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Role of cyclins in cAMP inhibition of glomerular mesangial cell


									                                                                                   Clinical Science (2004) 107, 81–87 (Printed in Great Britain)   81

Role of cyclins in cAMP inhibition of glomerular
                     mesangial cell proliferation

Chiharu ITO∗ , Hisashi YAMAMOTO∗ , Yusuke FURUKAWA†, Shin-ichi TAKEDA∗ ,
Tetsu AKIMOTO∗ , Osamu IIMURA∗ , Yasuhiro ANDO∗ , Yasushi ASANO∗
and Eiji KUSANO∗
 Division of Nephrology, Department of Internal Medicine, Jichi Medical School, Tochigi, Japan, and †Division of Molecular
Hematopoiesis, Department of Internal Medicine, Jichi Medical School, Tochigi, Japan

A      B     S     T     R     A      C     T

           MC (mesangial cell) proliferation is closely linked to the progression of glomerular disease. It
           has been reported that cAMP effectors suppress MC proliferation, inhibiting activation of MAPK
           (mitogen-activated protein kinase). In fibroblasts, activation of MAPK induces the expression of
           type D cyclin, whereas, in MCs, this induction has not been shown. In the present study, we
           explored the effects of cAMP on MAPK and expression of cell-cycle-regulated proteins. PDGF
           (platelet-derived growth factor) stimulated MAPK activity, up-regulated protein levels of cyclin D1,
           CDK2 (cyclin-dependent kinase 2) and PCNA (proliferating cell nuclear antigen), decreased the
           protein level of p27 and increased DNA synthesis. Fsk (forskolin) or PD98059 suppressed PDGF-
           induced DNA synthesis. Both agents inhibited PDGF-stimulated mRNA and protein expression
           of cyclin D1 and CDK2. Fsk or PD98059 also inhibited protein expression of PCNA and blocked
           a decrease in p27 protein. Fsk induced the phosphorylation of Raf-1 at Ser259 , which was
           inhibited by KT5720. These data suggest that cAMP inhibits MC proliferation through inhibition
           of MAPK activity, and this mechanism partly involves alteration in the levels of cell-cycle-regulated

INTRODUCTION                                                            MAPK (mitogen-activated protein kinase), in particu-
                                                                     lar the p42/p44 MAPK pathway, affects MC proliferation
MC (mesangial cell) proliferation is one of the character-           closely [6]. Multiple factors can activate MAPKs [7,8],
istics of progressive glomerular disease. MC proliferation           especially PDGF [9,10] in MCs. The relationship between
is affected by many factors, including growth factors.               MAPK cascade and expression of cell-cycle-regulated
One of these, PDGF (platelet-derived growth factor), is              proteins, such as cyclins or CDKs (cyclin-dependent
a major potent mitogen for MCs [1].                                  kinases), still remains unclear. Several studies have shown
   Previous studies performed by several groups have                 that activation of MAPK leads to cyclin D1 expression
shown that an increase in intracellular cAMP caused by               [11–13].
prostaglandin E2 [2], adrenomedullin [3], high glucose                  D-type cyclins are involved in controlling G1 phase
[4] or phosphodiesterase inhibitors [5] suppresses MC                progression and entry into S-phase. Increased cyclin
proliferation.                                                       D1 protein expression is required for MC proliferation

Key words: cyclin D1, cyclin-dependent kinase 2 (CDK2), forskolin, mitogen-activated protein kinase (MAPK), mesangial cell,
Abbreviations: BrdUrd, bromodeoxyuridine; CDK2, cyclin-dependent kinase 2; FBS, fetal bovine serum; Fsk, forskolin; MC,
mesangial cell; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular-signal-regulated kinase kinase; PCNA,
proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; PKA, cAMP-dependent
protein kinase; VSMC, vascular smooth muscle cell.
Correspondence: Dr Chiharu Ito (e-mail

