclin D1 levels against TGF-β1-stimulated S-phase entry were examined.
Materials and Methods
Cells were cultured according to Materials and Methods, Chapter 2. MCA-10T½ fibroblasts were observed to have lost the property of contact inhibition of growth (Reznikoff et al, 1973b).
Calculation of Generation Time
Generation time was calculated according to Materials and Methods, Chapter 2.
Whole cell extracts were isolated and immunoblot analyses implemented according to Materials and Methods, Chapter 2.
Rapamycin inhibited TGF-β1-mediated DNA synthesis
Cell cycle inhibitors rapamycin and wortmannin were observed to arrest cell cycle progression at G1 phase. To determine whether rapamycin and wortmannin inhibited TGF-β1-mediated DNA synthesis, incorporation of tritiated thymidine into DNA were monitored in radiolabeling assays. TGF-β1 significantly stimulated DNA synthesis when compared with control cells receiving no growth factor. TGF-β1-mediated stimulation was inhibited by nanomolar concentration (10 nM) of rapamycin, Figure 4-1. Wortmannin, on the other hand, did not significantly affect TGF-β1-stimulated DNA synthesis. Therefore rapamycin, but not wortmannin, interfered with TGF-β1-mediated S-phase entry.
TGF-β1 downregulation of p27kip1 and cyclin D1 was not affected by either rapamycin or wortmannin
P27kip1 was a common endpoint in measuring growth factor-mediated effects in various cellular models, including those of mesenchymal origin such as C3H-10T½ murine fibroblasts. P27kip1 down-regulation was examined in these fibroblasts to confirm correlation with S-phase entry at 16 hours. The time-point chosen was based on radiolabeling assay kinetics of cell cycle progression (please refer to Figure 6-2.) Rapamycin and wortmannin were also examined to determine whether these drugs inhibited TGF-β1-mediated p27kip1 down-regulation.
C3H-10T½ fibroblasts were grown to confluence and staged overnight in low-serum media before pre-incubation with rapamycin or wortmannin. Transforming growth factor-b1 was added to drug-treated cells for incubation at 37°C for 16 hours before harvesting for whole cell extracts. Cell extracts obtained for each experimental condition were normalized for equal protein loading and resolved by SDS-PAGE according to Materials and Methods, Chapter 2. Electrophoretic transfer onto nitrocellulose or PVDF membranes was followed with immunoblot analysis with polyclonal antibodies against p27kip1 and visualized via enhanced chemiluminescence.
As expected, when compared with control cells receiving no growth factor or compared with contact-inhibited cells, TGF-β1 down-regulated p27kip1 levels, Figure 4-2. This confirmed previous observations that cell cycle progression under TGF-β1 treatment corresponded with p27kip down-regulation. Surprisingly, rapamycin did not accumulate p27kip1 levels against TGF-β1, Figure 4-2, suggesting that the inhibitory effects of rapamycin observed in biochemical analyses must be due to p27kip1-independent mechanisms. Compensating mechanisms against rapamycin-sensitive p27kip1-accumulation may also contribute to these observations. While rapamycin had been demonstrated to accumulate p27kip1 levels to inhibit the mitogenic effects of various growth factors including insulin and interleukin-2, this may be a cellular context-specific effect.
Rapamycin and wortmannin did not affect TGF-β1-mediated p27kip1 down-regulation
Cyclin D1-cdk 4 complex is an important component in the cell cycle regulatory circuit, both for hyperphosphorylating pRB in G1 phase and for titrating the inhibitory activity of p27kip1 from cyclin E-cdk 2 complexes. Due to the stoichiometric relationship in the accelerating and braking components of the cell cycle, changes in key proteins such as p27 and cyclin D1 profoundly affected the activities of interacting partners. For this reason, the effects of TGF-β1, rapamycin and wortmannin on cyclin D1 protein levels were examined.
Epidermal growth factor and TGF-α (a growth factor that signals through the EGF-receptor) exhibited optimal stimulation at 12-16 hours post-addition in contrast to the TGF-β1 stimulation window at 32 hours, please see Figure 6-2. Since a delay in S-phase entry characteristic to TGF-β1 treatment may correspond with cyclin D1 levels in these fibroblasts, C3H fibroblasts were examined for cyclin D1 levels at 16 hours. Cyclin D1 was observed to be down-regulated by TGF-β1, Figure 4-2. Rapamycin and wortmannin did not affect TGF-β1-mediated down-regulation of cyclin D1, Figure 4-2. This suggests that down-regulation of cyclin D1 by TGF-β1 was rapamycin- and wortmannin-independent.
Rapamycin alone induced p27kip1 level but did not affect TGF-β1-stimulated p27kip1 down-regulation
Rapamycin alone appeared to induce p27kip1 levels even when concomitant incubation with TGF-β1 did not accumulate p27kip1 levels, Figure 4-3. This intriguing observation underscored the dispensability of p27kip1 accumulation in rapamycin-mediated inhibition of TGF-β1 effect on cell cycle progression. Rapamycin-induction of p27kip1 may also suggest the presence of a pathway that maintains stress-like signals leading to p27kip1 up-regulation. This pathway may be inhibited by positive signaling events mediated by TGF-β1.
