Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Abstract

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 2, 134-143, April 2006

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Ik-Soon Jang1*, Ji-Heon Rhim1*, Sang Chul Park1 and Eui-Ju Yeo2,3

1Department of Biochemistry and Molecular Biology Aging and Apoptosis Research CenterSeoul National University College of Medicine Seoul 110-799, Korea

2Department of Biochemistry, Gachon Medical School Incheon 405-760, Korea

3Corresponding author: Tel, 82-32-460-3519;

Fax, 82-32-930-5088; E-mail, [email protected] *These authors contributed equally to this work.

Accepted 3 March 2006

Abbreviations: AC, adenylyl cyclase; COX, cyclooxygenase; CaMK, calmodulin kinase; CREB, cAMP-responsive element binding protein; EDGs, endothelial cell differentiation genes; ERK, extracellular signal-regulated kinase; LPA, lysophosphatidic acid; MAPK, mitogen- activated protein kinase; MEK, MAPK/ERK kinase; MSK1, mitogen- and stress-activated kinase1; PKA, protein kinase A; PKC, protein kinase C

Abstract

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit of PKA and of the major regulatory subunit I. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased

following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-depen -dent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

Keywords: aging; cyclic AMP response element-binding protein; cyclic AMP-dependent protein kinases; cyclooxygenase 2; lysophosphatidic acid; proto-oncogene proteins c-fos

Introduction

Lysophosphatidic acid (LPA) is a phospholipid growth factor that elicits a variety of cellular responses, such as changes in cell shape, chemotaxis, proliferation, and differentiation, depending on the cell type (Moolenaar et al., 1997; An et al., 1998b; Moolenaar, 2000). The intracellular biochemical signaling events that mediate these effects of LPA include an increase in cytoplasmic calcium concentration, stimulation of phospholipases, activation of phosphatidylinositol 3-kinase and the Ras-Raf-MAP kinase cascade, and inhibition of adenylyl cyclase (AC) (Moolenaar et al., 1997; Moolenaar, 2000). Recently, the cell surface G-protein-coupled receptors for LPA were identified as products of a family of endothelial cell differentiation genes (EDGs) (Contos et al., 2000; Fukushima et al., 2001). The major members of this EDG family of proteins were shown to be EDG-2 (Hecht et al., 1996), EDG-4 (An et al., 1998a), and EDG-7 (Bandoh et al., 1999), recently renamed as LPA1, LPA2, and LPA3, respectively. Additional LPA receptors are being discovered. The specific responses of LPA receptors are manifested only when the G-proteins

LPA-induced CREB phosphorylation increase in senescent HDFs135

are coupled to an appropriate receptor. For example, LPA1 is coupled to pertussis toxin-sensitive Gi, LPA2 to both Gi and Gq (An et al., 1998a), and LPA3 to a pertussis toxin-insensitive G-protein, Gq (Bandoh et al., 1999). LPA1 and LPA2 inhibit all types of ACs through Gi activation, resulting in the reduction of cAMP (Taussig et al., 1993). cAMP binds to the regulatory subunits of protein kinase A (PKA), eliciting the release of the catalytic subunits which diffuse through the cytosol and phosphorylate various substrates (Clegg et al., 2000; Hansson et al., 2000; Skalhegg and Tasken, 2000). The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is a substrate of the activated PKA and becomes activated when phosphorylated on Ser133 (Yamamoto et al., 1988; Gonzalez and Montminy, 1989). Phosphorylation of CREB at Ser133 was induced not only by PKA but also by a variety of other signaling cascades that include protein kinase C (PKC) (Gonzalez et al., 1989; Cardenas et al., 2004), calmodulin kinase (CaMK) (Wu et al., 2001), ERK1/2 (Roberson et al., 1999; Shaywitz and Greenberg, 1999; Wu et al., 2001), p38 mitogen-activated protein kinase (p38MAPK) (Togo, 2004), and mitogen- and stress-activated kinase1 MSK1 (Lee et al., 2003; Schuck et al., 2003). Phosphorylated CREB drives the transcription of a large number of genes having CRE through interaction with its nuclear partner CREB binding protein (Chrivia et al., 1993). One of these CRE-regulated genes is c-fos, a member of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth. Activation of ERK1/2 is required for c-fos expression (Cook et al., 1999). LPA also stimulates CREB and subsequently induces c-fos via MAPK signaling components, ERK1/2, p38MAPK, and MSK1, in Rat-2 fibroblasts (Lee et al., 2003) and in mouse embryonic stem cells (Schuck et al., 2003). Cyclooxygenase (COX), the key rate-limiting enzyme in the prostaglandin synthetic pathway, is another CRE-regulated gene. Two isoforms of COX have been identified: a constitutive COX-1 and an inducible COX-2. Activation of ERK1/2 and phosphorylation of CREB are also upstream steps in COX-2 induction (Hahn et al., 2002). We observed in a previous study, that production of LPA-induced cAMP was altered during the aging of human diploid fibroblasts (HDFs) (Jang et al., 2003). In contrast to increased cAMP production in senescent HDFs, LPA inhibited cAMP production in young HDFs. LPA-induced cAMP accumulation correlated well with the stimulation of AC. Age-related quantitative and qualitative alterations in LPA receptors and downstream signaling molecules including inhibitory Gi proteins and PKC-dependent AC isoforms contribute to cAMP accumulation by LPA

