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(The FASEB Journal. 2006;20:1242-1244.)
© 2006 FASEB

Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress

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

* Istituto Dermopatico dell’Immacolata-IRCCS, Rome;

{dagger} Centro Cardiologico Monzino-IRCCS, Milan, Italy; and

{ddagger} 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

ABSTRACT

The understanding of endothelial cell responses to oxidative stress may provide insights into aging mechanisms and into the pathogenesis of numerous cardiovascular diseases. In this study, we examined the regulation and the functional role of cyclin D1, a crucial player in cell proliferation and survival. On H2O2 treatment, endothelial cells showed a rapid down-modulation of cyclin D1. Other D-cyclins were similarly regulated, and this decrease was also observed after exposure to other oxidative stress-inducing stimuli, namely 1,3-bis (2 chloroethyl)-1 nitrosourea treatment and ischemia. H2O2 treatment induced cyclin D1 ubiquitination followed by proteasome degradation. Phospholipase C inhibition prevented cyclin D1 degradation, and its activation triggered cyclin D1 down-modulation in the absence of oxidative stress. Activated phospholipase C generates inositol-1,4,5-trisphosphate (IP3) and Ca2+ release from internal stores. We found that both IP3-receptor inhibition and intracellular Ca2+ chelation prevented cyclin D1 degradation induced by oxidative stress. Furthermore, Ca2+ increase was transduced by Ca2+/calmodulin-dependent protein kinase (CaMK). In fact, H2O2 stimulated CaMK activity, CaMK inhibitors prevented H2O2-induced cyclin D1 down-modulation, and CaMK overexpression induced cyclin D1 degradation. Finally, overriding of cyclin D1 down-modulation via its forced overexpression or via CaMK inhibition increased cell sensitivity to H2O2-induced apoptotic cell death. Thus, cyclin D1 degradation enhances endothelial cell survival on oxidative stress.—Fasanaro, P., Magenta, A., Zaccagnini, G., Cicchillitti, L., Fucile, S., Eusebi, F., Biglioli, P., Capogrossi, M. C., Martelli, F. Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress.


Key Words: endothelium • ubiquitin-proteasome pathway • calcium signaling • apoptosis

ENHANCED OXIDATIVE DAMAGE to endothelial cells (EC) is a prominent feature of many physiological and pathological 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 (1 , 2) . Therefore, it is of pivotal importance to understand ROS-activated mechanisms leading to EC apoptosis, as much as the adaptive responses inducing enhanced EC survival on oxidative stress.

Phospholipase C-{gamma} (PLC-{gamma}) has a prominent role in determining EC fate on ROS exposure (3) . PLC-{gamma} is activated by a mechanism relying on tyrosine phosphorylation after stimulation of growth factor receptor tyrosine kinases (4) . Interestingly, several laboratories have demonstrated that PLC-{gamma} undergoes tyrosine phosphorylation in response to oxidant exposure as well (3 , 5 , 6) .

Activated PLC-{gamma} cleaves the membrane phospholipid phosphatidylinositol-4,5-bisphosphate, generating two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The former activates protein kinase C (PKC) (7) ; the latter stimulates Ca2+ release from the internal stores (8) . Intracellular Ca2+ concentration ([Ca2+]i) regulates numerous physiopathological events, and the Ca2+/calmodulin-dependent protein kinase family (CaMK) is frequently involved (9) .

Several proteins that stimulate cells 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 (10) . D-type cyclins activate CDK4/6, which, in turn, phosphorylates pRb. This event triggers the derepression of a subset of proliferation-associated E2F target genes and induces the progression through the G1 phase of the cell cycle (10) .

Along with this role as a regulator of cell proliferation, increasing evidence indicates that cyclin D1 is a crucial player in certain cases of neuronal apoptosis (11) . High levels of cyclin D1 expression also elicit apoptotis in non-neuronal cells, (12 13 14) , indicating that uncoordinated cyclin D1 expression presents a survival challenge to the cell.

D-cyclins protein levels are tightly regulated at transcriptional and post-transcriptional concentration. Growth factor stimulation induces cyclin D1 mRNA synthesis (10) . Moreover, cyclin D1 turnover is regulated by phosphorylation of specific threonine residues (Thr286 and Thr288), polyubiquitination, and subsequent degradation by the 26S proteasome (15) . Thr286 phosphorylation of cyclin D1 is catalyzed by glycogen synthase kinase-3ß (GSK-3ß), on growth factor withdrawal (16) and by p38Sapk2, after osmotic stress (17) . Thr288 has been shown to be substrate of Mirk/dyrk1B kinase (18) . Furthermore, alternative mechanisms of cyclin D1 ubiquitination have been described previously: a destruction box RxxL motif is necessary for cyclin D1 degradation induced by UV (19) and free-cyclin D1 (unbound to CDKs) is ubiquitinated independently of its phosphorylation on Thr286 and Thr288 (20) .

Thus, although further studies are needed, it appears that different pathways are activated in different pathophysiological situations. In this study, we investigated how oxidative stress regulates cyclin D1 turnover and the functional relevance of cyclin D1 degradation for EC survival.

MATERIALS AND METHODS

Cell cultures
Human umbilical vein EC (HUVEC; Clonetics) and U2OS osteosarcoma cell line (American Type Culture Collection) were grown in EGM-2 (Bio-Whittaker) and Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, respectively (21 , 22) . Drug treatments, transfections, and simulated ischemia (23) are described in Supplementary Materials.

Hind-limb ischemia
Surgical and immunohistochemical procedures are described in Supplementary Materials.

Northern blot analysis
RNA extraction and Northern blotting were performed as described previously (22) using a specific cyclin D1 probe (20) .

