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*
Second Department of Internal Medicine, University of Occupational and
Environmental Health, School of Medicine, Kitakyushu, Japan.
Department of Biochemistry, University of Occupational and Environmental
Health, School of Medicine, Kitakyushu, Japan.
Department of Anatomy, University of Occupational and Environmental
Health, School of Medicine, Kitakyushu, Japan.
Correspondence to Dr. Hiroshi Tanaka, Second Department of Internal Medicine, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahata-nishi, Kitakyushu 807-8555, Japan. Phone: +81 93 603 1611; Fax: +81 93 691 6913; E-mail: h-tanaka{at}med.uoeh-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1. This study investigated the expression
of profilin in rat anti-Thy-1.1 mesangial proliferative glomerulonephritis
(GN) and examined the effect of growth factors on its expression in cultured
rat mesangial cells. Profilin mRNA was constitutively expressed in isolated
glomeruli of untreated rats. However, in glomeruli of anti-Thy-1.1 GN rats,
its expression was upregulated beginning on day 1, reaching a peak level on
day 4 (3.9-fold versus control glomeruli), and decreased on day 14,
as determined by competitive reverse transcription-PCR. Increased expression
of profilin protein was confirmed using immunoblotting and
immunohistochemistry. Immunoelectron microscopy revealed the presence of
profilin in plasma membrane and the rough endoplasmic reticulum of mesangial
cells, indicating that profilin was produced in mesangial cells. In cultured
rat mesangial cells, expression of profilin mRNA and protein was upregulated
by basic fibroblast growth factor but not by platelet-derived growth factor or
transforming growth factor-ß. Suppression of profilin expression using an
antisense oligonucleotide against profilin inhibited [3H]thymidine
uptake. These findings indicated the involvement of profilin in anti-Thy-1.1
GN and suggest that the upregulation of profilin might be involved in the
progression of anti-Thy-1.1 GN possibly by affecting cell growth. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The dynamics of actin polymers are regulated by actin-binding proteins. To date, more than 100 of these proteins have been identified (11,12,13). Profilin, one such actin-binding protein measuring 12 to 15 kD, is conserved and widely distributed in eukaryotic cells (14). Loss of its expression causes defects in filamentous (F)-actin content, cytokinesis, cell growth, and development in Dictyostelium amoebae (15), and loss of profilin in mice is lethal at very early stages of development (16). Under in vitro conditions, profilin inhibits actin polymerization by sequestrating actin monomer from F-actin or, inversely, promotes polymerization in appropriate circumstances (17,18,19). Therefore, profilin is considered an important regulator of the actin-based cytoskeleton. It also interacts with PIP2, a component of the phosphatidylinositol cycle, and serves as a link through which the signaling pathways communicate with the dynamics of the actin cytoskeleton (20,21). These observations support the hypothesis that profilin is directly associated with cell motility such as migration or proliferation.
Anti-Thy-1.1 glomerulonephritis (GN) is a rat model experimentally induced by injection of an antibody to the Thy-1.1 antigen present on MC. It is characterized by initial severe MC injury and mesangiolysis, followed by MC proliferation and subsequent ECM deposition (22,23). We previously cloned rat profilin cDNA and reported increased profilin expression in glomeruli isolated from the kidneys of anti-Thy-1.1 antibody-treated rats at 7 d after disease induction (24). However, the involvement of cytoskeletal and actin regulatory proteins in glomerular diseases remains unclear at present.
To further understand the significance of profilin in anti-Thy-1.1 GN, we investigated the expression patterns of profilin protein and mRNA, compared the effects of several growth factors on profilin expression in cultured rat MC, and examined the role of altered profilin expression levels on cell growth. Our study revealed that profilin was expressed in glomerular MC of anti-Thy-1.1-treated rats, which coincided with the glomerular proliferation phase. The expression of profilin mRNA and protein could be regulated by basic fibroblast growth factor (bFGF) in cultured MC. Furthermore, suppression of profilin expression resulted in inhibition of DNA synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MC Cultures and Stimulation of Profilin Expression by Growth
Factors
Glomerular MC were isolated from male Wistar rats by the differential
sieving method and characterized as reported previously
(26,27).