                                                                                                                C   2004 The Biochemical Society
82   C. Ito and others

     [14]. However, some other proteins, such as CDKs,              kit for detecting BrdUrd incorporation by ELISA
     which control the cell cycle, are also required to induce      (Cell Proliferation ELISA BrdU calorimetric kit; Roche
     mitogenesis.                                                   Diagnostics, Tokyo, Japan). Briefly, cells in RPMI 1640
        In the present study, we have examined the effects of       and 20 % FBS were plated at 5 × 103 cells on 96-well
     cAMP on MAPK activity and expression of cell-cycle-            plates and incubated for 72 h. After serum-starvation
     regulated proteins in rat MCs in an attempt to investigate     for 48 h in RPMI 1640, cells were incubated for 30 min
     the critical interaction between the antimitogenic cAMP/       in RPMI 1640 containing agents, such as Fsk, prior
     PKA (cAMP-dependent protein kinase) pathway, MAPK              to stimulation with PDGF. Control cells were also in-
     pathway and cell cycle.                                        cubated for 30 min with RPMI 1640 containing DMSO
        Part of this work was presented at the 35th Annual          vehicle. The medium was then replaced with fresh me-
     Meeting of the American Society of Nephrology held in          dium, and the cells were stimulated with PDGF in the
     Philadelphia, PA, U.S.A., in November 2002, and sub-           absence or presence of the agents for 36 h. BrdUrd
     sequently published in abstract form [14a].                    (10 µM) was then added to the medium, and the cells
                                                                    were incubated further for 12 h within the last incub-
                                                                    ation period. The incorporation of BrdUrd was then
                                                                    terminated and quantified according to the manufac-
                                                                    turer’s instructions.
     The p44/42 MAPK assay kit, including anti-p44/42
     MAPK and anti-phospho-p44/42 MAPK (Thr202 /Tyr204 )
     antibodies, MEK1/2 (MAPK/ ERK kinase, where ERK is             Determination of intracellular cAMP
     extracellular-signal-regulated kinase) kinase assay kit, in-   and protein content
     cluding anti-MEK1/2 and anti-phospho-MEK1/2 (Ser217 /          cAMP was measured by enzyme immunoassay, as des-
     Ser221 ) antibodies, and anti-phospho-Raf-1 (Ser259 ) were     cribed previously [16]. Briefly, after serum deprivation
     purchased from Cell Signaling Technology (Beverly,             for 24 h, cells were stimulated with medium containing
     MA, U.S.A.). Anti-Raf-1 antibody and anti-cyclin D1,           Fsk. Medium was aspirated and cells were immediately
     cyclin E, CDK2, CDK4 and p27Kip1 antibodies were               immersed in 0.2 ml of 0.1 M HCl to stop the reaction.
     purchased from Santa Cruz Biotechnology (Santa Cruz,           Cells were then collected into glass tubes and boiled
     CA, U.S.A.). An antibody against PCNA (proliferating           for 3 min. After 15 min of centrifugation at 2500 g, the
     cell nuclear antigen) was obtained from DAKO (Kyoto,           supernatant was decanted and 50 µl of 50 mM sodium
     Japan). Fsk (forskolin) was dissolved in DMSO at a             acetate was added for the assay of cyclic nucleotides. The
     concentration of 10 mM. KT5720, a potent specific cell-         pellet was used for protein measurements. cAMP content
     permeant inhibitor of PKA, was purchased from Alexis           of the supernatant was assayed by enzyme immunoassay
     Biochemicals (San Diego, CA, U.S.A.) and dissolved             (Amersham Biosciences, Tokyo, Japan).
     in DMSO. All other materials, including PD98059, a
     selective and cell-permeant inhibitor of MEK inhibitor,
     were purchased from Sigma (St. Louis, MO, U.S.A.)              Western blotting
     unless otherwise stated.                                       Cells lysates were prepared as described previously [17].
                                                                    The supernatants were then subjected to an immune
     MC culture                                                     complex protein kinase assay or total cell lysate immuno-
     Glomerular MC preparations and primary MC cultures
                                                                    blotting. After incubation with the primary antibody,
     were prepared from rat kidneys of male Sprague–
                                                                    membranes were probed with a secondary antibody, fol-
     Dawley rats (200 g) with standard sieving methods, as
                                                                    lowed by detection using ECL (enhanced chemilumin-
     reported previously [15]. Cells were grown in RPMI
                                                                    escence) Western blotting detection system (Amersham
     1640 medium supplemented with 20 % FBS (fetal bovine
                                                                    Biosciences). The intensities of the bands were quantified
     serum), 100 units/ml penicillin, 100 mg/ml streptomy-
                                                                    using a GT-9000 scanner (Epson, Tokyo, Japan), and
     cin, 10 µg/ml insulin, 5.5 µg/ml transferrin and 6.7 ng/
                                                                    densitometric analysis was performed using NIH Image
     ml sodium selenite (Life Technology, Carlsbad, CA,
                                                                    1.62 software. The data were obtained from three or four
     U.S.A.). In each experiment, cells were plated at 5 ×
     105 cells in 100-mm diameter dishes (Falcon, Franklin
     Lakes, NJ, U.S.A.). They were made ‘quiescent’ by in-
     cubation for 48 h in RPMI 1640 medium in the absence
     of serum.                                                      Statistics
                                                                    The results are expressed as means + S.E.M. Data were
     Measurement of DNA synthesis                                   analysed by ANOVA combined with Fisher’s pro-
     Incorporation of the thymidine analogue BrdUrd (bro-           tected least-significant difference test. Differences with
     modeoxyuridine) was measured using a commercial                P < 0.05 were considered to be significant.

