Increased cell senescence contributes to the pathogenesis of aging and aging-related disease. Senescence of human fibroblasts
Human diploid fibroblasts exhibit a progressive decreased replication rate in culture, eventually reaching a maximum cumulative number of cell replications called the limiting population doubling level or Hayflick limit [
Senescence of human fibroblasts
A long-held concept in theories of cell senescence is the increased wear-and-tear of cell proteins leading to increased formation of reactive oxygen species (ROS) and related increased steady-state levels of oxidative damage to proteins and DNA – with telomere attrition associated with the latter [
Human fetal lung MRC-5 fibroblasts and human foreskin BJ fibroblasts were purchased from the European Collection of Animal Cell Cultures (ECACC, Porton Down, London, UK). MRC-5 and BJ cells were grown in Eagle’s Minimum Essential Medium (MEM; Invitrogen Paisley, Scotland) supplemented with 10% fetal bovine serum (FBS) (Labtech International Ltd., Uckfield, UK), 1% penicillin-streptomycin (Sigma-Aldrich, Poole, Dorset, UK), 2 mM L-glutamine, and 100 mM sodium pyruvate (Invitrogen). R-Sulforaphane (SFN, ≥95%), L-lactic dehydrogenase (rabbit muscle), pepsin (porcine stomach mucosa), pronase E (
MRC-5 and BJ fibroblasts were passaged every 7 days: seeding density - 4000 cells/cm2 for MRC-5 fibroblasts and 3000 cells/cm2 for BJ fibroblasts. Cell viability was assessed by the Trypan Blue dye exclusion technique. Cells were cultured under 5% CO2 in air, 100% humidity at 37°C with Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, and 100
MRC-5 fibroblasts at passage 4 were seeded in 6-well plates (200,000 cells per well). After 24 h, 1
Reverse transcriptase reaction was performed with 100 ng (BJ cells) or 50 ng (MRC-5 cells) total RNA and using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™). The reaction was incubated at 25°C for 10 min, then 37°C for 2 h, and then 85°C for 5 min. After 2-fold dilution, 2
The cell protein extract was prepared with RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM, Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM
Protein glycation, oxidative damage and N
Experiments were performed using ≥3 biological replicates. Data are presented as
When human MRC-5 fibroblasts were incubated in primary culture with SFN, growth arrest and toxicity was induced at ≥2
Delay of replicative senescence in MRC-5 and BJ fibroblasts by sulforaphane and link to suppression of glucose consumption. (a) Concentration-response curve for the effect of SFN on the growth of MRC-5 fibroblasts
SFN is an activator of transcription factor Nrf2 [
Changes in gene expression induced by sulforaphane in early passage, nonsenescent MRC-5 cells
We next compared the expression of ARE-linked genes and senescent-related genes in nonsenescent and senescent MRC-5 fibroblasts – cells from passages 3 and 11, respectively – and the effect of SFN treatment. Relative mRNA copy number assessment showed decreased expression of redox-related genes, NQO1, GSR, TXN, and TXNRD1, in senescence with increased expression by SFN in both nonsenescent and senescent fibroblasts, correcting the decrease in senescence (Figures
Effect of sulforaphane on gene expression in young and senescent MRC-5 fibroblasts
Considering extracellular matrix-related genes, SFN surprisingly increased the expression of matrix metalloproteinase- (MMP-) 1 and pro-
To explore the mechanism of SFN-induced decrease in glucose metabolism in the delay of fibroblast senescence, we studied the expression of genes regulating glycolysis and cellular uptake of glucose. The expression of the master regulator of glycolytic enzyme expression Mondo A (MLXIP) was decreased in MRC-5 fibroblast senescence, and this was unchanged by SFN treatment; the expression of its functional complexing partner, max-like protein X (MLX), was unchanged in senescence and by SFN treatment (Figures
Mechanism of decreased glucose consumption by sulforaphane in delay of fibroblast senescence