MC were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 µg/ml
streptomycin at 37°C in 5% CO2 incubator. MC from passages 5 to
10 were used in the experiments. For growth factor stimulation, 1 x
105 cells were plated on a 10-cm culture dish and incubated until
they reached subconfluence. The growth of MC was arrested for 48 h in
serum-free DMEM, and then the cells were incubated with DMEM with or without
10 ng/ml bFGF (Bachem California, Torrance, CA), 10 ng/ml platelet-derived
growth factor-AA (PDGF-AA) (Upstate Biotechnology, Lake Placid, NY), 10 ng/ml
PDGF-BB (Sigma, St. Louis, MO), or 1 ng/ml transforming growth factor-ß1
(TGF-ß1) (Austral Biologicals, San Ramon, CA) for 2, 6, or 18 h for mRNA
extraction or for 24 or 48 h for protein extraction. We also investigated the
dose-dependent effect of bFGF (0.01 to 100 ng/ml) on profilin mRNA expression
in MC incubated for 18 h.
RNA Extraction and RT-PCR
RNA was extracted from isolated glomeruli or cultured rat MC with acid
guanidinium thiocyanate (28)
and applied to an oligo(dT) cellulose column (Pharmacia Biotech, Uppsala,
Sweden) to purify mRNA. RT was performed with 50 ng of poly(A)+ RNA
using a first-strand cDNA synthesis kit (Pharmacia Biotech). Samples were
amplified by PCR using specific primers for rat type I collagen 
1 chain
cDNA (sense, 5'-CCAATCTGGTTCCCTCCCACC; antisense,
5'-GTAAGGTTGAATGCACTTTTG; product size, 213 bp), rat type III collagen
1 chain cDNA (sense, 5'-CAGAGGCCCAGTTGGTCCACA; antisense,
5'-TGAAGACATGATCTCCTCAGT; product size, 292 bp), rat type IV collagen
1 chain cDNA (sense, 5'-ACTCCTGGTCACCCTGTGGAG; antisense,
5'-TTCTCCTTTGATGACATCGTA; product size, 399 bp), or G3PDH (sense,
5'-TGAAGGTCGGTGTCAACGGATTTGGC; antisense,
5'-CATGTAGGCCATGAGGTCCACCAC; product size, 983 bp) (Clontech, Palo Alto,
CA) in separate tubes with an automatic thermal controller (PC-800; Astec,
Fukuoka, Japan). The samples were then subjected to agarose gel
electrophoresis and stained with ethidium bromide to visualize DNA bands,
followed by scanning densitometry (IBAS; Kontron, Munich, Germany). PCR was
performed using a specific number of cycles for each protein (22 to 28 cycles
for type I and III collagens and G3PDH and 26 to 34 cycles for type IV
collagen) or with increasing amounts of glomerular mRNA to examine dose- and
cycle-dependent increases in PCR products
(24,29).
Based on the results of these procedures, we selected the following number of
PCR cycles to achieve submaximal amplification: 24 PCR cycles for G3PDH, 26
cycles for type I and III collagens, and 32 cycles for type IV collagen. We
also sequenced the PCR products and confirmed their sequences with a DNA
sequencer (373A; Applied Biosystems, Tokyo, Japan).
Competitive RT-PCR for Profilin
To quantify target profilin mRNA, we used a competitor DNA that contained
the same primer template sequences as the target DNA
(30,31).
The recombinant competitor DNA was obtained by amplifying a foreign DNA
fragment using two composite primers
(32), each of which contained
the target profilin and prokaryotic chloramphenicol acetyltransferase (CAT)
primer sequences. One primer
(5'-ATGGCCGGGTGGAACGCCTACCACGACGATTTCCGGCAG)
consisted of the sense primer of rat profilin cDNA (positions 1 to 21,
underlined) and the antisense primer of the pSV2CAT gene (positions 4645 to
4625, italics). The other primer
(5'-AGGTCAGTACTGGGAACGCCGACATGGAAGCCATCACA)
consisted of the antisense primer of profilin cDNA (positions 426 to 406,
underlined) and the sense primer of the pSV2CAT gene (positions 4364 to 4384,
italics). PCR was performed with 1 µg of pSV2CAT DNA and 40 pmol of each
primer. The obtained DNA fragment had profilin primer template sequences on
opposite sides of the fragment, but a completely different intervening
sequence and length. The amplified fragments were recovered
electrophoretically, purified with phenol/chloroform and ethanol
precipitation, and used as the standard for competitive PCR. Competitive PCR
was performed by incubating 50 ng of reverse-transcribed poly(A)+
RNA purified from isolated glomeruli or MC and 5 pg of competitor DNA with the
primer set for profilin (sense, 5'-ATGGCCGGGTGGAACGCCTAC; antisense,
5'-AGGTCAGTACTGGGAACGCCG; product size, wild type, 426 bp; competitor,
317 bp) in the same tube. PCR was performed for 20 cycles. After agarose gel
electrophoresis, the amplification bands stained by ethidium bromide were
quantified from the negative film by scanning densitometry (IBAS Kontron). The
ratio of wild-type to mutant band density was calculated for each lane
(30,31).
