This Article Abstract Full Text (PDF) Services Download to citation manager Citing Articles Citing Articles via HighWire PubMed PubMed Citation

Altered Mitogen-Activated Protein Kinase Signal Transduction in Human Skin Fibroblasts During In Vitro Aging: Differential Expression of Low-Density Lipoprotein Receptor

¹ Donald W. Reynolds Department of Geriatrics
² Department of Biochemistry and Molecular Biology
³ Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Medical Research, Central Arkansas Veterans Healthcare System, Little Rock.

	Abstract

Top Abstract Methods Results Discussion References

 
The purpose of the study was to investigate the correlation of low-density lipoprotein receptor (LDLr) and mitogen-activated protein kinases (MAPK) in fibroblasts after serial passage in vitro. We used early-passage (20 mean population division, MPD) and late-passage (60 MPD) human skin fibroblasts to study the LDLr expression and MAPK at basal and after interleukin-1ß (IL-1ß) stimulation. We found a reduced LDLr expression in late-passage fibroblasts in comparison with early-passage fibroblasts, and late-passage fibroblasts showed a delayed induction of MAPK after IL-1ß stimulation, confirmed by the delay in translocation of MAPK from cytoplasmic to nuclear fraction. Using two specific inhibitors of MAPK, we could show a reduced LDLr expression in early-passage fibroblasts, indicating a direct relationship between MAPK signaling and LDLr expression. We conclude that one of the reasons for reduced LDLr gene expression in late passage fibroblast is related to MAPK signaling.

MAPK mediates extracellular signals that regulate cell growth, differentiation, survival, and death (15,16). At present, three major subfamilies of mammalian MAPK pathways have been studied in detail. All three MAPK are activated by phosphorylation of tyrosine and threonine residues and catalyzed by the dual specificity of MAPK. The ERK-1/2 pathway is strongly activated by mitogenic signals. Moreover, activated ERK-1/2 promotes cell proliferation. It has been further demonstrated that active ERK stimulates DNA synthesis (17) and enhances the activity of AP-1 transcription factor through Elk-1 and c-fos (18–20). Other members of the MAPK, SAPK/JNK1/2/3, and p38^MAPK (p38, p38ß, p38, and p38), are primarily activated by proinflammatory cytokines (21), cellular stress, tumor necrosis factor-, and ultraviolet irradiation (22,23). Upstream activators of these three kinases may function both specifically and with cross-specificity, resulting in activation of more than one MAPK. For example, in response to epidermal growth factor, ERK is activated as much as 20-fold, while JNK (c-jun NH₂ terminal kinase) is activated up to 6-fold (24–26). The nuclear translocation of MAPK appears to be a prerequisite for proper cellular response (27). ERK-1/2 MAPK nuclear translocation has been shown to be essential for growth factor-induced DNA replication and cell transformation (28,29).

Interleukin-1ß (IL-1ß), a central proinflammatory mediator, induces the expression of multiple genes involved in the inflammatory process and malignant diseases often associated with abnormal cholesterol metabolism (30–36). The signaling mechanism leading to the stimulation of LDLr expression by IL-1ß has been elucidated in HepG2 cells (6), but this mechanism is not well described in fibroblasts. However, it is known that growth stimuli such as insulin and platelet-derived growth factor augment LDLr activity in fibroblasts (37,38).

In this study, we investigated changes, in the induction of LDLr expression and stimulation of MAPK in early- and late-passage human diploid fibroblasts, after IL-1 stimulation. In addition, we investigated the activation and localization of ERK-1/2 in these cells, because the ERK pathway provides a critical link between the extracellular environment and cellular nuclear regulatory changes. Our results demonstrated that early- and late-passage fibroblasts respond differently to the cytokine. Maximum expression of LDLr mRNA was seen after 16 hours of cytokine treatment in early-passage cells, while in late-passage cells it was between 16 and 24 hours, in addition, IL-1ß-mediated induction was fourfold lower in late-passage cells compared with early-passage cells. Late passage cells required more time to reach the maximum stimulation of ERK, and the stimulation-induced ERK-1/2 molecules in late-passage cells were lower than in early-passage cells under similar conditions when compared with their corresponding controls. On the other hand, late-passage cells showed higher amounts of stress-activated MAPK p46^JNK1. Taken together, our results demonstrate an association between decline in nuclear translocation of ERK-1/2 and induction of LDLr following IL-1ß treatment in early- and late-passage fibroblasts. Our results also suggest that LDLr expression in these cells is partly controlled by MAPK.

