Research ArticleCell Biology

Increased MKK4 Abundance with Replicative Senescence Is Linked to the Joint Reduction of Multiple MicroRNAs

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Science Signaling  27 Oct 2009:
Vol. 2, Issue 94, pp. ra69
DOI: 10.1126/scisignal.2000442

Abstract

MKK4 (mitogen-activated protein kinase kinase 4) is a pivotal upstream activator of c-Jun N-terminal kinase and p38. Here, we report that the abundance of MKK4 increases in senescent human diploid fibroblasts through enhanced translation. We identified four microRNAs (miR-15b, miR-24, miR-25, and miR-141) that target the MKK4 messenger RNA (mRNA); the abundance of these microRNAs decreased during replicative senescence. Individually modulating the amount of each microRNA did not modify MKK4 abundance, but their concomitant overexpression decreased and their joint reduction increased MKK4 abundance. Reporter analyses indicated that these microRNAs acted through the MKK4 5′ and 3′ untranslated regions. Elevated MKK4 abundance inhibited cell proliferation and increased the phosphorylation and activity of p38 and PRAK (p38-regulated/activated protein kinase). Thus, multiple microRNAs acting on a single target, the MKK4 mRNA, collectively influence MKK4 abundance during replicative senescence.

Introduction

Replicative senescence is a process whereby cells halt proliferation irreversibly after completing a finite number of divisions but remain metabolically active (1, 2). It is believed to represent a tumor-suppressive mechanism and a contributing factor in aging (3, 4). Although several transcription factors are responsible for modulating protein abundance during replicative senescence, the contribution of posttranscriptional events, including altered messenger RNA (mRNA) stability and translation, is increasingly recognized (5). These processes are efficiently modulated by specific RNA-binding proteins (RBPs) and microRNAs (miRNAs) that bind to specific mRNA sequences (6, 7). miRNAs are ~22-nucleotide (nt)-long noncoding RNAs that associate with a target mRNA in a sequence-specific manner and typically repress gene expression by lowering the stability of the mRNA, decreasing its translation rate, or performing both functions (8). miRNAs interact with target mRNAs through the seed region, which spans residues 2 to 9 of miRNAs and typically complements the target mRNA at 7 or 8 nt (8). They are involved in the posttranscriptional regulation of genes implicated in complex cellular processes such as aging, cancer, proliferation, differentiation, and apoptosis (9, 10).

The global patterns of mRNA and protein abundance change during replicative senescence. Although the abundance of proliferative and stress-response proteins is typically reduced in senescent cells, several proteins are elevated [such as p16, p21, p27, p53, and cyclin D1 (11, 12)] and play important biological roles in senescence and aging. While investigating age-related changes in gene expression, we discovered that the abundance of the signaling protein MKK4 [mitogen-activated protein kinase (MAPK) kinase 4] increased with age in several human tissues (fig. S1). In response to external stimuli such as environmental stress or inflammation, the dual-specificity kinase MKK4 [also known as JNKK1 (c-Jun N-terminal kinase kinase 1), MEK4 (MAPK kinase 4), and SEK (stress-activated protein kinase/extracellular signal-regulated kinase kinase 1)] phosphorylates and thereby activates JNK (c-Jun N-terminal kinase) and p38, which in turn elicit biological responses such as cell proliferation, differentiation, and apoptosis (13, 14). Clinical correlative studies have identified a putative tumor suppressor function for MKK4 in various tumor types (15). Functional in vivo studies have identified a metastasis suppressor activity for MKK4 in prostate and ovarian cancers (16, 17), and other clinical and experimental studies suggest that MKK4 may have pro-oncogenic properties, indicating that the role of MKK4 is dependent on cellular and environmental contexts (18, 19).

Here, we demonstrate in an in vitro model of human replicative senescence that MKK4 abundance is regulated by the joint action of four miRNAs. The increase in MKK4 abundance with replicative senescence is elicited by the combined decline in multiple miRNAs—miR-15b, miR-24, miR-25, and miR-141—that suppress MKK4 translation by binding to sites in its 3′ and 5′ untranslated regions (UTRs).