treatment in senescent cells (Jang et al., 2003).

In this study, we have shown that LPA differentially modulates the activation of PKA and phosphorylation of CREB, and cause differential expression of CRE-regulated genes such as c-fos and COX-2, in young and senescent fibroblasts. We also confirmed the involvement of other protein kinases in the expression of CRE-regulated genes by using in vivo CRE cis-reporting system with luciferase activity and protein kinase inhibitors: bisindolylmaleimide (bIM) for PKC, PD98059 and U0126 for ERK1/2, SB203580 for p38MAPK, H89 for MSK1 as well as PKA.

Materials and Methods

Materials

The following reagents were used in this study: Dulbeccos modified Eagles medium, LPA and a monoclonal anti-beta actin antibody, from Sigma (St. Louis, MO), fetal bovine serum (FBS), penicillin, streptomycin, and superscript II reverse transcriptase kit, from Gibco/BRL Life Technologies, Inc. (Carlsbad, CA), PCR reagents including Taq polymerase, from Takara Shiga (Japan); [-32P]ATP, from

Amersham Pharmacia Biotech (Buckinghamshire, UK), PathDetect in vivo CRE cis-reporting system with luciferase activity, from Stratagene (La Jolla, CA), polyclonal antibodies against total CREB, and COX-2, from Santa Cruz Biotechnology, Inc (Santa Cruz, CA), a monoclonal antibody against phosphorylated CREB, from Cell Signaling Technology (Beverly, MA), an anti-c-fos monoclonal antibody (Clone G54-9.9), from BD Bioscience Pharmingen (Mountain View, CA), PD98059, U0126, SB203580, and H89, from Calbiochem (San Diego, CA).

Cell culture

Primary human fibroblasts were isolated from newborn foreskins as described previously (Boyce and Ham, 1983), and maintained in Dulbeccos modified Eagles medium containing 10% fetal bovine serum and antibiotics. Intracellular components of young cells from the early stage of culture, with population doubling (PD) of less than 25, were compared to those of senescent cells with PD 65-70 cultures. Senescent cells were characterized by morphology, enhanced -galactosidase activity, and a reduced rate of proliferation (Yeo et al., 2000a). Prior to LPA treatment, the cells were grown for 2-3 days to 60-70% sub-confluency in the culture medium, and then serum-starved to quiescence by incubation with serum free medium containing 0.1% BSA (SFM) for 2 days.