	METHODS

Top Abstract Methods Results Discussion References

 
Cell Culture and IL-1ß Stimulation
Fibroblasts used in this study were of strain A8; they were obtained from the foreskin of a 26-year-old healthy male donor and established by Dr. S. Goldstein (39). They were kindly provided to us by Dr. Beata Lecka-Czernik (Department of Geriatrics, University of Arkansas for Medical Sciences) at 17 MPD. A8 cells have a maximum life span of 68 MPD (40), after which they become senescent. In all experiments, early-passage fibroblasts were in their first third of replicative life span when they required <6 days to become confluent after a 1:8 split. Late-passage fibroblasts had completed 75%–90% of the life span and they grew more slowly, requiring 6–8 days to become confluent after 1:4 split. Early- (20 MPD) and late-passage (60 MPD) fibroblasts were grown in monolayers in 225 cm² stock flasks containing 25 ml of growth medium, which consisted of Eagle's minimum essential medium (BioWhittaker, Walkersville, MD), 24 mM NaHCO3, vitamin 1%, nonessential amino acid 0.1%, glutamine-1.2%, and antibiotic-antimycotic-1.2% supplemented with 10% fetal bovine serum (FBS). For experiments, confluent monolayers of cells from the stock flasks were dissociated with 0.25% trypsin and 1 mM of ethylenediaminetetraacetic (EDTA) acid solution. For each experiment, 7 x 10⁵ cells were plated on day 0 in 150 mm² culture plates containing 10 ml of growth medium with 10% FBS. On day 2, cells were refed with fresh medium. On day 5, when cells were about 60%–70% confluent, they were changed to a media containing 0.5% FBS, and 24 hours later they were treated with 10 ng/ml IL-1ß (this and other chemicals were obtained from Sigma, St. Louis, MO), for the indicated time. For inhibitor studies, cells were pretreated for 1 hour with MAPK inhibitors, PD098059 (Calbiochem Biosciences, Inc., La Jolla, CA) or UO126 (Cell Signaling Technology, Inc., Beverly, MA).

RNA Isolation
RNA was isolated from early- and late-passage cells utilizing the RNeasy kit (Qiagen, Inc., Valencia, CA). Briefly, cells were washed with phosphate buffered solution (PBS), trypsinized, and centrifuged at 300 x g for 5 minutes. Pellets were collected and immediately dissolved in RLT lysis buffer (Qiagen) containing ß-mercaptoethanol by pipetting up and down. Lysates were immediately processed for RNA preparation. The complete process was followed according to the instructions from Qiagen.

cDNA Probes
The cDNA probes were made by polymerase chain reaction amplification of a 400-base pair (bp) region upstream of the human LDLr cDNA, cloned in plasmid pcDV1 (ATCC #39966) using the sense, 5'-CAG GAC GAG TTT CGC TGC CAC G-3', and antisense, 5'-GCA GTT TCC ATC AGA GCA CTG-3', primers. The fragment was labeled with (-³²P)dCTP (ICN Biomedicals, Irvine, CA) by random priming (Megaprime DNA Labeling System, RPN1604, Amersham Biosciences, Piscataway, NJ), purified through QIA quick silica gel spin column (Qiagen), and denatured. High specific activity probes were used for hybridization.

Northern Blot Analysis
Twenty µg of RNA from each sample was denatured at 65°C for 15 minutes in sample denaturing loading buffer. The RNA was then subjected to overnight electrophoresis through a 1% agarose and 6.5% formaldehyde gel at 20 V in 20 mM 1x MOPS (20 mM 3-[N-morpholino propanesulfonic acid], 5 mM sodium acetate, and 1 mM EDTA acid [EDTA], pH 7.0) as a running buffer. The RNA was transferred to Zetaprobe GT nylon membrane (Bio-Rad Laboratories, Hercules, CA) by capillary blotting in 10 x sodium saline citrate (SSC) and ultraviolet cross-linked with Stratalinker (Stratagene, La Jolla, CA). The blots were blocked for 1 hour at 68°C in express-hyb hybridization solution (Clontech Laboratory, Inc., Palo Alto, CA) and hybridized at 68°C for 1 hour in a hybridization chamber (Thermo Hybaid, Franklin, MA) in fresh solution containing a human 400 bp LDLr probe. Membranes were rinsed with 0.2XSSC (15 mM sodium citrate and 150 mM NaCl; pH 7.0) and 0.05% sodium dodecyl sulfate (SDS) at room temperature for 30 minutes with two changes of wash buffer. Membranes were then washed with 0.1 x SSC and 0.1% SDS for 40 minutes at 60°C and 20 minutes at 65°C with continuous agitation and several changes of wash buffer. The values were corrected for equal RNA loading by stripping each blot in 0.5% SDS for 10 minutes in boiling water, blocked as above, and rehybridized with a ³²P-labeled 800 bp Eco R1 fragment from human ribosomal protein cDNA (ribosomal S3, a generous gift from Piotr Zimniak, PhD, University of Arkansas for Medical Sciences at Little Rock). Bands were visualized by exposing the membranes to storage phosphor screen. The intensity of each band and background were quantified with Image Quanta program (Molecular Dynamics, Inc., Sunnyvale, CA).