Results

MKK4 abundance increases with replicative senescence

In several human tissues, including pancreas and ovary, MKK4 abundance increased with age, as determined with a panel of tissue biopsies from human donors (fig. S1). We used human fetal lung diploid fibroblasts (HDFs) WI-38 and IMR-90 as in vitro models of aging. We defined late-passage [50 to 55 population doublings (Pdl)], nonproliferating HDFs as senescent and early-passage (typically 20 to 30 Pdl), proliferating HDFs as “young.” Senescent HDFs contained 3- to 15-fold more MKK4 than did young HDFs (Fig. 1A). Lack of correlation between MKK4 abundance and the amount of MKK4 mRNA was previously shown in prostate cancer cells, suggesting a role for altered translation rates (19). Accordingly, MKK4 mRNA abundance (Fig. 1B) and the stability of MKK4 protein (Fig. 1C) did not differ between senescent and young HDFs. Instead, the amount of MKK4 protein in senescent cells appeared to increase by translational mechanisms. Analysis of the association of MKK4 mRNA with translating ribosomes (polysomes) indicated that MKK4 mRNA was more actively translated in senescent HDFs compared with young HDFs (Fig. 1D). After fractionation of the cytoplasm through sucrose gradients and isolation of RNA in each fraction, MKK4 mRNA in young cells was found mostly in light fractions of the gradient, where mRNAs are not translated or are minimally translated (fractions 1 to 5) (Fig. 1D). In senescent cells, however, MKK4 mRNA was found in heavier parts of the gradient where the polysomes are located [low (LMWP) and high molecular weight (HMWP) polysomes, respectively], supporting the notion that MKK4 was more actively translated in senescent cells. The distribution of individual transcripts [MKK4 and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNAs] was determined by reverse transcription (RT) followed by quantitative polymerase chain reaction (qPCR) analysis of RNA prepared from each fraction of the sucrose gradients (Fig. 1D) [see (20) for details of polysome analysis]. A survey of RBPs did not identify specific regulators of MKK4 translation in prostate cancer or WI-38 cells (fig. S2). Thus, we hypothesized that miRNAs might contribute to the differential abundance of MKK4 during replicative senescence.

Fig. 1

Senescent HDFs show elevated abundance of MKK4 and reduced abundance of MKK4-directed miRNAs. (A) Representative Western blot analysis of MKK4 abundance in young (Y; Pdl 11) and senescent (S, Pdl 51) IMR-90 fibroblasts, as well as in young (Pdl 24) and senescent (Pdl 54) WI-38 fibroblasts. β-Actin was included as loading control. MKK4 signals from at least three Western blots were quantified by densitometry and normalized to β-actin signals. (B) MKK4 mRNA, as measured by RT-qPCR analysis of total RNA isolated from IMR-90 and WI-38 fibroblasts. (C) MKK4 protein stability was determined in young (Pdl 24) and senescent (Pdl 54) WI-38 cells that had been treated with cycloheximide (CHX, 50 μg/ml) and immunoblotting for MKK4 and GAPDH as a loading control. (D) Association of MKK4 mRNA (and GAPDH mRNA as a control) with cellular polysomes. After centrifugation of cytoplasmic components through linear 10 to 50% sucrose gradients, mRNA amounts in each fraction were measured by RT-qPCR and plotted as a percentage of the total mRNA in the sample. Arrow, direction of sedimentation; “-”, fractions lacking ribosome components; 40S and 60S, small and large ribosomal subunits, respectively; 80S, monosome; LMWP and HMWP, low and high molecular weight polysomes, respectively. (E) Schematic of the MKK4 mRNA, including the predicted target sites for miR-15b, miR-24, miR-25, and miR-141. (F) miR-15b, miR-24, miR-25, and miR-141 in young (Pdl 24) and senescent (Pdl 54) WI-38 cells were quantified by RT-qPCR analysis, normalized to 18S rRNA. As controls, U6 (which was unchanged between young and senescent cells) and let7a miRNA (which was elevated in senescent cells) were also measured. Data represent the means and SEM from three independent experiments. *P < 0.05. Statistical significance was determined by paired t test.

We previously found differences in miRNA profiles of young and senescent WI-38 fibroblasts (20). Among the miRNAs predicted to associate with the MKK4 mRNA, four miRNAs—miR-15b, miR-24, miR-25, and miR-141—were decreased in senescent HDFs, as measured by RT-qPCR of each individual miRNA (Fig. 1, E and F) (20). Moreover, the abundance of these four miRNAs was generally reduced in tissues from older compared to those from younger human donors (fig. S3). Only miR-15 was predicted to target the MKK4 3′UTR at one site; miR-25 and miR-141 each were predicted to target the MKK4 3′UTR at two sites; and miR-24 was predicted to target one site in the 5′UTR, one in the coding region, and one in the 3′UTR (Fig. 1, E and F, and fig. S4). Therefore, we tested the ability of miR-15b, miR-24, miR-25, and miR-141 to regulate the translation of MKK4.