     C   2004 The Biochemical Society
                                                                                                                                                 Mesangial proliferation and cAMP       83

                                                                                          55kD                                                                             p-p44 MAPK
                         1.6                                                                                                                                               p-p42 MAPK

           450nm/650nm                                                                    36kD
                         1.2          #
                                                                                   B      55kD
                                                                                                                                                                           p44 MAPK

                         0.8                                                                                                                                               p42 MAPK
                                               *                                          36kD
                         0.4                                                      C       55kD
                               (-)   (+)      (+)     (+)     (+)      (-)                36kD
PDGF    (10 ng/ml)
Fsk     (µM)                   (-)   (-)       1      10      (-)      (-)        D       55kD
                               (-)   (-)      (-)     (-)     (+)      (-)                                                                                                 MEK
PD98059 (30 µM)
FBS     (20 %)                 (-)   (-)      (-)     (-)     (-)      (+)
                                                                                  E       55kD
Figure 1 Effect of cAMP on PDGF-induced DNA synthesis in
rat MCs                                                                           PDGF    (10 ng/ml) (-)                           (+)     (+)           (+)
                                                                                  Fsk     (10 µM)    (-)                           (-)     (+)           (-)
Subconfluent MCs were rendered quiescent by serum deprivation for 48 h. Cells
                                                                                  PD98059 (30 µM)    (-)                           (-)     (-)           (+)
were pretreated in the absence or presence of Fsk or PD98059 for 30 min
before being stimulated with PDGF for 36 h. PDGF was added in the absence or                         F

                                                                                                     Arbitrary unit (%)
presence of Fsk or PD98059. As a positive control, FBS was added to MCs. BrdUrd
was added for the last 12 h. Incorporation of BrdUrd was measured by ELISA,                                                                      #
as described in the Materials and methods section. Values are means + S.E.M. of
                                                                    −                                                      50                                          #
eight wells. Data are representative of three separate experiments. #P < 0.01                                                                            #     #

compared with control (no treatment), ∗ P < 0.05 and ∗∗ P < 0.01 compared
with PDGF alone.                                                                                                                * * *
                                                                                  PDGF    (10 ng/ml)                            (-)      (+)     (+)               (+)
                                                                                                                                (-)      (-)     (+)               (-)
                                                                                  Fsk     (10 µM)
RESULTS                                                                           PD98059 (30 µM)
                                                                                                                                (-)      (-)     (-)               (+)