Preparation of Whole-Cell Lysate
Following treatment with IL-1ß as described above, cells were washed with ice-cold 1x PBS and lysed with RIPA buffer (1x PBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS with freshly added protease and phosphate inhibitors) on ice. Cells were collected and homogenized in a hand homogenizer for 5 seconds, passed through a syringe fitted with a 21-gauge needle, and incubated on ice for 40 minutes. Cell lysates were microcentrifuged at 10,000 x g at 4°C for 10 minutes, the supernatant was collected, and protein was estimated by the Bradford method (41). Equal amounts of protein were used to run the SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Western Blot Analysis
SDS-PAGE was carried out on 12% separating gel. Equal amounts of protein were loaded on each lane. Fractionated protein was electroblotted onto nitrocellulose membranes at 100 V for 1 hour at 4°C. Membranes were blocked in 5% (w/v) nonfat dry milk in Tris-buffered saline and Tween 20 (TBST) (20 mM Tris-HCl, pH 7.6; 137 mM NaCl, and 0.2% [v/v] Tween20), washed five times (5 min each) with TBST, and probed with primary antibodies. Antiphosphoindependent antibodies of ERK-1/2, p46/54^JNK, and p38^MAPK, diluted 1:2000 in TBST containing 5% bovine serum albumin, were incubated at room temperature for 1 hour with gentle shaking. Phosphospecific antibodies were diluted 1:1000 and incubated with membrane overnight at 4°C with gentle shaking. After proper washings, membranes were incubated with horseradish peroxidase-coupled anti-IgG (secondary antibody, dilution 1:3000) for 1 hour. All antibodies were purchased from Cell Signaling Technology. Enhanced chemiluminescence (ECL+, Amersham Biosciences) and fluorescence detection were used as per manufacturer's instructions for visualization of the bands. They were quantified on a Strom 860 Imager (Molecular Dynamic, Inc., Sunnyvale, CA).

Preparation of Cytoplasmic Fraction and Nuclear Fraction and Translocation
Semiconfluent cultures of early- and late-passage cells were treated with 10 ng/ml of IL-1ß for 10, 30, and 60 minutes. Cytoplasmic and nuclear fractions were prepared via the method used by Levkau and colleagues (42) and Boyle and colleagues (43), with minor modifications. In brief, treated cells were trypsinized, centrifuged at 500 x g for 10 minutes at 4°C, washed twice by resuspending the pellets in ice-cold 1x PBS, and centrifuged at 500 x g for 10 minutes at 4°C. The supernatant was discarded, and the cell pellets were lysed in buffer (150 mM NaCl, 150 mM sucrose, 20 mM Hepes [pH 7.4], 5 mM KCL, 2 mM DTT, 1 mM MgCl₂, 0.05 mM CaCl₂, 0.1% digitonin, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg /ml leupeptin) by gentle mixing with a pipette and incubation on ice with gentle agitation for 30 minutes. Cell suspensions were centrifuged at 1000 x g for 10 minutes at 4°C. The supernatant was collected as a cytoplasmic fraction. The pellets obtained from cytoplasmic extraction were suspended in lysis buffer, incubated on ice with gentle mixing for 60 minutes, and centrifuged at 30,000 x g for 30 minutes at 4°C. The clear supernatant contained a nuclear fraction. Protein was quantified as above. Equal amounts of protein were loaded on each lane, and SDS-PAGE was carried out, followed by immunoblotting. Membranes were incubated with antiphosphoindependent and antiphosphodependent ERK-1/2^MAPK primary antibodies followed by incubation with a peroxidase-linked secondary antibody. Immunoreactive proteins were detected with enhanced chemiluminescence according to the supplier's protocol.

Statistical Analysis
Values are expressed as the arithmetic mean ± 1 standard error of the mean (SEM). Analysis of the data was performed using a one-tailed Student's t test. Differences in means having p <.05 were considered to be statistically significant.

	RESULTS

Top Abstract Methods Results Discussion References

 
Influence of IL-1ß on LDLr mRNA Expression in Serially Passaged Normal Human Fibroblasts
Early- and late-passage cells were incubated in the presence of 10 ng/ml of IL-1ß for various periods of time up to 32 hours. Northern blot analysis of 10 studies demonstrated that expression of LDLr mRNA reached peak stimulation 16 hours after IL-1ß treatment in early-passage cells (all studies), while in late-passage cells, the peak was attained at 16 hours in 7 studies and 24 hours in 3 studies, after treatment, and the fold inductions were almost similar at 16 hours and 24 hours in late-passage cells (Figure 1A). Increases in LDLr mRNA levels were significantly different, 6.8- and 3-fold in early- and late-passage cells, respectively (p <.001, Figure 1B). Enhanced LDLr mRNA expression in early-passage cells dropped to 1.90- and 1.86-fold after 24 hours and 32 hours, respectively. However, in late-passage cells, the LDLr mRNA level remained elevated approximately 2.89-fold even at 24 hours and gradually decreased to 1.49-fold by 32 hours (Figure 1B). Early-passage cells showed a slightly elevated basal LDLr mRNA level over late-passage cells, but the difference was not significant.