Western blot analysis
Western blotting was performed as described previously (21 , 23) . Antibodies used are described in Supplementary Materials.

Immunoprecipitation and pulse-chase analysis
Immunoprecipitation and pulse-chase experiments were performed as described previously (24) (see also Supplementary Materials).

Calcium measurement
The variations of [Ca2+]i were measured using a conventional fluorescence microscopy system as described previously (25 ; see also Supplementary Materials).

CaMK assay
CaMK activity was measured using SignaTECT CaMK assay system (Promega) as described in Supplementary Materials.

Cell cycle and apoptosis analysis
Cell cycle and apoptosis were measured using a Becton-Dickinson flow cytometer and CellQuest and ModFit softwares (21 ; see also Supplementary Materials). Alternatively, apoptosis was measured by cell death detection ELISA kit (Roche) according to manufacturer’s instructions.

Adenoviral infection
Adenoviral infection was performed as described previously (21 ; see also Supplementary Materials).

Small interfering RNA-mediated gene silencing
Small interfering RNAs (siRNA) targeting cyclin D1 or CD4 (Santa Cruz) were transfected as described in Supplementary Materials.

Statistical analysis
Variables were analyzed by Student’s t test and one-way ANOVA. A value of P ≤ 0.05 was deemed statistically significant. Values are indicated ± SE.

RESULTS

D-cyclins are down-regulated in response to oxidative stress
To investigate cyclin D1 role in EC apoptosis on oxidative stress, HUVEC were treated with 400 µmol/l H2O2 for 1–24 h. Figure 1 A shows that after 2 h of H2O2 treatment, cyclin D1 accumulation significantly decreased (23±2% of control, P<0.001) and proteins levels were maintained low until 8 h after treatment. Afterward, cyclin D1 protein started to recover and returned to control levels 24 h after H2O2 addition. Cyclin D3 protein was similarly modulated but to a lower extent (at 2 h it was 44±7% of the control, P<0.001), whereas cyclin D2 expression was not detectable.


Figure 1
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  		Figure 1. H2O2 treatment induces D-cyclin down-modulation. A) HUVEC were treated with 400 µmol/l H2O2 for indicated time, followed by Western blotting. Graph shows cyclin D1 protein expression normalized for {alpha}-tubulin levels and expressed as % 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).

In the adopted culture conditions, HUVEC treatment with 400 µmol/l induced negligible levels of cell death (21) . We found that D-cyclins down-modulation was also observed after lethal doses (800 µmol/l) of H2O2 (21 ; Fig. 1B ) and occurred with similar kinetics in other endothelial (bovine artery EC and porcine artery EC, not shown) and nonendothelial cell types (U2OS osteosarcoma cell line, see Supplementary Materials, Fig. S1A). Interestingly, U2OS expressed cyclin D2, which was negatively modulated by H2O2 as much as cyclin D1. It is worth noting that H2O2 concentrations >800 µmol/l H2O2 were necessary to induce cell death in U2OS (not shown), confirming that sublethal doses of H2O2 are sufficient to trigger D-cyclins down-modulation.

Other cyclins were not modulated by oxidative stress: up to 8 h of treatment with 400 µmol/l H2O2 did not affect the expression of cyclins A, B, and E (see Supplementary Materials, Fig. S1B). Similarly, CDK 1, 2, 4, and 6 protein levels were not modulated, while p21Waf1/Cip1/Sdi was up-regulated 6–8 h after H2O2 treatment (not shown).

We also tested whether other interventions causing intracellular red/ox imbalance induced D-cyclin down-modulation.

EC are relevant targets of chemiotherapics (26) . Specifically, the alkylating agent 1,3-bis (2 chloroethyl)-1 nitrosourea (BCNU, Carmustine), a widely used anticancer chemotherapy drug, is an inhibitor of glutathione (GSH) reductase that blocks the conversion of oxidized to reduced GSH (27) . Incubation of HUVEC with 0.25 mmol/l BCNU for 2 h caused cyclin D1 down-regulation, and this phenomenon was prevented by preincubation with 10 mmol/l NAC. Treatment with NAC prevents the decrease of reduced GSH induced by BCNU (27) , indicating a direct relationship between BCNU induced red/ox imbalance and cyclin D1 down-modulation (Fig. 2 A). We found that cyclin D3 was similarly regulated.


Figure 2
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  		Figure 2. Oxidative stress induces D-cyclin down-modulation. A) BCNU induces cyclin D1 down-modulation. HUVEC were either preincubated with 10 mmol/l NAC (+) or sham treated (-) for 30 min, followed by incubation with indicated concentrations of BCNU for 2 h and immunoblotting. Graph shows cyclin D1 protein expression levels (n=3). B) HUVEC were incubated for 8 h with glucose-free medium in normoxic conditions or in an hypoxic camera (hypox) in the presence or absence of 5000 U/ml of catalase (cat) or 2.5 mmol/l 4-hydroxy-TEMPO, followed by immunoblots. Graph shows cyclin D1 protein expression levels (n=3). C) Ischemia induces cyclin D3 down-modulation. Representative cyclin D3 immunostainings (top) of normoperfused (n) and ischemic (i) adductor muscles. EC were identified in serial sections by CD34 immunostaining (bottom). Magnification x1000; bar = 15 µm. Black arrowheads indicate cyclin D3 positive nuclei of EC, and red arrowheads indicate cyclin D3 positive nuclei most likely belonging to myofibers. D) Representative (n=5–9) Western blotting of adductor muscle extracts derived from normo-perfused (normo) or ischemic (isch 8h) p66ShcA wt or -/- mice, 8 h after femoral artery dissection. Cyclin D3 was detected using a specific Ab. Red Ponceau staining indicates uniform protein loading.