Effect of cAMP on PDGF-induced
                                                                                  Figure 2 Effect of cAMP on MAPK pathway
DNA synthesis                                                                     Subconfluent MCs were rendered quiescent by serum deprivation for 24 h. Cells
As shown in Figure 1, addition of PDGF to the culture
                                                                                  were pretreated in the absence or presence of Fsk or PD98059 for 30 min before
medium resulted in an increase in the amount of DNA
                                                                                  being stimulated with PDGF for 5 min. PDGF was added in the absence or presence
synthesis. Incubation with Fsk (10 µM) and PD98059
                                                                                  of Fsk or PD98059. Equal protein quantities of cell lysates (20–30 µg) were
(30 µM), a MEK inhibitor, suppressed the increase in the
                                                                                  electrophoresed and immunoblotted with phospho-MAPK (A), MAPK (B), phospho-
amount of DNA synthesis induced by PDGF. This effect
                                                                                  MEK (C) and MEK (D) antibodies. An immune complex protein kinase assay was
of Fsk was dose-dependent (Figure 1).
                                                                                  performed with a phospho-Elk-1 (E) antibody. A representative experiment of three
                                                                                  separate experiments is shown. (F) Quantification of the phosphorylation of p42/
Changes of intracellular cAMP                                                     p44 MAPK, MEK and Elk-1. Values are expressed as the relative activity of
content by Fsk                                                                    phosphorylated p42/44 MAPK (open bars), MEK (hatched bars) or Elk-1 (stippled
Fsk (10 µM) increased intracellular cAMP levels (con-                             bars) induced by PDGF. Values are means + S.E.M. of three separate experiments.
trol, 0.2 + 0.10; 5 min, 13.6 + 0.74; 15 min, 6.0 + 0.24;
          −                   −                   −                               #P < 0.05 and ∗ P < 0.01 compared with PDGF alone.
30 min, 0.4 + 0.06 pmol/mg of protein; n = 4). PDGF did
not affect the level of intracellular cAMP (results not                           time-dependent manner. However, changes in cyclin E
shown).                                                                           (Figures 3B and 3F) and CDK4 (Figures 3D and 3H)
                                                                                  protein levels by PDGF were minor or not affected.
Effect of cAMP on MAPK pathway                                                       Both Fsk and PD98059 attenuated the induction of
We then explored the effects of Fsk and PD98059 on the                            cyclin D1 (Figures 4A and 4G) and CDK2 (Figures 4C
p42/p44 MAPK pathway. Both agents decreased MEK                                   and 4I) proteins by PDGF, whereas both inhibitors did
phosphorylation (Figures 2A and 2F), MAPK phosphor-                               not affect the expression of cyclin E (Figures 4B and
ylation (Figures 2C and 2F) and activation (Figures 2E                            4H) and CDK4 (Figures 4D and 4J) proteins. However,
and 2F) induced by PDGF.                                                          PDGF decreased p27 protein expression, whereas Fsk
                                                                                  blocked this suppression (Figures 4E and 4K). Addition
Expression of cell-cycle-regulated proteins                                       of PDGF resulted in an increase in PCNA expression,
PDGF increased the expression of cyclin D1 (Figures 3A                            which was decreased by Fsk and PD98059 (Figures 4F
and 3E) and CDK2 (Figures 3C and 3G) proteins in a                                and 4L).

                                                                                                                                                 C       2004 The Biochemical Society
84   C. Ito and others

                 A                                                                                              E                          100

                                                                                                                     Arbitrary unit (%)

                                                                                         cyclin D1                                                        50                                                              #
                         36kD                                                                                                                                                               *
                 B                                                                                                                                                                 0


                                                                                                                      Arbitrary unit (%)
                         50kD                                                             cyclin E

                 C                                                                                                                                                                 0

                         36kD                                                              CDK2
                                                                                                                G                          100

                                                                                                                     Arbitrary unit (%)
                 D                                                                                                                                   50
                         36kD                                                             CDK4                                                                                     0


                                                                                                                     Arbitrary unit (%)
                 PDGF (10 ng/ml)                                                                                H
                                              0       12       24       36
                 time (hr)                                                                                                                 100

                                                                                                   PDGF (10 ng/ml)                                                                          0         12       24         36
                                                                                                   time (hr)

     Figure 3 Effect of PDGF on expression of cell-cycle-regulated proteins
     Subconfluent MCs were rendered quiescent by serum deprivation for 48 h. Cells were then stimulated with PDGF. Equal protein quantities of cell lysates (20–30 µg)
     were electrophoresed and immunoblotted with cyclin D1 (A), cyclin E (B), CDK2 (C) and CDK4 (D) antibodies. A representative experiment of three or four separate
     experiments is shown. (E–H) Quantification of the protein levels of cell-cycle-regulated proteins. The band density of cyclin D1 (E), cyclin E (F), CDK2 (G) and CDK4
     (H) was determined and expressed relative to the band at 24 h. Values are means + S.E.M. of three or four separate experiments. #P < 0.05 and ∗ P < 0.01
     compared with the value at 24 h.