MicroRNAs miR-15b, miR-24, miR-25, and miR-141 jointly, but not individually, repress MKK4 translation

Individual reduction in the concentration of each miRNA was accomplished by transfecting young WI-38 cells with antagomirs, short transcripts complementary [antisense (AS)] to each miRNA. Individual knockdown of each miRNA had negligible influence on MKK4 protein amounts, as assessed by Western blot analysis (Fig. 2A). However, concomitant knockdown of the four miRNAs increased MKK4 protein abundance (Fig. 2B). These interventions lowered each miRNA concentration between 2-fold and >20-fold over basal miRNA concentrations (Fig. 2C). MKK4 mRNA abundance was unchanged after transfection of all four (AS)miRNAs (Fig. 2D), indicating that the increased concentration of miRNAs directly affected the MKK4 protein abundance. To test if miRNA overexpression had the opposite effect, precursor (Pre) miRNAs were transfected into senescent WI-38 cells. Transfection of (Pre)miRNAs individually had little or no effect on MKK4 abundance (Fig. 2E), but combined transfections of (Pre)miRNAs lowered MKK4 abundance, especially when all four (Pre)miRNAs were transfected together (Fig. 2, E and F, and fig. S5). Transfection of senescent WI-38 cells with (Pre)miRNAs increased the abundance of each miRNA by 3- to >10-fold (Fig. 2G). MKK4 mRNA expression remained elevated after transfection of the four (Pre)miRNAs (Fig. 2H), indicating that the (Pre)miRNAs specifically reduced MKK4 abundance at the protein level.

Fig. 2

miRNAs directed against MKK4 mRNA cooperatively reduce MKK4 protein abundance. (A to D) Young WI-38 cells were transfected with control (Ctrl) siRNA or with different combinations of (AS)miRNAs (100 nM final). Forty-eight hours later, the individual (A) or combined (B) effects of the (AS)miRNAs on MKK4 abundance was determined by Western blot analysis, with β-actin as a loading control. RT-qPCR was used to quantify each miRNA individually (C), as well as MKK4 mRNA in young WI-38 cells transfected with control siRNA or with all four (AS)miRNAs, with 18S rRNA for normalization (D). (E to H) Senescent WI-38 cells were transfected with either control (Ctrl) siRNA or different combinations of (Pre)miRNAs (100 nM final). The individual (E) or combined (F) effect of the (Pre)miRNAs on MKK4 abundance was determined by Western blot analysis, with β-actin as a loading control. RT-qPCR was used to quantify each miRNA (G), and MKK4 mRNA in senescent WI-38 cells transfected with control siRNA or all four (Pre)miRNAs, with 18S rRNA for normalization (H). MKK4 signals were quantified by densitometry from three independent Western blots (A, B, E, and F). Data represent the means and SEM from three independent experiments (C, D, G, and H). *P < 0.05. Statistical significance was determined by paired t test.

To determine the action of these miRNAs, we used HeLa (human cervical carcinoma) cells because subsequent analyses required larger volumes of cells, which were easier to obtain from this cell line. In HeLa cells, transfection of all four (Pre)miRNAs also reduced MKK4 abundance, although transfection of all four (AS)miRNAs only modestly elevated MKK4 abundance compared with the control transfection group (Fig. 3A). Sucrose gradient fractionation of cytoplasmic lysates of transfected cells, followed by RT-qPCR analysis of MKK4 mRNA (and housekeeping GAPDH mRNA), showed that transfection of all four (Pre)miRNAs moderately shifted MKK4 mRNA distribution to fractions with less active translation (lower molecular weight polysomes) of the gradient compared with the control transfection group (Fig. 3, B and C). In contrast, transfection of all four (AS)miRNAs only slightly increased the percentage of transcripts in the actively translating (higher molecular weight) polysomes (Fig. 3, B and C).

Fig. 3

miRNAs directed against MKK4 mRNA cooperatively repress MKK4 association with polysomes. (A) HeLa cells were transfected with control siRNA, with four pooled (Pre)miRNAs [(Pre)miR-15b, (Pre)miR-24, (Pre)miR-25, and (Pre)miR-141] or with four pooled (AS)miRNAs [(AS)miR-15b, (AS)miR-24, (AS)miR-25, and (AS)miR-141]. Forty-eight hours later, MKK4 and GAPDH as a loading control were assessed by Western blot analysis. (B) Analysis of polysome profiles in cells transfected as described in (A). (C) Distribution of the MKK4 mRNA (left) and GAPDH mRNA (right) on polysome gradients prepared from the transfection groups described in (A). (D and E) WI-38 cells of intermediate passage (Pdl 39) were transfected as described in (A). Forty-eight hours later, MKK4 was assessed by Western blot analysis (D) and MKK4 mRNA distribution on polysome gradients was determined (E). MKK4 signals were quantified by densitometry from three Western blots (A and D).