Competitive RT-PCR assays were performed at least in triplicate.
Immunoblotting of Profilin
Total protein was prepared by sonication of 1 vol of isolated glomeruli or
MC for 60 s in 5 vol of ice-cold buffer consisting of 5 mM Tris, pH 7.2, 0.1
mM ATP, 0.5 mM dithiothreitol, 1% Triton X-100, 1% DMSO, and 10 µg/ml each
of leupeptin and pepstatin
(18). The extract was
clarified by centrifugation at 12,000 x g for 30 min at
4°C. Samples (10 µg/ml) each of the glomerular extract were
electrophoresed on 15% sodium dodecyl sulfate-polyacrylamide gels
(32) and blotted onto a
polyvinylidene fluoride membrane (Millipore, Bedford, MA). The blotted
membrane was incubated with 0.5 µg/ml anti-rat profilin antibody, an
affinity-purified rabbit polyclonal antibody raised against synthesized rat
profilin oligopeptide (Glu-83-Ser-92), and visualized with
peroxidase-conjugated anti-rabbit IgG F(ab')2 (diluted
1:1000; Cappel, Durham, NC) and enhanced chemiluminescence reagents (ECL,
Amersham, Buckinghamshire, United Kingdom)
(24).
Immunohistochemistry
Renal specimens were fixed with 4% paraformaldehyde and embedded in
paraffin. Indirect immunoperoxidase staining of 5-µm thick sections was
performed using the antibody to profilin. After a 2-h incubation with 0.5
µg/ml anti-profilin antibody at room temperature, sections were reacted for
45 min with biotinylated goat anti-rabbit Ig (Vector, Burlingame, CA),
followed by treatment with streptavidin-conjugated peroxidase (Dako, Glostrup,
Denmark). In addition, tissues were stained with a murine IgG monoclonal
antibody (mAb) against the proliferating cell nuclear antigen (PCNA) (Dako) or
a murine IgG mAb against -smooth muscle actin (-SMA) (Progen
Biotechnik, Heidelberg, Germany). For negative controls, the primary antibody
was deleted or an equivalent concentration of an irrelevant murine mAb or
preimmune rabbit IgG was used. Glomerular expression of profilin and
-SMA was graded semiquantitatively to reflect changes in the area and
intensity of glomerular staining
(33), using the following
five-point system: 0, very weak or no staining; 1+, weak staining with <25%
of the glomerular tuft showing focally increased staining; 2+, 25 to 49% of
the glomerular tuft with focally increased staining; 3+, 50 to 75% of the
glomerular tuft demonstrating increased staining; and 4+, >75% of the
glomerular tuft showing strong staining. Sections adjacent to those used for
immunostaining were stained with Delafield's hematoxylin and eosin and used
for counting the number of cells. We examined 20 to 43 cross sections of
glomerular tuft in each specimen in a blinded manner.
Immunoelectron Microscopy
Specimens were fixed in a solution of periodate-lysine-paraformaldehyde for
6 h at 4°C (34). Indirect
immunoperoxidase staining of approximately 40-mm-thick sections was performed
using antibody to profilin. Sections were post-fixed in 0.1% osmium tetraoxide
in 0.1 M phosphate-buffered saline (PBS) for 2 min, embedded in epoxy resin,
cut into ultrathin sections, and examined with an electron microscope (JEM
1210, JEOL Ltd., Tokyo, Japan) without counterstaining. The specificity of
immunoreactions was confirmed by substituting the normal rabbit serum or PBS
for the primary antibody.
Transfection with Antisense Oligonucleotides
Sense and antisense oligonucleotide sequences were 5'-ATGGCCGGGTGGAAC
and 5'-GTTCCACCCGGCCAT, respectively. Antisense profilin oligonucleotide
was complementary to rat profilin mRNA at the translation initiation region.