We also tested whether D-cyclin levels were modulated on EC exposure to ischemia, a condition associated with increased ROS formation and oxidative stress ((23) and references therein). We found that 8 h of simulated ischemia induced a threefold decrease of cyclin D1 levels (P<0.001), and this phenomenon was prevented by cell treatment with catalase and 4-hydroxy-TEMPO ROS scavengers (Fig. 2B ). Similar results were obtained with cyclin D3.

To assess whether D-cyclin expression was also regulated by ischemia in vivo, we tested their expression in a mouse model of hind-limb ischemia (23) . Unilateral hind-limb ischemia was induced by femoral artery removal, and adductor muscles were analyzed after 8 h. In keeping with previous results (23) , femoral artery removal induced a 5.4 ± 0.5 increase of apoptosis, as assessed by TUNEL assay (see Supplementary Materials, Fig. S2). D-cyclins expression was assessed by immunohistochemistry in adductor muscle sections: Whereas cyclin D1 expression was barely detectable (not shown), cyclin D3 antibody (Ab) clearly marked EC nuclei in normoperfused mice (Fig. 2C ). Some nuclei of myofibers were positively stained as well. In ischemic muscles, cyclin D3 expression decreased in both EC and myofibers. This event was quantified by Western blotting of normoperfused and ischemic muscles (Fig. 2D ). On ischemia, cyclin D3 levels were 49.8 ± 6.8% of the control (P<0.001). To assess whether cyclin D3 down-modulation was indeed due to ischemia-induced oxidative stress, we took advantage of p66ShcA null (–/–) mouse model. Femoral artery excision induces a similar decrease of blood perfusion in p66ShcA –/– and wt mice. However, oxidative stress is not increased when p66ShcA –/– mice are exposed to ischemia (23) . Figure 2D shows that p66ShcA deletion prevented cyclin D3 down-modulation induced by ischemia.

We concluded that D-cyclin expression is down-modulated by a variety of oxidative stress inducing stimuli. The molecular mechanisms underlying this event were investigated in H2O2-treated cells for the sake of simplicity.

H2O2 induces cyclin D1 degradation
Northern blotting analysis showed that cyclin D1 mRNA was present throughout the time course of H2O2 treatment (see Supplementary Materials, Fig. S3). Therefore, we analyzed whether H2O2 increased cyclin D1 protein turnover. Easily transfectable U2OS cells were chosen as experimental system. A plasmid encoding hemagglutinin (HA) epitope-tagged human cyclin D1 was transfected, and cells were treated with 600 µmol/l H2O2 for 2 h. Thereafter, they were pulse labeled for 30 min with [35S]methionine and chased with medium containing cold methionine. Figure 3 shows that the turnover of both ectopically expressed (black arrow) and endogenous (gray arrow) cyclin D1 significantly increased in pro-oxidant conditions.


Figure 3
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  		Figure 3. H2O2 induces cyclin D1 degradation. U2OS cells were transfected with a plasmid encoding HA-tagged human cyclin D1. Thirty six hours later, cells were treated with 600 µmol/l H2O2 for 2 h, pulse labeled with [35S]methionine, and chased with medium containing cold methionine. Then, cells were lysed and immunoprecipitated with an Ab to cyclin D1 (CyD1) or to an irrelevant control (IgG), followed by SDS-PAGE. A) H2O2 induces an increase of turnover of both ectopically expressed (black arrow) and endogenous (gray arrow) cyclin D1. HA-tag caused an electrophoretic mobility slow-down of the overexpressed cyclin D1 allele. B) Cyclin D1 signals are expressed as % value at time 0. Dotted lines indicate cyclin D1 half-life.

Pharmacological alterations in the glycosylation machinery or in calcium levels can disrupt normal endoplasmic reticulum (ER) protein biogenesis and trigger a complex chain of events termed the unfolded protein response (UPR) (28) . Activation of the mammalian UPR is characterized by increased transcription of a series of molecular chaperones, such as GRP78, as well as by the block of cyclin D1 translation (28) .

No significant decrease of cyclin D1 translation was observed in our experimental conditions. Indeed, Fig. 3A shows that a similar rate of 35S-labeled cyclin D1 was expressed both in H2O2- and mock-treated cells.

Moreover, H2O2 treatment failed to induce GRP78 expression (see Supplementary Materials, Fig. S4).

We concluded that, on H2O2 treatment, cyclin D1 degradation was induced and that UPR played no major role in cyclin D1 negative regulation.

Cyclin D1 is degraded by the ubiquitin-proteasome pathway after H2O2 treatment
To test whether cyclin D1 down-modulation induced by H2O2 could be prevented by proteasome inhibition, HUVEC were cultured in the presence of either lactacystin or N-acetyl-L-leucinyl-L-leucinyl-N-norleucinal (LLnL), two potent cell permeant inhibitors of the proteasome. Both lactacystin (Fig. 4 A) and LLnL (see Supplementary Materials, Fig. S5) completely prevented H2O2-induced down-modulation of cyclin D1. Conversely, cell treatment with N-acetyl-leucyl-leucyl-methioninal, a Calpain inhibitor, failed to do so (not shown). Similar results were obtained when lactacystin was added after H2O2 treatment (not shown), indicating that proteasome inhibition can not only prevent but can also revert cyclin D1 degradation induced by oxidative stress.


