Abstract
Purpose.:
To investigate the alterations in microRNA (miRNA) expression during replicative senescence (RS) in human trabecular meshwork (HTM) cells.
Methods.:
Two HTM cell lines were serially passaged until they reached RS. Changes in expression of 30 miRNAs were assessed by real-time quantitative (q)-PCR. The effects of miR-146a on gene expression were analyzed with gene arrays and the results confirmed by real-time q-PCR. Protein levels of IRAK1 and PAI-1 were analyzed by Western blot and those of IL6 and IL8 by ELISA. Senescence-associated markers were monitored by flow cytometry and cell proliferation by BrdU incorporation.
Results.:
RS of HTM cells was associated with significant changes in expression of 18 miRNAs, including the upregulation of miR-146a. miR-146a downregulated multiple genes associated with inflammation, including IRAK1, IL6, IL8, and PAI-1, inhibited senescence-associated β-galactosidase (SA-β-gal) activity and production of intracellular reactive species (iROS), and increased cell proliferation. Overexpression of either IRAK1 or PAI-1 inhibited the effects of miR-146a on cell proliferation and iROS production in senescent cells.
Conclusions.:
RS in HTM cells was associated with changes in miRNA expression that could influence the senescent phenotype. Upregulation of the anti-inflammatory miR-146a may serve to restrain excessive production of inflammatory mediators in senescent cells and limit their deleterious effects on the surrounding tissue. Among the different proteins repressed by miR-146a, the inhibition of PAI-1 may act to minimize the effects of senescence on the generation of iROS and growth arrest and prevent alterations of the extracellular proteolytic activity of the TM.
The trabecular meshwork (TM) from glaucoma donors is characterized by chronic activation of a stress response that leads to increased production of inflammatory markers.
1–3 Chronic activation of a similar inflammatory response has been found during aging and certain age-related conditions in other tissues.
4–12 One of the factors proposed to contribute to such a response is the increased presence of senescent cells.
Senescent cells have been shown to accumulate with age and in certain pathologic conditions in several tissues and organs,
5,9,13 including the TM in glaucoma.
14 The senescent response is associated with a series of phenotypic changes that have been proposed to disrupt the tissue microenvironment and contribute to pathologic alterations associated with aging.
15,16 An important alteration observed in senescent cells that may contribute to tissue malfunction is the presence of a characteristic senescence-associated secretory phenotype (SASP).
17–19 Such a secretory phenotype involves an increase in the release of inflammatory mediators and growth factors that can affect the function of adjacent cells and lead to a chronic activation of a stress response
18 similar to that observed in the TM of glaucoma donors.
3
The regulatory mechanisms that mediate the phenotypic changes in senescent cells and, in particular, those involved in the chronic activation of inflammatory mediators have not been completely elucidated.
MicroRNAs (miRNAs) are important regulators of gene expression and have been implicated in a variety of cellular functions, including differentiation, apoptosis, and cancer progression.
20–22 miRNAs are transcribed as primary transcripts or pri-miRNAs that are converted in the nucleus into 70-nucleotide, stem-loop structures known as pre-miRNAs. These pre-miRNAs are then processed in the cytoplasm to mature miRNAs, 21 to 23 nucleotides in length, by the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC).
23 After integration into the active RISC, miRNAs bind to target sites in the 3′ untranslated region (UTR) of the specific mRNA transcripts and inhibit translation or induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC.
24 There is some experimental evidence suggesting that miRNAs play a role in cellular senescence. Several miRNAs such as miR-34 and -20a have been shown to induce senescent growth arrest.
25,26 Ablation of Dicer in mouse embryonic fibroblasts also induces senescence by upregulating p53.
27 Furthermore, we have recently shown that stress-induced premature senescence (SIPS) is associated with significant alteration in expression of several miRNAs in both human fibroblasts and TM cells.
28 It has been hypothesized that miRNAs play both positive and negative roles in regulating the senescent response.
29–31 Specifically, it has recently been reported that the anti-inflammatory miR-146 is upregulated in senescent fibroblasts in response to increased levels of inflammatory cytokines induced by the senescent response, generating a negative feedback loop that restrains excessive production of inflammatory mediators in senescent cells and limits the deleterious effects of the SASP on the surrounding tissues.