View larger version (30K): [in this window] [in a new window]   Figure 1. Kinetics of low-density lipoprotein receptor (LDLr) mRNA induction in early- and late-passage fibroblasts following interleukin-1-beta (IL-1ß) treatment. Cells were grown and treated for indicated time as described under Methods. Total cellular RNA was extracted and blotted as described under Methods. The membrane was hybridized with a 32P-labeled LDLr cDNA probe. A: LDLr mRNA and ribosomal S3 bands. Intensity of the bands was quantitated using the Image Quanta program. LDLr mRNA levels were normalized to mRNA encoding the ribosomal S3 protein (loading control) levels. B: Intensity of bands expressed as fold inductions in early-passage (circles and solid line) and late-passage (triangles and dashed line) fibroblasts above respective controls. Mean ± SEM (standard error of mean) were calculated from 10 separate experiments. *p <.001

View larger version (38K): [in this window] [in a new window]   Figure 2. Activation of phosphorylated extracellular signal-regulated kinase 1/2 (ERK-1/2) in early- and late-passage fibroblasts following interleukin-1-beta (IL-1ß) treatment. Cells were plated exactly the same as described in Methods and treated with 10 ng/ml of IL-1ß for indicated intervals. After these intervals, cells were washed with 1x PBS (phosphate-buffered saline) and lysed in RIPA (ristocetin-induced platelet agglutination) buffer. SDS (sodium dodecyl sulfate) sample buffer was added to each sample. Equal amounts of whole-cell lysate were separated by 12% gels. Phosphorylation levels of ERK-1/2 were analyzed by Western blotting using phospho-specific antibodies, following electrotransfer of total proteins to a nitrocellulose membrane. Values are expressed as fold inductions in early-passage (circles and solid line) and late-passage (triangles and dashed line) fibroblasts above respective controls. The insert shows the bands of ERK-1/2 and phospho ERK-1/2 on representative blots. Mean ± SEM (standard error of mean) were derived from 7 separate experiments, and bands were normalized for loading by reprobing the membrane with ß-Actin antibody. *p <.02

View larger version (47K): [in this window] [in a new window]   Figure 3. Effect of 10 ng/ml of interleukin-1-beta (IL-1ß) on the induction of phospho c-jun NH2 terminal kinase (p54/p46JNK-1/2) in early-passage (circles and solid line) and late-passage (triangles and dashed line) fibroblasts. The samples used in Figure 2 were analyzed with phospho-specific anti-p46/54JNK antibodies. Values were expressed as the fold induction of phosphorylated p46/54JNK compared to unstimulated cells. A: Phospho JNK1. B: Phospho JNK2. The insert shows the bands of JNK-1/2 and phospho JNK-1/2 on representative blots. Mean ± SEM (standard error of mean) were derived from 4 separate experiments, and all bands were normalized for loading by reprobing the membrane with ß-Actin antibody. *p <.01

View larger version (36K): [in this window] [in a new window]   Figure 4. Induction of phospho p38 in early-passage (circles and solid line) and late-passage (triangles and dashed line) fibroblasts after exposure to 10 ng/ml of interleukin-1-beta (IL-1ß) for the indicated times. Cell lysates used in Figure 2 were analyzed with the phospho-specific anti-p38MAPK antibody. The Western blotting protocol was the same as that in Figure 2. Values were expressed as the fold induction in antiphospho-p38MAPK compared to noninduced cells. The insert shows the bands of p38 and phospho p38 on representative blots. Mean ± SEM (standard error of mean) were derived from 5 separate experiments. All bands were normalized for loading by reprobing the membrane with ß-Actin antibody

View larger version (72K): [in this window] [in a new window]   Figure 5. Nuclear translocation of extracellular signal-regulated kinase 1/2 (ERK-1/2) in early- and late-passage fibroblasts after interleukin-1-beta (IL-1ß) treatment. Cells were treated with 10 ng/ml of IL-1ß for 10, 30, and 60 minutes. Nuclear and cytoplasmic fractions were prepared and analyzed by Western blotting with phospho-independent and phospho-specific ERK-1/2 antibodies as described under Methods. Top panel shows the bands on representative bolts of early- and late-passage cell samples for phospho-independent anti-ERK-1/2 and anti-phospho ERK-1/2. C = cytoplasmic fraction; N = nuclear fraction. Bottom panel shows mean ± SEM (standard error of mean) of intensity of nuclear phospho-ERK-1/2 bands in relative units on Western blots of 5 separate experiments. Bands from early-passage (circles and solid line) and late-passage (triangles and dashed line) fibroblasts were normalized for loading by reprobing the membranes with ß-Actin for cytoplasmic fractions and Histone-H1 for nuclear fractions. *p <.001

View larger version (120K): [in this window] [in a new window]   Figure 6. Effect of mitogen-activated protein kinase (MAPK) inhibitors on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK-1/2), p46/54JNK (c-jun NH2 terminal kinase), and p38MAPK in early-passage fibroblasts after stimulation with interleukin-1-beta (IL-1ß). Cells were grown as described in Methods and were treated with 10 ng/ml of IL-1ß for 10 minutes in the presence or absence of PD098059 or UO126, added 1 hour before IL-1ß. Cell extracts were prepared, and equal amounts were loaded onto gels and immunoblotted with antiphospho-ERK-1/2, antiphospho-46/54JNK, or antiphospho-p38MAPK antibodies. All bands were normalized for loading by reprobing the membrane with ß-Actin antibody. The blots shown are representative of 3 separate experiments