We also used WI-38 cells that had undergone 39 population doublings (Pdl 39) and were thus intermediate in Pdl between young and senescent cells to study both increases and decreases in the polysomal association of MKK4 mRNA. Transfection of (Pre)miRNAs into Pdl-39 WI-38 cells reduced MKK4 protein abundance and the association of MKK4 mRNA with actively translating polysomes compared with the control transfection group (Fig. 3, D and E). Conversely, transfection of Pdl-39 WI-38 cells with (AS)miRNAs elevated MKK4 abundance and moderately increased the association of MKK4 mRNA with actively translating polysomes (Fig. 3, D and E). Even small shifts in distribution of a given mRNA by one or two gradient fractions can have a strong effect on the translation of the mRNA, as seen previously for p16 mRNA (20).

Both the 3′UTR and the 5′UTR of MKK4 mRNA contribute to the translational repression by miRNAs

To assess the contribution of the individual miRNA sites within the MKK4 mRNA, we prepared reporter constructs. For the analysis of 3′UTR sites, the 1.3-kb region spanning the predicted miRNA target sites was cloned at the 3′ end of the Renilla luciferase (RL) reporter complementary DNA (cDNA) in the psiCHECK-2 plasmid. A second reporter protein [firefly luciferase (FL)] expressed from the same plasmid by a separate promoter served as internal control for transfection. Several reporters were prepared, including an “empty” plasmid (pLuc), a plasmid expressing the intact MKK4 3′UTR region, pLuc-MKK4(WT), and additional reporters in which 2 or 3 nt within the seed region of the miRNA sites were mutated (figs. S6 and S7). The ratios of RL/FL activities in transfected WI-38 cells were measured to quantify the contribution of the MKK4 3′UTR (wild type or with mutations in various miRNA target sites) to miRNA-mediated translation repression of MKK4. Insertion of the MKK4(WT) 3′UTR region reduced the activity of reporter pLuc-MKK4(WT) to 45% of that seen for pLuc, whereas the sequential addition of mutations in the miRNA sites (24, 25, 141, and 15; figs. S6 and S7) progressively elevated RL/FL ratios, eventually reaching those seen with pLuc (Fig. 4A). Moreover, reduction in the concentration of miRNAs by transfection of (AS)miRNAs increased the expression of all of the reporters (Fig. 4A, gray bars), indicating that the decrease in RL/FL activity in reporters bearing the MKK4 3′UTR (wild type and mutated at two or three sites) required the presence of the miRNAs. The loss in RL/FL activity was not due to a selective reduction in the abundance of RL mRNA because the relative abundance of RL and FL mRNAs was unchanged, as shown for the pLuc-MKK4(WT) plasmid under conditions of normal and reduced miRNA concentrations (Fig. 4B). A similar analysis in HeLa cells yielded comparable results (fig. S7).

Fig. 4

MKK4 reporter analysis. (A) psiCHECK-2–derived reporter constructs used are shown in fig. S7. pLuc, control vector expressing RL and FL. pLuc-MKK4(WT) is a fusion of RL and the 1.3-kb proximal MKK4 3′UTR segment bearing all six predicted miRNA target sites. pLuc-MKK4(24/25) was derived from pLuc-MKK4(WT) by introducing mutations in the seed regions of miR-24 and miR-25 sites. In pLuc-MKK4(24/141), the mutations were introduced in miR-24 and miR-141 sites. In pLuc-MKK4(24/25/141), the mutations were introduced in miR-24, miR-25, and miR-141 sites. In pLuc-MKK4(15b/24/25/141), all six miRNA sites on the MKK4 3′UTR were mutated (fig. S6). WI-38 cells that were transfected with either control siRNA or pooled (AS)miRNAs were subsequently transfected with reporter plasmids (fig. S7), and RL activity and FL activity were measured 24 hours later. RL/FL values are shown. (B) RL and FL mRNAs were measured in WI-38 cells (with normal or reduced amounts of miRNAs) by RT-qPCR analysis 24 hours after transfection of pLuc-MKK4(WT). (C) Cells were transfected with pLuc, pLuc-MKK4(5) (which expresses an RL chimeric RNA with the wild-type MKK4 5′UTR) and pLuc-MKK4(5m) (which expresses an RL chimeric RNA with the mutated MKK4 5′UTR (fig. S7). Twenty-four hours later, RL and FL activities were measured. (D) The plasmid pcDNA-FLAG-MKK4 and the miR-24 site within the MKK4 coding region are shown in fig. S7. WI-38 cells were cotransfected with either control siRNA or (Pre)miR-24 along with pFLAG-MKK4. Twenty-four hours later, MKK4 was quantified by Western blot and densitometry analysis of the FLAG signals. Data are representative of three experiments. Data represent the means and SEM from three independent experiments (A to C). *P < 0.05. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey multiple comparisons test.