136 Exp. Mol. Med. Vol. 38(2), 134-143, 2006

Semi-quantitative RT-PCR

Total RNA was extracted from young and senescent cells using an acid guanidinium thiocyanate phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). In order to compare the amounts of mRNA in young and senescent cells, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed, as described previously (Nicoletti and Sassy-Prigent, 1996). A series of mixtures of RNA from young and senescent cells was prepared as indicated, so that each mixture had the same total amount of RNA (2 g) in a constant volume (12 l). The RT reaction was carried out in a final volume of 20 l using superscript II reverse transcriptase according to the manufacturers protocol, and 4 l of the final RT product mix was then

PCR amplified. The primer sets used were: 5'-AGG CAA AAC AGA TTC AG-3' and 5'-CTC CTT CCC CAA CAC TG-3' for PKA RI; 5'-GAA GGT GAT

TCC CAA GGA CTA CAA-3' and 5'-TCC CGG TCG ATC CCC CAG AG-3' for RI; 5'-CGA CGC CAA

AGG GGA CAG-3' and 5'-GGG GCA CAG ACT CAA TAA ATG-3' for RII; 5'-CTG AAC GCC TGA

AAG TAG TAG-3' and 5'-GCA GTG GGT TCA ACA ATA TCC-3' for RII; 5'-AGT CCC GCT CAG AAC

ACA G-3' and 5'-GAT AAT CTC AGG GGC CAG G-3' for PKA C; 5'-TAA TGC CGG ACT TGA AC-3' and 5'-TCT GCC TTT AAC TCT TT-3' for PKA C; and 5'-TAC GCC GAC CAG CCC ATC CAG-3' and 5'-AAC CAC AGG GGC ACA AGC ACA-3' for PKA C.

Measurement of PKA activity

LPA-treated cells were harvested and homogenized in an ice-cold homogenization buffer (0.25 M suc-rose, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM EGTA, and protease inhibitors), centrifuged at 20,000 g at 4oC for 20 min, and PKA assays were performed as described previously (Yang et al., 1999). The kinase reaction mixture contained 10 l of substrate cocktail (500 M kemptide and 10 M cAMP), 10 l of inhibitor cocktail (20 M PKC inhibitor peptide and 20 M compound R24571), 10 l of tissue homogenate, and 10 l of a mixture containing 0.5 mM ATP, 75 mM MgCl2 and 10 Ci of [-32P]ATP (3,000 Ci/mmol). After incubation for 10 min at 30oC, 25 l of the mixture was blotted onto a P81 paper square. The paper was washed once with 0.75% phosphoric acid and the radioactivity incorporated into Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) was measured using a scintillation counter (Model Tri-Carb 1600 CA, Packard Instrument Company), in triplicate. PKI-inhibitable kinase activity was calculated as percentage of total PKA activity.

CRE binding luciferase assay

Quiescent cells were transfected with PathDetect in vivo CRE cis-reporting system with luciferase activity and a plasmid pcDNA with -galactosidase activity as a control for transfection efficiency. Cells were pretreated with 10 M of inhibitors of PKC, MEK-1, and p38MAPK, and PKA/MSK1 for 1 h and treated with 30 M of LPA for the indicated times. Nuclear extracts were assayed for CRE binding luciferase activity as described in the manufacturers instruction.

Western blot analysis

Expression levels of signaling proteins were examined by Western blot analysis, as described previously (Yeo et al., 2000b). Total cell lysates were prepared in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25 g/ml leupeptin, 25 g/ml aprotinin, 5 mM benzamidine, and 1% Igepal CA630. Protein concentrations of the lysates were determined using a BCA protein assay kit, as described by the manufacturer. Cell lysates containing equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon PVDF membranes. Blots were blocked with a solution containing 5% non-fat dried milk and 0.1% Tween 20, and treated with antibodies in the blocking solution overnight. Blots were then washed and further incubated with horseradish peroxidase-conjugated anti-rabbit IgGs (1:5,000). The immune complexes were visualized using an enhanced chemiluminescence (ECL) system, as described by the manufacturer.

Statistical analysis

The Graph-Pad Prism (GraphPad, San Diego, CA) was used for statistical analysis. Student's t-test was used to assess differences between LPA-treated and untreated (data at time 0) groups for the indicated times. Data are presented as the mean standard deviation and P values of less than 0.05, 0.01, and 0.001 were marked as *, **, and ***, respectively, in some figures. P values of less than 0.05 were considered to be significant.