View larger version (40K): [in this window] [in a new window]   Figure 7. Inhibition of interleukin-1-beta (IL-1ß)-induced low-density lipoprotein receptor (LDLr) expression in early-passage fibroblasts after treatment of mitogen-activated protein kinase (MAPK) inhibitors. Young fibroblasts were grown as described under Methods. Cells were treated with 10 ng/ml of IL-1ß for 16 hours with or without the inhibitors PD098059 (50 and 100 µM) or UO126 (5 and 10 µM), added 1 hour before IL-1ß treatment. Total RNA was isolated, and 20 µg RNA of each sample, including a control, were subjected to Northern blot analysis. Membranes were hybridized with a 32P-labled cDNA probe for LDLr. The insert shows the representative blot for LDLr mRNA and mRNA encoding of the ribosomal S3 protein (loading control). Bars indicate mean ± SEM (standard error of mean) of LDLr mRNA derived from 5 separate experiments. Values were normalized to ribosomal S3 loading control. *p <.02 when compared with IL-1ß alone

	DISCUSSION

Top Abstract Methods Results Discussion References

IL-1ß, a well-known cytokine, triggers a signaling cascade that leads to activation of MAPK and that has been extensively used to induce LDLr and MAPK in different cell lines (6,21,44,45). Other mediators, including several growth factors, have been found to up-regulate LDLr activity, presumably by their mitogenic activity. In our study, induction time in LDLr transcript expression was similar to MAPK in young and old cells. The kinetics of LDLr induction and ERK-1/2 activation are closely related in both cell types. Correlation between LDLr expression and ERK-1/2 activation has already been shown in several human cultured cell lines (6–8). Numerous studies have demonstrated that the proliferative capacity of cells declines with aging (46). Growth activation of quiescent cells leads to enhanced LDLr expression at the cell surface via LDLr gene transcription (47). Our results show decreased expression of LDLr in late-passage cells after IL-1ß treatment, which is related to the growth receptors and proliferative capacity of cells. We have also observed increased ERK protein and baseline phosphorylated ERK in late-passage cells compared with early-passage cells. Consequently, fold induction of ERK is decreased and its activation is prolonged after IL-1ß treatment in late-passage cells. Thus in late-passage cells, a higher basal phosphorylated ERK-1/2, lower inducibility, and delayed translocation of ERK-1/2 are seen. Phosphorylated ERK-1/2 levels are supposed to indicate proliferative status. However several reports have shown that the quantity of nuclear ERK and the length of time ERK spends in the nucleus determine whether ERK signaling is proliferative or antiproliferative [reviewed in (48)]. It has been shown in hamster fibroblasts that the phospho ERK protein pool, accumulated in the nucleus, is inactivated with time and remains in the nucleus in an inactive form (49). In hepatocytes, prolonged activation of the phospho ERK level inhibits DNA synthesis (50). An altered profile of the transcription factor, which binds with DNA to initiate the synthesis and cell proliferation, is known to decrease in human late-passage fibroblasts (51). Thus, further investigation on this line is needed to determine whether phospho ERK-1/2 present in late-passage cells are still active and functional.

The ERK^MAPK group regulates multiple targets in response to growth factors via a RAS-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to proinflammatory cytokines and following exposure of cells to several forms of environmental stresses. The p38^MAPK that shares sequence similarity with other MAPK pathways is also activated by proinflammatory cytokines and environmental stress. The mechanism of p38^MAPK activation is mediated by dual phosphorylation on threonine-180 and tyrosine-182. Thus, p38^MAPK, like JNK, may be regulated in part by a stress-activated signal transduction pathway. However, in our experiments, early- and late-passage fibroblasts show differences in the time course and extent of activation in p38^MAPK and JNK, indicating that these pathways may be distinct. Indeed, p38^MAPK and JNK may represent parallel stress-activated signal transduction pathways (52). JNK and p38^MAPK may activate a number of transcription factors reported to contribute to c-jun promoter activity (53).

In most cell types, the mitogenic signal is relayed from the cytoplasm to the nucleus by nuclear translocation of phosphorylated ERK-1/2, resulting in phosphorylation and activation of a range of transcription factors (54). Thus, subcellular localization of ERK-1/2 is an important determinant of their functions. ERK-1/2 is activated and translocated into the nucleus, while it is largely cytoplasmic in unstimulated cells (55,56). Mitogenic capacity and responsiveness to the mitogen is lost in aging and leads to cellular senescence (57). In view of the importance of the ERK pathway to cellular proliferation and response to growth factors, it was in our interest to confirm the notion of delayed activation of IL-1ß-induced MAPK in late-passage fibroblasts. We examined the ERK-1/2 nuclear translocation in these cells, which is vulnerable to aging in a variety of experimental models in vivo and in vitro [reviewed in (57)]. We used 10 ng/ml of IL-1ß for 10, 30, and 60 minutes to activate the cells. This enabled us to demonstrate a very clear difference in the movement of phosphorylated ERK-1/2 from the cytoplasm to the nucleus in these cells. Early-passage cells showed a rapid entry of phosphorylated ERK-1/2 to the nucleus. However in late-passage cells, nuclear translocation of active ERK-1/2 required 30 minutes. From these results, we concluded that IL-1ß triggered a rapid entry of phosphorylated ERK-1/2^MAPK into the nucleus of early-passage cells, but entry was delayed in late-passage cells. In our experiments, the ERK-1/2 baseline protein level in whole-cell lysates of late-passage cells was higher than in early-passage cells, as reported by others (26,53), and in our results, late-passage cells also showed more basal phosphorylated ERK1/2, possibly due to higher constitutive MEK in late-passage cells (58). Moreover, fold induction revealed a decreased ERK-1/2 activation in late-passage cells. It is still unclear whether the accumulation of unphosphorylated ERK molecules in late-passage cells affects the nuclear translocation of phosphorylated molecules. Adachi and colleagues (59) recently found that nuclear translocation of ERK occurs via an active transport mechanism requiring formation of an ERK dimer. Furthermore, it has been shown that, although phosphorylation is important for dimer formation, microinjected phosphorylated ERK-1/2 molecules could dimerize with unphosphorylated molecules, resulting in translocation and accumulation of inactive ERK proteins in the nucleus (55). This is consistent with our results that showed equal distribution of total ERK-1/2 in cytoplasmic and nuclear fractions of late-passage cells. Alternatively, the nuclear import of phosphorylated ERK molecules may not be inhibited, but ERK may be rapidly dephosphorylated by nuclear phosphatase (s), in late-passage cells, as shown by other researchers (47).