31 However, there is still little information about the role that miRNAs play in modulating the senescent response.
We investigated changes in expression of miRNAs during the process of replicative senescence (RS) in human TM (HTM) cells. Since one of the miRNAs significantly upregulated during this process was miR-146a, which has been implicated in the modulation of the SASP in fibroblasts,
31 we further investigated the effects of this miRNA on gene expression in HTM cells and the mechanisms by which miR-146a may contribute to the senescent response in the TM.
Donor human eyes or cornea rings were obtained from the New York Eye Bank within 7 days after death, according to the tenets of the Declaration of Helsinki. HTM from a single individual was dissected from surrounding tissue and digested in 10 mg collagenase/20 mg bovine serum albumin (BSA)/5 mL phosphate-buffered saline (PBS). The cells were seeded on collagen I-coated 3-cm Petri dishes and maintained at 37°C in a humidified atmosphere of 5% CO2 in TM culture medium containing 20% fetal bovine serum (FBS). The TM culture medium was low-glucose Dulbecco's modified Eagle's medium (DMEM) with l-glutamine and 110 mg/L sodium pyruvate, supplemented with 100 μM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. All reagents were obtained from Invitrogen Corp. (Carlsbad, CA). The HTM636 and HTM1073 cell lines were generated from 22- and 26-year-old donors. These cells lines were used for experiments involving comparisons between late-passage (p15 or p11) and early-passage (p4–6) cells. The HTM682, HTM113, and HTM714 cell lines were generated from 47-, 49- and 42-year-old donors and were used at p4 to p7, to study the effects of miR-146a on gene and protein expression. None of the donors had a history of POAG or ocular diseases.
Transfection of miRNAs or plasmids was performed with a nonviral transfection system (Nucleofector System; Amaxa Inc. Gaithersburg, MD), according to the manufacturer's instructions. miR-146a mimic (146aM) or negative miRNA control mimic (ConM, 120 pmol per 5 × 105 cells; Dharmacon, Inc. Chicago, IL), or PAI-1 plasmid (pPAI-1) (pCMV-SPORT6; Open Biosystems, Huntsville, AL), recombinant IRAK1 plasmid (pIRAK1; pENTR221, OHS4559-99869058; Open Biosystems), or psiCHECK2 vector (2 μg per 5 × 105 cells, pCon; Promega Corp., Madison, WI) were transfected into HTM cells (program T23; Amaxa). For experiments involving cotransfection of expression plasmids and miRNA mimics, 2 μg of plasmid (pPAI-1, pIRAK1, or pCon) and 120 pmol of miRNA mimic (either 146aM or ConM) were cotransfected into 5 × 105 HTM cells by using the same program. The culture medium was replaced with fresh DMEM 24 hours after transfection, and cell culture supernatant or cells were collected 72 hours after transfection.
Cell growth was quantified with a BrdU cell proliferation assay (Calbiochem, San Diego, CA) according to the manufacturer's instructions. Briefly, 100 μL of cells (nontransfected p4 and p15, or transfected with miR-146a mimic [146aM] or control mimic [ConM]) at concentration of 4 × 104 cells/mL were seeded into a 96-well culture dish and incubated for 24 (p4 and p15) or 48 (146aM and ConM) hours. Culture medium was then replaced with 100 μL fresh DMEM containing 10% FBS and BrdU 1:10,000 dilution. After overnight incubation, the cells were fixed, and BrdU incorporation was measured by using anti-BrdU antibody and reconstituted peroxidase goat anti-mouse IgG HRP conjugate. The color was then developed by adding substrate solution to each well. After 15 minutes of incubation in the dark at room temperature (RT), blocking solution (Stop; Cell Signaling, Danvers, MA) was added to each well, and absorbance was measured with a spectrophotometric plate reader at dual wavelengths of 450 to 540 nm.