Results

Altered expression of catalytic and regulatory subunits of PKA in senescent cells

Previously, we reported that levels of cellular components of cAMP signaling systems variously

LPA-induced CREB phosphorylation increase in senescent HDFs137

C RI RII GAPDH

Y S Y S Y S Y S Y S

1 1.7 1 1.4 1 1.1 1 0.7 1 1.0

Figure 1. mRNA levels of PKA catalytic and regulatory subunits in young and senescent HDFs. Total RNA was isolated from young (PD 23, Y) and senescent cells (PD 70, S) using an acid guanidinium thiocyanate phenol-chloroform extraction method and the mRNA levels were compared by semi-quantitative RT-PCR. RT-PCR products of the PKA catalytic subunit (C) and the regulatory subunits (RI, RI, RII) were separated on 1.2% agarose gel, photographed. The density of each band was analyzed using a densitometer and their relative expressions in senescent HDFs were compared to those in young HDFs. This experiment was performed three times and similar results were observed. The result of a typical experiment is shown.

Figure 2. LPA-induced PKA activation profiles in young and senescent HDFs. Subconfluent young (PD 20) and senescent (PD 67) fibroblasts were serum starved for 2 days and treated with 30 M of LPA for the indicated times. The PKA activities in the total cell lysates were measured as described in Materials and Methods. The radioactivity incorporated into the kinase substrate was measured using a beta-counter, and the enzyme activity was expressed as pmol/g/min. This experiment was performed three times and similar results were observed, and the result of a typical experiment is shown. The results shown represent the means standard deviation of a representative experiment. **P 0.05 and ***P 0.001 in comparison with the control values (at time 0) in each group.

Altered LPA-induced PKA activation in senescent HDFs

Cellular cAMP decreased in a time- and dose-dependent manner after incubation with LPA in young fibroblasts, whereas cAMP increased in senescent fibroblasts (Jang et al., 2003). To test whether PKA activation by LPA also altered with age, we determined the PKA activity after LPA treatment. As shown in Figure 2, basal PKA activity is much lower in senescent cells than in young cells. Total PKA activity decreased in a time-dependent manner in LPA-treated young cells, but increased in senescent cells.

Altered LPA-induced CREB phosphorylation in senescent HDFs

Once PKA is activated, phosphorylation of CREB, one of substrates of PKA, increases inside the nucleus. We measured the phosphorylation level of CREB by immunoblot analysis with a phospho-specific antibody that recognizes CREB on Ser133. As shown in Figure 3, basal CREB phosphorylation is much higher in young cells than in senescent cells. LPA, at a dose of 30 M, reduced phosphorylation of CREB at Ser133 after 30 min in young fibroblasts, but stimulated it in senescent fibroblasts in a time-dependent manner. This CREB phosphorylation pattern correlated well with the pattern of cAMP accumulation and the PKA activity profile. These results suggest that the senescence-associated increase of cAMP levels after LPA treatment causes increased CREB phosphorylation.

LPA-induced early gene expression in young and senescent HDFs

Transcription of early genes such as c-fos increases after p-CREB activation of a CRE box located upstream. We tested the effect of CREB phosphorylation on CRE-regulated early response transcription

change in HDFs. In this report, we examined the changes in catalytic and regulatory subunits of PKA. We detected specific mRNA transcripts of the human PKA catalytic subunits C and C, regulatory subunits RI, RI, RII, RII, and GAPDH as a control, by semi-quantitative RT-PCR with specific primers (Figure 1). Young HDFs contained mainly catalytic subunit C and regulatory subunit RI, and to a minor degree RI and RII. We found that the mRNA levels of major catalytic subunit C and the regulatory subunit RI increased in the aged cells, whereas the levels of RII mRNA decreased slightly and that of minor regulatory subunit RI did not change (Figure 1).