Although most miRNAs are believed to function through 3′UTR sites, examples are emerging of miRNAs acting through the 5′UTR and coding region (10, 2022). Because putative miR-24 target sites were also found in the MKK4 5′UTR and coding region, the contribution of these sites was also tested. pLuc-MKK4(5) contained the wild-type MKK4 5′UTR (5′), and pLuc-MKK4(5m) had a mutation that disrupted miR-24 binding (5′m) to the 5′ end of RL (fig. S7). pLuc-MKK4(5) showed 49% RL/FL activity compared with pLuc activity, whereas the 5′ mutation in pLuc-MKK4(5m) abrogated this repression (Fig. 4C), indicating that the miR-24 site on the MKK4 5′UTR could independently repress the RL reporter. In each case, transfection with (AS)miRNAs blocked this effect (Fig. 4C). The contribution of the miR-24 site in the coding region to the translation repression of MKK4 was assessed by measuring the abundance of a FLAG-tagged MKK4 protein, expressed from a transcript devoid of MKK4 3′ or 5′UTR sequences (fig. S7). Unlike the endogenous MKK4 protein, the abundance of which was reduced by (Pre)miRNAs (Fig. 3A), FLAG-MKK4 was refractory to the presence of transfected (Pre)miR-24, suggesting that the site in the coding region did not contribute to modulating MKK4 abundance (Fig. 4D). Together, these findings support the notion that the 3′UTR-targeting miRNAs function jointly with those targeting the 5′UTR to suppress MKK4 production.

MKK4 mRNA-directed miRNAs influence proliferation, senescence, and senescence-associated cell signaling

DNA replication, as assessed by [3H]thymidine incorporation, was used as a measure of proliferation. [3H]Thymidine incorporation was higher in HeLa cells transfected with (Pre)miRNAs to reduce MKK4 abundance compared to control small interfering RNA (siRNA)–transfected HeLa cells (Fig. 5, A to C). Both transfection groups showed reduced [3H]thymidine incorporation when MKK4 was overexpressed and increased [3H]thymidine incorporation when MKK4 was silenced by siRNA (Fig. 5, A to C). The growth-inhibitory influence of MKK4 was also investigated in HDFs. In young WI-38 cells, MKK4 abundance was lowered by siRNA, an effect that was reversed by cotransfection of (AS)miRNAs. As observed in HeLa cells, modulation of MKK4 in WI-38 cells was accompanied by changes in DNA replication, with [3H]thymidine incorporation increasing when MKK4 abundance was reduced and decreasing when MKK4 abundance was elevated (Fig. 5D). The increase in cell proliferation seen in WI-38 cells with lower MKK4 amounts contrasts with the slight reduction in proliferation reported for MKK4-null mouse embryo fibroblasts (MEFs) (23). Differences in the degree of reduction of MKK4 abundance, the species studied (human versus mouse), and the time periods of MKK4 loss or silencing could explain the distinct effect of MKK4 on proliferation seen in our study and that of Tournier et al. (23). WI-38 cell morphology was altered, with high MKK4-expressing cells becoming enlarged, a characteristic feature of presenescent and senescent cells (Fig. 5E). No apoptosis was detected after short-term silencing of MKK4 (fig. S8).

Fig. 5

MKK4 reduces proliferation. [3H]Thymidine incorporation was measured 48 hours after cotransfection of HeLa cells with control siRNA or Pre(miRNAs) and (A) pFLAG or pFLAG-MKK4 plasmids or (B) control or MKK4 siRNA. The abundance of MKK4, FLAG-MKK4, and the loading control GAPDH was determined by Western blot analysis and densitometry (C). (D) Quantification of MKK4 by Western blot analysis and densitometry (top) and [3H]thymidine incorporation (graph) 48 hours after transfection of WI-38 cells that were intermediate between young and senescent cells (Pdl 39) with the small RNAs shown. (E) Phase-contrast micrographs of cells from the transfection groups described in (D). Scale bar, 50 μm. Data represent the means and SEM from three independent experiments (A, B, and D). Western blots are representative of three independent experiments (C and D). *P < 0.05. Statistical significance was determined by ANOVA followed by Tukey multiple comparisons test.

