Cutting edge: Calcium/Calmodulin-dependent protein kinase type IV is essential for mesangial cell proliferation and lupus nephritis.
ABSTRACT Renal involvement in systemic lupus erythematosus remains a major cause of morbidity and mortality. Although immune parameters that instigate renal damage have been characterized, their link to local processes, which execute tissue damage, is poorly understood. Using genetic-deletion and pharmacological-inhibition approaches, we demonstrated that calcium/calmodulin-dependent protein kinase type IV, which contributes to altered cytokine production in systemic lupus erythematosus patients, controls spontaneous and platelet-derived growth factor-stimulated mesangial cell proliferation and promotes IL-6 production through AP-1. Our studies identified calcium/calmodulin-dependent protein kinase type IV as a valuable treatment target for lupus nephritis and point out the importance of local kidney factors in the expression of tissue damage that, if properly targeted, should enhance clinical benefit and limit toxicity.
- SourceAvailable from: sciencedirect.com[show abstract] [hide abstract]
ABSTRACT: Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional protein kinase expressed abundantly in the central nervous system. Because changes in intracellular Ca(2+) concentrations affect progression through the mitotic cell cycle, enhanced expression of CaMKIV has been reported in small cell lung carcinoma and hepatocellular carcinoma. To elucidate the involvement of CaMKIV in epithelial ovarian carcinogenesis, we analyzed serial frozen sections for CaMKIV protein expression in 26 patients with ovarian epithelial carcinoma and ten patients with benign cystadenoma of the ovary by fluorescent immunohistochemistry. We analyzed the relationship between the percentages of CaMKIV-stained cells and the patient's characteristics, including histological classification, clinical stage, histological grade, and clinical outcome. In the benign ovarian cystadenoma, CaMKIV was detected in none of the cases examined. Most of the CaMKIV proteins were found in the nucleus of epithelial ovarian cancer tissue. CaMKIV expression was significantly associated with clinical stage (P<0.01), histological grade (P<0.01), and clinical outcome (P<0.01). Survival data were available for all patients, and univariate Cox regression analysis showed that CaMKIV expression was significantly associated with poor prognosis (P<0.05). Our results demonstrate that CaMKIV expression in epithelial ovarian cancer correlates with the malignant potential of this tumor.Cancer Letters 09/2002; 183(2):185-93. · 4.26 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from dysregulation of the immune system. Interleukin-6 (IL-6) is a multifunctional cytokine produced by macrophages, monocytes and T and B cells. It stimulates B-cell differentiation/maturation, immunoglobulin secretion, and T-cell functions. Elevated levels of IL-6 in serum, urine and renal glomeruli were detected in patients with active SLE and in murine models of SLE. Our study investigated the role of IL-6 in an SLE-like disease in New Zealand Black/White (NZB/W) F1 mice by administration of an anti-murine IL-6 monoclonal antibody (mAb). Intraperitoneal administration of the anti-IL-6 mAb suppressed the production of anti-dsDNA autoantibody. B-cell proliferation induced by anti-IgM and anti-CD40 was lower in the anti-IL-6 mAb-treated mice, ex vivo studies demonstrated that anti-IL-6 mAb treatment inhibited anti-dsDNA production. Anti-CD3-induced T-cell proliferation and mixed lymphocyte reactions were inhibited by anti-IL-6 mAb treatment, indicating a partial down-regulation of T cells. Histological analysis showed that treatment with anti-IL-6 mAb prevented the development of severe kidney disease. These results suggest that treatment with anti-IL-6 mAb has a beneficial effect on autoimmunity in murine SLE and that autoreactive B cells may be the primary target for anti-IL-6 mAb treatment; its effect on autoreactive T cells is also indicated.Immunology 12/2006; 119(3):296-305. · 3.71 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The