Figure 4
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  		Figure 4. Cyclin D1 is degraded by ubiquitin-proteasome pathway. A) The proteasome inhibitor Lactacystin prevented H2O2-induced cyclin D1 down-modulation. HUVEC were incubated with 25 µmol/. lactacystin for 2 h. Then, H2O2 (400 µmol/l) was added and 2 h later cells were lysed and immunoblots were performed. Graph shows cyclin D1 protein expression levels (n=3). B) Cyclin D1 is polyubiquitinated on H2O2 treatment. U2OS were incubated with 50 µmol/l LLnL for 2 h. Afterward, cells were treated with 400 µmol/l H2O2 and 2 h later were lysed. Immunoprecipitations with irrelevant ({alpha}-MIg) or cyclin D1 specific Ab ({alpha}-CyD1) were performed, followed by Western blotting with a ubiquitin Ab (top). Asterisks indicate aspecific bands, probably due to immunoglobulins. Brackets indicate specific smears attributable to high molecular weight ubiquitinated species of cyclin D1. Then, membrane was stripped and reprobed with a cyclin D1 Ab to to assess equal efficiency of immunoprecipitation (bottom).

To further investigate cyclin D1 degradation, we tested whether cyclin D1 was polyubiquitinated in vivo. U2OS cells were treated with either LLnL alone or LLnL and H2O2. Then, cyclin D1 was immunoprecipitated followed by immunoblotting to ubiquitin (Fig. 4B , lanes 2 and 3). Figure 4B shows that slower-migrating polyubiquitinated forms of cyclin D1 were enriched in the immunoprecipitates derived from cells treated with LLnL and H2O2 (lane 3, brackets). The same extract immunoprecipitated with an irrelevant Ab failed to detect any specific signal (lane 1). Similar results were obtained with a different cyclin D1 Ab (not shown).

We concluded that the proteolysis of cyclin D1 on H2O2 treatment is controlled by the ubiquitin-proteasome pathway.

To assess whether cyclin D1 phosphorylation at Thr286 and Thr288 was necessary for cyclin D1 degradation after H2O2 treatment, a specific cyclin D1 allele bearing mutations of both sites (cyclin D1-mut) was assayed (20) . U2OS cells overexpressing HA-tagged cyclin D1-mut showed a decrease of cyclin D1 accumulation comparable to that obtained with cyclin D1 wt after H2O2 treatment (Fig. 5 A). Similar results were obtained using mouse cyclin D1 mutated at Thr286 (15) (not shown).


Figure 5
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  		Figure 5. Cyclin D1 degradation is PLC-{gamma} dependent. A) Thr286 and Thr288 are not necessary for cyclin D1 degradation. U2OS cells were transfected with a plasmids encoding HA-tagged cyclin D1 wt, HA-tagged cyclin D1 mutated at Thr286, and Thr288 (CyD1-mut) or with backbone plasmid. Thirty-six hours after transfection, cells were treated with 400 µmol/l H2O2 followed by immunoblot with antibodies to cyclin D1 (top) or HA-tag (bottom). In first case, HA-tag caused an electrophoretic mobility slow-down of the overexpressed cyclin D1 allele (black arrowhead) that allowed its separation from endogenous cyclin D1 (gray arrowhead). On H2O2 treatment, overexpressed cyclin D1 wt levels were 41 ± 0.2% of control (P<0.015; n=3); cyclin D1 mut levels were 62 ± 0.1% of control (P<0.008; n=3). Difference between overexpressed cyclin D1 wt and mut levels in H2O2 treated samples was not significant. B) PLC inhibition prevents cyclin D1 degradation induced by H2O2. HUVEC were incubated with 40 µmol/L U73122 for 10 min; then cells were treated with 400 µmol/l H2O2 for 2 h followed by immunoblotting. In our experimental conditions, U73122 treatment induced a minimal retard in electrophoretic mobility of cyclin D1 and, to a lower extent, of tubulin. Graph shows cyclin D1 protein expression levels (n=3). C) PLC activation triggers cyclin D1 degradation in the absence of H2O2. HUVEC were treated with indicated doses of m-3M3FBS for 2 h, followed by immunoblotting. Graph shows cyclin D1 protein expression levels (n=3–6).

These data suggest that cyclin D1 phosphorylation is not necessary for cyclin D1 demise induced by H2O2.

Cyclin D1 degradation induced by oxidative stress is PLC dependent
Oxidants trigger PLC-{gamma} activation by a tyrosine phosphorylation dependent mechanism (3 , 5 , 6 , and data not shown). Thus, we tested whether PLC-{gamma} was involved in H2O2-dependent degradation of cyclin D1. To determine whether PLC-{gamma} activity was necessary to induce cyclin D1 degradation by H2O2, HUVEC were pretreated with a selective inhibitor of phosphoinositide-specific PLC function, U73122. Figure 5B shows that U73122 completely prevented H2O2-induced cyclin D1 down-modulation. The inactive analog of U73122, known as U73433, was not assayed because of its toxicity in the experimental conditions used, possibly due to activities unrelated to PLC inhibition (6) .

Then, we tested whether PLC-{gamma} activation was sufficient to trigger cyclin D1 degradation in the absence of H2O2. To this aim, we used 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a compound known to directly activate all PLC isoforms (29) . HUVEC treatment with m-3M3FBS caused a dose-dependent down-modulation of cyclin D1 (Fig. 5C ). As expected, U73122 prevented m-3M3FBS induced cyclin D1 negative modulation (not shown).

Although a role of other PLC isoforms cannot be ruled out, the most likely interpretation of these results is that H2O2-dependent degradation of cyclin D1 is mediated by PLC-{gamma}.