                                                                                                                                           Arbitrary unit (%)

                         A                                                                                                                                                         100
                                                                                       cyclin D1                                                                                       50
                              36kD                                                                                                                                                                *

                                                                                                                    H                                                              150
                                                                                                                                           Arbitrary unit (%) Arbitrary unit (%)

                         B                                                                                                                                                         100
                              50kD                                                     cyclin E                                                                                        50
                         C                                                             CDK2                            I                                                           100
                              36kD                                                                                                                                                     50                           *     *
                         D    36kD                                                                                   J                                                             150
                                                                                                                                           Arbitrary unit (%) Arbitrary unit (%)


                          E 29kD                                                                                                                                                       0
                              21kD                                                                                  K                                                              100                                        #
                                                                                                                                                                                       50                           #

                                                                                                                                                                                        0                  *
                              36kD                                                     PCNA
                                                                                                                                                Arbitrary unit (%)

                                                                                                                    L                                                              100
                                                                                                                                                                                                  *                       *
                  PDGF    (10 ng/ml)           (-)      (+)     (+)      (+)                                                                                                            0
                  Fsk     (10 µM)              (-)      (-)     (+)      (-)
                  PD98059 (30 µM)              (-)      (-)     (-)      (+)                      PDGF    (10 ng/ml)                                                                            (-)    (+)     (+)      (+)
                                                                                                  Fsk     (10 µM)                                                                               (-)    (-)     (+)      (-)
                                                                                                  PD98059 (30 µM)                                                                               (-)    (-)     (-)      (+)

     Figure 4 Effect of cAMP on PDGF-induced expression of cell-cycle-regulated proteins
     Subconfluent MCs were rendered quiescent by serum deprivation for 48 h. Cells were pretreated in the absence or presence of Fsk or PD98059 for 30 min before
     being stimulated with PDGF for 24 h. PDGF was added with or without Fsk or PD98059. Equal protein quantities of cell lysates (20–30 µg) were electrophoresed
     and immunoblotted with cyclin D1 (A), cyclin E (B), CDK2 (C), CDK4 (D), p27 (E) and PCNA (F). A representative experiment of three or four separate experiments is
     shown. (G–L) Quantification of the protein levels of cell-cycle-regulated proteins. Band density of cyclin D1 (G), cyclin E (H), CDK2 (I), CDK 4 (J) and PCNA (L) was
     determined and expressed relative to PDGF alone. Band density of p27 (K) was determined and expressed relative to control. Values are means + S.E.M. of three or
     four separate experiments. In (G), (I) and (L), ∗ P < 0.05 compared with PDGF alone. ∗ P < 0.05 compared with control, #P < 0.05 versus PDGF alone in (K).

     C   2004 The Biochemical Society
                                                                                                                                         Mesangial proliferation and cAMP   85

          A                                                                                           sistent with previous studies reporting that the cAMP/
                                                                                                      PKA pathway can interfere with the stimulation of
                                                                                                      MAPK pathway in MCs [3,5,21]. Although the precise
KT5720 (1 µM) (-)                          (-)   (-)   (-)   (+)      (+)       (+)   (+)             mechanism of this inhibition is still unclear, we [13] and
Fsk    (10 µM) (+)                         (+)   (+)   (+)   (+)      (+)       (+)   (+)
time   (min)    0                           5    15    30     0        5        15    30              others [22] have reported recently the phosphorylation
                                                                                                      of Ser259 on Raf-1 by cAMP, which is known to be a
                B                        100                                                          negative regulator of Raf-1 activity. Ser259 has also been
                    Arbitrary unit (%)