therapeutic effects of a neutralizing monoclonal antibody (mAb) to the interleukin-6 receptor (IL-6R) were examined in the MRL-lpr/lpr murine lupus nephritis model. Animals (15 wk old) were treated with ip mAb IL-6R for 5 wk. GFR in these mice at the end of this treatment period were comparable to those of congenic strain disease-resistant MRL-(+)/+ controls treated with rat immunoglobulin G (IgG) (254 +/- 61 versus 285 +/- 26 microL/min; P = not significant). GFR was significantly (P < 0.05) lower in lpr/lpr mice receiving ip rat IgG (disease controls) at the same time (165 +/- 76 microL/min). The fractional mesangial volume (Mv) and surface density of open glomerular capillaries (Sv) in mAb II-6R-treated lpr/lpr and IgG-treated +/+ mice (Mv, 0.21 +/- 0.04 and 0.19 +/- 0.04 micron3/micron3; Sv, 0.18 +/- 0.01 and 0.20 +/- 0.01 micron/micron2, respectively) were similar. However, Mv (0.40 +/- 0.04) was significantly higher (P < 0.001) and Sv (0.13 +/- 0.04) was lower (P < 0.01) in IgG-treated lpr/lpr animals. Treatment with mAb IL-6R significantly reduced anti-dsDNA antibody levels after Week 2 of treatment, but these values rebounded at Week 4. The late development of neutralizing antibodies and the increased secretion of IL-6 at Week 4 were likely responsible. Despite these events, neutralizing mAb to the IL-6R proved to be effective therapeutically, as demonstrated by preserved glomerular function and structure.Journal of the American Society of Nephrology 07/1993; 4(1):58-61. · 8.99 Impact Factor
Cutting Edge: Calcium/Calmodulin-Dependent Protein
Kinase Type IV Is Essential for Mesangial Cell Proliferation
and Lupus Nephritis
Kunihiro Ichinose,* Thomas Rauen,*,†Yuang-Taung Juang,* Katalin Kis-Toth,*
Masayuki Mizui,* Tomohiro Koga,* and George C. Tsokos*
Renal involvement in systemic lupus erythematosus
remains a major cause of morbidity and mortality. Al-
though immune parameters that instigate renal damage
have been characterized, their link to local processes,
which execute tissue damage, is poorly understood.
Using genetic-deletion and pharmacological-inhibition
approaches, we demonstrated that calcium/calmodulin-
dependent protein kinase type IV, which contributes
to altered cytokine production in systemic lupus erythe-
matosus patients, controls spontaneous and platelet-
derived growth factor-stimulated mesangial cell proli-
feration and promotes IL-6 production through AP-1.
Our studies identified calcium/calmodulin-dependent
protein kinase type IV as a valuable treatment target
for lupus nephritis and point out the importance of
local kidney factors in the expression of tissue damage
that, if properly targeted, should enhance clinical benefit
and limit toxicity.
The Journal of Immunology, 2011,
gial cells (MCs) and was reported to orchestrate the cytokine
network of glomerular inflammation. There is clear evidence
that IL-6 is involved in mesangial proliferation and patho-
genesis of lupus nephritis (2–5). The expression of various
transcription factors, such as AP-1, CREB, and NF for IL-6,
was reported to control IL6 promoter activity (6–8). Among
them, AP-1 has been implicated in transcriptional regulation
of a wide range of genes participating in cell survival, prolif-
eration, and apoptosis (9–11). The multifunctional calcium/
calmodulin-dependent protein kinase type IV (CaMKIV)
belongs to a family of serine/threonine protein kinases that
regulates autoimmunity and cell proliferation (12–14). A
small molecule inhibitor of CaMKIV, KN-93 mitigates dis-
upus nephritis is still the major cause of morbidity
and mortality in patients with systemic lupus erythe-
matosus (SLE) (1). IL-6 can be produced by mesan-
ease development in lupus-prone mice by suppressing cyto-
kine production and costimulatory molecule expression in
lymphocytes (15). In this report, we provide evidence that
pharmacologic inhibition or genetic depletion of CaMKIV in
lupus-prone MRL/lpr mice results in decreased mesangial
IL-6 production, reduced MC proliferation, and less kidney
damage. Our data suggest a prominent role for CaMKIV in
the expression of systemic autoimmunity, as well as that of
local renal damage.