H2O2-induced cyclin D1 degradation is Ca2+ dependent
PLC-{gamma} activity catalyzes the generation of IP3 and DAG second messengers (3 , 4) : The former binds to specific receptors (IP3R) on the ER and provokes an increase of [Ca2+]i (8) , whereas the latter is a direct activator of PKC (7) . HUVEC treatment with Go6850, calphostin C, and H-7 dihydrochloride, three PKC inhibitors (7) , failed to prevent cyclin D1 down-modulation induced by H2O2 (not shown). Moreover, HUVEC treatment with phorbol 12-myristate 13-acetate (PMA), a specific activator of PKC (7) , at concentrations ranging from 10 nmol/l to 50 µmol/l for 2 h, did not trigger cyclin D1 degradation (not shown). These data strongly suggest that PKC is not involved in H2O2-induced cyclin D1 degradation.

Then, we determined whether Ca2+ mobilization was involved in cyclin D1 degradation induced by H2O2.

To assess whether H2O2 and m-3M3FBS treatment induced an increase of [Ca2+]i in the adopted experimental conditions, Fura 2-acetoxymethyl ester loaded cells were assayed using time-resolved fluorescent microscopy (25) . HUVEC incubation with both 400 and 800 µmol/l H2O2 induced a slow developing rise of [Ca2+]i that started ~10 min after H2O2 addition and lasted more than 40 min from the beginning of the experiment (Fig. 6 A). These results are in agreement with previous reports showing that, while EC exposure to low doses of H2O2 triggered repetitive [Ca2+]i oscillations, H2O2 >500 µmol/l induced a steady [Ca2+]i increase without oscillations (30) . Cell treatment with 25 µmol/l m-3M3FBS induced a rapid increase of [Ca2+]i that was sustained throughout the time course (see Supplementary Materials, Fig. S6).


Figure 6
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  		Figure 6. H2O2-induced cyclin D1 degradation is Ca2+ dependent. A) H2O2 treatment induces a slow developing increase in [Ca2+]i. HUVEC were loaded with FURA 2-acetoxymethyl ester and [Ca2+]i variations were measured using time-resolved fluorescent microscopy. Concentration of [Ca2+]i was expressed as the time-resolved ratio between fluorescence images obtained at 340 and 380 nM excitation wavelengths. A representative trace for both H2O2 concentrations is shown. B) H2O2-induced cyclin D1 degradation is Ca2+ dependent. HUVEC were incubated for 30 min with the intracellular Ca2+-chelator BAPTA-acetoxymethyl ester and then incubated with 400 µmol/l H2O2 for 2 h. Afterward, cells were harvested and immunoblottings performed. Graph shows cyclin D1 protein expression levels (n=3). C) IP3Rs inhibitor prevents cyclin D1 down-modulation induced by H2O2. HUVEC were pretreated for 10 min with 300 nmol/l 2-APB. Then, H2O2 (400 µmol/l) was added and 2 h later immunoblots were performed. Graph shows cyclin D1 protein expression levels (P<0.001; n=3).

Then, it was assessed whether H2O2-induced cyclin D1 degradation was [Ca2+]i dependent. To this aim, we tested whether cell treatment with BAPTA-AM, an intracellular Ca2+ chelator, prevented cyclin D1 down-modulation. Figure 6B shows that BAPTA-AM significantly prevented cyclin D1 down-modulation induced by H2O2 (at 10 and 20 µmol/l, P<0.017).

To gain further insight into [Ca2+]i role in cyclin D1 degradation, HUVEC were pretreated with either 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C, two IP3Rs inhibitors, before incubation with H2O2. Both 2-APB (Fig. 6C ) and xestospongin C (see Supplementary Materials, Fig. S7) significantly attenuated cyclin D1 down-modulation induced by H2O2.

In conclusion, although it is possible that other pathways contribute to cyclin D1 degradation induced by H2O2, the present results demonstrated that H2O2-induced [Ca2+]i increase plays a pivotal role in cyclin D1 degradation.

H2O2-induced cyclin D1 degradation is CaMK dependent
CaMK transduces [Ca2+]i elevation signals to a number of target proteins (9) . Thus, we assayed whether CaMK activity was modulated after H2O2 stimulation. Thapsigargin, a SERCA inhibitor that elicits [Ca2+]i increase (8) , was used as a positive control. In keeping with previous data (31) , after HUVEC incubation with 400 µmol/l H2O2, CaMK activity was strongly enhanced up to 1 h and declined later on (Fig. 7 A). As expected, HUVEC treatment with 25 µM m-3M3FBS stimulated CaMK activity as well (see Supplementary Materials, Fig. S8).


Figure 7
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  		Figure 7. CaMK plays a crucial role in H2O2-induced cyclin D1 degradation. A) CaMK activity was positively modulated after H2O2 stimulation. HUVEC were treated either with 1 µmol/l thapsigargin (Tg, positive control) or with 400 µmol/l H2O2 for indicated periods of time. CaMK activity was expressed as % untreated cells (*P<0.002). B) CaMK inhibitor KN93 prevented cyclin D1 degradation induced by H2O2. HUVEC were incubated with 10 µmol/l KN93 for 30 min; then, cells were treated with 400 µmol/l H2O2 for 2 h followed by immunoblotting. Graph shows cyclin D1 protein expression levels (n=4). C) Overexpression of constitutively activated CaMK is sufficient to induce cyclin D1 degradation. HUVEC were infected with 50 MOI of adenoviruses encoding either CaMKII-D3 (AdCaMK) or LacZ (AdLacZ). Fourteen hours later, cells were incubated with 50 µmol/l LLnL or solvent alone for 2 or 4 h, followed by immunoblotting. Graph shows cyclin D1 protein expression levels (n=4).

