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Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress

Pasquale Fasanaro^*,1, Alessandra Magenta^*,1, Germana Zaccagnini, Lucia Cicchillitti, Sergio Fucile, Fabrizio Eusebi, Paolo Biglioli, Maurizio C. Capogrossi^* and Fabio Martelli^*,2

^* Istituto Dermopatico dell’Immacolata-IRCCS, Rome;

Centro Cardiologico Monzino-IRCCS, Milan, Italy; and Dipartimento Fisiologia Umana e Farmacologia,

Universita’ di Roma La Sapienza, Rome, Italy

²Correspondence: Laboratorio Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, Via dei Monti di Creta 104, Rome 00167, Italy. E-mail: f.martelli{at}idi.it

SPECIFIC AIMS

Enhanced oxidative damage to endothelial cells (EC) is a prominent feature of many physiopathological conditions. Indeed, reactive oxygen species (ROS) have been shown to mediate EC apoptosis in a variety of cardiovascular diseases, including ischemia/reperfusion injury, diabetic vasculopathy, hypertension, atherosclerosis, and heart failure.

Several proteins that stimulate cell cycle progression have been demonstrated to induce apoptosis when deregulated. Specifically, the cyclin D1 gene (CCND1/BCL1/PRAD1) plays an integral part in cell growth and survival control. In this study, we investigated how oxidative stress regulates cyclin D1 turnover and the functional relevance of cyclin D1 degradation for EC survival.

PRINCIPAL FINDINGS

1. D-cyclins are down-regulated in response to oxidative stress
Human umbelical vein EC (HUVEC) were treated with 400 µmol/l H₂O₂ for 1–24 h: after 2 h of H₂O₂ treatment, cyclin D1 accumulation decreased significantly (23±2% of control; P<0.001; Fig. 1 A) and low levels of this cyclin were maintained up to 8 h after treatment. Thereafter, cyclin D1 expression recovered, returning to control levels 24 h after H₂O₂ addition. Cyclin D3 protein was similarly modulated, albeit to a lower extent, while cyclin D2 was undetectable.

View larger version (28K): [in this window] [in a new window]   Figure 1. H2O2 treatment induces D-cyclins down-modulation. A) HUVEC were treated with 400 µmol/l H2O2 for the indicated time, followed by Western blotting. Graph shows cyclin D1 protein expression normalized for -tubulin levels and expressed as % of untreated cells (*P<0.001 vs. control; n=6–16). B) Dose-dependent down-modulation of D-cyclins. HUVEC were incubated for 2 h with H2O2, followed by immunoblots. Graph shows cyclin D1 protein expression levels (*P<0.001 vs. control; n=3–7).

D-cyclin down-modulation was observed both at sublethal (200–400 µmol/l) and lethal (800 µmol/l) doses of H₂O₂ (Fig. 1B ) and occurred with similar kinetics in other EC (bovine artery EC and porcine artery EC) and non-EC types (U2OS osteosarcoma cell line). Interestingly, up to 8 h of H₂O₂ treatment did not modulate protein levels of cyclins A, B, and E.

We also tested whether other interventions causing oxidative stress induced D-cyclins down-modulation.

Cell treatment with the alkylating chemotherapic drug 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU) causes glutathione (GSH) reductase inhibition and the decrease of reduced-to-oxidized GSH ratio. HUVEC incubation with 0.25 mmol/l BCNU for 2 h caused cyclin D1 down-regulation, and this phenomenon was inhibited by the ROS-scavenger NAC. These data indicate a direct relationship between BCNU-induced red/ox imbalance and cyclin D1 down-modulation.

Then, we assessed whether D-cyclins levels were modulated by EC exposure to ischemia, a condition associated with increased ROS formation and oxidative stress. Eight hours of in vitro simulated ischemia induced a threefold decrease of cyclin D1 levels (P<0.001); this phenomenon was prevented by cell treatment with ROS scavengers.

We previously reported that hind-limb ischemia is associated with a sharp increase of oxidative stress. Thus, unilateral hind-limb ischemia was induced by femoral artery dissection and the D-cyclin expression in adductor muscle was assessed by immunohistochemistry and Western blotting after 8 h. Although cyclin D1 was barely detectable, cyclin D3 was clearly expressed in both EC and myofibers. In ischemic muscles, cyclin D3 expression levels decreased to 49.8±6.8% of the control (P<0.001). We previously demonstrated that in p66^ShcA null mice, induction of ischemia by femoral artery excision does not increase oxidative stress. Likewise, p66^ShcA deletion prevented cyclin D3 down-modulation induced by ischemia.