Materials and Methods
Female MRL/lpr, Camk4tm1Tch/J, and MRL/MPJ mice were purchased from
Jackson Laboratory. MRL/lpr.Camkiv2/2mice were generated on an MRL/
lpr background. Experiments were approved by the Institutional Animal Care
Committee of Beth Israel Deaconess Medical Center. Measurement of anti-
dsDNA Ab levels were performed, as described previously (15). Proteinuria
was measured in a semiquantitative manner, as described before (15). Briefly,
mice in each group (n = 4) were placed together overnight in a Nalgene
metabolic cage to collect urine. This procedure was repeated in two inde-
pendent experiments, so that the presented data display the average from
a total of eight mice/group. Kidneys from 16-wk-old mice were fixed in
formalin, paraffin sections were stained with periodic acid-Schiff, and renal
lesions were evaluated according to previously described criteria (16, 17).
Scoring was performed blindly by a nephropathologist.
Primary culture of MCs
Primary MCs were isolated according to Allam et al. (18), and purity of
isolated MCs was assessed by morphologic characteristics, positivity for
smooth muscle actin (.99%), and negativity for cytokeratin 18 (.99%).
Cultured MCs were used for experiments between passages three and seven.
MCs were plated in 12- or 6-well plates and serum starved for 24 h before
experiments were performed. Cells were treated with 20 ng/ml platelet-
derived growth factor (PDGF)-BB (PeproTech) for 24 h. As indicated, cells
were treated with KN-93 (20 mM) for 48 h prior to addition of PDGF-BB.
For RNA and protein analyses, EMSAs, and luciferase experiments, MCs
were pooled from five mice/group, and each experiment was performed in
two or three independent replicates.
Briefly, MCs were homogenized in radio immunoprecipitation assay buffer
at 4˚C for 30 min. After centrifugation (14,000 rpm; 30 min; 4˚C), super-
natants were collected. The following polyclonal rabbit Abs were used for
*Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, MA 02215; and†Department of Nephrology
and Clinical Immunology, University of Aachen, 52074 Aachen, Germany
Received for publication August 17, 2011. Accepted for publication September 27,
This work was supported by Public Health Service, National Institutes of Health Grant
RO1 AI 49954 (to G.C.T.).
Address correspondence and reprint requests to Dr. George C. Tsokos, Division of
Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Har-
vard Medical School, 330 Brookline Avenue, CLS 937, Boston, MA 02115. E-mail
The online version of this article contains supplemental material.
Abbreviations used in this article: CaMKIV, calcium/calmodulin-dependent protein
kinase type IV; MC, mesangial cell; PDGF, platelet-derived growth factor; SLE, systemic
immunoblotting: anti-CDK2, anti-cyclin D1, anti-CaMKIV (all from Cell
Signaling), anti–c-Jun (Santa Cruz), anti–histone-H3 (Abcam), and anti-actin
RNA extraction and PCR
Primary MCs were homogenized, total RNA was extracted using the RNeasy
Mini Kit (Qiagen), and cDNA was generated using the Reverse Transcription
kit (Promega). PCR primers were as follows: IL-6: 59-CCGGAGAGGA-
GACTTCACAG-39 (forward) and 59-CCAGTTTGGTAGCATCCATC-39
(reverse) and CaMKIV: 59-TCACATGGACACTGCTCAGA-39 (forward)
and 59-TGCATCTTTCTCCACCTCCT-39 (reverse). 18S rRNA primers were
reported previously (15).
A total of 400,000 primary MCs was plated on six-well plates and serum
starved for 24 h. Then, cells were pretreated with KN-93 (20 mM) for 48 h
before the addition of PDGF-BB (20 ng/ml; 24 h). IL-6 concentrations were
detected with a commercial ELISA kit (R&D Systems).