Then, we assessed whether CaMK activity was necessary to induce cyclin D1 degradation. We found that HUVEC treatment with the CaMK inhibitor KN93 prevented H2O2-induced cyclin D1 down-modulation (Fig. 7B ). Similar results were obtained with phenoxybenzamine, a calmodulin (CaM) inhibitor, whereas KN92, an inactive analog of KN93, was inert (not shown).

Finally, we asked whether forced expression of activated CaMK was sufficient to induce cyclin D1 degradation. HUVEC were infected with a recombinant replication-defective adenovirus expressing a CaMKII allele that is locked into the activated state by site-directed mutagenesis (CaMKII-D3) (32) . Although the overexpression of a control protein (LacZ) was inert, CaMKII-D3 overexpression triggered cyclin D1 down-modulation. This negative regulation was due to proteasomal degradation, since it was prevented by treatment with both LLnL (Fig. 7C ) and lactacystin (not shown).

Although the adopted experimental approach does not allow us to distinguish among the different CaMK isoforms, the present results demonstrate that CaMK plays a crucial role in cyclin D1 degradation.

The ERK arm of the MAPK signaling pathway plays an important role in cell survival control. Increase of [Ca2+]i promote ERK activation either directly (33 , 34) or by the intervention of CaMK (35 , 36) . We found that HUVEC treatment with as much as 50 µM PD038059, a specific ERK inhibitor, did not prevent H2O2-induced cyclin D1 down-modulation. These data indicate that ERK is not necessary for this event (see Supplementary Materials, Fig. S9).

Cyclin D1 overexpression increases cell susceptibility to H2O2-induced apoptosis
We attempted to investigate the physiological relevance of cyclin D1 down-modulation. To this aim, we assessed whether the override of cyclin D1 down-regulation via its forced over-expression affected cell proliferation and apoptosis in response to H2O2.

HUVEC were infected with adenoviruses encoding either cyclin D1 (AdCyD1) or GFP (AdGFP), as a negative control. Then, cells were treated with 400 µmol/l H2O2 or solvent alone and both cell cycle phase distribution and apoptotic cell death were quantified in propidium iodide stained cells. Cyclin D1 over-expression was demonstrated by Western blot analysis (see Supplementary Materials, Fig. S10A). Interestingly, the higher levels of over-expression achieved with adenoviral vectors were sufficient to overcome cyclin D1 degradation, yielding a complete override of cyclin D1 down-modulation on H2O2 treatment.

We found that H2O2 treatment increased the portion of cells in G2/M phases and inhibited DNA synthesis. However, no significant difference was found after override of cyclin D1 down-modulation (see Supplementary Materials, Fig. S10B and C).

Then, cell death was assessed calculating the percentage of cells displaying subdiploid DNA content (Fig. 8 A). In untreated AdGFP-infected cells, the apoptotic subdiploid fraction was less than 4.5% at 12 and 24 h; at 48 h some cell death was present (14.2±0.6%), possibly as a secondary effect of adenoviral infection. At this H2O2 dosage, no significant increase in the subdiploid fraction was observed within 48 h of H2O2 treatment. As previously reported (11 12 13 14) , cyclin D1 overexpression in the absence of H2O2 increased the percentage of cells exhibiting subdiploid DNA content (P<0.001). After H2O2 incubation, however, the apoptotic subdiploid fraction was 17.5±1.3% and significantly higher than both untreated cyclin D1 overexpressing cells (P<0.005) and AdGFP-infected cells treated with H2O2 (P<0.001).


Figure 8
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  		Figure 8. Cyclin D1 over-expression increases cell susceptibility to H2O2-induced apoptosis. Asynchronously growing HUVEC were infected with 50 MOI of adenoviruses encoding either cyclin D1 (AdCyD1) or GFP (AdGFP). Eighteen hours after infection, 400 µmol/l H2O2 or solvent alone were added and apoptosis rates were determined at the indicated times (n=3). A) % Propidium iodide stained cells displaying subdiploid DNA content was evaluated by flow cytometry. Alternatively (B), apoptosis was measured determining fragmentation of cellular DNA. After H2O2 incubation, apoptosis significantly increased over both untreated AdCyD1 infected cells (P<0.005) and Ad-GFP infected cells treated with H2O2 (P<0.001). C, D) HUVEC were infected with AdCyD1 or AdGFP, synchronized in early S phase and treated with either 400 µmol/l H2O2 or solvent alone for the indicated times. Apoptosis was measured (n=4) calculating cell %displaying subdiploid DNA content (C) or apoptotic fragmentation of cellular DNA (D). As expected, H2O2 treatment induced a small but significant (P<0.03) increase of apoptosis in AdGFP-infected cells. More importantly, apoptosis of H2O2-treated AdCyD1-infected cells significantly increased over both untreated cells (P<0.004) and Ad-GFP infected cells treated with H2O2 (P<0.002).

To confirm that the observed increase of the subdiploid fraction was due to apoptosis, the amount of cytoplasmic nucleosomes generated during the apoptotic fragmentation of cellular DNA was assessed (Fig. 8B ). Since this is an earlier parameter compared to the appearance of the subdiploid population, the assay was conducted at 12 and 24 h after H2O2 treatment. Only minimal levels of nucleosomal fragmented DNA were found in both H2O2-treated and -untreated cells infected with AdGFP. In contrast, in cyclin D1 overexpressing cells, apoptosis significantly increased after treatment with H2O2 over both untreated cyclin D1 overexpressing cells (P<0.007) and AdGFP-infected cells treated with H2O2 (P<0.001).