Cells were washed twice in cold PBS. Total protein was extracted with RIPA buffer (150 mM NaCl, 10 mM Tris [pH 7.2], 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA [pH 8.0]) containing 1× protease inhibitor cocktail (Roche, Inc., Indianapolis, IN). Protein concentration was determined with a protein assay (Micro BCA Protein Assay Kit; Pierce, Rockford, IL). Total protein extracts (40 μg) were separated by 8% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). The membranes were blocked with 5% nonfat dry milk and incubated overnight with the primary antibodies anti-IRAK1 (Cell Signaling, Inc.) or anti-PAI-1/serpine-1 (Santa Cruz Biotechnology, Santa Cruz, CA). Then they were incubated with a secondary antibody conjugated to HRP for 1 hour at RT. Immunoreactive proteins were visualized by chemiluminescence (ECL Plus; GE Healthcare, Pittsburgh, PA). For detection of endogenous control, the membrane was stripped with stripping buffer (25 mM glycine ([pH 3.0] plus 1% sodium dodecyl sulfate [SDS]) and then incubated with anti-β-tubulin (SC-9935; Santa Cruz Biotechnology).
HTM cells were transfected with miR-146aM or ConM. Three days after transfection, cell culture supernatant was collected, and 25 μL culture medium was used to quantify protein levels of IL6 and IL8 (Human TH1/Th2 plex FlowCytomix kit; Render MedSystems, Inc., Burlingame, CA) according to the manufacturer's instructions. Briefly, 25 μL of samples or standards were mixed with 25 μL of bead mixture and 50 μL of biotin-conjugate mixture. After 2 hours' incubation at RT, the tubes were centrifuged at 200g for 5 minutes. The pellet was washed twice with assay buffer and then 50 μL of streptavidin-PE was loaded to each tube. After a 1-hour incubation, the pellet was spun down and washed twice with assay buffer. The samples were then analyzed on a flow cytometer in the FL3 channel.
Effects of miR-146a on the Expression of Cellular Senescence Markers in HTM Cells
We observed significant changes in miRNA expression in replicative scenescent HTM cells. There were several differences between the two cell lines analyzed, suggesting some level of variability in the alterations of miRNA expression induced by RS. However, 18 microRNAs of the 30 analyzed were consistently up- or downregulated in both cell lines.
Similar to what was previously observed in SIPS, RS of TM cells was associated with the downregulation of several members of the miR-106b family (miR-17–5p, -18a, -20a, -20b, -106a, and -106b) that are located in the oncogenic miRNA polycistronic clusters 17–5p-92a1, 106b-25, and 106a-363. These clusters are frequently upregulated in cancer, and their oncogenic effects are mediated at least in part by members of the miR-106b family that are known to promote cell cycle progression.
40–42 The consistent downregulation of these miRNAs observed in both stress-induced and RS appears to be a common feature of senescent cells that could contribute to their permanent growth arrest.
A downregulation of members of the miR-15 family, similar to that previously observed in SIPS, was found in one of the cell lines (HTM1073-07-26), whereas only miR-15a was significantly downregulated in HTM636-07-22. Given the proapoptotic role of miR-15a, the downregulation of this miRNA in senescent cells could contribute to the increased resistance to the apoptosis characteristic of senescent cells.
RS was also associated with changes in miRNAs that have not been observed in SIPS. These changes included a particularly notable upregulation of several miRNAs: miR-146a, -329, -369, -409–5p, -432, -493, and -495.
One of the miRNAs more clearly upregulated in senescent HTM cells, miR-146a, has also been found to be upregulated in senescent human fibroblasts and is believed to be implicated in modulating the inflammatory response
29 and in particular the senescence-associated inflammatory mediators
IL6 and
IL8.
43 miR-146a is known to target IRAK1, a key activator of the innate immune system signaling cascade that leads to the induction of inflammatory target gene expression.
43 Upregulation of miR-146a/b in senescent fibroblasts has been hypothesize to serve as a mechanism aimed at preventing excessive production of inflammatory mediators by senescent cells, thus limiting the impact of the SASP on adjacent cells.
31
Although in senescent fibroblasts it has been reported that both miR-146a and -146b were upregulated during RS,
31 HTM cells showed a notable upregulation only of miR-146a, whereas they showed a relatively low upregulation of miR-146b (3.4-fold in HTM1073 cells and 1.67-fold in HTM636 cells). These results suggest cell-type–specific differences in the changes in expression of these miRNAs during RS.