Cell cycle analyses
MCs were trypsinized, washed twice with PBS, fixed in cold 95% ethanol, and
stored at 4˚C until use. Before flow cytometric analysis, cell pellets were
washed and resuspended in a solution of RNase (0.5 mg/ml) in PBS and
incubated at 37˚C for 20 min. Then, propidium iodide (40 mg/ml) was
added for 30 min. Stained cells were analyzed on FACScan (BD Biosciences).
(C) from MRL/MPJ, MRL/lpr, and MRL/lpr.Camkiv2/2mice. D, Proteinuria was quantified weekly starting from 8 wk of age. Anti-dsDNA IgG Abs from 8-wk-
old (E) and 16-wk-old (F) mice of each group were detected by ELISA (n = 4–8 mice/group).
CaMKIV deficiency improves lupus pathology. Mean scores for glomerular injury (A), tubular damage (B), and perivascular lymphocyte infiltration
cytometry. A total of 400,000 MCs derived from MRL/MPJ and MRL/lpr mice was treated or not with KN-93 for 48 h and stimulated with or without PDGF-
BB for 24 h. Cumulative flow cytometry data (percentage of cells in each cell cycle phases) are shown for unstimulated (A) and PDGF-BB–stimulated (B) MCs.
Values are the mean 6 SD (n = 4–5 mice/group) of three independent replicates. C, A total of 400,000 MCs derived from MRL/MPJ, MRL/lpr, and MRL/lpr.
Camkiv2/2mice was stimulated or not with PDGF-BB for 24 h. Cumulative flow cytometry data (percentage of cells in each cell cycle phases) are shown. Values
represent mean 6 SD (n = 4–5 mice/group) performed in three independent replicates.
CaMKIV suppresses cell cycle arrest of lupus MCs. A and B, MCs were stained by propidium iodide, and cell cycle analysis was performed by flow
The Journal of Immunology 5501
Data were acquired using CellQuest software (BD Biosciences), $10,000
events were collected for each graph. Data analysis was performed with
FlowJo version 7.6.1 (Tree Star).
Mouse IL6 promoter luciferase plasmid (in pGL3-Basic vector; Invitrogen)
was kindly provided by Dr. David L. Allen (University of Colorado, Denver,
CO). Transient transfections were performed in primary MCs (seeded at 1 3
105cells/well) by using 1 mg reporter DNA, 10 ng pRTK plasmid/trans-
fection, and 2 ml Lipofectamine 2000 (InvivoGen). Twenty-four hours after
transfection, cells were incubated or not with PDGF-BB (20 ng/ml) for an-
other 24 h. Luciferase activities in the cell lysates were measured using the
Dual-Luciferase Reporter Assay System (Promega). Experiments were re-
peated at least four times. Values in the bar graphs are given as mean 6 S.D.
A total of 500,000 MCs was used for preparation of nuclear protein extracts,
as described before (21). A dsDNA probe harboring the AP-1 site (2327) of
the murine IL6 promoter (59-AGTGCTGAGTCACTTTTAAAG-39) was
radiolabeled with g-[32P]-ATP using a T4-polynucleotide kinase. EMSA
was performed, as described before, using 5 mg nuclear protein and 1 mg
poly(deoxyguanylic-deoxycytidylic) acid sodium salt per reaction (19). Un-
labeled AP-1 probe was used for competition assays in 50- and 100-fold molar
excess, as indicated.
Densitometries and statistical analyses
Densitometries of Western blot and PCR images were performed using
Image J software (National Institutes of Health). The Kruskal–Wallis test
was used to determine statistical significance (*p # 0.05, **p # 0.01).
Results and Discussion
In our efforts to prove the importance of CaMKIV in the
pathogenesis of SLE and lupus nephritis, we transferred the
Camkiv null locus into the lupus-prone mouse strain MRL/lpr
(20) (Supplemental Fig. 1A, 1B). Total numbers of spleno-
cytes, peripheral lymph node cells, and major B and T cell
compartments were not different between MRL/lpr and
MRL/lpr.Camkiv2/2mice (data not shown). However, at 16
wk of age, kidney damage was largely diminished in MRL/lpr.