S-phase cells display an exquisitely high sensitivity to H2O2 (37 , 38) . Thus, one may expect that the override of cyclin D1 down-modulation may have particularly dramatic effects in cells undergoing DNA synthesis. To test this hypothesis, cyclin D1 or GFP overexpressing HUVEC were synchronized in early S phase by aphidicolin and then released. With the use of this synchronization procedure, S-phase fraction increased ~7 fold (AdGFP, from 6.8±0.4% to 52.9±3.1%, P<0.001; AdCyD1 from 8.3±1.1 to 56.6±4.0%, P<0.001). S-phase cells were then treated with either H2O2 or solvent alone for 12, 24, or 48 h, and apoptosis was measured calculating the percentage of cells displaying subdiploid DNA content by flow cytometry (Fig. 8C ). S-phase synchronization enhanced cell death induced by H2O2 in control cells and even more significantly in cyclin D1 overexpressing cells: indeed, 24 h after treatment 43.9 ± 1.0% of the cells showed subdiploid DNA content. Similar results were obtained measuring the amount of cytoplasmic nucleosomes generated during the apoptotic fragmentation of cellular DNA (Fig. 8D ).

In conclusion, overcoming cyclin D1 down-modulation by its overexpression makes cells more sensitive to H2O2-induced apoptosis.

Overriding cyclin D1 degradation via CaMK inhibition enhances H2O2-induced apoptosis
We asked whether the override of cyclin D1 down-modulation elicited by CaMK inhibition increased cell sensitivity to H2O2-induced apoptosis.

HUVEC were synchronized in early S phase and then treated for 24 h with H2O2, in the presence or absence of CaMK inhibitor KN93. Figure 9 A shows that KN93 treatment significantly enhanced cell death induced by H2O2, as assessed calculating both the percentage of cells that had subdiploid DNA content (Fig. 9A , left, P<0.001) and measuring the amount of cytoplasmic nucleosomes generated during the apoptotic fragmentation of cellular DNA (Fig. 9A , right, P<0.001).


Figure 9
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  		Figure 9. CaMK inhibition increases H2O2-induced apoptosis. A) HUVEC were synchronized in early S phase and treated with either CaMK inhibitor KN93 (10 µmol/l) or solvent alone. One hour later, H2O2 or solvent alone were added and cells were incubated for additional 24 h. Apoptosis was measured calculating % of cells displaying subdiploid DNA content by flow cytometry (left, n=11) or apoptotic DNA fragmentation (right, n=4). Effect of KN93 alone was negligible, while as expected in synchronized cells, H2O2 treatment induced a 3-fold increase of apoptosis. In cells incubated with KN93 and H2O2, apoptosis significantly increased over both cells treated with H2O2 alone or KN93 alone. B) H2O2 sensitivity induced by CaMK inhibition is mediated by the deregulation of cyclin D1 levels. Low density cultures of U2OS cells were transfected with either CD4 (negative control) or cyclin D1-specific siRNAs. After 18 h, cells were treated with either CaMK inhibitor KN93 (10 µmol/l) or solvent alone. One hour later, H2O2 or solvent alone was added and cells were incubated for additional 24 h. Apoptosis was measured assessing the apoptotic fragmentation of cellular DNA. In cells transfected with cyclin D1 siRNA and incubated with both KN93 and H2O2, apoptosis was significantly decreased (n=3). In cells transfected with cyclin D1 siRNA, the difference between H2O2 and H2O2+KN93 treated cells is not significant. Bottom) Western blotting showing cyclin D1 down-modulation by siRNA.

To assess whether this phenomenon was mediated by the deregulation of cyclin D1 levels, cell sensitivity to KN93 and H2O2 was tested in cells where cyclin D1 expression was silenced via siRNA strategy. U2OS cells were transfected with either control or cyclin D1-specific siRNA, yielding a 64.3±4.7% inhibition of cyclin D1 expression (P<0.001). To achieve optimal levels of siRNA transfection, cells were seeded at low density. Cell density has been shown to be a crucial parameter for cell viability: thus, to minimize toxicity, cells were not synchronized. Figure 9B shows that H2O2 treatment of low-density cultures of U2OS induced a 2.1 ± 0.4-fold increase of apoptosis that was further increased (3.5±0.1-fold) by the CaMK inhibitor KN93. We found that cyclin D1-specific siRNA 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.

DISCUSSION

This study aimed at understanding the adaptive responses inducing enhanced EC survival in pro-oxidant conditions. Specifically, we found that oxidative stress induced rapid cyclin D1 down-modulation and that this event contributed to decrease EC death (Fig. 10 ).


Figure 10
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  		Figure 10. Diagram summarizing signal transduction pathway activated by H2O2 and leading to cyclin D1 degradation.

In keeping with previous studies, in this study we used H2O2 concentrations within the submillimolar range. Although H2O2 concentrations measured in vivo are generally lower, one should consider the specificity of both the cell culture system and the H2O2 administration method. Indeed, we show that under the adopted conditions of cell density and growth factors presence, the apoptosis induced by 400 µmol/l H2O2 in asynchronous EC is negligible and 800 µmol/l H2O2 was insufficient to induce apoptosis in U2OS. To corroborate the relevance of our findings, we show that chemotherapics and ischemia, two red/ox imbalance inducing stimuli, both trigger cyclin D1 down-modulation.

Cyclin D1 down-modulation induced by H2O2 was due to its degradation via the ubiquitin-proteasome pathway. This finding is in agreement with previous studies indicating that cyclin D1 degradation is induced by several stress stimuli, including growth factor deprivation (15 , 16) , osmostic stress (17) , UV (19 , 39) , and cisplatin damage (40) .