Our report demonstrates that IL-1ß activates the MAPK cascade and stimulates LDLr gene expression at the transcriptional level in early- and late-passage fibroblasts. Inhibitor studies suggest that ERK-1/2 activation and LDLr expression in these cells are correlated. It also suggests that MAPK activation is not the only factor to induce the LDLr expression in early- and late-passage fibroblasts, as our results showed that less LDLr expression, more basal and less activation of ERK in late-passage cells is more likely the age effect. It remains to be determined whether the activation of ERK-1/2^MAPK is a basis of the regulation of LDLr gene in these cells. As aging is associated with an increased risk for atherosclerosis, our studies could provide a potential mechanism for reduction in serum lipid levels that contribute to changes in cholesterol homeostasis and lead to atherosclerosis. Furthermore, our report could create an opportunity to design novel therapeutic compounds that would be useful in treating inflammatory disorders in aging.

	Acknowledgments

 
This work was partly supported by funds from the Central Arkansas Veterans Healthcare System. We thank Dr. Beata Lecka-Czernik for providing the A8 cell line, Dr. Piotr Zimniak of the University of Arkansas for Medical Sciences at Little Rock for his generous gift of human ribosomal protein cDNA (ribosomal S3), Drs. Usha Ponnappan and Masood Shammas for critically reviewing this manuscript, and Drs. Piotr Zimniak, Ludwika Zimniak, Sharda P. Singh, and Mark R. Engle for helping with numerous suggestions throughout the study. We also thank the Office of Grants and Scientific Publications for their editorial assistance during the preparation of this manuscript.

Coauthor Dr. Chidambaram Bhuvaneswaran is deceased.

Address correspondence to Kodetthoor B. Udupa, PhD, Medical Research (151/LR), Central Arkansas Veterans Healthcare System, 4300 W. 7th St., Little Rock, AR 72205. E-mail: udupakodetthoor{at}uams.edu

	Footnotes

 
Decision Editor: Estela Medrano, PhD

Received September 4, 2003

Accepted December 3, 2003

	References

Top Abstract Methods Results Discussion References

 

1. Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res.. 1965;37:614-636.[Medline]
2. Goldstein S, Moreman EJ, Jones AR, et al. Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts. Proc Natl Acad Sci U S A.. 1991;88:9680-9684.[Abstract/Free Full Text]
3. Cristofalo VJ, Volker C, Francis MK, et al. Age-dependent modifications of gene expression in human fibroblasts. Crit Rev Eukaryotic Gene Express.. 1998;8:43-80.[Medline]
4. Wheaton K, Sampsel K, Francois-michel B, et al. Loss of functional caveolae during senescence of human fibroblasts. J Cell Physiol.. 2001;187:226-235.[Medline]
5. Cristofalo VJ, Beck J, Allen RG. Cell senescence: an evaluation of replicative senescence in culture as a model for cell aging in situ. J Gerontol Biol Sci.. 2003;58A:776-781.
6. Kumar A, Middleton A, Chambers TC, Mehta KD. Differential roles of extracellular signal-regulated kinase-1/2 and p38^MAPK in interleukin-1ß- and tumor necrosis factor--induced low density lipoprotein receptor expression in HepG2 cells. J Biol Chem.. 1998;273:15742-15748.[Abstract/Free Full Text]
7. Singh RP, Dhawan P, Golden C, Kapoor GS, Mehta KD. One-way cross-talk between p38^MAPK and p42/44^MAPK, inhibition of p38^MAPK induces low density lipoprotein receptor expression through activation of the p42/44^MAPK cascade. J Biol Chem.. 1999;274:19593-19600.[Abstract/Free Full Text]
8. Nakahara M, Fujii H, Maloney PR, et al. Bile acids enhance low density lipoprotein receptor gene expression via a MAPK cascade-mediated stabilization of mRNA. J Biol Chem.. 2002;277:37229-37234.[Abstract/Free Full Text]
9. Goldstein JL, Brown MS. The low density lipoprotein receptor and its relationship to atherosclerosis. Ann. Rev Biochem.. 1977;46:897-930.[Medline]
10. Goldstein JL, Brown MS. Familial hypercholesterolemia: identification of defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol. Proc Nat Acad Sci U S A.. 1973;70:2804-2808.[Abstract/Free Full Text]
11. Brown MS, Dana SE, Goldstein JL. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesteroleia. J Biol Chem.. 1974;249:789-796.[Abstract/Free Full Text]
12. Goldstein JL, Brown MS. The LDL pathway in human fibroblasts: a receptor mediated mechanism for the regulation of cholesterol metabolism. Curr Top Cell Regul.. 1976;11:147-181.[Medline]
13. Li S, Song KS, Lisanti, MP. Baculovirus-based expression of mammalian caveolin in Sf21 insect cells. A model system for the biochemical and morphological study of caveolae biogenesis. J Biol Chem.. 1996;271:568-573.[Abstract/Free Full Text]
14. Woong-yang P, Jeong-soo P, Kyung-A C, et al. Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells. J Biol Chem.. 2000;275:20847-20852.[Abstract/Free Full Text]
15. Davis R. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem.. 1993;268:14553-14556.[Free Full Text]
16. Cobb M, Goldsmith EJ. How MAP kinases are regulated. J Biol Chem.. 1995;270:14843-14846.[Free Full Text]
17. Graves LM, Guy HI, Kozlowski P, et al. Regulation of carbamoyl phosphate synthetase MAP kinase. Nature.. 2000;403:328-332.[Medline]
18. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem.. 1995;270:16483-16486.[Free Full Text]
19. Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell.. 1995;80:199-211.[Medline]
20. Whitmarsh AJ, Shore P, Sharrocks AD, Davis RJ. Integration of MAP kinase signal transduction pathways at the serum response element. Science.. 1995;269:403-407.[Abstract/Free Full Text]
21. McDermott EP, O'Neill LA. Ras participates in the activation of p38 MAPK interleukin-1 by associating with IRAK, IRAK2 TRAF6, and TAK-1. J Biol Chem.. 2002;277:7808-7815.[Abstract/Free Full Text]
22. Kyriakis JM, Avruch J. Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem.. 1996;271:24313-24316.[Free Full Text]
23. Freshney NW, Rawlinson I, Guesdon F, et al. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell.. 1994;78:1039-1049.[Medline]
24. Minden A, Lin A, McMahon M, et al. Differential activation of ERK and JNK mitogen activated protein kinases by Raf-1 and MEKK. Science.. 1994;266:1719-1723.[Abstract/Free Full Text]
25. Liu Y, Guyton KZ, Gorospe M, et al. Age-related decline in mitogen-activated protein kinase activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem.. 1996;271:3604-3607.[Abstract/Free Full Text]
26. Logan SK, Falasca M, Hu P. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N terminal kinase signaling pathway. Mol Cell Biol.. 1997;17:5784-5790.[Abstract]
27. Brunet A, Roux D, Lenordmand P, et al. Nuclear translocation of p42/44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J.. 1999;18:664-674.[Medline]
28. Kim-Kaneyama J, Nose K. Shibanima M. Significance of nuclear relocalization of ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent fibroblasts. J Biol Chem.. 2000;275:20685-20692.[Abstract/Free Full Text]
29. Robinson MJ, Stippec SA, Goldsmith E, et al. A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Curr Biol.. 1998;8:1141-1150.[Medline]
30. O'Neill LA. Interleukin-1 signal transduction. Int J Clin Lab Res.. 1995;25:169-277.[Medline]
31. Saklatvala J, Davis W, Guesdon F. Interleukin 1 (IL1) and tumor necrosis factor (TNF) signal transduction. Phil Trans R Soc Lond... 1996;351:151-157.[Medline]
32. Evans R, Argiles J, Williamson D. Metabolic effect of tumor necrosis factor-alpha (cachectin) and interleukin-1. Clin Sci.. 1980;77:357-364.
