(Received for publication, January 10, 1997, and in revised form, March 18, 1997)
From the A potentially important cross-talk characteristic
of transforming growth factor- Transforming growth factor (TGF)1- Studies in a variety of cell types have demonstrated a modulatory
capacity of TGF- A common pathway of raising [Ca2+]i with PDGF,
isoproterenol, and angiotensin II is the activation of phospholipase C Our present study demonstrates that TGF- [32P]Inorganic phosphate,
[ An SV40-transformed murine glomerular
mesangial cell line (MMC) that has been previously described (24) was
primarily used in these studies. These cells retain many of the
differentiated characteristics of mesangial cells in primary culture
(24). To assure that our findings were not influenced by
transformation, we performed a limited series of experiments in rat
glomerular mesangial cells that were conducted between passages 4 and
10. Isolation of rat mesangial cells was performed as detailed
previously (25).
MMC grown in Dulbecco's modified Eagle's medium (DMEM)
(Life Technologies, Inc.) with 10% fetal calf serum were harvested and
plated onto 100-mm dishes with growth media. After reaching 80%
confluence, cells were incubated in serum-free DMEM for 24 h.
During the subsequent final 8 h of incubation, cells were treated with TGF- First strand cDNA was prepared from
total RNA isolated from mouse cerebellum, kidney, and MMC using Moloney
murine leukemia virus reverse transcriptase and oligo(dT) primers.
Polymerase chain reaction (PCR) was performed using specific primers
for the type I IP3R isoform (5 To assess whether IP3R mRNA was affected by TGF- MMC incubated in
serum-free DMEM for 24 h were washed with phosphate-free DMEM and
incubated in the same buffer for 10 min. [32P]Inorganic
phosphate was added to give a final concentration of 0.3 mCi/ml, and
the incubation was continued for a further 90 min. The cells were
exposed to various agonists for the desired times, and the incubations
were then quenched by the addition of 4 ml of ice-cold PBS containing
200 µM sodium orthovanadate. Cells were washed twice with
the PBS/vanadate solution and scraped off the dish. Following
centrifugation at 150 × g for 15 s, the cells
were washed twice in PBS/vanadate and finally resuspended in lysis
buffer that contained 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% (w/v) Triton X-100, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM tetrasodium
pyrophosphate, 100 mM NaF, 5 nM okadaic acid, 1 mM PMSF, and 5 µg/ml each of aprotinin and leupeptin. The
cells were solubilized on ice for 30 min. Insoluble material was
removed by centrifugation for 10 min at 25,000 × g.
All of the extracts were precleared with 25 µl of a 50% (v/v) slurry
of Staphylococcus aureus cell wall (Pansorbin, Calbiochem).
IP3R was immunoprecipitated from the labeled extracts by
overnight incubation with 100 µl of 20% protein A-Sepharose beads
and 100 µg of the antibody to the type I IP3R. The
immunoprecipitates were washed three times in lysis buffer, and the
phosphorylated proteins were analyzed by SDS-PAGE. The polypeptides in
the gel were transferred to nitrocellulose and then autoradiographed.
Membranes were subsequently immunoblotted with antibody to
IP3R to localize the phosphorylated IP3R.
Back phosphorylation of IP3R immunoprecipitates
was performed as detailed previously (26). MMC were rested in
serum-free DMEM for 24 h and then incubated with various agonists
for 15 min. The cells were centrifuged and solubilized in lysis buffer, and the extracts were obtained were immunoprecipitated with
IP3R antibody as described above. The immunoprecipitates
were washed three times in a phosphorylation buffer that contained 120 mM KCl, 50 mM Tris-HCl (pH 7.2), 0.1% Triton
X-100 (w/v), 0.3 mM MgCl2, 0.5 mM
PMSF, and 10 µg/ml each aprotinin and leupeptin. Aliquots of protein
A-Sepharose beads were incubated in 50 µl of the phosphorylation
buffer containing 100 units/ml of the catalytic subunit of PKA and 1 µCi of [ The
peptides RPSGRRESLTSFGNP and ARRDSVLAAS, which
include the major phosphorylation site for PKA (serine 1755 and serine 1589) (14, 15) were selected for in vitro kinase assays. MMC incubated in serum-free DMEM were treated with agonists for various periods of time, washed with PBS, harvested with cold extraction buffer
(25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM An antibody raised to the C terminus of the rat brain
type I IP3R recognized a 240-kDa polypeptide from the
SV40-transformed MMC (Fig. 1A). The same
sized band was noted from protein derived from mouse and rat cerebellum
(data not shown). The addition of TGF-
To determine if the effects of chronic exposure to
TGF-
It has been previously demonstrated that the
cerebellum contains the long form (insertion of the SII segment) of the
type I IP3R, whereas peripheral tissues may contain
exclusively the alternatively spliced short form (SII
It has
been previously demonstrated that phosphorylation of the
IP3R may affect its function; therefore, we evaluated
whether TGF-
Given that the IP3R has been previously
demonstrated to be phosphorylated by PKA (15, 17), and based on our
prior data that cyclic nucleotide dependent kinases may mediate
TGF-
The sites on the type I
IP3R from cerebellum that have been demonstrated to be
phosphorylated by PKA are serine 1755 and serine 1589 (14, 15, 17).