Analysis of the changes in gene expression induced by miR-146a in HTM cells was consistent with the anti-inflammatory role proposed for this miRNA. In addition to downregulation of the well-characterized target IRAK1 and the concomitant downregulation of IL6 and IL8 observed in senescent fibroblasts, miR-146a also decreased the expression of multiple genes involved in the inflammatory response including: IL11, IL8, CXCL3 (GRO3), CXCL6 (GCP2), CCL2, CCL20, PTGS1, and IL6. None of these genes contains any predicted target sequences or miR-146a and should be considered secondary targets of this miRNA.
Pathway analysis indicated that several these changes in gene expression could result from direct targeting of
IRAK1 by miR-146a through activation of
NF-ê
B.
44
Several of the genes downregulated by miR-146a were found to be significantly upregulated in senescent TM cells compared with their expression in the low-passage control cells. This upregulation could contribute to the SASP and the activation of an inflammatory response in senescent HTM cells. These genes included
IL6,
IL8,
PAI-1,
CXCL3,
CXCL6, and
CCL2, which have been reported to be part of the SASP in other cell types,
18,45 as well as the additional member of the chemokine signaling pathway
CCL20.
Of interest, the overall levels of upregulation of these genes in senescent cells were lower in HTM1073 cells, where miR-146a was upregulated 111-fold, compared with expression in HTM636 cells, in which this miRNA was upregulated sevenfold. This observation is consistent with the concept that the increase in expression of miR-146a observed in senescent cells antagonizes with the increased expression of inflammatory mediators associated with the senescence response.
The observed increased expression of
PAI-1 could contribute to the pathogenesis of primary open-angle glaucoma. PAI-1 is the principal inhibitor of tissue plasminogen activator (tPA) and urokinase (uPA), the activators of plasminogen. Therefore, an increase in PAI-1 may lead to decreased fibrinolysis and extracellular proteolysis in aqueous humor and the trabecular meshwork, which could result in a decrease in aqueous humor outflow facility by increasing protein deposition and obstruction.
46,47 In addition, increased expression of
TGF-B2, which leads to increased outflow resistance and elevated intraocular pressure, has been reported to result in increased
PAI-1 production in cell culture and organ culture models.
48,49 Similarly, corticosteroid treatment, which also results in increased outflow resistance, has been shown to induce a decrease in activity of tissue plasminogen activator.
50 Therefore, the inhibitory effect of miR-146a on the upregulation of PAI-1 observed in senescent cells could contribute to preventing a decrease in the activity of the plasminogen system and a concomitant increase in outflow resistance.
Some of the components of the SASP, such as
PAI-1, are believed to contribute to the reinforcement of the senescence phenotype by autocrine and paracrine mechanisms.
38,39 Our results showed that transfection of presenescent cells with miR-146a mimic had significant inhibitory effects on SA-β-gal activity, production of ROS, and cell proliferation. The effects of miR-146a on ROS generation and cell proliferation were prevented by overexpression of
IRAK1, suggesting that the changes in expression of multiple genes involved in the SASP induced by the inhibition of
IRAK1 contribute to increased ROS production and growth arrest in senescent cells. Since overexpression of
PAI-1 was enough to prevent the effects of miR-146a on cell proliferation, our results also point to
PAI-1 as an important component of the SASP of HTM cells that is negatively modulated by miR-146a.
In conclusion, RS in HTM cells was associated with significant changes in miRNA expression that may contribute to the modulation of the senescent response. Specifically, the upregulation of miR-146a antagonized the induction of inflammatory mediators: the increased production of ROS and the decrease in proliferative capacity characteristic of the SASP. The observed inhibition of PAI-1 by miR-146a may limit the alterations in the extracellular proteolytic activity of the TM during aging and minimize the autocrine and paracrine effects of PAI-1, including increased generation of iROS and decreased cell proliferation. These results are consistent with the concept that upregulation of miR-146a in senescent HTM cells may serve to prevent an excessive increase in the production of inflammatory mediators and limit some of the potentially deleterious effects of the SASP on the physiology of the TM.
Supported by National Eye Institute Grants EY01894, EY016228, and EY05722, and Research to Prevent Blindness.
Disclosure:
G. Li, None;
C. Luna, None;
J. Qiu, None;
D.L. Epstein, None;
P. Gonzalez, None
The authors thank the Flow Cytometry Core Facility at the Duke Cancer Center.