Cardiac fibroblast hyperplasia associated with enhanced matrix deposition is a major determinant of tissue remodeling in several disease states of the heart. However, mechanisms controlling cell cycle progression in cardiac fibroblasts remain unexplored. Identification of cell cycle regulatory elements in these cells is important to develop strategies to check adverse cardiac remodeling under pathological conditions. This study sought to probe the mechanisms underlying ERK1/2-mediated p27Kip1 regulation in mitogenically stimulated cardiac fibroblasts. Addition of 10% fetal calf serum to quiescent cultures of adult rat cardiac fibroblasts promoted ERK1/2 activation, as evidenced by its phosphorylation status. Reduction in [3H]thymidine incorporation into DNA increased population doubling time, flow cytometry, and Western blot analysis showing reduced levels of cyclins D and A, p27Kip1 induction, and retinoblastoma protein (Rb) hypophosphorylation in ERK1/2-inhibited cells indicated ERK1/2 dependence of G1-S transition in cardiac fibroblasts. Lack of p27Kip1 protein in serum-stimulated, ERK1/2-active cells was associated with increased levels of Skp2, an E3 ubiquitin ligase for p27Kip1, whose knockdown by RNA interference induced p27Kip1 expression. Further, forced expression of Skp2 in ERK1/2-inhibited cells downregulated p27Kip1. Transcriptional upregulation of p27Kip1 mRNA in ERK1/2-inhibited cells, demonstrated by real-time PCR, correlated with forkhead box O 3a (FOXO3a) transcription factor activation, shown by gel shift assay. FOXO3a knockdown attenuated p27Kip1 mRNA and protein expression in ERK1/2-inhibited cells. We provide evidence for the first time that, in cardiac fibroblasts, activated ERK1/2 regulates p27Kip1 expression transcriptionally and posttranslationally via FOXO3a- and Skp2-dependent mechanisms. Additionally, this study uncovers interesting interactions between critical cell cycle regulatory elements that are only beginning to be understood.
- cardiac fibroblasts
the recognition in recent years that cardiac fibroblasts are importantly involved in myocardial remodeling associated with conditions such as hypertension, myocardial infarction, and cardiomyopathies marks a paradigm shift in our understanding of the pathogenesis of heart failure (23, 39, 43, 46). The vertebrate myocardium is a highly organized structure consisting of two interdependent compartments, parenchyma and stroma. Cardiomyocytes represent the contractile component of the parenchyma whereas fibroblasts, vascular cells, and a structural network of matrix proteins represent the stroma. In the normal heart, the quantitative relationship between these two compartments is well maintained, which ensures optimal pump function. However, myocyte hypertrophy and apoptosis, fibroblast hyperplasia, and interstitial fibrosis that occur as a result of pathological processes disturb the ratio of functional parenchymal cells to connective tissue and promote adverse remodeling of the heart (23, 39, 43, 46). In particular, cardiac fibroblast proliferation associated with enhanced collagen deposition, the relative resistance of cardiac fibroblasts to apoptosis, and their persistence in the infarct scar result in stromal accumulation, which contributes substantially to the pathogenesis and progression of heart failure (30, 41). Surprisingly, while recent investigations (30, 36, 41) have provided insights into the molecular basis of cardiac fibroblast resistance to apoptosis, the regulation of cell cycle progression in these cells remains unexplored.
Identification of the factors and mechanisms that determine cardiac fibroblast turnover in a setting of wound repair and tissue remodeling postinjury is an important clinical goal. In this regard, our recent investigations implicate extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 or p44/42 MAPK) in the survival pathway in cardiac fibroblasts, showing that it mediates apoptosis resistance in these cells via nuclear factor-κB (NF-κB)-dependent cellular inhibitor of apoptosis proteins-2 (cIAP-2) induction (36). ERK1/2, together with p38 MAPK and c-Jun NH2-terminal kinase (JNK), belongs to a family of serine/ threonine kinases that are critically involved in the cell-signaling network. Unlike p38 MAPK and JNK, which are activated in response to environmental stress, ERK1/2 is activated by growth factors and is reported to be a key player in distinct cellular functions such as proliferation, differentiation, and apoptosis (31, 34, 48). Several reports stress the role of ERK1/2 activity in cell cycle progression through the G1 phase. In fact, cardiac fibroblast hyperplasia following mitogenic stimulation has been shown to be mediated by ERK1/2 (13) but the cell cycle regulatory elements and mechanisms that link ERK1/2 signaling to G1-S transition in cardiac fibroblasts have not been identified.