                                                              *                                       described as a target site of Raf-1 inhibition mediated by
                                                       *                                              Akt/PKB (protein kinase B) [23], although this cross-
                                                 #                #
                                                                            #                         talk in MCs is obscure. It has been reported that Ser43
                                           0                                                          and Ser621 are target sites for PKA, which inhibit Raf-1
              time (min)                         0     5     15        30
                                                                                                      activity [24,25]. Further investigations are necessary to
                                                                                                      determine the effects of cAMP on phosphorylation or
Figure 5 Effect of cAMP on Raf-1 (Ser259 ) phosphorylation                                            dephosphorylation mechanisms of these residues. It is
Subconfluent MCs were rendered quiescent by serum deprivation for 24 h. Cells                          crucial to determine whether the PKA catalytic subunit
were stimulated with Fsk in the absence or presence of pretreatment with KT5720                       affects phosphorylation of these residues.
for 30 min. Equal protein quantities of cell lysates (20–30 µg) were electro-                             One of the biological effects of MAPK is cell pro-
phoresed and immunoblotted with an phospho-Raf-1 antibody (A). A representative                       liferation, but the relationship between MAPK and mito-
experiment of three separate experiments is shown. (B) Quantification of the                           genesis or cell division is still unclear. Upon activation
phosphorylation of Raf-1. Band density was determined and expressed relative                          in a fibroblast cell line, MAPK translocates to nucleus
to 30 min of Fsk alone. Pretreatment with vehicle ( ) or KT5720 ( ). Values                           where it phosphorylates transcription factors and induces
are means + S.E.M. of three separate experiments. ∗ P < 0.01 compared with
           −                                                                                          early gene translocation [26]. Activation of the MAPK
30 min, #P < 0.05 compared with vehicle.                                                              pathway at the G0 /G1 transition has been shown to in-
                                                                                                      duce expression of cyclin D1 protein [12]. In the present
Phosphorylation of Raf-1 (Ser259 )                                                                    study, we observed that PDGF induced cyclin D1 protein
We have reported previously [13] the phosphorylation                                                  expression, which can be inhibited by both Fsk and
of Raf-1 (Ser259 ) by Fsk in rat VSMCs (vascular smooth                                               PD98059. There are few reports investigating the ef-
muscle cells). Therefore we explored whether Fsk can                                                  fects of MAPK on other cell-cycle-regulated proteins.
induce Raf-1 phosphorylation in MCs. As shown in                                                      However, others groups have revealed that cAMP modu-
Figure 5, Fsk phosphorylated Raf-1 (Ser259 ), and this                                                lates expression of these factors and inhibits cell growth,
phosphorylation was inhibited by KT5720.                                                              especially in VSMCs [27,28]. The cell cycle molecules
                                                                                                      induced by cAMP in MCs vary from those in VSMCs
                                                                                                      [28]. This dissociation may occur not only due to a dif-
DISCUSSION                                                                                            ference in cell types, but also in culture conditions, such
                                                                                                      as variations in culture media.
In the present study, we have shown that cAMP inhibits                                                    In the present study, we used PD98059 as a well-known
MC proliferation induced by PDGF by attenuating the                                                   MEK inhibitor. PD98059 can inhibit both activation and
MAPK cascade and modulating the expression of some                                                    phosphorylation of MEK by c-Raf. The concentration of
cell-cycle-regulated proteins.                                                                        PD98059 used in the present study (30 µM) is sufficient
   MAPK is an important mediator that is involved in the                                              to block the MAPK pathway, as shown by the inhibitory
intracellular network of interacting proteins that trans-                                             effect of PD98059 on MEK phosphorylation (Figure 2D).
duce extracellular stimuli to intracellular responses.                                                PD98059 itself has no effect on the kinase activity of
PDGF is a potent stimulator of MAPK in MCs, and the                                                   PKA [29], but the possibility that PD98059 inhibits other
MAPK cascade is a central pathogenic pathway in experi-                                               protein kinases at the concentration used cannot be ruled
mental mesangioproliferative glomerulonephritis [18,19].                                              out.
In glomeruli of rats with diabetes mellitus, a surfeit of                                                 Recently Lin et al. [30] have shown that cAMP can
MAPK activation has also been confirmed [20]. Although                                                 suppress MC growth by inhibiting the PI3K (phospho-
the role of the MAPK cascade in glomeruli is unclear,                                                 inositide 3-kinase)/Akt signalling pathway. It is specu-
excessive MAPK activation in glomeruli-composed cells                                                 lated that (i) cAMP inhibits Raf-1/ERK and PI3K/Akt
seems to be the main pathological trigger. It is tempting                                             pathways, both of whose downstream targets are pre-
to speculate that the effect of MAPK inhibition on MC                                                 requisites for the induction of cell-cycle-regulating pro-
proliferation might be attributable, in part, to the inhi-                                            teins, such as cyclin D1; or (ii) cAMP suppresses another
bition of PDGF-induced intracellular signalling events                                                pathway, other than Raf-1/ERK and PI3K/Akt, which
in vivo.                                                                                              is more critical for cell growth. Considering the data
   In the present study, we have demonstrated the inhib-                                              generated by many groups studying the interaction be-
itory effects of cAMP effectors on MC mitogenesis, con-                                               tween the cAMP/PKA pathway and growth-related