Camkiv2/2mice compared with age-matched MRL/lpr mice
(Supplemental Fig. 1C). MRL/lpr.Camkiv2/2mice developed
significantly less glomerular, tubulointerstitial, and perivascular
MRL/MPJ and MRL/lpr mice was treated or not with KN-93 for 48 h and stimulated with or without PDGF-BB for 24 h. IL-6 mRNA expression was assessed by
PCR (A) and quantified by densitometry of agarose gels (B). C, IL-6 levels in the supernatants were measured by ELISA. D–F, A total of 400,000 MCs derived
from MRL/MPJ, MRL/lpr, and MRL/lpr.Camkiv2/2mice were stimulated or not with PDGF-BB for 24 h. IL-6 mRNA expression was assessed by PCR (D) and
quantified by densitometry of agarose gels (E). F, IL-6 levels in the supernatants were measured by ELISA. G, MCs from MRL/lpr and MRL/lpr.Camkiv2/2mice
were transfected with empty pGL3 luciferase vector, IL6 promoter construct (mIL6p-WT), or a mutated IL6 promoter construct with a disrupted AP-1 site
(mIL6p-AP1 mut); incubated or not with PDGF-BB for 24 h; and assayed for relative promoter activity (n = 4–5 mice/group; performed in three independent
experiments). *p # 0.05, **p # 0.01.
PDGF-BB–mediated IL6 gene transcription is inhibited in MCs from MRL/lpr.Camkiv2/2mice. A–C, A total of 400,000 MCs derived from
5502CUTTING EDGE: CaMKIV IN LUPUS NEPHRITIS
lesions than did MRL/lpr mice (Fig. 1A–C). As shown in
Fig. 1D, proteinuria was also robustly decreased in MRL/lpr.
Camkiv2/2mice. Furthermore, MRL/lpr.Camkiv2/2mice
displayed significantly lower serum titers of anti-dsDNA Abs
at 8 and 16 wk of age compared with MRL/lpr mice (Fig.
Mesangial proliferation is a hallmark of lupus nephritis.
Incubation of MCs from MRL/MPJ and MRL/lpr mice with
PDGF-BB for 24 h upregulated protein levels of cell cycle
regulators CDK-2 and cyclin D1; however, pretreatment with
CaMKIV inhibitor KN-93 suppressed these levels (Supple-
mental Fig. 2A–C). In line with this, these proteins were
downregulated in MRL/lpr.Camkiv2/2MCs in the presence or
absence of PDGF-BB stimulation (Supplemental Fig. 2D–F).
MCs from MRL/MPJ and MRL/lpr mice were G1syn-
chronized by serum starvation for 24 h and labeled with
propidium iodide, and DNA content was analyzed by flow
cytometry. The percentage of G1-synchronized MCs entering
the G2/M phase was higher in MRL/lpr mice compared with
MRL/MPJ mice (Fig. 2A). This may indicate that MCs from
MRL/lpr mice actively divide, even in the absence of exoge-
nous stimuli, suggesting increased proliferation abilities of
MRL/lpr MCs. Pretreatment with KN-93 clearly diminished
the percentage of MCs entering the G2/M phase, with a
concomitant increase in the percentage of MCs arrested in the
G0/G1phase in PDGF-BB–stimulated MCs from MRL/lpr
mice (Fig. 2B). In addition, KN-93 pretreatment reduced the
frequency of cells entering the S phase (Fig. 2B). Next, we
analyzed the cell cycle status of G1-synchronized MCs from
MRL/lpr.Camkiv2/2mice in the absence or presence of
PDGF-BB. A large percentage of MCs from the MRL/lpr.