Progression through the mammalian cell cycle is dependent on the activation of cyclin-dependent kinases (CDKs) through association with cyclins, which results in the phosphorylation of the retinoblastoma protein (Rb) and consequent E2F-dependent transcription of S-phase genes to facilitate G1-S transition, which is a critical event in the eukaryotic cell cycle. Inhibition of CDK activity by cyclin-dependent kinase inhibitors (CDKIs) results in Rb hypophosphorylation and cell cycle arrest (17, 29). Timed regulation of CDKIs plays a crucial role in cell-cycle progression (3). The abundance of p27Kip1, an important member of the Cip/Kip family of CDKIs and critical regulator of G1-S transition, is regulated predominantly by a posttranslational mechanism mediated by S-phase kinase-associated protein 2 (Skp2), an F-box protein of the SCF ubiquitin ligase complex (7, 15). Apart from posttranslational regulation, p27Kip1 is also regulated transcriptionally (21, 26). Forkhead box O 3a (FOXO3a), a member of the Forkhead box O (FOXO) family of transcription factors, is reported to enhance p27Kip1 gene expression, and phosphorylation-dependent suppression of the activity of FOXO3a has been shown to facilitate cell-cycle progression in response to mitogenic signals (42, 47, 49, 51). Further, it has been demonstrated that ERK1/2 signaling contributes to the downregulation of p27Kip1 (4, 10, 14, 22, 24) but the exact mechanisms have not been fully understood. It has been reported that ERK1/2 upregulates Skp2 expression, which, in turn, can downregulate p27Kip1 through proteasomal degradation (8, 45). However, downregulation of p27Kip1 expression by ERK1/2 via an Skp2-independent mechanism has also been reported (10, 33), which suggests context- and cell type-related mechanistic heterogeneity in ERK1/2-mediated regulation of p27Kip1 expression. ERK1/2 is also reported to downregulate p27Kip1 transcriptionally through negative regulation of FOXO3a (18, 50). However, such reports are sporadic and the mechanisms by which ERK1/2 regulates p27Kip1 and influences G1-S transition in eukaryotic cells in general and cardiac fibroblasts in particular warrant further investigation.
Against this backdrop, we probed the mechanisms that link ERK1/2 activation to G1-S transition in serum-stimulated adult rat cardiac fibroblasts. Using loss-of-function and gain-of-function approaches, we provide evidence for the first time of a novel pathway in the proliferative growth of cardiac fibroblasts that involves FOXO3a- and Skp2-dependent transcriptional and posttranslational regulation of p27Kip1 expression by ERK1/2.
All fine chemicals were purchased from Sigma Chemical (St. Louis, MO). [3H]thymidine (specific activity: 18 Ci/mmol) was obtained from Bhabha Atomic Research Center, India. Primary antibodies for cyclin D, cyclin E, cyclin A, p27Kip1, Skp2, phospho-Rb, and Total-Rb were purchased from Santa Cruz Biotechnology. Cell lysis buffer and primary antibodies for FOXO3a, phospho ERK1/2, and total ERK1/2 were obtained from Cell Signaling Technology. PureLink RNA mini kit for RNA isolation was obtained from Ambion. Reagents for cDNA synthesis including RT buffer, RNase inhibitor, random primers, dNTPs, and M-MLV reverse transcriptase were from Promega (Madison, WI). BCA protein assay kit and ECL kit were from Pierce. p27- and Skp2-specific primers for real-time PCR analysis were obtained from Applied Biosystems (TaqMan Gene Expression). Nitrocellulose membrane was from Millipore. Small interfering (si)RNA for Skp2 and Foxo3a was obtained from Ambion. siRNA for p42 MAPK was obtained from Cell Signaling Technology. OPTI-MEM-I was obtained from GIBCO. Lipofectamine 2000 was obtained from Invitrogen. Native ORF Skp2 clone in a pCMV-6 Entry vector with COOH-terminal Myc-DDK tag, anti-DDK antibody, and turbofectin were purchased from Origene (Rockville, MD). Animal care and use were as per the guidelines and approval of the Sree Chitra Tirunal Institute for Medical Sciences and Technology Institutional Animal Ethics Committee.