The sense rat profilin oligonucleotide at the same region was used as the
control oligonucleotide. We synthesized 15-base deoxyribonucleotides on an
automated solid-phase synthesizer (Sawady Technology, Tokyo, Japan). Before
use, the oligomers were purified by gel filtration, precipitated with ethanol,
lyophilized to dryness, and dissolved in the culture medium. Oligonucleotides
were introduced into MC using the cationic liposome-mediated transfection
method (35). Oligonucleotides
dissolved in 100 µl of serum-free DMEM were mixed with 5 µl of
LipofectinTM reagent, a 1:1 (wt/wt) formation of
N-[1-(2,3-dioleoyloxy)propyl]-n,n,n-trimethylammonium
chloride and dioleoyl phosphotidylethanolamine (BRL Life Technologies,
Gaithersburg, MD) in the same volume of serum-free DMEM, and incubated for 10
min at room temperature. To MC plated in 6-well culture dishes (1 x
104 cells/well), oligonucleotide/liposome complex was added. After
6 h of incubation, it was replaced with a 10% FCS-containing medium for 24 h.
The concentration of oligonucleotides was 2.2 µM (10 µg/ml). Suppression
of profilin mRNA expression levels by the antisense oligonucleotide was
confirmed by competitive RT-PCR.
Measurement of [3H]Thymidine Incorporation
[3H]Thymidine incorporated into cells was measured as described
previously (36). Briefly,
after 48 h of serum deprivation, transfected MC (1 x 104
cells/well) were exposed to a medium containing 10% FCS. After a 24-h
incubation with serum, 1 µCi/ml [3H]thymidine (Amersham) was
added to each well for 1 h. The cells were then washed three times with
ice-cold PBS and detached from plates with a solution of 0.25% trypsin in PBS.
The radioactivity of the samples was determined in a liquid scintillation
counter (n = 6). Cell proliferation was expressed as counts per
minute of [3H]thymidine incorporated into cells.
Statistical Analyses
Data are expressed as the mean ± SD. Differences between groups were
examined for statistical significance using ANOVA or t test. A
P value <0.05 denoted a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By immunoblotting, expression of profilin protein was hardly detectable in control glomeruli (Figure 2). The expression of profilin protein in anti-Thy-1.1 antibody-treated rats increased on day 1, peaked on day 4, and gradually decreased by day 14. A band detected at around 58 kD was confirmed by immunoblotting for actin to be a profilin-actin complex (data not shown). Thus, the increase in profilin mRNA expression generally corresponded with profilin protein accumulation during the course of the disease. The expression of both mRNA and protein reached a peak level on day 4.
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Localization of Profilin in Diseased Glomeruli
Immunohistochemistry showed that profilin was hardly detectable in the
glomeruli of normal rats (Figure
3A). On day 1 after treatment, profilin was preferentially
expressed in parts of the mesangial area
(Figure 3B). On day 4, profilin
staining was markedly increased and extended to the entire glomerulus,
including the mesangial area and capillary loop
(Figure 3C). By day 14, the
intensity of profilin staining diminished and was hardly detectable; very weak
staining was noted at certain parts of the capillary loop
(Figure 3D). No profilin
staining was observed in sclerotic areas of diseased glomeruli.
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Immunoelectron microscopy showed immunoreaction for profilin at the plasma membrane of MC on day 4 (Figure 4, arrows). Immunostaining was also observed in the rough endoplasmic reticulum of MC (Figure 4B, arrowheads) but not in the rough endoplasmic reticulum of endothelial or epithelial cells, suggesting that profilin was produced, at least in part, in mesangial cells. There were also predominant profilin depositions in the extracellular space around the plasma membrane of MC (Figure 4B, asterisks).
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Upregulation of Profilin during Glomerular Cell Proliferation in
Anti-Thy-1.1 GN
Next, we examined the relationship between profilin over-expression and
cellular events observed during the course of anti-Thy-1.1 GN. The results of
semiquantitative analysis of glomerular profilin expression were consistent
with the results obtained by immunoblotting and RT-PCR for profilin. The level
of profilin expression paralleled the time course of glomerular cell
proliferation as determined by PCNA staining and the number of glomerular
cells counted per glomerular cross section; both reached peak values on day 4
(Table 1). The expression of
-SMA was different from that of profilin: Its expression was increased
on day 1 and reached a peak on day 7, 3 d after the peak on day 4 of profilin
induction, and at a time when MC proliferation had already begun to decrease
(Table 1). These results
suggested that profilin was upregulated consistent with the mesangial
proliferative phase.