Overexpression of FLAG-MKK4 triggered a senescent phenotype in young WI-38 cells, as determined by the increased activity of a broadly used marker, senescence-associated β-galactosidase [SA-β-gal (24)]; in comparison, cells expressing the empty vector (FLAG) were SA-β-gal negative (Fig. 6A). Likewise, the senescence-associated proteins p21 and cyclin D1 were more abundant in MKK4-overexpressing cells (Fig. 6B). To study the influence of miRNAs on the senescence phenotype, MKK4-directed (Pre)miRNAs and (AS)miRNAs were retransfected every four days for 5 weeks. At the end of this period, the cells in the (AS)miRNA-transfected group contained higher amounts of MKK4, were larger and divided more slowly than control cells, were positive for SA-β-gal activity, and showed elevated abundance of the senescence marker p16. In contrast, the cells transfected with the (Pre)miRNAs were smaller, proliferated more rapidly, and showed lower abundance of p16 and MKK4 than control cells (Fig. 6, C and D).

Fig. 6

Altering MKK4 abundance changes markers of replicative senescence. (A and B) 48 hours after transfection of young WI-38 cells with pcDNA-FLAG or pcDNA-FLAG-MKK4, SA-β-gal activity and FLAG signals were detected by phase-contrast microscopy and fluorescence microscopy, respectively (A). Scale bars, 50 μm (A and C). The abundance of MKK4, the loading control GAPDH, and the senescence-associated proteins p21 and cyclin D1 was assessed by Western blot analysis (B). (C) Young WI-38 cells (Pdl 24) were sequentially transfected (every 4 days for 5 weeks) with the small RNAs indicated. Cellular morphology and SA-β-gal activity were assessed by phase-contrast microscopy. Images are representative of two independent experiments. (D) Western blot analysis of the abundance of MKK4, the senescence marker p16, and the loading control GAPDH in the populations described in (C). (E and F) Western blot analysis of total PRAK and p-PRAK, total and p-p38, p16, and the loading control GAPDH, as assessed 48 hours after transfection of WI-38 cells (Pdl 39) with control siRNA or (Pre)miRNAs. Protein abundance was also assessed in untransfected young (Pdl 24) and senescent (Pdl 54) WI-38 cells. (G) MKK4 was quantified by Western blot analysis and densitometry (top) and [3H]thymidine incorporation (graph) in senescent WI-38 (Pdl 54) cells 48 hours after transfection of the small RNAs is shown. Data are shown as the means and SEM. The signals in three independent Western blot analyses were quantified by densitometry (B, D, E, F, and G). **P < 0.001. Statistical significance was determined by ANOVA followed by Tukey multiple comparisons test.

Sun and co-workers showed that the p38 substrate PRAK (p38-regulated/activated protein kinase) is critical for the induction of senescence (25). Accordingly, cells transfected with (Pre)miRNAs, and hence containing less MKK4, also showed reduced abundance of p16 and phosphorylated PRAK at Ser93 (Fig. 6E). Conversely, phosphorylated PRAK, phosphorylated p38, and p16 were higher overall in senescent compared to young WI-38 cells (Fig. 6F). In contrast, the JNK pathway did not appear to be affected by alterations in MKK4 abundance (fig. S9). Finally, in senescent WI-38 cells, transfection of MKK4 siRNA or (Pre)miRNAs reduced MKK4 abundance and promoted [3H]thymidine incorporation (Fig. 6G).

Discussion

Together, our data indicate that MKK4 abundance is increased during replicative senescence, largely because of the reduced abundance of miRNAs miR-15b, miR-24, miR-25, and miR-141. Furthermore, increased MKK4 contributes to decreased cell proliferation, a hallmark characteristic of senescent cells. Ongoing studies are aimed at elucidating the mechanisms by which the abundance of miR-15b, miR-24, miR-25, and miR-141 are decreased during replicative senescence and how increased MKK4 affects the responsiveness of senescent cells to stress-causing agents.