Cardiac fibroblasts were isolated from young adult male Sprague-Dawley rats (2–3 mo) and characterized as described earlier (25). Subconfluent cultures of cardiac fibroblasts from passage 2 or 3, stimulated to proliferate with 10% fetal calf serum, were used for the experiments. ERK1/2 in serum-stimulated cells was inhibited using PD98059 (PD) or, where indicated, by RNA interference to corroborate the data with the pharmacological inhibitor.
Measurement of DNA Synthesis
Subconfluent cultures of cardiac fibroblasts in M199, synchronized by serum deprivation for 24 h, were subjected to mitogenic stimulation with 10% FCS in the presence of PD in DMSO along with 1 μCi/ml [3H]thymidine for 24 h. The inhibitor was added 45 min before addition of 10% FCS. An equivalent concentration of DMSO was added to the control groups. Radioactivity associated with acid-insoluble fraction was determined by Liquid Scintillation Spectrometry (Wallac 1409).
Determination of Cell Number and Population Doubling Time
Subconfluent cultures of cardiac fibroblasts in M199, synchronized by serum deprivation for 24 h, were subjected to mitogenic stimulation with 10% FCS in the presence of PD. Cell counts were determined before and after 24 h of treatment using a Neubauer counting chamber. Population doubling time was calculated using the formula: doubling time, Td = 0.693/k, where k = (2.3 log N2/N1)/Δt, and N1 and N2 represent the number of cells per dish at the start and end of the treatment, respectively, and Δt = the treatment duration in an hour.
Flow Cytometric Analysis of Cell Cycle Phase Distribution
Subconfluent cultures of cardiac fibroblasts in M199, synchronized by serum deprivation for 24 h, were subjected to mitogenic stimulation with 10% FCS in the presence of PD for 24 h. The cells were trypsinized with trypsin/EDTA, washed with Ca2+/Mg2+-free PBS, and fixed in 70% ethanol in PBS for 1 h at 4°C. The fixed cells were resuspended in 0.25 ml PBS and treated with 5 μl of 10 mg/ml RNase A for 10 min at 37°C. Cellular DNA was stained with 10 μg/ml propidium iodide (PI), and samples were filtered through a 70-mm nylon mesh to remove cell clumps. Flow cytometric analysis was done using a BD FACS Aria bench top flow cytometer (Becton and Dickinson). Single cell populations were gated using forward scatter, an indicator of cell size, vs. side scatter, an indicator of cell granularity. The FL2 detector measures fluorescent light from PI, which emits a red color at a 650-nm wavelength of the FACS can laser, and PI intensity is proportional to the DNA content of the cell. The FL2-PI area vs. width plots distinguished true cycling G2/M cells from doublets or aggregates of G0/G1 cells. Based on DNA content, the cells were sorted into G0/G1, S, and G2/M populations. At least 20,000 cells were collected per sample.
Cells were seeded into 12-well plates at 80 × 103 cells/well. After 24 h, the cells were incubated in Opti-MEM with Ambion predesigned Silencer-Select siRNA (5 pmol Skp2, 5 pmol Foxo3a, and 100 nmol ERK2 MAPK) and Lipofectamine (2 μl) for 19 h. Following an additional incubation in M199 with 10% FCS for 12 h, the cells were incubated with or without PD in M199 containing 10% FCS for the indicated duration. Cell lysate was prepared in SDS lysis buffer, denatured, and used for Western blot analysis.