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Profilin Expression Precedes that of Matrix Proteins in Anti-Thy-1.1
GN
We also examined the relationship between profilin expression and time
course of ECM protein expression. The expression of ECM mRNA glomeruli that
were up- or downregulated during anti-Thy-1.1 GN was examined by RT-PCR. The
expression levels of type I, III, and IV collagens increased from day 4 to day
14 and were at peak levels on day 7 (Figure
5). These results indicated that peak profilin expression preceded
that of ECM components. The expression of G3PDH mRNA, used as the internal
control for RT-PCR, showed no change throughout the study.
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Profilin mRNA and Protein Are Induced in Response to bFGF But Not
PDGF or TGF-ß in Cultured MC
We also investigated the mechanisms that regulate the expression of
profilin in cultured rat MC. In growth-arrested MC, profilin mRNA and protein
were constitutively expressed at low levels (Figures
6 and
7). We used growth factors
involved in the disease course of anti-Thy-1.1 GN to stimulate profilin
expression. Incubation of quiescent MC with bFGF significantly enhanced
profilin mRNA expression in MC from 6 to 18 h; the peak expression was noted
at 6 h and was 2.9-fold higher than the control level (P < 0.01)
(Figure 6). PDGF-AA, PDGF-BB,
and TGF-ß1 had no stimulatory effects on profilin mRNA expression. The
bFGF-induced increase in profilin mRNA expression was accompanied by increased
protein expression from 24 to 48 h (Figure
7). Furthermore, the bFGF-induced increase in profilin mRNA was
concentration-dependent and saturable as determined by RT-PCR
(Figure 8) with a median
effective dose of 0.1 ng/ml.
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Inhibition of MC Proliferation by Profilin Antisense
Oligonucleotide
We examined the effects of altered expression levels of profilin on DNA
synthesis in cultured rat MC. Transfection of profilin antisense
oligonucleotide into MC effectively reduced the expression of profilin mRNA
compared with the untransfected control and sense oligonucleotides
(Figure 9A). Transfection of MC
with an antisense oligonucleotide significantly reduced
[3H]thymidine incorporation in response to serum stimulation
compared with untransfected MC and sense-transfected MC
(Figure 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Profilin expression was increased on day 1, peaked on day 4, and decreased by day 14. The maximal expressions of profilin protein and mRNA were noted to occur at the same time when the peak number of PCNA-positive cells, a marker of MC proliferation (37), was observed. Interestingly, under our culture conditions, profilin synthesis was induced upon stimulation by bFGF but not with PDGF or TGF-ß. bFGF is expressed in injured glomeruli and induces MC proliferation in the early phase of anti-Thy-1.1 GN (38,39). Our findings in cultured MC therefore suggest that bFGF may be associated with overexpression of profilin in anti-Thy-1.1 GN. We also evaluated the expression of ECM proteins throughout the course of anti-Thy-1.1 GN to determine the role of profilin in glomerulosclerosis, an important event in the progression of glomerular disease. In treated rats, the expression of mRNA of type I, III, and IV collagens was markedly increased from days 7 to 14, consistent with previous reports (40,41,42). However, profilin mRNA expression was downregulated in this phase of glomerular sclerosis. In addition, TGF-ß, an important regulator of ECM synthesis, failed to stimulate the expression of profilin mRNA under culture conditions. These results indicate that profilin was not directly involved in mesangial sclerosis.
Immunohistochemical staining of profilin in glomeruli of anti-Thy-1.1 antibody-treated rats was detected in the mesangial area on day 1, and further extended to the capillary loop on day 4. Glomerular endothelial cells are also known to proliferate in response to cellular injury in anti-Thy-1.1 GN (43). We further performed immunoelectron microscopy to identify the sites of upregulated profilin synthesis, because profilin staining in capillary loops may suggest that not only MC but also endothelial and/or epithelial cells overexpress profilin on day 4. Immunoelectron microscopy revealed that profilin was mainly present at the plasma membrane and the rough endoplasmic reticulum of mesangial cells, but not on endothelial or epithelial cells. These results suggest that profilin is expressed in MC and that endothelial and epithelial cells are not likely the major sources of profilin synthesis in anti-Thy-1.1 GN rats. Profilin was also present at the extracellular space around mesangial cells including capillary loops. We speculate that mesangiolysis followed by anti-Thy-1.1 antibody treatment may have caused profilin deposition by the release and concentration of endogenous profilin into the extracellular space. Other possible sources of profilin include profilin-rich platelets or monocytes/macrophages (44), which infiltrate injured glomeruli in the initial stages of anti-Thy-1.1 GN (from day 1 to day 3) (3,45). Upregulation of profilin on day 1 does not role out profilin production or release from monocytes/macrophages, because mesangial cells do not proliferate and are destroyed during this initial phase. However, because the infiltrating phase of these cells did not coincide with the peak of profilin expression within glomeruli, these plasma-derived cells do not appear to be the major source of profilin in the proliferating phase of anti-Thy-1.1 GN.