In contrast to MKK4, evidence suggests that MKK7, which can also phosphorylate JNK, plays a role in delaying senescence (26). The premature senescence observed in MKK7−/− MEFs could not be rescued by MKK4, which is likely due to differences in the substrate specificity of these two MAPK kinases. Unlike MKK7, MKK4 can phosphorylate JNK and p38 MAPKs, the latter of which promotes replicative senescence (27). Sun et al. reported that the p38 substrate PRAK was critical for inducing senescence (25). PRAK−/− mice displayed enhanced tumorigenesis, and PRAK−/− cells exhibited impaired senescence; in addition, tumorigenesis and senescence were linked to the phosphorylation of the transcription factor p53, which is also a tumor suppressor and a senescence-associated protein. p53 was proposed to be a key effector of the Ras pathway that leads to PRAK activation (25). According to our analysis of the senescence markers SA-β-gal and p16, reducing MKK4 abundance by either siRNA or (Pre)miRNAs inhibited the senescence phenotype, whereas elevating MKK4 enhanced cellular senescence (Fig. 6, A to D).

MKK4 abundance in HDFs was dependent on the combinatorial influence of at least four miRNAs acting through the MKK4 3′- and 5′UTRs. The elevated amounts of miR-15b, miR-24, miR-25, and miR-141 contributed to the reduced abundance of MKK4 in proliferating fibroblasts, whereas their reduction led to increased MKK4 production in senescent fibroblasts. A similar pattern was observed in tissues from human donors (figs. S1 and S3), suggesting that increased MKK4 may also occur in vivo in senescent cells.

Together, these findings support a model in which individual miRNAs may only have an incremental effect on target mRNA abundance, whereas the combined action of several miRNAs on a given target mRNA affects protein abundance. The combined effect of miRNAs affords the cell with a versatile means of increasing and decreasing the amount of a protein with a high degree of accuracy. This ability to fine-tune protein abundance by collections of miRNAs is increasingly recognized in pathophysiological processes that require adaptive gene expression patterns (6, 8). As shown here for MKK4, miRNAs ensure a flexible, potent, and precise control of protein abundance during key cellular processes—such as replicative senescence—underlying cancer and aging.

Materials and Methods

Cell culture and transfections

Early-passage, proliferating (young, ~20 to 30 Pdl) and late-passage, senescent (50 to 55 Pdl) WI-38 HDFs (Coriell Cell Repositories) were cultured as described (20). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Control (Ctrl) siRNA, 2′-O-methyl (AS)miR-15b, (AS)miR-24, (AS)miR-25, (AS)miR-141, (Pre)miR-15b, (Pre)miR-24, (Pre)miR-25, and (Pre)miR-141 were from Ambion. RNAs were transfected at a final concentration of 100 nM; oligonucleotides and plasmids were transfected with Lipofectamine 2000 (Invitrogen). Plasmids pcDNA3.1-FLAG-MKK4 and pcDNA3.1-FLAG were transfected at 2 μg per 60-mm plate. siRNA directed against MKK4 (siMKK4, Santa Cruz Biotechnology) was transfected into cells at a final concentration of 100 nM with Oligofectamine (Invitrogen).

Plasmids for luciferase analysis were derived from the dual luciferase reporter vector psiCHEK-2 (referred to as pLuc; Promega). The plasmid pLuc-MKK4(WT) comprised the MKK4 3′UTR spanning the first 1.3 kb where all six predicted miRNA target sites are located. Additional plasmids were derived from pLuc-MKK4(WT) by mutating the six sites individually or in combination (fig. S7) with a site-directed mutagenesis kit (Stratagene) (fig. S6). The mutated miRNA seed recognition sequences for each miRNA recognition site are as follows: GC to TA for miR-15b, GG to TT for miR-24, GGC to TTA and GC to TA for miR-25, and CG to AT and GG to TT for miR-141. HeLa cells (and WI-38 or IMR-90 cells; fig. S7) were transfected with 200 ng of plasmid DNA with Lipofectamine 2000. Cells were lysed 24 hours after transfection, and the ratios of RL and FL activities were measured with a dual-luciferase assay (Promega).

RNA and protein analysis

Total cellular RNA was isolated directly from cells or from gradient fractions with Trizol (Invitrogen). After RT with random hexamers and SSII reverse transcriptase (Invitrogen), real-time qPCR analysis was performed with gene-specific primer pairs (below) and SYBR Green PCR master mix (Applied Biosystems), as described (20). The following primers were used: CTCTTCACTCCCAACAAT and ACATGGGAGAGCTGGGAGTA for MKK4 mRNA, TGCACCACCAACTGCTTA and GGCATGGACTGTGGTCATGAG for GAPDH mRNA, CCCTGATCAAGAGCGAAGAG and GTCTAACCTCGCCCTTCTCC for RL mRNA, TTCGCTAAGAGCACCCTGAT and GTAATCAGAATGGCGCTGGT for FL mRNA, and CCCTATCAACTTTCGATGGTAGTCG and CCAATGGATCCTCGTTAAAGGATTT for 18S ribosomal RNA (rRNA).