A further level at which glucose metabolism is regulated is hexokinase (HK) which catalyzes the formation of glucose-6-phosphate (G6P) from glucose – the initial entry of glucose into glycolysis and pentose phosphate pathway metabolism. HK1 and HK2 isozymes are expressed in fibroblasts. HK1 and HK2 mRNA levels were increased in senescent MRC-5 fibroblasts, and this was not corrected by SFN, although there was a time × treatment effect where SFN decreased mRNA of HK1 and HK2 in senescence (Figure
An additional level at which SFN imposes a CR response was through increased expression of thioredoxin-interacting protein (TXNIP). The most profound effect of senescence on the gene expression of MRC-5 cells was the 8-fold increase in the expression of TXNIP. This was increased further to 21-fold by treatment with SFN (Figure
There were multiple indications of early-stage protection from oxidative damage by SFN treatment: decreased flux of 8-oxodG DNA (Figure
SFN also decreased the cell protein content of transglutaminase-produced cross-link GEEK by ca. 45%: 3.15 ± 0.71 versus 1.74 ± 0.29 mmol/mol lys. This likely reflected increased proteolysis and clearance of GEEK-modified proteins since the flux of formation of GEEK was increased 5-fold on approach to senescence and was not decreased by SFN (Figures
Transcriptomic analysis of nonsenescent and senescent MRC-5 fibroblasts showed change in the expression of 106 of 20,773 genes assessed: 59 increased and 47 decreased (Table
Gene expression changes in senescence of MRC-5 fibroblasts
No. | Gene ID | Name | ΔLog2 expression | Comment | |
---|---|---|---|---|---|
1 | NPY | Pro-neuropeptide Y | 0.022 | 6.50 | Regulates cell cycle, proliferation. |
2 | TRIM63 | E3 ubiquitin-protein ligase | 0.012 | 5.26 | Promotes ubiquitination and degradation of misfolded proteins. |
3 | BANK1 | B-cell scaffold protein with ankyrin repeats | 0.003 | 3.53 | Lyn-mediated tyrosine phosphorylation of inositol 1,4,5-trisphosphate receptors |
4 | LRRC69 | Leucine-rich repeat-containing protein 69 | 0.005 | 2.97 | Unknown function. |
5 | PPP1R3G | Protein phosphatase 1 regulatory subunit 3G | 0.013 | 2.78 | Stimulates glycogen synthase activity. |
6 | TMEM47 | Transmembrane protein 47 | 0.044 | 2.69 | Senescence associated gene. |
7 | SLC14A1 | Urea transporter 1 | 0.009 | 2.57 | Facilitates transmembrane urea transport down a concentration gradient. |
8 | DAP | Death-associated protein 1 | 0.048 | 2.49 | Negative regulator of autophagy |
9 | INA | Alpha-internexin | 0.031 | 2.18 | Intermediate filament protein. |
10 | GMFG | Glia maturation factor gamma | 0.007 | 2.08 | Target of senescence-associated microRNA, hsa-miR-369-5P. |
11 | MATN3 | Matrilin-3 | 0.037 | 1.97 | Extracellular matrix protein |
12 | PSG1 | Pregnancy-specific beta-1-glycoprotein 1 | 0.040 | 1.96 | Senescence-associated protein. |
13 | DAAM2 | Disheveled-associated activator of morphogenesis 2 | 0.028 | 1.95 | Regulator of the Wnt signaling pathway. |
14 | PDK2 | Pyruvate dehydrogenase kinase, isoenzyme 2 | 0.038 | 1.73 | Linked to increased glycolysis in senescence. |
15 | SORT1 | Sortilin 1 | 0.035 | 1.69 | Sorting receptor in the Golgi compartment and clearance receptor on the cell surface. |
16 | PSG9 | Pregnancy-specific beta-1-glycoprotein 9 | 0.048 | 1.68 | Senescence associated protein. |
17 | MAP7D3 | MAP7 domain-containing protein 3 | 0.049 | 1.66 | Promotes the assembly and stability of microtubules. |
18 | GPC4 | Glypican-4 | 0.016 | 1.60 | Cell surface heparan sulfate proteoglycan involved in Wnt signaling. |
19 | KCTD12 | Pfetin | 0.016 | 1.58 | Potassium channel protein. |
20 | UHMK1 | Serine/threonine-protein kinase Kist | 0.007 | 1.50 | Controls CDKN1B subcellular location and cell cycle progression in the G1 phase. |
21 | RCAN3 | Calcipressin-3 | 0.039 | 1.44 | Negative regulator of calcineurin-linked transcriptional regulation. |
22 | PDP2 | Mitochondrial pyruvate dehydrogenase-phosphatase 2 | 0.031 | 1.34 | Drives mitochondrial dysfunction in senescence. |
23 | MALSU1 | Mitochondrial assembly of ribosomal large subunit protein 1 | 0.049 | 1.33 | Involved in mitochondrial ribosome function and mitochondrial translation. |
24 | UFD1L | Ubiquitin recognition factor in ER-associated degradation protein 1 | 0.038 | 1.25 | Upregulated in stress-induced senescence. |