Synthetic peptides that contain these two serine sites,
RPSGRRESLTSFGNP and ARRDSVLAAS, respectively, were used as substrate in an in vitro kinase assay with
crude cell lysates prepared from control and TGF-
Table I.
Stimulation of IP3R peptide phosphorylation by TGF-
TGF-
The main conclusion based on the results of our study is that the
type I IP3R is a target of regulation by TGF- Our results differ somewhat from those of Wojcikiewicz et
al. (19) in that carbachol decreased IP3R protein
expression due mainly to enhanced degradation of the IP3R
in human neuroblastoma cells. However, this group also reported a
decrease in mRNA levels after 3 h of exposure to carbachol,
which may have contributed to the decreased protein levels. The
presence of several ATTTA sites in the 3 Our observation that TGF- The implication of decreased IP3R protein expression is
that [Ca2+]i mobilization would be impaired, as
demonstrated previously (18) in neuroblastoma cells. Theoretically, the
TGF- Short term (15-30 min) treatment of TGF- The interaction of TGF-
Department of Medicine,
(TGF-
) is to inhibit
platelet-derived growth factor-induced intracellular calcium rise
(Baffy, G., Sharma, K., Shi, W., Ziyadeh, F. N., and Williamson, J. R. (1995) Biochem. Biophys. Res. Commun. 210, 378-383) in
murine mesangial cells. The present study examined the possible basis
for this effect by evaluating the regulation of the type I inositol
1,4,5-trisphosphate receptor (IP3R) by TGF-
. TGF-
1
down-regulates IP3R protein expression by >90% with
maximal and half-maximal effects after 8 and 2 h, respectively.
TGF-
1 also decreased IP3R mRNA expression by 59% after 1 h. Phosphorylation of the IP3R was also
demonstrated as early as 15 min after TGF-
1 exposure. Back
phosphorylation assays of IP3R from TGF-
1-treated
mesangial cells with protein kinase A (PKA), indicated that
TGF-
1-induced phosphorylation of the IP3R occurs at
similar sites as for PKA. In vitro kinase assays using the
known IP3R peptide substrates for PKA, RPSGRRESLTSFGNP and
ARRDSVLAAS, demonstrated that TGF-
1 induces phosphorylation of both
peptides (158 and 123% of control values, respectively). TGF-
1-induced phosphorylation was prevented by the addition of the
PKA inhibitor peptide in the in vitro kinase assay. It is proposed that TGF-
-mediated effects on the IP3R may be
an important characteristic of its ability to modulate the response of
cells to factors that employ IP3R-mediated calcium
release.
1
has been implicated in a variety of inflammatory and noninflammatory
kidney diseases (1). Our prior studies have demonstrated that TGF-
1
is up-regulated in animal models of diabetic kidney disease (2) and
inhibition of TGF-
activity by neutralizing antibodies reduces
diabetic renal hypertrophy and gene expression of type IV collagen and fibronectin (3). Apart from its well described effects to stimulate matrix production and regulate cell growth, TGF-
also has a
characteristic effect to modulate the phenotypic actions of other
factors. In particular, TGF-
can inhibit the proliferative ability
of PDGF and other mitogens in human mesangial cells (4). This is of particular relevance to many glomerular diseases in that up-regulation of multiple growth factors is observed concomitantly (5, 6).
to affect the cellular response to exogenous factors. TGF-
inhibits PDGF-induced proliferation and inositol 1,4,5-trisphosphate (IP3) production in human bone marrow
fibroblasts (7). In studies with cardiac fibroblasts (8) and vascular smooth muscle cells (9), pretreatment with TGF-
1 for at least 30 min
inhibits [Ca2+]i release in response to
isoproterenol and angiotensin II, respectively. In our studies with
transformed murine mesangial cells, we demonstrated that TGF-
markedly inhibits PDGF-BB-induced increase in
[Ca2+]i (10).
or phospholipase C
and the generation of IP3 from
phosphatidylinositol 4,5-bisphosphate (11). The raised
IP3 levels bind to IP3 receptors (IP3Rs) in the endoplasmic reticulum to release stored
calcium into the cytoplasmic space (11). This is thought to be a
crucial step in allowing the cell to respond to these agonists. There are at least three isoforms of the IP3 receptor, derived
from three distinct genes (reviewed in Ref. 12). The type I isoform is
abundant in cerebellum but is also present in many peripheral tissues.
