CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

<div><p>We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.</p></div

p27 is required for myofibroblast differentiation of cardiac fibroblasts.

<p>Cardiac fibroblasts were isolated from hearts of wild-type (“wt”) mice and p27-deficient (“p27 −/−”) littermates. Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours in the presence or absence of 50 μM caffeine. Induction of αSMA was detected by immunoblot and immunostaining. <b>(A)</b> Representative immunoblots, Vimentin served as loading control. <b>(B)</b> Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, <i>n</i> = 8: wt untreated, wt +TGFβ1, p27 −/− untreated, p27 −/− +TGFβ1; <i>n</i> = 5: all others, *<i>p</i> < 0.05 versus wt untreated, ^#<i>p</i> < 0.05 versus wt +caffeine (one-way ANOVA). <b>(C)</b> Representative immunostainings: αSMA was stained in red and Vimentin in green, nuclei were counterstained with DAPI (blue), shown are the overlays of all fluorescence channels. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. αSMA; α smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; n.s., not significant; TGFβ1, transforming growth factor β1.</p

The N- and C-terminus of p27 are required for endothelial cell migration and ATP content.

<p><b>(A)</b> Schematic representation of mitochondrially targeted p27 deletion mutants lacking the N-terminus (“ΔN”), the C-terminus (“ΔC”), or both (“ΔNΔC”). The full-length protein (“fl”) and all mutants contain an N-terminal mitochondrial targeting sequence (“MTS,” red) and a C-terminal myc tag (green). Numbers indicate the deletion endpoints within p27. <b>(B-E)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for the mitochondrially targeted p27 mutants depicted in (A). <b>(B, C)</b> Expression and localization of the mitochondrially targeted mutant p27 proteins were analyzed by immunoblot and immunofluorescence. <b>(B)</b> Representative immunoblot, tubulin served as loading control. <b>(C)</b> Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the targeted p27 mutants by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. <b>(D)</b> Migratory capacity was measured in a scratch wound assay by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 6, *<i>p</i> < 0.05 versus EV, ^#<i>p</i> < 0.05 versus fl mito p27 (one-way ANOVA). <b>(E)</b> ATP content was measured with a luminometric assay. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus EV, ^#<i>p</i> < 0.05 versus fl mito p27 (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. ATP, adenosine triphosphate; CDI, cyclin-dependent kinase inhibitor; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high power field; n.s., not significant; TIM23, translocase of inner mitochondrial membrane 23.</p

Mitochondrial p27 is sufficient to induce endothelial cell migration.

<p><b>(A, B)</b> Endothelial cells were treated with 50 μM caffeine for 18 hours, and mitochondrial (“mito”) and nonmitochondrial (“non-mito”) fractions were separated. p27 and the closely related p21 protein were detected by immunoblot; TIM23 and Trx-1 served as purity controls for the fractions. <b>(A)</b> Representative immunoblots. <b>(B)</b> Semiquantitative analysis of mitochondrial p27 normalized to TIM23. Data are mean ± SEM, <i>n</i> = 6, *<i>p</i> < 0.05 (two-tailed unpaired <i>t</i> test). <b>(C)</b> Proteinase K digestion of mitochondria. The different digestion conditions yield intact mitochondria (1), mitochondria stripped of attached proteins (2), and mitoplasts (3); 4 denotes complete digestion. p27 and marker proteins for the outer (TOM40) or inner (TIM23) mitochondrial membrane and the mitochondrial matrix (GRP75) were detected by immunoblot. <b>(D, E)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Expression and localization of the organelle-targeted p27 proteins were analyzed by immunoblot and immunofluorescence. <b>(D)</b> Representative immunoblot, Tubulin served as loading control. Because of the presence of a trimeric nuclear localization signal at the C-terminus, the nuclear-targeted protein has a larger molecular weight. <b>(E)</b> Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the targeted p27 variants by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. <b>(F)</b> Endothelial cells were transfected with the siRNAs used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.g001" target="_blank">Fig 1</a>. Forty-eight hours later, cells were transfected with an empty vector (“EV”) or the expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Three hours later, a wound was set. Migratory capacity was assessed 18 hours later by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 5: p27, p27 siRNA-1/EV, p27 siRNA-2/EV, p27 siRNA-1/nuc p27 siRNA-1/mito p27; <i>n</i> = 6: all others, *<i>p</i> < 0.05 versus corresponding scr, ^#<i>p</i> < 0.05 versus scr/mito p27, ^§<i>p</i> < 0.05 versus p27 siRNA-1/mito p27, ^$<i>p</i> < 0.05 versus p27 siRNA-2/mito p27 (one-way ANOVA). <b>(G)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Three hours later, a wound was set, and cells were treated with 50 μM caffeine for 18 hours or left untreated. Migratory capacity was assessed by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 5–7, *<i>p</i> < 0.05 versus EV −caffeine (Mann-Whitney pairwise comparison with Bonferroni-corrected <i>p</i>-values). <b>(H)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Twenty-four hours after transfection, the mitochondrial membrane potential was measured with JC1 using flow cytometry. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus EV, ^#<i>p</i> < 0.05 versus nuc p27 (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. DAPI, 4′,6-diamidino-2-phenylindole; HPF, high power field; n.s., not significant; TIM23, translocase of inner mitochondrial membrane 23; TOM40, translocase of outer mitochondrial membrane 40; Trx-1, thioredoxin-1.</p

Caffeine effects in the heart depend on p27.