25 | HLA-E | HLA class I histocompatibility antigen, |
0.002 | 1.25 | Senescent fibroblast marker. |
26 | NDST2 | Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 2 | 0.016 | 1.23 | Involved in biosynthesis of heparan sulfate. |
27 | ZMAT2 | Zinc finger matrin-type protein 2 | 0.001 | 1.09 | Regulated by sirtuin-1. |
28 | MUC1 | Mucin-1 | 0.015 | 1.06 | Transmembrane glycoprotein participating in growth factor receptor signaling. |
29 | CTBS | Di-N-acetylchitobiase (EC 3.2.1.-) | 0.037 | 1.04 | Involved in the degradation of asparagine-linked glycoproteins |
30 | ABTB2 | Ankyrin repeat and BTB/POZ domain-containing protein 2 | 0.002 | 0.99 | Substrate adaptor for cullin-3 ubiquitin ligase; may increase Nrf2 degradation in senescence. |
31 | LYNX1 | Ly-6/neurotoxin-like protein 1 | 0.024 | 0.85 | Negative regulator of nicotinic receptor signaling. |
32 | DNAJC19 | Mitochondrial import inner membrane translocase subunit TIM14 | 0.008 | 0.82 | Mitochondrial co-chaperone. |
33 | COL4A3BP | Collagen type IV alpha-3-binding protein | 0.001 | 0.81 | Controls mitochondrial fission, fusion, dysfunction, and mitophagy. |
34 | TMEM165 | Transmembrane protein 165 | 0.014 | 0.80 | Target of miR-181a in fibroblast senescence. |
35 | TAX1BP1 | Tax1-binding protein 1 | 0.011 | 0.76 | Ubiquitin-binding adaptor protein; negative regulation of the NF- |
36 | HSD11B1L | Hydroxysteroid 11-beta-dehydrogenase 1-like protein | 0.004 | 0.75 | Involved in glucocorticoid metabolism. |
37 | TMX2 | Thioredoxin-related transmembrane protein 2 | 0.045 | 0.66 | Disulfide isomerase enriched on the mitochondria-associated membrane of the endoplasmic reticulum. |
38 | ZNF525 | Zinc finger protein 525 | 0.045 | 0.65 | May be involved in transcriptional regulation. |
Genes listed had expression changes between passages 3 and 11 in MRC-5 fibroblasts which were corrected by SFN treatment. Gene expression changes are rank-ordered by log2 expression change (highest to lowest).
There were 15 genes with expression that changed between SFN-treated senescent and SFN-treated nonsenescent fibroblasts (Table
Gene expression changed in senescence of MRC-5 fibroblasts treated with SFN
No. | Gene ID | Name | ΔLog2 expression | |
---|---|---|---|---|
1 | TXNIP | Thioredoxin-interacting protein | 0.039 | 6.8 |
2 | ACKR4 | Atypical chemokine receptor 4 | 0.024 | 5.6 |
3 | C11orf87 | Neuronal integral membrane protein 1 | 0.039 | 3.8 |
4 | PRRX2 | Paired related homeobox 2 | 0.046 | 3.3 |
5 | FZD4 | Frizzled class receptor 4 | 0.005 | 2.5 |
6 | ACAA2 | Mitochondrial 3-oxoacyl-coenzyme A thiolase | 0.024 | 2.5 |
7 | MOB4 | Mps one binder kinase activator-like 3 | 0.007 | 1.8 |
8 | SLC46A1 | Proton-coupled folate transporter | 0.035 | 1.7 |
9 | CSNK1G1 | Casein kinase 1 gamma 1 | 0.023 | 1.5 |
10 | ACTR2 | Actin-related protein 2 homolog | 0.014 | 1.1 |
11 | MAEA | Macrophage erythroblast attacher | 0.016 | 1.1 |
12 | GALC | Galactosylceramidase | 0.024 | 1.1 |
13 | VAT1 | Vesicle amine transport 1 | 0.019 | 1.1 |
14 | ARSJ | Arylsulfatase family member J | 0.004 | 0.9 |
15 | SUN1 | Sad1 and UNC84 domain containing 1 | 0.045 | −0.7 |
Genes listed had expression changes between passages 3 and 11 in MRC-5 fibroblasts treated with SFN. Gene expression changes are rank-ordered by log2 expression change (highest to lowest).
We show herein for the first time that SFN, a health beneficial isothiocyanate formed from a glucosinolate precursor in broccoli, delays fibroblast senescence through a CR mimetic-like response. This was achieved by treatment with 1
The response was associated with glucose and glycolytic restriction mediated by a multitier mechanism limiting the cellular availability and metabolism of glucose: increased expression of TXNIP, curbing the entry of glucose into cells; decreased HK2, curbing the entry of glucose into cellular metabolism; and decreased PFKFB2 and increased G6PD, downregulating glycolysis. Benefits accrued were protection from increased protein and DNA oxidative damage and GEEK protein cross-linking, which are otherwise part of increased macromolecular wear-and tear driving senescence.
