MicroRNA-15b regulates mitochondrial ROS production and the senescence-associated secretory phenotype through sirtuin 4/SIRT4

Mammalian sirtuins are involved in the control of metabolism and life-span regulation. Here, we link the mitochondrial sirtuin SIRT4 with cellular senescence, skin aging, and mitochondrial dysfunction. SIRT4 expression significantly increased in human dermal fibroblasts undergoing replicative or stress-induced senescence triggered by UVB or gamma-irradiation. In-vivo, SIRT4 mRNA levels were upregulated in photoaged vs. non-photoaged human skin. Interestingly, in all models of cellular senescence and in photoaged skin, upregulation of SIRT4 expression was associated with decreased levels of miR-15b. The latter was causally linked to increased SIRT4 expression because miR-15b targets a functional binding site in the SIRT4 gene and transfection of oligonucleotides mimicking miR-15b function prevented SIRT4 upregulation in senescent cells. Importantly, increased SIRT4 negatively impacted on mitochondrial functions and contributed to the development of a senescent phenotype. Accordingly, we observed that inhibition of miR-15b, in a SIRT4-dependent manner, increased generation of mitochondrial reactive oxygen species, decreased mitochondrial membrane potential, and modulated mRNA levels of nuclear encoded mitochondrial genes and components of the senescence-associated secretory phenotype (SASP). Thus, miR-15b is a negative regulator of stress-induced SIRT4 expression thereby counteracting senescence associated mitochondrial dysfunction and regulating the SASP and possibly organ aging, such as photoaging of human skin.

Repetitive UV treatment of primary human skin fibroblasts. Human dermal fibroblasts were isolated from the foreskin of four different donors (age: 1 to 3 years). Approval had been obtained from the Ethics Committee of the Heinrich-Heine-University. The study has been conducted according to the ethical rules stated in the Declaration of Helsinki Principles and the ICH GCP guideline was followed as applicable. Cells were cultivated in Eagle's minimum essential medium with Earle's salts (MEM, PAA Laboratories, Pasching, Austria) and supplemented with 10% fetal bovine serum (Invitrogen, Karlsruhe, Germany), 1% antibiotics/ antimycotics (penicillin, streptomycin, amphotericin B), and 1% glutamine (Invitrogen) and then cultivated on 100 mm plastic culture dishes (Greiner, Solingen, Germany) at 37°C in humidified air with 5% CO 2 . Cells were used between passages five and ten and grown to confluency before treatment. Twenty four hours before UV irradiation media were changed to serum-free MEM. Cells were exposed once daily to a single dose of 100 mW/m² ultraviolet radiation from TL20W/12RS SLV (Phillips, Hamburg, Germany) fluorescent bulbs, which primarily emit in the range of 290-315 nm (= UVB) for a period of five days. For irradiation, medium was replaced by phosphate-buffered saline (37°C, Invitrogen). Control cells (sham treated) were held on room temperature under similar conditions, but without irradiation. Following the last treatment, cells were cultivated for 24 and 72 hours with serum-free MEM medium at 37°C.
Quantification of SIRT4 protein levels by ELISA. Cells were lysed in buffer containing 1% Nonidet P-40, 50 mM Tris/HCl, pH 8.0, 150 mM NaCl, and 2 mM EDTA and protein concentrations were measured using the Bio-Rad protein assay kit. Ninety-six well plates (M9410, Sigma) were coated overnight at 4°C with 20 µg protein lysate in 100 µl coating buffer (0.03 M Na 2 CO 3 , 0.07 M NaHCO 3 , pH 9.6). Plates were washed with PBS-T (0.05% Tween) and blocked with 1% BSA for 2 h at room temperature followed by successive incubation steps with a primary antibody against SIRT4 Calculation of the SCINEXA TM Score. Calculation of the SCINEXA TM Score was performed as described previously [4] except for the following adaptations: In this study the different skin parameters were scored from 0 to 5, not 0 to 3. Regarding extrinsic skin aging the following parameters were not included: Yellowness and dryness of the skin, pseudo scars, permanent erythema as well as the pathological skin conditions (carcinomas, melanoma, actinic pre-cancerosis) as these were exclusion criteria of the study. Instead, we included sunburn and lentigines solaris at two different body sites and evaluated coarse wrinkles at six different locations. Regarding intrinsic skin aging we evaluated lax skin appearance at two different body sites instead of judging the overall appearance of the skin. In addition coarse wrinkles formation was evaluated at six different locations while reduced fat tissue was not included as a scoring parameter. The overall number of parameters remained the same as in the original publication.
Analysis of SMC4 and DLEU2 expression in human skin samples. Expression levels of SMC4 and DLEU2 were determined by real-time quantitative RT-PCR www.impactaging.com analysis as described in the Material and Methods section of the manuscript. PCR primer for SMC4 and DLEU2 were derived from the sequences: NM_05496.3 (SMC4: 5´-TCG AAG CCA TGG AAT TGA CTT-3´ and 5´-TGT TCA GTC TGG-CCT TTT GGT-3´)  Technologies) was employed. Washing of the arrays were performed according to the manufacturer's recommendation. The Cy3 fluorescence intensities were detected by scanning the arrays using the Agilent DNA microarray scanner (G2505B). The resulting image files were analyzed with Feature extraction software. Raw data files were processed and analysed using the GeneSpring Software Version 11 (Agilent Technologies) and were deposited at GEO (Gene Expression Omnibus) (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE45 729).
Enzymatic glutamate dehydrogenase (GDH) activity assay. GDH activity was determined colorimetrically employing an assay kit (Sigma-Aldrich, MAK099) essentially as described by the manufacturer. Cells were lysed in GDH assay buffer and protein concentration of the lysates was measured using the protein assay kit II (Bio-Rad, #500-0002). Enzymatic activities were determined from 25 µg of lysate by calculating the rates of enzymatic activity from the slope of the linear part of the reaction curve.
Immunoblot analysis. Total cell lysates were prepared in lysis buffer containing 1% Nonidet P-40, 50 mM Tris/HCl, pH 8.0, 150 mM NaCl, and 2 mM EDTA. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond C, GE Healthcare, Freiburg, Germany). Blots were probed with an E2F1-specific antibody (#3742; Cell Signaling). Following incubation with the secondary antibody signals were visualized using a LI-COR imaging system. GAPDH levels (antibody from Abcam, Cambridge) served as loading control.