Cyclin D1 degradation is regulated in specific circumstances by phosphorylation of Thr286 and Thr288 (15 , 18 , 20) . We found that the expression of a specific cyclin D1 allele bearing mutations of both Thr286 and Thr288 was efficiently down-modulated by H2O2 treatment, suggesting that cyclin D1 phosphorylation by the kinases targeting these sites, namely GSK-3ß, p38Sapk2, and Mirk/dyrk1b, are not necessary for cyclin D1 demise induced by H2O2. In keeping with these data, cell treatment with LiCl, a potent inhibitor of GSK-3ß, failed to prevent cyclin D1 down-modulation induced by H2O2 (L. Cicchillitti and F. Martelli, unpublished observations).

We also found that cyclin D1 turnover was regulated by PLC-dependent mobilization of intracellular Ca2+. We did not formally prove that PLC isoforms different from PLC-{gamma} were not involved in this event. However, to our knowledge, PLC-{gamma} is the only PLC isoform described as activated by oxidative stress.

Both growth factors and ROS are capable of triggering PLC-{gamma} activity and Ca2+ mobilization, eliciting markedly different responses. Indeed, when quiescent cells are induced to proliferate by serum stimulation, cyclin D/CDK4 activity is stimulated by calmodulin (CaM) (41) and CaMK activities (42) . Another relevant target on growth factors stimulation of quiescent cells is calcineurin, a Ca2+/calmodulin-dependent phosphatase that positively regulates cyclin D1 translation (43) . Modulation of responses to [Ca2+]i increase can occur by coincident Ca2+-independent signals, but there is also growing evidence that the strength, frequency, source, and location of the Ca2+ signal are determinants for specific regulatory events. Athough further studies are needed to elucidate the differences between proliferative and oxidant stimuli, one distinguishing parameter is the duration of the stimulus: Unlike growth factors, H2O2 elicited a slowly developing and sustained Ca2+ mobilization.

We also found that EC treatment with H2O2 induced CaMK activity and that this activity was necessary for cyclin D1 degradation. The most likely interpretation of our data is that Ca2+ mobilization from internal stores leads to Ca2+/calmodulin-dependent activation of CaMK. However, a Ca2+/calmodulin-independent activation pathway of CaMK by H2O2 has been described, although in a different experimental system and on exposure to much higher concentrations of H2O2 (10 mmol/l) (44) .

In a previous study, we showed that cell treatment with H2O2 induces a rapid arrest of S-phase progression that is dependent on the dephosphorylation of pRb by the PP2A phosphatase (21) . A certain concentration of variability was observed between different batches of primary EC in the time course of both pRb dephosphorylation and cyclin D1 degradation; however, in all circumstances, cyclin D1 down-modulation always followed pRb dephosphorylation (21) . Thus, it is unlikely that the main functional implication of H2O2-induced cyclin D1 degradation is pRb dephosphorylation. In keeping with this interpretation, we and others (45) found that re-expression of cyclin D1 in H2O2-treated cells was not sufficient to reactivate DNA synthesis.

Growing evidence indicates that cyclin D1 is involved in certain cases of neuronal apoptosis (11) . High levels of cyclin D1 expression elicit an apoptotic response (12 13 14) , indicating that increased cyclin D1 expression can present a survival challenge to the cell. We found that sublethal doses of H2O2 triggered significant apoptosis in cells over-expressing cyclin D1. Similarly, when cyclin D1 degradation was prevented by CaMK inhibition, cells were significantly sensitized to H2O2-induced apoptosis. Therefore, the reduction of endogenous cyclin D1 concentration observed after H2O2 treatment of EC must be mechanistically important in establishing cell survival. In agreement with these data, sensitivity to H2O2-induced cell death is increased in PLC-{gamma}1 null cells, where cyclin D1 degradation in response to oxidants is likely disabled (6) . Furthermore, cyclin D1 overexpression sensitizes cancer cells to fenretinide and adriamycin, two drugs inducing intracellular oxidative stress (46 , 47) . As expected, when cells were exposed to a lethal dose of H2O2, cell viability was not altered by the down modulation of cyclin D1 by RNAi (P. Fasanaro and F. Martelli, unpublished observations). These data indicate that cyclin D1 degradation has an antiapoptotic function that is physiologically relevant within the sublethal range of H2O2 dosage.

Both CDK-dependent and CDK-independent activities might contribute to cyclin D1 pro-apoptotic activity. Cyclin D1 expression may lead to unscheduled pRb phosphorylation and reactivation of certain E2F responsive genes that in turn may activate apoptosis (11) . However, although CaMK inhibition prevented cyclin D1 degradation induced by H2O2, it failed to prevent pRb dephosphorylation (P. Fasanaro and F. Martelli, unpublished observations), suggesting that pRb may not be implicated in cyclin D1 sensitization to H2O2.

An alternative scenario is proposed by the evidence that cyclin D1 regulates DNA repair: cyclin D1 can bind to PCNA inhibiting its functions (39 , 48) , and this may directly affect the ability of the cell to cope with DNA oxidative damage. In keeping with this observation, S-phase cells display an exquisitely high sensitivity to H2O2 on cyclin D1 re-expression.

Finally, cyclin D1 has been shown to affect the activity of various cellular transcription factors without the participation of CDKs and it is conceivable that the expression of apoptotic genes may be directly regulated by cyclin D1 (49) .

ACKNOWLEDGMENTS

This work was partly supported by Ministero della Salute (RC/2.15, RF02/conv.228, and RF04/conv.102) and MURST (PNR-T12 n.66084).

FOOTNOTES

1 These authors contributed equally to this work.

Received for publication July 23, 2005. Accepted for publication January 12, 2006.

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