Real-Time PCR Analysis
Total RNA was isolated from serum-stimulated, PD-treated cardiac fibroblasts using TRI reagent as per the manufacturer's instructions. Following DNase I treatment, 2 g of total RNA were reverse transcribed to cDNA with random hexamer primers and M-MLV reverse transcriptase. Taqman quantitative real-time polymerase chain reaction analysis was carried out using the ABI Prism 7500 Sequence Detection System (Applied Biosystems) with specific FAM-labeled probes. PCR reactions were performed, as per the manufacturer's instructions, with cDNA (90 ng), TaqMan Universal PCR Master Mix, and oligonucleotide primers and probes for p27Kip1 (Assay ID: Rn00582195 m1), Skp2 (Assay ID: s148803), or 18S (4333760T) under the following thermal cycling conditions: 95°C for 10 min followed by denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min for each of 40 cycles. p27Kip1 and Skp2 expression levels were normalized to 18S rRNA.
DNA-binding activity of FOXO3a in cardiac fibroblasts was assessed by EMSA using Light Shift Chemiluminescent EMSA Kit. Subconfluent cardiac fibroblasts cultures in M199 with 10% FCS were treated with PD for the indicated durations, and nuclear extracts were prepared using the NEPER nuclear extraction kit. Protein concentration of the nuclear extracts was determined using the Bradford protein assay method. The probes for the FOXO3a DNA binding region were as follows: 5′-CTAGCAAGCAAACAAACTTATTTTGAACACGGG-3′ (forward) and 5′-CCCGTGTTCAAAATAAGTTTGTTTGCTTGCTAG-3′ (reverse). Probes for FOXO3a were biotinylated using Thermo Scientific 3′-end biotin labeling kit. The nuclear extracts were incubated with the biotinylated probes and components of the LightShift Chemiluminescent kit at 37°C for 60 min, electrophoresed on a 6% nondenaturing gel, transferred to nylon membrane, and immobilized by ultraviolet cross-linking at 254 nm for 10 min. The bands were visualized by enhanced chemiluminescence using streptavidin-conjugated horseradish peroxidase. Specificity of binding was confirmed by competition with excess unlabeled FOXO3a oligonucleotides (200-fold).
Overexpression of Skp2 in ERK1/2-Inhibited Cardiac Fibroblasts
Constitutive expression of Skp2 was achieved using the Native ORF clone of Skp2 in a pCMV-6 Entry vector with COOH-terminal Myc-DDK tags, following the manufacturer's instructions (Origene). The ready-to-transfect Skp2 plasmid (RR207717) consisted of the Skp2 clone (1,272 bp) between Sgf-1 and Mlu-1 restriction sites in a pCMV plasmid (4,929 bp). The Skp2 clone size was verified by PCR amplification of the primers provided in the kit. The size of the plasmid (6,201 bp) was further analyzed by single site restriction digestion at the Mlu-1 site. Skp2 overexpression plasmid cocktail was prepared with 1 μg plasmid and 3 μl turbofectin in 100 μl Opti-MEM, which was added to the cultures for incubation in M199 with 10% FCS for 12 h. The cells were then incubated for another 12 h with M199 containing 10% FCS in the presence or absence of PD. The cell lysates were used for protein expression analysis by Western blotting.
Western Blot Analysis
Serum-stimulated, subconfluent cardiac fibroblast cultures were treated with PD for the indicated durations, and Western blot analysis was carried out by standard protocols using β-actin as loading control. Protein expression was quantified by densitometric scanning (Bio-Rad Laboratories).
Cell Viability Assay
Hoechst 33342/PI staining or 4′,6-diamidino-2-phenylindole (DAPI)/PI staining methods were used to determine loss of viability after transfection or exposure of cells to PD.
Statistical significance was assessed using Student's t-test. P ≤ 0.05 was considered significant.