33. Foa R, Guarini A, Francia Decelle P, et al. Constitutive production of tumor necrosis factor alpha in hairy cell leukemia: possible role in the pathogenesis of the cytopenia(s) and effect of treatment with interferon-alpha. J Clin Oncol.. 1992;10:954-959.[Abstract]
34. Grunfeld C, Feingold KR. Metabolic disturbances and wasting in the acquired immunodefiency syndrome. N Engl J Med... 1992;327:329-337.[Medline]
35. Grunfeld C, Kotler D, Shigenaga J, et al. Circulating interferon-alpha levels and hypertriglyceridemia in the acquired immunodeficiency syndrome. Am J Med... 1991;327:329-337.
36. Riikonen P, Soarinen U, Teppo A, et al. Cytokine and acute-phase reactant levels in serum of childern with cancer admitted for fever and neutropenia. J Infect Dis.. 1992;166:432-436.[Medline]
37. Chait A, Ross R, Albers JJ, Bierman EL. Platelet-derived growth factor stimulates activity of low density lipoprotein receptors. Proc Natl Acad Sci U S A.. 1980;77:4084-4088.[Abstract/Free Full Text]
38. Witte LD, Cornicelli JA, Miller RW. Goodman DS Effect of platelet-derived and endothelial cell- derived growth factors on the low density lipoprotein receptor pathway in cultured human fibroblasts. J Biol Chem.. 1982;257:5392-5401.[Abstract/Free Full Text]
39. Goldstein S, Moerman JE, Porter K. High-voltage electron microscopy of human diploid fibroblasts during aging in vitro. Exp Cell Res.. 1984;154:101-111.[Medline]
40. Lecka-Czernik B, Moreman EJ, Jones RA, Goldstein S. Identification of gene sequence overexpression in senescent and Warner syndrome human fibroblasts. Exp Gerontol.. 1996;31:159-174.[Medline]
41. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.. 1976;72:248-254.[Medline]
42. Levkau B, Herren B, Koyama HR, et al. Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesion in human endothelial cell apoptosis. J Exp Med.. 1998;187:579-586.[Abstract/Free Full Text]
43. Boyle WJ, Lampert MA, Lipsick JS, Baluda MA. Avian myeloblastosis virus and E26 virus oncogene products are nuclear proteins. Proc Natl Acad Sci U S A.. 1984;81:4265-4269.[Abstract/Free Full Text]
44. Stopeck AT, Nicholson AC, Mancini FP, Hajjar DP. Cytokine regulation of low density lipoprotein receptor gene transcription in HepG2 cells. J Biol Chem.. 1993;268:17489-17494.[Abstract/Free Full Text]
45. Hamanaka R, Kohno K, Seguchi T, et al. Induction of low density lipoprotein receptor and a transcription factor SP-1 by tumor necrosis factor in human microvascular endothelial cells. J Biol Chem.. 1992;267:13160-13165.[Abstract/Free Full Text]
46. Hensler PJ, Pereira-Smith OM. Human replicative senescence. A molecular study. Am J Pathol.. 1995;147:1-8.[Medline]
47. Mazzone T, Basheeruddin K, Duncan H. inhibitors of translation induce low density lipoprotein receptor gene expression in human skin fibroblasts. J Biol Chem.. 1989:;264;:15529-15534.
48. Brian S, Zuckerbraun MD, Richard AS, et al. RhoA influences the nuclear localization of extracellular signal-regulated kinases to modulate p21^Waf/Cip1 expression. Circulation.. 2003;108:876-881.[Abstract/Free Full Text]
49. Jacques P, Phillipe L. Fidelity and spatio-temporal control in MAP kinase (ERKs) signaling. Eur J Biochem.. 2003;270:3291-3299.[Medline]
50. Dent P, Jarvis WD, Birrer MJ, et al. The role of signaling by the p42/p44 mitogen-activated protein (MAP) kinase: a potential route to radio-chemo-sensitization of tumor cells in the induction of apoptosis and loss of clogenicity. Leukemia.. 1998;12:1843-1850.[Medline]
51. Goberdhan PD, Judith C. Altered profile of transcription factor-binding activities in senescent human fibroblasts. Exp Cell Res.. 1994;212:132-140.[Medline]
52. Derijard B, Raingeaud J, Barrett T, et al. Independent human MAP-kinase signal transduction pathway defined by MEK and MKK isoforms. Science.. 1995;267:682-685.[Abstract/Free Full Text]
53. Coeffey ET, Smiciene G, Hongisto V, et al. C-Jun N-terminal protein kinase (JNK) 2/3 is specifically activated by stress, mediating c-Jun activation, in the presence of constitutive JNK1 activity in cerebellar neurons. J Neurosci.. 2002;22:4335-4345.[Abstract/Free Full Text]
54. Ridley SH, Sarsfield SJ, Lee JC, et al. Action of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of progestaglandin H synthase-2, metalloproteinases and IL-6 at different levels. J Immunol.. 1997;158:3165-3173.[Abstract]
55. Fukuda M, Gotoh Y, Nishida E. Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J.. 1997;16:1901-1908.[Medline]
56. Khokhlatechev AV, Canagarajah B, Wilsbacher J, et al. Phosphorylation of the MAP kinase ERK promotes its homodimerization and nuclear translocation. Cell.. 1998;93:605-615.[Medline]
57. Tresini M, Lorenzini A, Frisoni L, et al. Lack of elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signalling pathway in senescent human fibroblast. Exp Cell Res.. 2001;269:287-300.[Medline]
58. Lim IK, Hong KW, Kwak IH, et al. Translocation inefficiency of intracellular proteins in senescence of human diploid fibroblasts. Ann N Y Acad Sci.. 2001;928:176-181.[Medline]
59. Adachi M, Fukuda M, Nishida E. Nuclear export of MAPkinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J Cell Biol.. 2000;148:849-856.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. Bose, H. Zhang, K. B. Udupa, and P. Chowdhury Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion Am J Physiol Gastrointest Liver Physiol, November 1, 2005; 289(5): G926 - G934. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Download to citation manager Citing Articles Citing Articles via HighWire PubMed PubMed Citation