138 Exp. Mol. Med. Vol. 38(2), 134-143, 2006

A

LPA

C 10 30 60 Time (min)

c fos

-

Young

Senescent

p-CREB CREB

-actin

Young

Senescent

-actin

p-CREB CREB -actin

0 10 30 60 Time

(min)

Figure 4. LPA-induced c-fos expression in young and senescent HDFs. Subconfluent young and senescent fibroblasts were treated with 30 M of LPA for the indicated times. The lysates containing the same amount of protein (45 g) were analyzed by immunoblotting using polyclonal anti-c-fos antibodies. Four isoforms of c-fos were indicated by arrows. The content of -actin was also monitored as a control. The experiment was performed three times with similar results.

c os

-f

-actin

B

Young

1.6 Senescent

Ratio of pCREB/CREB

1.2

0.8

Young Senescent

-

A B

***

***

0.4

** ***

***

* *

***

0.0

0 10 30 60

Incubation time (min)

Figure 3. LPA-induced CREB phosphorylation in young and senescent HDFs. Subconfluent young (PD 20) and senescent (PD 67) fibroblasts were serum starved for 2 days and treated with 30 M of LPA for the indicated times. The lysates containing the same amount of protein (45 g) were analyzed by immunoblotting using polyclonal anti-CREB antibodies, monoclonal anti-phospho-CREB and anti--actin antibodies (A). The density of each band was analyzed using a densitometer and the relative density of phosphorylated CREB (p-CREB) to that of total CREB were calculated and plotted (B). The experiment was performed three times with similar results.

Figure 5. LPA-induced COX-2 expression in young and senescent HDFs. Subconfluent young and senescent fibroblasts were treated with 30 M of LPA for the indicated times. The lysates containing the same amount of protein (45 g) were analyzed by immunoblotting using polyclonal anti-COX-2 antibodies. The content of a constitutive isoform COX-1 and -actin was also monitored as controls. The experiment was performed three times with similar results.

factor c-fos after LPA treatment. Many isoforms of c-fos (e.g., 55 kDa, 57 kDa, 60 kDa, and 62 kDa) have been described, and they could also undergo posttranslational modification (Curran et al., 1985). The purified mouse anti-fos monoclonal antibody recognized c-fos as multiple bands in the area of 50-65 kDa in young and senescent HDFs (Figure 4). Results from three experiments show that 30 M of

LPA significantly increased c-fos protein levels within 30 min in young HDFs. In senescent cells, the expression of c-fos decreased initially until 30 min but increased at 1 h after LPA treatment. We also examined the expression of the key enzyme in

prostaglandin synthesis: COX-2, which was shown to be induced by LPA in the other cell systems (Ershov and Bazan, 1999; Hahn et al., 2002; Symowicz et al., 2005). A constitutive isoform COX-1 was also examined as a control. In young HDFs, the inducible isoform COX-2 protein level increased rapidly within 10 min but COX-1 was unaltered (Figure 5). The expression of COX-2 decreased initially until 30 min but increased 1 h after LPA treatment in senescent HDFs.

Effect of kinase inhibitors on LPA-induced CRE-luciferase activity in young and senescent HDFs

In order to confirm the changes in CRE-dependent gene expression induced by LPA, we measured the luciferase activity in HDFs transfected with Path-

LPA-induced CREB phosphorylation increase in senescent HDFs139

Figure 6. Effect of protein kinase inhibitors on CRE-luciferase activity. Quiescent young (A) and senescent fibroblasts (B) were transfected with PathDetect in vivo CRE cis-reporting system with luciferase activity and a plasmid pcDNA with -galactosidase activity as a control for transfection efficiency. Cells were pretreated with 10 M of inhibitors to PKC (bisindolylmaleimide, bIM), MEK-1 (U0126 and PD98059), p38MAPK (SB203580), and PKA (H89) for 1 h and then treated with 30 M of LPA for the indicated times. The activities of luciferase and -galactosidase were measured in the cell nuclear extract as described in Materials and Methods. The results shown represent the means standard deviations of a representative experiment. *P 0.01, **P 0.05 and ***P 0.001 in comparison with the control values (at time 0) in LPA group.