ERK1/2 Is Required for G1-S Transition in Mitogen-Stimulated Cardiac Fibroblasts
Western blot analysis showed a significant increase in the levels of the phosphorylated form of ERK1/2 in cardiac fibroblasts following different durations of mitogenic stimulation of serum-starved cells, pointing to its activation (Fig. 1A). In subsequent experiments, we examined whether cell proliferation in response to serum is dependent on ERK1/2 activation. For this, we evaluated the effect of ERK1/2 inhibition using PD, a widely used pharmacological inhibitor, on serum-induced cardiac fibroblast proliferation by comparing serum-stimulated subconfluent cultures of cardiac fibroblasts in the presence and absence of the inhibitor. PD at different concentrations (5, 10, and 20 μM) significantly reduced DNA synthesis, as shown by a reduction in [3H]thymidine incorporation into DNA (Fig. 1B). A concentration of 10 μM PD was used in all other experiments. We also ascertained inhibition of serum-induced ERK1/2 activation by PD at this concentration (Fig. 1C). The inhibitor did not compromise cell viability at these concentrations (data not shown). ERK1/2 inhibition increased the population doubling time from 28.9 to 104.7 h (Fig. 1B), clearly indicating that ERK1/2 is required for serum-induced proliferation.
Consistent with the observations with PD, RNA interference-based inhibition of ERK1/2 activity significantly attenuated cell proliferation in serum-stimulated cardiac fibroblasts, which further confirmed the involvement of ERK1/2 in the regulation of cardiac fibroblast proliferation (Fig. 1D). Western blot analysis confirmed siRNA-based ERK inhibition (Fig. 1D, inset).
To identify the ERK1/2-regulated cell cycle checkpoint, we examined the time dependence of the PD effect on DNA synthesis. PD was added to cultures at 0, 5, 10, and 14 h after addition of 10% FCS. Cells stimulated with 10% FCS, without the inhibitor, served as control. At the end of 24 h, cells were lysed and DNA synthesis was measured. Addition of PD at 0, 5, or 10 h but not 14 h after serum significantly reduced DNA synthesis, providing preliminary evidence that the regulatory role of ERK1/2 is in the early phase of the cell cycle, possibly the G1-S transition (Fig. 2A). The inhibitory effect of PD, added 10 h after mitogenic stimulation, suggested that ERK activity is sustained at diminished levels and is required for progression through even late G1. Further, flow cytometric analysis showed a significant increase in the number of cells in G0/G1 and a corresponding reduction in the S phase, confirming that ERK1/2 is critical for G1-S transition in cardiac fibroblasts (Fig. 2B).
Expression Profile of Cell Cycle Regulatory Proteins in Mitogen-Stimulated Cardiac Fibroblasts upon ERK1/2 Inhibition
To characterize the G0/G1 block further, we analyzed the expression of G1-S regulators in serum-stimulated subconfluent cultures of cardiac fibroblasts with or without PD. Western blot analysis revealed significantly reduced protein levels of cyclin D1 and cyclin A (Fig. 3A) in ERK1/2-inhibited cells. Cyclin E (Fig. 3A), however, remained unaffected upon ERK1/2 inhibition, pointing to differential regulation of the cyclins in serum-stimulated cardiac fibroblasts. A critical event in G1-S transition is the phosphorylation of Rb, which is a prerequisite for cell cycle progression. In the present study, we found significant hypophosphorylation of Rb in ERK1/2-inhibited cells (Fig. 3B), consistent with impaired G1-S progression.
As hypophosphorylation of Rb is brought about by CDKIs, we examined the effect of ERK1/2 inhibition on p27Kip1 protein expression in serum-stimulated cells and found striking induction of p27Kip1 in ERK1/2-inhibited cells (Fig. 4A). As regulation of p27Kip1 expression was a major focus of this study, we examined p27Kip1 protein induction in cells following siRNA-mediated ERK1/2 knockdown as well. Consistent with the observation with PD, significant induction of p27Kip1 protein was observed upon ERK inhibition by siRNA (Fig. 4B). Inhibition of proliferation and induction of p27Kip1 upon specific inhibition of ERK1/2 using siRNA indicated a regulatory role for ERK1/2 rather than other MAPKs.