Thus, D-cyclins expression is down-modulated by a variety of stimuli inducing oxidative stress. For the sake of simplicity, the molecular mechanism underlying this event was investigated only in H₂O₂-treated EC.

2. Cyclin D1 is degraded by the ubiquitin-protease pathway after H₂O₂ treatment
Northern blotting analysis showed that cyclin D1 mRNA was present throughout the time course of H₂O₂ treatment. Therefore, we analyzed whether H₂O₂ increased cyclin D1 protein turnover. Pulse-chase analysis of ³⁵S-labeled cells revealed that after cell treatment with 600 µmol/l H₂O₂ for 2 h, cyclin D1 degradation rate almost doubled.

To assess proteasome involvement, HUVEC were treated with either lactacystin or N-acetyl-L-leucinyl-L-leucinyl-N-norleucinal (LLnL). These two cell permeant inhibitors of the proteasome completely prevented H₂O₂-induced down-modulation of cyclin D1. Then, we investigated whether H₂O₂ treatment induced cyclin D1 polyubiquitination. U2OS cells were treated with either LLnL alone or LLnL and H₂O₂. Afterward, cyclin D1 was immunoprecipitated, followed by immunoblotting to ubiquitin. H₂O₂ treatment induced an increase of the slower-migrating polyubiquitinated forms of cyclin D1.

3. Cyclin D1 degradation induced by oxidative stress is phospholipase C dependent
Since oxidants trigger phospholipase C (PLC)- activation, we tested whether PLC- was involved in H₂O₂-dependent degradation of cyclin D1. We found that PLC- activity was necessary to induce cyclin D1 degradation by H₂O₂. Indeed, HUVEC treatment with U73122, a selective inhibitor of PLC function, completely prevented H₂O₂-induced cyclin D1 down-modulation.

Moreover, PLC- activation was also sufficient to trigger cyclin D1 degradation in the absence of H₂O₂. HUVEC treatment with the PLC activator 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) caused a dose-dependent down-modulation of cyclin D1 that was prevented by U73122 treatment.

4. H₂O₂-induced cyclin D1 degradation is Ca²⁺ and CaMK dependent
PLC- activity catalyzes the generation of IP3 messenger that, in turn, binds to specific receptors (IP3R) on the ER and provokes an increase of intracellular calcium concentration ([Ca²⁺]_i).

Kinetics of Ca²⁺ mobilization measured in Fura-2-loaded cells showed that H₂O₂ treatment induced a steady [Ca²⁺]_i increase that correlated with cyclin D1 degradation dynamic. We found that cyclin D1 degradation was [Ca²⁺]_I dependent, since the intracellular Ca²⁺ chelator BAPTA-AM prevented cyclin D1 down-modulation induced by H₂O₂ (P<0.017). In keeping with these results, HUVEC treatment with either 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C, two IP3R inhibitors, significantly attenuated cyclin D1 down-modulation induced by H₂O₂.

CaMK transduces [Ca²⁺]_i elevation signals to a number of target proteins. Thus, we assayed whether CaMK activity was modulated after H₂O₂ stimulation. In keeping with previous data, HUVEC treatment with 400 µmol/l H₂O₂ for 30 min induced CaMK activity more than fivefold. We also found that CaMK activity was necessary to induce cyclin D1 degradation, since HUVEC treatment with CaMK inhibitor KN93 prevented H₂O₂-induced cyclin D1 down-modulation.

Finally, we asked whether forced expression of activated CaMK was sufficient to induce cyclin D1 degradation. In HUVEC overexpressing a constitutively active form of CaMKII, cyclin D1 down-modulation was induced. This negative regulation was due to degradation, since it was prevented by treatment with proteasome inhibitors.