Detect in vivo CRE cis-reporting system and a plasmid pcDNA with -galactosidase activity as a

control for transfection efficiency. The luciferase assay revealed a high level of CRE-dependent gene expression in the absence of LPA, and LPA treatment slowly increased CRE-dependent gene expression in young HDFs (Figure 6A, --). In senescent cells, though the basal luciferase activity was weaker, LPA-stimulated luciferase activity increased more rapidly compared to young cells, indicating an increase of CRE-dependent gene expression (Figure 6B, --).

The observed discrepancy between PKA-associated CREB phosphorylation and CRE-dependent gene expression suggests an involvement of other protein kinases. In addition to PKA, PKC, CaMK, ERK1/2 and p38 MAPK have been implicated in CREB phosphorylation and its activation. LPA-stimulated expression of luciferase under the control of the CRE box was examined after pretreatment of the cells with PKC inhibitor, bisindolylmaleimide (Toullec et al., 1991), MAPK/ERK kinase (MEK) inhibitor, PD98059 (Alessi et al., 1995), and U0126 (Duncia et al., 1998), and p38MAPK inhibitor, SB203580 (Clerk and Sugden, 1998), as well as PKA inhibitor, H89 (Engh et al., 1996). Pretreatment of cells with the protein kinase inhibitors for 1 h inhibited the CRE-luciferase activity induced by LPA both in young and senescent HDFs (Figure 6A and 6B). Our data suggest that the cooperative effect of the above kinases may be required for the expression of CRE-responsive genes.

Discussion

Correlation of cAMP status with LPA-induced PKA activation and CREB phosphorylation in young and senescent HDFs

In our previous study, we demonstrated an altered profile of LPA-induced cAMP accumulation in the senescent state (Jang et al., 2003). In this study, we found that the mRNA levels of both the major catalytic subunit C, and of the regulatory subunit RI, also increased in senescent fibroblasts (Figure 1). Our results are in agreement with the findings of Liu et al. (Liu et al., 1986), who showed increased levels of PKA in extracts of senescent IMR-90 lung fibroblasts compared to young cells. Blumenthal et al. (Blumenthal et al., 1993) also demonstrated a higher PKA activity in parallel with increased PKA protein content in senescent cells, than both in replicating or quiescent cells. Since the regulatory subunit, RI, also increased in senescent HDFs, it would bind to the catalytic subunits of PKA, resulting in lower PKA activity (Figure 2) and lower CREB phosphorylation (Figure 3) in the absence of LPA. The increased level of PKA holoenzyme may allow the senescent HDFs to respond to LPA sensitively. We also found that PKA profiles after LPA treatment are similar to those of cAMP reported previously (Jang et al., 2003). Total PKA activity, following LPA treatment, decreases in a time-dependent manner in young cells, but increases in senescent cells (Figure 2).

140 Exp. Mol. Med. Vol. 38(2), 134-143, 2006

Though it was suggested that a diminished expression of CREB might contribute to the altered cAMP-mediated regulation of gene expression with senescence (Dimri and Campisi, 1994; Chin et al., 1996), we observed that the protein content of CREB was not altered, but its phosphorylation was enhanced, after LPA treatment in senescent cells (Figure 3). Therefore it is unlikely that the altered expression of CREB protein is responsible for the increased cAMP signaling of senescent cells. Our data clearly suggest that activation of cAMP signaling may give rise to robust CREB phosphorylation via PKA activation in senescent HDFs.