Posttranslational and Transcriptional Mechanisms Underlie p27Kip1 Induction in ERK1/2-Inhibited Cardiac Fibroblasts
Skp2 protein expression in cardiac fibroblasts is ERK1/2 dependent.
The induction of p27Kip1 in ERK1/2-inhibited cells led us to probe its posttranslational and transcriptional regulation by ERK1/2. To test this, we examined Skp2 protein and mRNA expression in serum-stimulated cardiac fibroblasts in the presence and absence of PD. ERK1/2 inhibition was found to inhibit Skp2 protein expression, showing that ERK1/2 is a positive regulator of Skp2 (Fig. 4A). Skp2 mRNA levels, however, were unaffected by ERK1/2 inhibition, which ruled out transcriptional regulation of Skp2 by ERK1/2 (Fig. 4C, bottom).
Skp2 knockdown by RNA interference induces p27Kip1 protein expression in mitogen-stimulated cells.
To determine whether the negative regulation of p27Kip1 protein expression by ERK1/2 is dependent on Skp2, we silenced Skp2 by RNA interference in serum-stimulated cardiac fibroblasts and examined p27Kip1 protein levels. Western blot analysis confirmed knockdown of Skp2 by Skp2-specific siRNA (Fig. 5). Skp2 knockdown restored p27Kip1 protein levels in serum-stimulated, ERK1/2-active cells, indicating that p27Kip1 regulation by ERK1/2 in cardiac fibroblasts is Skp2 dependent (Fig. 5).
Forced expression of Skp2 in ERK1/2-inhibited cells abrogates p27Kip1 protein expression.
As Skp2 knockdown restored p27Kip1 protein levels in serum-stimulated cells, we checked whether forced expression of Skp2 protein in ERK1/2-inhibited cardiac fibroblasts (that do not express Skp2) would attenuate p27Kip1 protein expression. Strong constitutive expression of Skp2 was achieved by transfecting ERK1/2-inhibited cells with Skp2 ORF clone in a ready-to-transfect pCMV-6 expression vector. Skp2 expression was confirmed using antibody against the COOH-terminal DDK Tag of the vector (Fig. 6). Forced expression of Skp2 in these cells prevented the upregulation of p27Kip1 protein (Fig. 6), affirming the centrality of Skp2 in p27Kip1 regulation by ERK1/2 in these cells.
ERK1/2 inhibition promotes activation of FOXO3a in cardiac fibroblasts.
Next, we checked if, in addition to posttranslational regulation, p27Kip1 expression in ERK1/2-inhibited cells is subject to transcriptional control as well. Consistent with the postulation, real-time PCR analysis showed a twofold increase in p27Kip1 mRNA in serum-stimulated cells upon ERK1/2 inhibition (Fig. 4C, top). Subsequent experiments focused on the involvement FOXO3a in the transcriptional upregulation of p27Kip1. EMSA confirmed that ERK1/2 inhibition in serum-stimulated cells induces FOXO3a activation (Fig. 7A).
FOXO3a knockdown in ERK1/2-inhibited cells attenuates p27Kip1 mRNA and protein expression.
To ascertain the role of FOXO3a in the transcriptional regulation of p27Kip1, FOXO3a was silenced in serum-stimulated but ERK1/2-inhibited cardiac fibroblasts by RNA interference and p27Kip1 mRNA and protein levels were analyzed. Western blot analysis confirmed FOXO3a knockdown by FOXO3a-specific siRNA (Fig. 7B). FOXO3a knockdown in ERK1/2-inhibited cells prevented induction of p27Kip1 mRNA and protein, indicating transcriptional regulation of p27Kip1 by FOXO3a (Fig. 7, B and C).
An integral component of the pathogenesis of heart failure is cardiac remodeling, which encompasses the responses of cardiac cells to conditions such as myocardial infarction, pressure and volume overload, and inflammatory heart muscle disease (5, 9, 35). Studies on mechanisms of heart failure have traditionally focused on the role of myocytes as the cause of heart failure. However, although myocyte hypertrophy may be associated with both adaptive and pathological remodeling, cardiac fibroblast hyperplasia is invariably associated with adverse myocardial remodeling. Such a distinction is particularly important since cardiac fibroblasts account for about two-thirds of the myocardial cell population and ∼90% of the nonmyocytes in the heart and are an important source of matrix proteins and several growth factors and cytokines that profoundly impact myocardial pathophysiology (23, 32, 39, 46). Against this backdrop, the present study sought to investigate the role of ERK1/2 and its potential downstream targets in the regulation of G1-S transition in cardiac fibroblasts.