5. Cyclin D1 overexpression increases cell susceptibility to H₂O₂-induced apoptosis
To investigate the physiological relevance of cyclin D1 down-modulation, we assessed whether override of cyclin D1 down-regulation via its forced overexpression affected cell proliferation and apoptosis in response to 400 µmol/l H₂O₂. HUVEC were infected with adenoviruses encoding either cyclin D1 (Ad-CyD1) or GFP (Ad-GFP, negative control), and both cell cycle phase distribution and apoptotic cell death were quantified by propidium iodide staining. Apoptosis was also determined measuring the amount of DNA apoptotic fragmentation.

On H₂O₂ treatment, the override of cyclin D1 down-modulation caused no significant differences in cell cycle. Conversely, Ad-CyD1-infected cells displayed significantly higher apoptosis after H₂O₂ incubation (P<0.001).

S-phase cells display increased sensitivity to H₂O₂. To test the role of cyclin D1 in cells undergoing DNA synthesis, HUVEC were synchronized in early S phase by aphidicolin block. Afterward, cells were allowed to re-enter S phase and treated with 400 µmol/l H₂O₂. We found that S-phase synchronization significantly enhanced apoptotic cell death induced by H₂O₂ in cyclin D1 overexpressing cells.

6. Overriding cyclin D1 degradation via CaMK inhibition enhances H₂O₂-induced apoptosis
To confirm the role of cyclin D1 down-modulation, we tested whether the override of cyclin D1 down-modulation elicited by CaMK inhibition increased cell sensitivity to H₂O₂-induced apoptosis (Fig. 2 ).

View larger version (20K): [in this window] [in a new window]   Figure 2. CaMK inhibition increases H2O2-induced apoptosis. S-phase synchronized HUVEC were treated with CaMK inhibitor KN93 (10 µmol/l) or solvent alone followed by incubation with or without 400 µmol/l H2O2 for additional 24 h. Apoptosis was measured by calculating percentage of cells displaying subdiploid DNA content (left, n=11) or the apoptotic DNA fragmentation (right, n=4). In cells incubated with KN93 and H2O2, apoptosis significantly increased over both cells treated with H2O2 alone or KN93 alone.

S-phase synchronized HUVEC were treated with H₂O₂, in the presence or absence of KN93 CaMK inhibitor. Treatment with 10 µmol/l KN93 significantly enhanced cell death induced by H₂O₂, as assessed measuring both the percentage of cells having subdiploid DNA content (P<0.001) and the apoptotic fragmentation of cellular DNA (P <0.001).

Then, we assessed whether this phenomenon was mediated by the deregulation of cyclin D1 levels. To this aim, KN93 and H₂O₂ were tested in cells where cyclin D1 expression was silenced via small interfering RNA (siRNA) strategy. Cyclin D1-siRNA significantly decreased cell sensitivity to H₂O₂ in KN93-treated cells (P<0.001), indicating that cyclin D1 down-modulation is the relevant target of CaMK inhibition.

CONCLUSIONS AND SIGNIFICANCE

Understanding EC responses to oxidative stress may provide useful insights into aging mechanisms and into the pathogenesis of a variety of cardiovascular diseases. We found that cyclin D1 is rapidly down-modulated after EC treatment with sublethal doses of H₂O₂. This negative modulation is also observed after exposure to other oxidative stress-inducing stimuli.

To further investigate the molecular mechanism underpinning cyclin D1 down-modulation after H₂O₂ treatment, we found that H₂O₂ increased cyclin D1 ubiquitination followed by proteasome degradation. The study of the signaling pathways activated by oxidative stress showed that cyclin D1 degradation was dependent on PLC-IP3-mediated mobilization of intracellular Ca²⁺ stores; Ca²⁺ increase was in turn transduced by CaMK. Finally, the functional role of cyclin D1 degradation was examined. We found that overriding of cyclin D1 down-modulation via its forced overexpression or via CaMK inhibition increased cell sensitivity to apoptotic cell death induced by H₂O₂ (Fig. 3 ).

View larger version (32K): [in this window] [in a new window]   Figure 3. Diagram summarizing signal transduction pathway activated by H2O2 and leading to cyclin D1 degradation.

To the best of our knowledge, this is the first study linking together the posttranscriptional regulation of cyclin D1 levels after oxidative stress, the signaling involved in this phenomenon, and the functional consequences of cyclin D1 degradation.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4695fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-4695fjev1 20/8/1242    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fasanaro, P. Articles by Martelli, F. Search for Related Content PubMed PubMed Citation Articles by Fasanaro, P. Articles by Martelli, F.