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Supplementary Figures 1-10 Table 1). Means ± s.d. from three independent experiments analysing TACC3depleted cells vs. controls are depicted. To evaluate statistical significances, Student's t-tests were performed (*p<0.05; **p<0.01).
Suppl. Figure 5. Differentiation between extrinsic (UV radiation) and intrinsic (chronological) skin aging through determination of the SCINEXA TM score. Human skin samples obtained from neck vs. buttock skin of the same individuals (age ranges: 18-25 years and 60-66 years; median ± SEM) were assessed for multiple parameters as previously described [4] in order to distinguish between extrinsic (score >2) and intrinsic (score <2) skin aging. To evaluate statistical significance, Wilcoxon signed rank tests were performed (***p<0.001).
Suppl. Figure 6. Quantitative analysis of members of the miR-15/16 family in primary human dermal fibroblasts. Determination of copy numbers of the indicated miRNAs of the miR-15/16 family in fibroblasts four days (A) and six days (B) after γIR (20 Gy) as compared to control (sham treated) cells. Means ± s.d. from six independent experiments are depicted. To evaluate statistical significance for each miRNA, ANOVA (or on ranks) SNK was performed. For clarity, results are depicted for each day separately and sorted by copy number ranking (*p<0.05).
Suppl. Figure 7. Increased expression of SMC4 and DLEU2 in human photoaged skin. Analysis of SMC4 and DLEU2 expression by real-time quantitative RT-PCR in human skin samples obtained from neck vs. buttock skin of the same individuals [age ranges: 18-25 years (A) and 60-66 years (B); n=13-14 per group; median ± SEM]. To evaluate statistical significance, Wilcoxon signed rank tests were performed (***p<0.001).
Suppl. Figure 10. Transfection of MicroRNA-15b inhibitors does not result in an inhibition of glutamate dehydrogenase (GDH) activity in human dermal fibroblasts. Fibroblasts were transfected with miR-15b inhibitors or control oligonucleotides and GDH activity was determined at the indicated days following. N=5-6, mean ± s.d.; to evaluate statistical significances, Mann-Whitney rank sum test were performed.