<p><b>(A)</b> The mouse cardiomyocyte cell line HL-1 was lentivirally transduced with an empty vector (“EV”) or an expression vector for mitochondrially targeted p27 (“mito p27”) and treated with 500 μM H₂O₂ for 48 hours. Apoptosis was measured as annexin V positive/7-PI negative cells by flow cytometry. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus EV −H₂O₂, ^#<i>p</i> < 0.05 versus EV +H₂O₂ (one-way ANOVA). <b>(B)</b> Respiration was determined in isolated heart mitochondria of adult wild-type mice (“wt”) and p27-deficient littermates (“p27ko”), who had received drinking water without caffeine or water supplemented with 0.05% caffeine for 10 days. Respiration was measured as O₂ consumption without the addition of substrates (“mito”) and after the successive addition of malate/glutamate (“M/G”), ADP, rotenone (“rot”), and succinate (“succ”) (left panel). The right panel shows a magnification of O₂ consumption after the addition of M/G and ADP, respectively. Data are mean ± SEM, <i>n</i> = 5–8 per group, *<i>p</i> < 0.05 versus wt without caffeine (one-way ANOVA). <b>(C)</b> Adult p27-deficient animals and their wild-type littermates received drinking water or water supplemented with 0.05% caffeine for 10 days. RNAs were isolated from the hearts of those mice, and microarray analyses were conducted. Data are represented as a Venn diagram. The numbers in the circles indicate the number of transcripts regulated in the two genotypes (<i>n</i> = 3 animals per genotype and treatment, <i>p</i> < 0.05). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. ADP, adenosine diphosphate; n.s., not significant; PI, propidium iodide.</p

Serine 10 and threonine 187 of p27 are required for endothelial cell migration and mitochondrial import.

<p><b>(A, B)</b> Endothelial cells were treated with caffeine for 18 hours or left untreated, and phosphorylation of serine 10 (“p27 P-S10”) and threonine 187 (“p27 P-T187), as well as total p27 (“p27”), were detected by immunoblot. <b>(A)</b> Representative immunoblots with the corresponding loading control (Tubulin) below the respective immunoblot. The asterisk denotes p27 phosphorylated on threonine 187. <b>(B)</b> Semiquantitative analyses of the ratio of phosphorylated p27 to total p27 for both phosphorylation events. Data are mean ± SEM, <i>n</i> = 7: p27 P-S10, <i>n</i> = 6: p27 P-T187, *<i>p</i> < 0.05 (two-tailed unpaired <i>t</i> test). <b>(C, D)</b> Endothelial cells were transfected with an empty vector (“EV”) and expression vectors for mitochondrially targeted p27 (“mito p27 wt”) or a mutant in which serine 10 and threonine 187 were replaced by alanine (“mito p27 S/T-A”). Expression and localization of the corresponding proteins were analyzed by immunoblot and immunofluorescence. <b>(C)</b> Representative immunoblot, tubulin served as loading control. <b>(D)</b> Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the targeted p27 mutants by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. <b>(E)</b> Endothelial cells were transfected as in (C), a wound was set, and migratory capacity was assessed by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus mito p27 wt (one-way ANOVA). <b>(F, G)</b> Endothelial cells were transfected with expression vectors for p27 wild type or the corresponding S/T-A mutant, both without a mitochondrial targeting sequence. Mitochondrial fractions (“mito”) were prepared, and the expressed proteins were detected by immunoblot. <b>(F)</b> Representative immunoblots: the p27 proteins were detected with an anti-myc antibody (“myc (p27)”), TIM23 served as a loading control, and Trx-1 as purity control for the mitochondrial fractions. Analysis of total cell lysates (“lysate”) ensures similar expression levels. <b>(G)</b> Semiquantitative analysis of mitochondrial p27 normalized to TIM23. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus p27 wt (two-tailed unpaired <i>t</i> test). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. DAPI, 4′,6-diamidino-2-phenylindole; HPF, high power field; n.s., not significant; TIM23, translocase of inner mitochondrial membrane 23; Trx-1, thioredoxin-1.</p

Caffeine improves outcomes after myocardial infarction in prediabetic mice and induces mitochondrial translocation of p27.