Discrepancy of CREB phosphorylation and CRE-regulated Gene expression by LPA

It was previously reported that LPA rapidly stimulated phosphorylation of CREB at Ser133 in a time-and dose-dependent manner in Rat-2 fibroblasts (Lee et al., 2003). LPA was also shown to increase TCF and CREB phosphorylation and subsequent activation of the c-fos promoter, resulting in an increase of c-fos mRNA about two fold at a concentration of 100 M in C6 glioma cells (Segura et al., 2005), and about 60 fold increase at a concentration of 20 M in mouse embryonic stem

cells (Schuck et al., 2003). LPA was also shown to increase COX-2 mRNA expression in retinal pigment epithelium (RPE) cells (Ershov and Bazan, 1999), in renal mesangial cells (Hahn et al., 2002), and in ovarian cancer cells (Symowicz et al., 2005). LPA was shown to contribute, in part, to the development, progression, and metastasis of ovarian cancer by inducing the expression of genes including COX-2 (Symowicz et al., 2005). However, unlike other cell systems, young HDFs showed a time-dependent decrease in CREB phosphorylation after treatment with 30 M LPA (Figure 3). Moreover, the CRE-regulated gene expression did not correlate well with CREB phosphorylation in young HDFs. CRE-regulated gene products, c-fos and COX-2, increased in young HDFs (Figure 4 and Figure 5, respectively). In contrast, a time-dependent increase in CREB phosphorylation was observed in senescent HDFs (Figure 3) and the induction profiles of c-fos (Figure 4) and COX-2 (Figure 5) are different from those of young cells-decreasing initially (at 30 min) but increasing later. This discrepancy between CREB phosphorylation and CRE-regulated gene expression suggest the possible operation of other mechanisms of age-dependent transcriptional control than the PKA system, depending on the type and age of the cell.

Protein kinases involved in CREB phosphorylation

and CRE-regulated gene expression

Sequence analysis of CREB cDNA predicts a cluster of PKA, PKC and casein kinase II consensus recognition sites near the N terminus of the protein (Gonzalez et al., 1989). The proximity of these potential phosphorylation sites indicates that they may interact either positively or negatively to regulate CREB bioactivity (Gonzalez et al., 1989). Indeed, involvement of several protein kinases including PKA, PKC, CaMK and MAPK signaling components on CREB phosphorylation and CRE-regulated gene expression has been reported.

It was suggested that MAPK cascade couples PKA and PKC to CREB phosphorylation in area CA1 of rat hippocampus (Roberson et al., 1999). Activation of ERK1/2 is required for c-fos expression (Cook et al., 1999) and is an upstream step in COX-2 induction (Hahn et al., 2002). LPA also stimulates CREB via MAPK signaling components, ERK1/2 and p38MAPK, in Rat-2 fibroblasts (Lee et al., 2003). The likely agent of coupling MAPK and CREB phosphorylation was suggested to be MSK1 (Schuck et al., 2003), which is activated by both ERK1/2 and p38MAPK and is required for induction of c-fos by LPA in mouse embryonic stem cells (Schuck et al., 2003) and Rat-2 fibroblasts (Lee et al., 2003). Phenylephrine stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38MAPK, MSK1 and PKA (Markou et al., 2004). Forskolin-induced CREB phosphorylation and activation in NIH 3T3 cells was shown to be mediated directly by PKA and by a delayed PKA-dependent p38/MSK-1 pathway (Delghandi et al., 2005). In our study, we also found that inhibition of MAPK signaling components (ERK1/2 with PD98059 and U0126, of p38MAPK with SB203580) efficiently blocked LPA-induced CRE-luciferase activity in both young and senescent HDFs (Figure 6). These data suggest that MAPK signaling components are required for CRE-regulated gene expression by LPA in HDFs.

Mitogenic doses of LPA induce sustained MAPK activation and DNA synthesis in Rat-1 fibroblasts (Cook et al., 1999). However, activation of the MAPK cascade alone is not sufficient to induce c-fos expression, which requires cooperation with other signals such as Ca2+ mobilization (Cook et al., 1999). In fact, the induction of c-fos observed in response to LPA was strongly inhibited by buffering the intracellular [Ca2+] (Cook et al., 1999). Calcium release from intracellular stores promotes calcium wave propagation into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors (Hardingham et al., 2001). Studies on the role of Ca2+/cAMP-dependent signal transduction