                                                                                                                                         C   2004 The Biochemical Society
86   C. Ito and others

     pathway, the former one predominates. To explore the                   7 Mondorf, U. F., Piiper, A., Herrero, M., Bender, M.,
     possibility of the latter, gene chip analysis will be helpful            Scheuermann, E. H. and Geiger, H. (1998) Lipoprotein (a)
                                                                              stimulates mitogen activated protein kinase in human
     in documenting the differences between gene expression                   mesangial cells. FEBS Lett. 441, 205–208
     patterns with or without cAMP effectors. In any case,                  8 Kawata, Y., Mizukami, Y., Fujii, Z., Sakumura, T.,
     comprehensive studies to examine the effect of cAMP on                   Yoshida, K. and Matsuzaki, M. (1998) Applied pressure
                                                                              enhances cell proliferation through mitogen-activated
     cell biological function will be necessary in in vitro and               activation in mesangial cells. J. Biol. Chem. 273,
     in vivo studies.                                                         16905–16912
        In vitro MCs or experimental mesangial proliferative                9 Huwiler, A., Stabel, S., Fabbro, D. and Pfleilschifter, J.
                                                                              (1995) Platelet-derived growth factor and angiotensin II
     models have shown that CDK2 and PCNA protein levels                      stimulate the mitogen-activated protein kinase cascade in
     are increased and p27 protein level is decreased during                  renal mesangial cells: comparison of hypertrophic and
                                                                              hyperplastic agonists. Biochem. J. 305, 777–784
     the proliferative phase [14,31,32]. The results of the pres-
                                                                           10 Choudhury, G. G., Kamitsos, C. and Hernandez, J.,
     ent in vitro study are in agreement with these reports.                  Gentilini, A., Bardgette, J. and Abboud, A. H. (1997)
     The alteration of cyclin E protein expression in a time-                 PI-3-kinase and MAPK regulate mesangial cell
                                                                              proliferation and migration in response to PDGF.
     dependent manner is consistent with the data from a
                                                                              Am. J. Physiol. 273, F931–F938
     previous report [33]. The difference in changes of CDK4               11 Weber, J. D., Raben, D. M., Phillips, P. J. and Baldassare,
     protein may depend on culture condition or the dose of                   J. J. (1997) Sustained activation of extracellular-signal-
                                                                              regulated kinase 1 (ERK1) is required for the continued
     PDGF used.                                                               expression of cyclin D1 in G1 phase. Biochem. J. 326,
        In conclusion, we have demonstrated that cAMP can                     61–68
     suppress MC proliferation, in part, by inhibiting the                 12 Terada, Y., Inoshita, S., Nakashima, O., Kuwahara, M.,
                                                                              Sasaki, S. and Marumo, F. (1999) Regulation of cyclin D1
     MAPK cascade. This inhibition may correlate with                         expression and cell cycle progression by mitogen-
     the suppression of cell-cycle-regulated protein expres-                  activated protein kinase cascade. Kidney Int. 56,
     sion.                                                                    1258–1261
                                                                           13 Ito, C., Kusano, E., Furukawa, Y. et al. (2002) Modulation
                                                                              of the erythropoietin-induced proliferative pathway by
                                                                              cAMP in vascular smooth muscle cells. Am. J. Physiol.
                                                                              Cell Physiol. 283, C1715–C1721
     ACKNOWLEDGMENTS                                                                                                         ¨
                                                                           14 Lang, S., Hartner, A., Sterzel, R. B. and Schocklmann,
                                                                              H. O. (2000) Requirement of cyclin D1 in mesangial cell
                                                                              mitogenesis. J. Am. Soc. Nephrol. 11, 1398–1408
     We thank Mrs Yuko Watanabe for her technical assistance.             14a Ito, C., Yamamoto, H., Furukawa, Y. et al. (2002) Cyclic
     This study was supported in part by a Grants-in-Aid                      AMP inhibits PDGF-induced mesangial cell proliferation
                                                                              through negative expression of cyclin D1 and Cdk2.
     13671125 for scientific research from the Ministry of                     J. Am. Soc. Nephrol. 13, F-P0157
     Education, Science and Culture, Japan, and by grants                  15 Akai, Y., Kusano, E., Amemiya, M. et al. (1996) PMA and
     from Jinseihinketsu-kenkyuukai and Jin-kenkyuukai.                       ionomycin differently affect atrial natriuretic peptide
                                                                              stimulated cyclic GMP production in rat mesangial cells.
                                                                              Tohoku J. Expe. Med. 178, 137–149
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                                                       Received 13 October 2003/23 February 2004; accepted 2 March 2004
                                                       Published as Immediate Publication 2 March 2004, DOI 10.1042/CS20030335

                                                                                                       C   2004 The Biochemical Society

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