Camkiv2/2mice was arrested at the G0/G1phase compared
with control MRL/lpr and MRL/MPJ mice (Fig. 2C). Inter-
estingly, PDGF-BB was not able to overcome the G0/G1
block imposed by the genetic deletion of CaMKIV. These
findings further supported the hypothesis that MCs from
MRL/lpr mice have an intrinsic ability to proliferate without
Mesangial IL-6 production was induced by PDGF-BB
stimulation and effectively suppressed by KN-93 treatment
in MRL/lpr MCs at mRNA and protein levels (Fig. 3A–C).
Similarly, MRL/lpr.Camkiv2/2MCs showed decreased IL-6
expression compared with MRL/MPJ and MRL/lpr MCs
upon stimulation with PDGF-BB (Fig. 3D–F). Additionally,
IL-6 mRNA expression in activated splenocytes was signifi-
cantly decreased in MRL/lpr.Camkiv2/2mice (Supplemental
After we demonstrated that IL-6 expression was diminished
in MRL/lpr.Camkiv2/2MCs, we sought to identify possible
transcriptional mechanisms underlying IL-6 expression in
response to PDGF-BB stimulation. IL6 promoter activity in
MRL/lpr.Camkiv2/2MCs was significantly decreased com-
pared with MRL/lpr MCs; this effect was even more pro-
nounced following stimulation with PDGF-BB. AP-1 has
been shown to be involved in IL6 gene transcription (21).
Site-directed mutagenesis of an AP-1 site within the murine
IL6 promoter (located 327 bp upstream of the first ATG)
limited its promoter activity significantly (Fig. 3G). Although
cytoplasmic protein levels of AP-1 member c-jun were in-
creased in MRL/lpr.Camkiv2/2MCs compared with MRL/
lpr MCs, it was decreased in the nuclei of these cells (Sup-
plemental Fig. 3B–D). DNA binding assays using nuclear
MC extracts and a synthetic double-stranded oligonucleotide
defining the AP-1 motif showed an increased binding of
nucleoprotein from MRL/lpr MCs stimulated with PDGF-
BB to this site, which was diminished when nucleoprotein
lysates from MRL/lpr.Camkiv2/2MCs were used (Supple-
mental Fig. 2E). The binding was specific because it was
competed out with the cold, unlabeled probe at a 10- and 50-
fold molar excess (Supplemental Fig. 2F).
Previously, CaMKIV was shown to contribute to decreased
IL-2 production in SLE T cells, and its inhibition in lupus-
prone MRL/lpr mice mitigates disease pathology by interfer-
ing with immune parameters (15, 19). Our studies in the
newly developed MRL/lpr.Camkiv2/2mice clearly demon-
strated that CaMKIV is important in the expression of both
autoimmunity and kidney pathology. They also reveal pro-
found effects on MC proliferation and production of IL-6,
which is known to be involved in the development of glo-
merulonephritis (5). It is undisputed that immune complexes,
autoantibodies, and autoreactive T cells are important in the
instigation of lupus nephritis (1), yet little emphasis has been
paid to the local kidney factors that eventually execute tissue
damage. Previously, it was shown that the kallikrein genes
contribute to lupus nephritis in mice and men and, therefore,
factors independent of the immune system are important in
the expression of kidney pathology (22). Along these lines,
a congenic mouse that was derived from the lupus-prone
NZM2328 mouse develops severe glomerulonephritis with-
out breaking tolerance to nuclear Ags (23). In this study, we
showed that MCs from MRL/lpr mice are able to proliferate
in vitro in the absence of exogenous stimuli and that this
proliferation, along with the increased IL-6 production, is
significantly suppressed in the genetic absence or pharmaco-
logic inhibition of CaMKIV. We have presented proof that
CaMKIV, known to be important in the suppression of the
production of IL-2, is important in MC proliferation and the
expression of lupus nephritis. As such, CaMKIV represents
a unique link between the immune system and the biology of
MC, and its targeting may prove to be of significant clinical
value as it will mitigate systemic autoimmunity and suppress
The authors have no financial conflicts of interest.
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