P34cdk4 and P34cdk2 were constitutively expressed under TGF-β1 treatment
Since the interaction between accelerating and braking components of the cell cycle machine occurs in a stoichiometric manner, altering the abundance of key components changes the distribution of interactive partners to exert inhibition or activation of G1 progression. P34cdk4 is the catalytic subunit in cyclin D1-cdk 4 complexes and hyperphosphorylates the protein product of the retinoblastoma gene, pRB.
These hyperphosphorylation events enable the release of transcription factors E2F-DP1 for S-phase critical transcription. Since rapamycin did not affect TGF-β1-mediated changes in p27kip1 and cyclin D1, alterations of cdk 4 and cdk 2 were examined. Similar to previous observations with cyclin E, the levels of cdk 4 and cdk 2 were constitutive under TGF-β1 treatment and were also constitutive under drug treatment, Figure 4-4. Thus the inhibitory effect of rapamycin against TGF-β1 mediated DNA synthesis observed in biochemical assays was not due to changes in the levels of cell cycle components examined.
These results demonstrated the importance of complementation of biochemical assays to protein abundance assays in examining the effects of extracellular signals in cell cycle progression. Down-regulation of specific cdk-inhibitors appeared to be context-specific. Recently, p57kip2 down-regulation was determined to be another end-point in TGF-β1-mediated stimulation in osteoblasts (Tomohiko et al, 1999). In human embryonic fibroblasts, TGF-β1-mediated stimulation in DNA synthesis corresponded with a down-regulation of p21cip1 (Miyazaki et al, 1998). Here, in the C3H-10T½ murine embryonic fibroblast system where TGF-β1 was stimulatory, down-regulation of p27kip1 was important but not a sole contributor to cell cycle progression.
Cyclin D1 down-regulation by TGF-β1 in these fibroblasts may explain the delay in S-phase entry characteristic of this threonine/serine-receptor kinase signaling growth factor. In addition, TGF-β1 delayed EGF-induced cyclin D1 in these fibroblasts and supported a role for cyclin D1 in determining onset of S-phase. Since cyclin D1 levels correlated with the duration of the G1 phase in various cells and since overexpression of cyclin D1 resulted in a shortened G1 phase, the delay of EGF-inducible cyclin D1 by TGF-β1 in this cell system may account for the delay in EGF-mediated S-phase entry kinetics exerted by TGF-β1. The effect of TGF-β1 on cyclin D1 profile on various early acting growth factors may therefore contribute to the modulation of EGF- and PDGF-specific cell cycle kinetics. However, Cyclin D1 regulation did not appear to be a driver of rapamycin-inhibition of TGF-β1 effects.
Macromolecular-synthesis significant to S-phase entry (DNA synthesis and protein synthesis) under TGF-β1- and rapamycin treatment showed inhibition of the TGF-β1 effect by rapamycin. Down-regulation of cell cycle-sensitive System A amino-acid transporter activity was also observed in independent experiments, Figure 6-14. On the other hand, wortmannin was ineffective against TGF-β1-mediated cell cycle progression into S-phase and this corresponded with the failure of wortmannin to significantly inhibit TGF-β1-stimulated DNA synthesis, protein synthesis, and system specific amino acid transport, Figures 5-1, 6-19, 6-20. Whereas biochemical data from radiolabeling assays demonstrated a divergence between rapamycin-sensitive- and wortmannin-sensitive pathways, immunoblot analysis showed that p27kip1 and cyclin D1 were not altered by rapamycin or wortmannin. Therefore in mechanistic dissection of signaling pathways, biochemical analyses are important complements to immunoblot analyses.
The observation that rapamycin-inhibition of TGF-β1 mediated S-phase entry was independent from p27kip1 accumulation or cyclin D1 down-regulation suggested cell-type specificity of these effects. In Swiss 3T3 fibroblasts, rapamycin-inhibition of bombesin-mediated cell cycle progression did not correlate with p27kip1 accumulation, but rather was incidental of cyclin D1 down-regulation (Withers et al, 1997). Uncoupling p27kip1 regulation from rapamycin-sensitive pathways was demonstrated in an immortalized NIH 3T3 line (N-3T3), where rapamycin inhibited G1/S progression but did not affect p27kip1 levels or changed cyclin D1 activity (Chen et al, 1996). This suggested that rapamycin-mediated inhibition of cell cycle progression rested elsewhere from hyperphosphorylation of the retinoblastoma protein (Chen et al, 1996). A dramatic illustration of p27kip1 dispensability in mediating both rapamycin- and TGF-β1 effects came from studies of p27kip1 knockout mice, where cells lacking functional p27kip1 retained sensitivity to rapamycin and TGF-β1 (Nakayama et al, 1996).
The lack of effect of rapamycin on p27kip1 against TGF-β1 accumulation did not preclude the possibility that this drug may affect the physical interaction between p27kip1 and targeted cell cycle complexes or the distribution of relevant cdk inhibitors. Rapamycin alone induced p27kip1 levels, suggesting that p27kip1 regulation was contributed in part by a rapamycin-mediated pathway. Future relevant investigations may focus on the distribution of interacting cell cycle components under rapamycin treatment as well as whether the conformation of individual components may be affected by this drug.
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