LPA-induced CREB phosphorylation increase in senescent HDFs141

and transcription factor CREB in SK-N-MC cells also demonstrated that the NPY Y(1) receptor induces the expression of CRE containing target genes through the CaM kinase-CREB pathway, and inhibits them when cellular cAMP levels are elevated in SK-N-MC cells (Sheriff et al., 2002). Wu et al., who focused on the kinetic contributions of various signaling pathways to the overall time course of CREB phosphorylation, reported that phosphorylated CREB formation was dominated by the CaMK pathway up to 10 min, and by the MAPK pathway at 60 min (Wu et al., 2001). The two pathways acted in concert at 30 min. It was also demonstrated that PKC mediates the stimulation of CREB-dependent gene expression through p38MAPK pathway upon membrane disruption (Togo, 2004). Previously, we reported that LPA-triggered cytosolic Ca2+ signals were partially reduced but conserved during the aging process (Yeo et al., 2002). Ca2+-dependent PKC / are activated with different time courses both in young and senescent HDFs (data in press). We now find that inhibition of PKC with bisindolylmaileimide efficiently blocked LPA-induced CRE-luciferase activity in both young and senescent HDFs (Figure 6). Activation of Ca2+ signaling in senescent cells will initially affect Ca2+-dependent protein kinases such as PKC/

and CaMK, and will in turn interact with other protein kinases such as ERK1/2, p38MAPK and MSK1 as well as PKA for the expression of CRE-regulated genes. The c-fos promoter contains several regulatory cis-elements, namely a serum response element (SRE) and AP-1-like site, in addition to a CRE. These promoter elements are interdependent and thus connected by protein complexes (Robertson et al., 1995). SRF with TCF phosphorylation is required for SRE-regulated c-fos induction (Treisman, 1995) and CREB and ATF1 phosphorylation and subsequent recruitment of the co-activator CREB binding protein (CBP) are required for CRE-regulated c-fos induction. The above mentioned protein kinases are also involved directly or indirectly in TCF phosphorylation for SRE activation, and ATF1 phosphorylation for CRE activation in addition to CREB phosphorylation. COX-2 was shown to be induced by LPA in the several cell systems (Ershov and Bazan, 1999; Hahn et al., 2002; Symowicz et al., 2005). We also observed LPA-induced COX-2 induction especially in young HDFs. The COX-2 induction mechanism is also complicated by the promoter with several regulatory elements in addition to CRE, which include nuclear factor kappaB (NF-B), CCAAT/

enhancer-binding protein (C/EBP), NF-IL-6, Pea-1, myb, xenobiotic-response element, Pea-3/SP-1 (Gen-

Bank #D28235). LPS was shown to induce activation of JNK-mediated AP-1 and ERK/p38 kinase-mediated CREB or C/EBPdelta, which bind the core elements of COX-2 promoter (Uto et al., 2005). UVB induces COX-2 expression via activations of p38MAPK and JNK, and thus DNA binding activity of AP-1 transcription factor in human keratinocytes HaCaT cells (Cho et al., 2005). It was suggested that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-B activation, because of a greater I B degradation in old macrophages (Wu et al., 2003). Aging can also modulate differentially these upstream kinases and transcription factors after LPA treatment, which might explain the discrepancy between LPA-induced CREB phosphorylation and COX-2 gene expression in the two age groups of HDFs.

Taken together, our study found a senescence-associated increase of cAMP after LPA treatment, which correlates well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. However, CREB phosphorylation does not correlate well with the CRE-responsive gene expression. Most of CRE-responsive genes such as c-fos and COX-2 contain a complex mix of promoters which interact with a variety of transcription factors in addition to CREB. These transcription factors could be modulated by various upstream protein kinases whose activities are also altered during aging. Further studies are needed to clarify the influence of age-related alteration of cAMP signaling at the transcriptional level.

Acknowledgement

This work was supported by grants from the Korea Science and Engineering Foundation (No. R04-2000-000-00041-0), and the Aging and Apoptosis Research Center (RII-2002-097-05001-0 and RII-2002-097-06003-0). We express our gratitude to Dr. Sri Ram for providing us with many valuable discussions and correcting the manuscript.

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