Another major finding of the present study was that reduction of profilin
expression by antisense transfection led to a marked suppression of
[3H]thymidine uptake in cultured MC. These results were consistent
with the previous report demonstrating that the lack of profilin expression by
either antisense or gene disruption techniques results in inhibition of cell
growth rate (15), although the
mechanisms by which profilin regulates cell growth are unclear. One possible
explanation is the importance of cell adhesion signaling in cell growth. Many
mammalian cell types are dependent on adhesion to the ECM for continued
survival. A variety of normal cell types undergo apoptosis when they lose
attachment to ECM in a process termed anoikis
(46,47).
These adhesive interactions are mediated by cell surface receptors called
integrins. Integrins form multimolecular complexes of cytoskeletal and
signaling proteins called focal adhesions, where integrins link to actin
filaments and regulate intracellular signaling
(4). In fact,
profilin-deficient cells show inhibition of actin cytoskeleton formation
(15), and loss of profilin in
mice is lethal at very early stages of development
(16). Therefore, suppression
of cell growth by downregulation of profilin expression might result from
disruption of actin cytoskeleton formation that links to cell adhesive
interactions. Another possible mechanism is profilin binding to phospholipids.
Profilin is present as a profilin-PIP2 complex in the plasma
membrane
(21,48).
Cleavage of PIP2 by activated phospholipase C1, a substrate
for receptor tyrosine kinases, releases profilin from the membrane, thereby
affecting the equilibrium between the monomer and filamentous actin
concentration
(20,21).
Thus, by participating in the pathway activated by receptor tyrosine kinases,
profilin may be related to the mechanism(s) by which receptor tyrosine kinases
control the reorganization of the actin cytoskeleton
(48), and may play a role in
cell proliferation.
Profilin is also involved in the regulation of cell migration. A lack of profilin changes actin distribution in cells and reduces the speed of cell motion (15). Cell migration is believed to be an important feature in GN. MC migrate in response to various growth factors such as PDGF (49) and thrombospondin (50). It is also known that other cytoskeletal linking proteins, moesin and radixin, which are critical for cell-cell adhesion and microvilli formation, are upregulated in rat anti-Thy-1.1 GN model (33). These data suggest that profilin may also be involved in the regulation of cell migration in anti-Thy-1.1 GN. bFGF, in addition to its role in proliferation, may have a role in cytoskeletal change via stimulation of profilin expression.
-SMA, an actin isoform that may interact with profilin in the cells,
is also induced by various forms of glomerular injury in experimental and
human GN including anti-Thy-1.1 GN
(51). It is also recognized as
a phenotype marker of myofibroblast-like activated MC
(52). Because the expression
of -SMA in diseased glomeruli is limited within MC and is different
from that of profilin, it is not certain whether coexpression of profilin and
-SMA is of significance in the rearrangement of the actin cytoskeleton.
However, the expression of ß/ actin mRNA is also increased in rats
with anti-Thy-1.1 GN at day 3 to 14
(53). These observations
suggest that profilin may participate in the regulation of actin filaments and
reorganization of the cytoskeleton in mesangial proliferative GN.
In summary, we showed in the present study using a rat model of anti-Thy-1.1 GN a marked increase of glomerular profilin expression during the phase of MC proliferation. Profilin expression in cultured MC was stimulated by bFGF, and the growth of these cells was suppressed by antisense oligonucleotide transfection. Our results suggest that profilin plays an important role as a regulatory molecule in the actin cytoskeleton and that it is important in the progression of mesangial proliferative GN by regulating cell growth.
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Acknowledgments |
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This work was supported in part by a research grant from the University of Occupational and Environmental Health, Japan.
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Footnotes |
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American Society of Nephrology
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, type I collagen; 
, type III collagen; [UNK], type IV
collagen. Data represent the mean ± SD values.