We observed a profound increase in glucose consumption by MRC-5 on the approach to senescence and countering of this by SFN. A similar response was found in BJ fibroblasts. Increased glucose metabolism was mediated by increased HK2 expression at mRNA and protein levels. Increased HK2 mRNA and total HK activity in senescent human fibroblasts was reported previously [
Increased cellular HK2 activity on the approach to senescence with cells replete with glucose leads to increased cellular G6P concentration. G6P is the first metabolite of glycolysis and pentose phosphate pathways of glucose metabolism. It is also an essential cofactor for functional activity of the Mondo A/Mlx complex that increases expression of glycolytic enzymes by complex translocation to the nucleus and binding to promoter carbohydrate response elements of target genes, including expression of TXNIP as an autoregulatory curb on glucose metabolism during periods of excess glucose availability [
A further feature of increased glycolysis mediated by HK2 is the accumulation of G6P and partial displacement of HK2 from mitochondria, impairing mitochondrial oxygen consumption similar to that found under conditions of inhibition of ADP recycling [
Increased glycolysis in fibroblast senescence may be also driven by increased PFKBP2 activity [
Entry of glucose into glycolysis, hexokinase-2 linked mitochondrial dysfunction, and transcriptional signaling for glycolytic overload. Text boxes indicate processes involved in senescence-associated glycolytic overload. These are reversed by SFN treatment except for TXNIP-mediated curb on glucose uptake which is enhanced. Key: green, transcriptional and allosteric effectors for increased glycolysis; blue, HK2 displacement from mitochondria with increased cellular G6P, with metabolic channelling to glycogen synthesis. VDAC: voltage-dependent anion channel.
In transcriptomic analysis, SFN treatment corrected 36% of gene expression changes found in senescence. Changes in gene expression related to suppression of increased glycolysis (PDK2, PDP2) improved mitochondrial function (COL4A3BP), increased glycogen synthesis (PPP1R3G), decreased inflammation (TAX1BP1), and increased proteolysis (TRIM63). These are likely linked to the glycolytic restriction and downstream effects of SFN contributing to delay of senescence. The apparent disparity between outcomes in microarray transcriptomics analysis and mRNA copy number array was due to the robust quantitative methodology of the latter technique. Where gene expression changes were large – such as for SFN treatment-associated increase in TXNIP expression in senescent MRC-5 cells – outcomes from the two methods were corroborative.
SFN-induced decrease in cell protein content of the major cellular protein cross-link, GEEK, formed by transglutaminase may also contribute to delay of senescence. Histone proteins are targets for cross-linking in senescence and may thereby impair replicative capacity [
Our experimental findings show evidence of cellular glucose and glycolytic restriction induced by SFN associated with delay of fibroblast senescence. Glucose is a major caloric nutrient source of human fibroblasts in primary culture [
The results of this study suggest the following conclusions:
SFN delays the senescence of human MRC-5 and BJ fibroblasts Cell senescence is associated with a progressive and marked increased rate of glucose metabolism through glycolysis, and this is countered by SFN treatment – glycolytic restriction previously found to delay senescence [ SFN decreased glucose metabolism on the approach to senescence by increasing the expression of TXNIP, curbing the entry of glucose into cells; decreasing HK2, curbing the entry of glucose into cellular metabolism; decreasing 6-phosphofructo-2-kinase, downregulating the formation of the allosteric enhancer of glycolysis F-2,6-P2; and increasing G6PD, downregulating the ChRE-mediated transcriptional enhancement of glycolysis by Mondo/Mlx/G6P SFN also enhanced the clearance of proteins cross-linked by transglutaminase which otherwise increased in senescence Screening of compounds to counter senescence-associated glycolytic overload may be an effective strategy to identify compounds with antisenescence activity
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflict of interest in relation to this work.
F.H. and M.X. performed most of the experiments and participated in the experimental design, data analysis, figure preparation, and discussion. M.F. supervised the transcriptomics analysis. N.R., M. F, and P.J.T. contributed to the experimental design, data analysis, discussion, and writing. P.J.T. and M.F. secured the funding. Florence Hariton and Mingzhan Xue are joint first authors.
F.H. thanks the Biotechnology and Biosciences Research Council (BBSRC) and Unilever for an Industrial PhD studentship. P.J.T. thanks the BBSRC for funding on the Selective Chemical Intervention in Biological Systems (SCIBS) programme. We thank Dr. J.D. Moore for guidance on microarray transcriptomics data analysis.
The supplementary materials include one table and 2 figures. Supplementary Table 1: transcriptomic analysis of genes with changed expression in senescence of MRC-5 fibroblasts in vitro. Supplementary Figure 1: changes in gene expression induced by sulforaphane in early passage, nonsenescent MRC-5 fibroblasts in vitro. Supplementary Figure 2: effect of sulforaphane on end-of-passage gene expression in young and senescent MRC-5 fibroblasts in vitro.