Mature miR-15b, miR-24, miR-25, and miR-141 were quantified with the Taqman miRNA Detection Assay (Applied Biosystems).

Prediction of MKK4 mRNA as a target of miR-15b, miR-24, miR-25, and miR-141

The MKK4 mRNA was predicted to be target of miR-15b, miR-24, miR-25, and miR-141 at several sites within the 3′UTR after performing computational analyses with prediction algorithms that included Pictar, TargetScan, RNAhybrid, and RNA22. Using RNA22 (http://cbcsrv.watson.ibm.com/rna22.html) and the sequences of MKK4 mRNA and miR-15b, miR-24, miR-25, and miR-141, the program returned the predicted target sites shown in fig. S4.

Immunohistochemistry, immunofluorescence, and SA-β-gal activity

For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde, then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS). For FLAG detection, samples were blocked in 5% bovine serum albumin for 1 hour and incubated with a 1:1000 dilution of the rabbit monoclonal antibody against FLAG (Sigma Aldrich) in the background-reducing antibody diluent (DAKO) for 16 hours at 4°C. Slides were washed with PBS-T and then incubated with a 1:1000 dilution of the fluorescein isothiocyanate–labeled monoclonal antibody against rabbit for 1 hour at room temperature. The cell preparations were counterstained with DAPI (4′,6-diamidino-2-phenylindole) in Prolong GOLD antifade (Invitrogen-Molecular Probes). SA-β-gal activity was detected with a kit (Cell Signaling) according to the manufacturer’s recommendations. SA-β-gal images were acquired with an inverted AX10 Observer D1 microscope (Carl Zeiss, Inc.), and images were analyzed with Axiovision software. Images of cells that exhibited SA-β-gal and overexpressed pcDNA-FLAG-MKK4 were acquired sequentially with the multidimensional acquisition platform and analyzed with Axiovision software.

Western blot analysis

Whole-cell lysates were prepared with RIPA buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% SDS, and 1 mM dithiothreitol] and proteins were resolved by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Invitrogen). After incubation with primary antibodies recognizing MKK4, GAPDH, phosphorylated p38 (p-p38), PRAK, phosphorylated PRAK (p-PRAK), p21, p16 or α-Tubulin (Santa Cruz Biotechnology), p38 or cyclin D1 (Cell Signaling), β-actin (Abcam), or FLAG (Sigma-Aldrich), blots were incubated with the appropriate secondary antibodies and signals were detected by ECL Plus (Amersham). Western blotting signals were quantified by densitometry with Image J software.

Polyribosome analysis

Cells were incubated with cycloheximide (100 μg/ml; Calbiochem) for 15 min. Cytoplasmic lysates (500 μl) were centrifuged through 10 to 50% linear sucrose gradients and divided into 10 fractions for RT-qPCR analysis, as previously described (20).

Acknowledgments

We thank M. Bernier, B. Damdinsuren, and M. Kaileh for providing reagents and N. N. Hooten for help with the preparation of the manuscript. This research was supported by the National Institute on Aging–Intramural Research Program, NIH.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/2/94/ra69/DC1

Methods

Reference

Fig. S1. MKK4 abundance increases with aging in a panel of tissue biopsies from young and old individual donors.

Fig. S2. Survey of putative MKK4 mRNA-interacting RNA-binding proteins.

Fig. S3. Higher expression of predicted human MKK4 mRNA-directed miRNAs in fetal tissues compared with adult tissues.

Fig. S4. Predicted microRNA-MKK4 mRNA hybrids.

Fig. S5. Unchanged MKK7 and Hsp27 abundance despite changes in MKK4.

Fig. S6. Point mutations introduced at each predicted miRNA site.

Fig. S7. MKK4 reporter analysis in HeLa cells recapitulates MKK4 reporter analysis in WI-38 HDFs.

Fig. S8. Activities of caspase-3 and caspase-7 remain unaltered after modulating MKK4.

Fig. S9. Analysis of cells treated with (Pre)miRNAs and (AS)miRNAs to modulate MKK4 reveals that MKK4 does not appear to activate the JNK pathway in S cells.

References and Notes

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