<p>Two-month-old wild-type mice were fed a diabetogenic diet for 11 weeks. For the last 10 days, one group of animals received drinking water supplemented with 0.05% caffeine. Afterward, myocardial infarction was induced by ligation of the left anterior descending coronary artery for 60 minutes followed by reperfusion. Twenty-one days after infarction, hearts were excised, sectioned, and the sections stained. <b>(A)</b> Representative Gomori stainings of sections of 3 different hearts for each dietary regimen. <b>(B)</b> Infarct size per left ventricle and <b>(C)</b> minimum left ventricular (“LV”) wall thickness in the infarcted myocardium. Data are mean ± SEM, <i>n</i> = 8: diabetogenic diet, <i>n</i> = 10: diabetogenic diet +caffeine, *<i>p</i> < 0.05 (one-way ANOVA). <b>(D)</b> Representative immunostainings of border zone sections for each dietary regimen. TIM23 is stained in red, p27 in green, nuclei were counterstained with DAPI (blue), merge shows an overlay of all fluorescence channels. The dotted rectangles indicate the sections shown in higher magnifications. <b>(E)</b> Heart mitochondria were isolated, and p27 was detected by immunoblot; GRP75 and TIM23 served as loading controls. To control for purity of the mitochondria, a total heart lysate (“lys”) was used in parallel, and Vimentin was detected. Shown is a representative immunoblot. <b>(F)</b> Semiquantitative analysis of mitochondrial p27; data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. DAPI, 4′,6-diamidino-2-phenylindole; GRP75, 75 KDa glucose-regulated protein; TIM23, translocase of inner mitochondrial membrane 23.</p

Caffeine enhances respiration, ATP content, and mitochondrial localization of p27 in old mouse hearts.

<p><b>(A-D)</b> Twenty-two-month-old wild-type mice received drinking water (“old”) or water supplemented with 0.05% caffeine for 10 days (“old+caffeine”). <b>(A)</b> O₂ consumption was measured in isolated heart mitochondria without the addition of substrates (“mito”) and after the successive addition of malate/glutamate (“M/G”), ADP, rotenone (“rot”), and succinate (“succ”) (left panel). The right panel shows a magnification of O₂ consumption after the addition of malate/glutamate and ADP, respectively. Data are mean ± SEM, <i>n</i> = 6 per group, *<i>p</i> < 0.05 (one-way ANOVA). <b>(B)</b> Mitochondrial ATP content was measured with a luminometric assay. Data are mean, <i>n</i> = 5 per group, *<i>p</i> < 0.05 (one-way ANOVA). <b>(C)</b> Heart mitochondria were isolated, and p27 was detected by immunoblot; GRP75 and TIM23 served as loading controls. To control for purity of the mitochondria, a total heart lysate (“lys”) was used in parallel, and Vimentin was detected. Shown is a representative immunoblot. <b>(D)</b> Semiquantitative analysis of mitochondrial p27; data are mean ± SEM, <i>n</i> = 7 per group, *<i>p</i> < 0.05 (one-way ANOVA). <b>(E, F)</b> Six-month-old wild-type mice received drinking water or water supplemented with 0.05% caffeine for 10 days. <b>(E)</b> Heart mitochondria were isolated, and p27 was detected by immunoblot; GRP75 and TIM23 served as loading controls. To control for purity of the mitochondria, a total heart lysate (“lys”) was used in parallel, and Vimentin was detected. Shown is a representative immunoblot. <b>(F)</b> Semiquantitative analysis of mitochondrial p27; data are mean ± SEM, <i>n</i> = 5 per group, *<i>p</i> < 0.05 (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. ADP, adenosine diphosphate; ATP, adenosine triphosphate; GRP75, 75 KDa glucose-regulated protein; TIM23, translocase of inner mitochondrial membrane 23.</p

p27 is required for endothelial cell migration.

<p><b>(A, B)</b> p27 was knocked down in endothelial cells by transfection with 2 different siRNAs targeting the p27 mRNA (“p27 siRNA-1,” “p27 siRNA-2”) or a scrambled siRNA (“scr”) as control, and p27 levels were determined by immunoblot. <b>(A)</b> Representative immunoblots, Actin served as loading control. <b>(B)</b> Knockdown efficiency was determined by semiquantitative analysis of immunoblots. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus scr (one-way ANOVA). <b>(C)</b> Endothelial cells were transfected with the same siRNAs as before, a wound was set 48 hours after transfection, and the cells were treated with 50 μM caffeine for another 18 hours or left untreated. Migratory capacity was assessed by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus scr −caffeine, ^#<i>p</i> < 0.05 versus scr +caffeine (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. HPF, high power field; siRNA, small interfering RNA.</p

Mitochondrial p27 restores the impaired αSMA up-regulation in p27-deficient cardiac fibroblasts.

<p>Fibroblasts isolated from the hearts of p27-deficient mice were lentivirally transduced with an expression vector for mitochondrially targeted p27 (“mito p27”) or a corresponding empty vector (“EV”). <b>(A)</b> Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the mitochondrially targeted p27 by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. <b>(B, C)</b> Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours, and αSMA was detected by immunoblot. <b>(B)</b> Representative immunoblots, Vimentin served as loading control. <b>(C)</b> Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus mito p27 +TGFβ1 (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. αSMA; α smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; n.s., not significant; TGFβ1, transforming growth factor β1; TIM23, translocase of inner mitochondrial membrane 23.</p