Protein kinases such as Akt, ERK1/2, and p38 MAPK are key components of the signaling network that respond to mitogenic and survival signals to control the cell cycle via cell cycle regulatory elements such as cyclins, CDKs, CDKIs, Skp2, and FOXO (2, 7, 12, 18, 19, 37). This study focused on ERK1/2, which is reported to regulate cell cycle progression upon mitogenic stimulation (4, 14, 20, 22, 48). In the present study, significant reductions in DNA synthesis and cell number (Fig. 1, B and D), the time-dependent PD effect (Fig. 2A), flow cytometry (Fig. 2B), significant reductions in cyclin D and cyclin A (Fig. 3A), hypophosphorylation of Rb (Fig. 3B), and induction of p27Kip1 (Fig. 4, A and B) in ERK1/2-inhibited cells show conclusively that ERK1/2 is required for G1-S transition in cardiac fibroblasts. Inhibition of ERK1/2 may regulate the activities of the cyclin D-Cdk4/6 and cyclin A-Cdk2 complexes by downregulating cyclins D and A. As cyclin E remained unaffected upon ERK1/2 inhibition (Fig. 3A), it is possible that inhibition of cyclin E-Cdk2 activity by p27Kip1 rather than by cyclin E downregulation may impact Rb phosphorylation and the G1 checkpoint in cardiac fibroblasts. Consistent with our earlier observation that cyclin E is unaffected during impaired G1-S transition in hypoxic cardiac fibroblasts (37), these findings suggest that G1-S arrest in cardiac fibroblasts may not be critically dependent on downregulation of cyclin E expression.
In a variety of cell types, alterations in the expression levels of CDKIs and/or their modification after de novo synthesis are reported to regulate cell proliferation (3). Generally, CDKIs are regulated predominantly at the posttranslational level (7, 15). SCF complex ligases are the largest family of E3 ligases and consist of core components (Skp1, Cul-1, and Rbx/Roc1 or Ro52) and the F-box protein Skp2 which is the substrate-recognizing component of the complex. The SCFSkp2 complex induces ubiquitination of several targets, including p27Kip1 (7, 15). Cell cycle-dependent changes in Skp2 levels involve posttranslational as well as transcriptional mechanisms (2, 19, 38). Selective degradation of Skp2 by the anaphase-promoting complex Cdh1 (APCCdh1) ubiquitin-proteasome system has recently been shown to be a major posttranslational regulatory mechanism (2). It has been reported that kinases such as Akt and Pim-1 can phosphorylate Skp2 to prevent APCCdh1-mediated degradation, which increases its stability and activity during G1-S transition (6, 16, 27). Apart from these kinases, a few recent reports indicate positive correlation between ERK1/2 activation and Skp2 induction (8, 45) but the underlying mechanisms remain unclear. In this study, we demonstrate that ERK1/2 inhibition significantly downregulates Skp2 protein (Fig. 4A) but not mRNA levels (Fig. 4C, bottom), which suggests posttranscriptional rather than transcriptional regulation of Skp2 by ERK1/2 in cardiac fibroblasts. Notably, the reduction in Skp2 levels was associated with upregulation of p27. Thus this study points to a clear link between ERK1/2 activation and Skp2 expression on the one hand and, on the other, an inverse relationship between Skp2 and p27Kip1, two critical regulators of the G1-S checkpoint. Furthermore, induction of p27Kip1 in serum-stimulated cells upon Skp2 knockdown (Fig. 5) and abrogation of p27Kip1 expression in ERK1/2-inhibited cells upon forced expression of Skp2 (Fig. 6) clearly suggest that ERK1/2-dependent downregulation of p27Kip1 protein in serum-stimulated cardiac fibroblasts involves a posttranslational mechanism mediated by the SCFskp2 complex.
There are reports that p27Kip1 is subject to transcriptional control as well (11, 21). In the present study, a significant increase in p27Kip1 mRNA levels upon ERK1/2 inhibition indicated transcriptional control of the p27Kip1 gene in relation to ERK1/2 status (Fig. 4C, top). In this regard, the FOXO transcription factors comprise a large family of functionally diverse transcription factors that control multiple target genes involved in cell cycle regulation. It has been demonstrated that FOXO1, FOXO3a, and FOXO4 regulate the CDKI and cyclin-D genes in many cell types (26, 40, 47, 49, 51), which has generated immense interest in the regulation of these transcription factors whose association with tumorigenesis is well known. Among the FOXO family members that are regulated through phosphorylation by several pathways, including the ERK pathway, the present study focused on the regulation of FOXO3a and its role in cardiac fibroblast proliferation (1, 12, 42, 44, 50). Notably, it has also been reported that Skp2 can ubiquitinate and degrade the phosphorylated form of FOXO3a (28, 42, 47). Moreover, a role for ERK1/2 in the negative regulation of FOXO3a is suggested by the recent observation that ERK1/2-dependent phosphorylation of FOXO3a facilitates its degradation by MDM2, an E3 ligase like Skp2 (50). It has been demonstrated that ERK1/2 regulates FOXO3a via p66shcA-mediated Akt phosphorylation (18). In the present study, EMSA demonstrated that FOXO3a, which is inactive in serum-stimulated, ERK1/2-active cells, is activated upon ERK1/2 inhibition, which indicates negative regulation of FOXO3a by ERK1/2 (Fig. 7A). Our data also show upregulation of Skp2 in ERK1/2-active cardiac fibroblasts, indicating inverse correlation between Skp2 and FOXO3a activation. In tandem with recent reports, the data suggest that, in cardiac fibroblasts, activated ERK1/2 can potentially regulate FOXO3a through direct phosphorylation as well as upregulation of Skp2, both of which can promote degradation of FOXO3a. Importantly, the direct relationship between p27Kip1 mRNA levels and the activation status of FOXO3a, and attenuation of p27Kip1 mRNA and protein expression in ERK1/2-inhibited cells upon siRNA-mediated FOXO3a knockdown (Fig. 7, B and C) suggest that FOXO3a transcriptionally upregulates p27Kip1.
To conclude, we provide evidence for the first time that activated ERK1/2 regulates p27Kip1 expression in cardiac fibroblasts both transcriptionally and posttranslationally via FOXO3a- and Skp2-dependent mechanisms (Fig. 8). Apart from delineating a novel mechanism that controls G1-S transition in cardiac fibroblasts, the findings point to potential interactions between critical cell cycle regulatory elements that are only beginning to be understood and need to be probed further. Future investigations should explore the exciting possibility that targeting one or more of these molecules would modify myocardial tissue response to injury.
K. Shivakumar gratefully acknowledges financial support from the Indian Council of Medical Research to perform these studies. S. Pramod thanks the Council of Scientific and Industrial Research, Government of India, for a research fellowship.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: S.P. and K.S. conception and design of research; S.P. performed experiments; S.P. and K.S. analyzed data; S.P. and K.S. interpreted results of experiments; S.P. prepared figures; S.P. and K.S. drafted manuscript; S.P. and K.S. edited and revised manuscript; S.P. and K.S. approved final version of manuscript.
S. Pramod is grateful to Dr. M. Nilanjana and L. Zhan for financial support and technical assistance, respectively, to perform the Skp2 knockdown experiment over a 4-mo stay in the Laboratory of Molecular Cardiology and Angiogenesis, University of Connecticut Health Center. We thank Dr. Jackson James of the Rajiv Gandhi Centre for Biotechnology, Trivandrum, for facilities for plasmid preparation. We acknowledge the facilities provided by Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST).
- Copyright © 2014 the American Physiological Society