Alternative splicing of the type I IP3R results in deletion of the SII segment in nonneural tissues (13, 14). The SII segment is
present between two serines (serine 1589 and serine 1755) (13, 14),
which are phosphorylated by protein kinase A (PKA) (14, 15).
Phosphorylation of the cerebellar type I IP3R by PKA
impairs [Ca2+]i mobilization by IP3
(16, 17). In addition, muscarinic receptor activation impairs
IP3-induced [Ca2+]i mobilization by
enhancing the degradation of the type I isoform in neuroblastoma cells
(18, 19). The type III isoform of the IP3R has been
recently found to be up-regulated during apoptosis of T-lymphocytes
(20). The type I isoform of the IP3R is present in the
glomerulus of the kidney (21), primarily in mesangial cells (22), as
well as in the renal vascular system (23). Other isoforms of the
IP3R have not been identified in the glomerulus (21). Since
TGF-
appears to modulate IP3-induced [Ca2+]i mobilization in a variety of cell types,
we postulate that TGF-
may mediate some of its effects via
regulation of the IP3R.
inhibits the protein
expression of the type I isoform of the IP3R in
mesangial cells and that this may be partly due to a decrease of
the steady-state mRNA level. In addition, TGF-
rapidly
phosphorylates the IP3R. The likely consequence of these
effects is to modulate IP3R function and thus affect the
cellular responsiveness to agents that act via activation of the
IP3R.
Materials
-32P]ATP, and [
-32P]CTP were from
DuPont NEN. An enhanced chemiluminescence kit was purchased from Amersham Corp. TGF-
1 was purchased from R & D Systems. All other reagents were from Sigma unless otherwise noted.
1 Regulation of IP3R
Protein
1 (10 ng/ml) for the last 1, 2, 4, and 8 h, washed
with PBS three times, and harvested in lysis buffer that contained 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% (w/v)
Triton X-100, 1 mM EDTA, 1 mM PMSF, and 5 µg/ml each of aprotinin and leupeptin. All samples, including the
control samples, were harvested after the same overall duration of
incubation. Protein concentrations of samples were quantitated, and
equal amounts of protein were run on a 7% SDS-PAGE gel, transferred to
nitrocellulose, and immunoblotted with an antibody raised to the C
terminus of the type I IP3R from brain (26). The primary
antibody was then removed, and the membrane was incubated with
horseradish peroxidase-conjugated secondary antibody. Immunoreactive
bands were detected using enhanced chemiluminescence (Amersham).
Densitometric analysis of scanned images was performed on a Macintosh
7600/132 computer using the public domain NIH Image program.
Measurements in control samples were assigned a relative value of
100%.
1
-CGT GGA TGT TCT ACA CAG ACC
AG-3
) and (5
-TTG GAA CTT CTT GAA GAG ACT A-3
) (13). These primers are on either side of the SII
splice domain of the type I
IP3R (13). Each reaction mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 3 mM MgCl2, oligonucleotide primers at 0.5 µM, all four dNTPs (each at 0.2 mM), 2.5 units of Taq DNA polymerase (Perkin-Elmer), and 15% of the
first strand cDNA products in a final volume of 50 µl. After an
initial cycle of 4 min at 94 °C, the reaction was cycled 40 times
for 60 s at 55 °C, 3 min at 72 °C, and 60 s at
94 °C. The reaction was completed with one cycle at 72 °C for 5 min. For initial analysis, the PCR product was run on 2% agarose gel
stained with ethidium bromide. The PCR product was cloned into the
pCRII TA cloning system (Invitrogen, La Jolla, CA) and sequenced to
confirm its identity.
1
treatment, MMC incubated in serum-free media was treated with TGF-
1
(10 ng/ml) for 1, 2, and 4 h, washed with ice-cold PBS and total
RNA isolated using acid guanidinium thiocyanate-phenol-chloroform (27).
Poly(A) mRNA was isolated from total RNA by an oligo(dT) affinity
column (Promega, Madison, WI). 3 µg of poly(A) mRNA were loaded
onto a 1.2% agarose gel containing 2.2 M formaldehyde, electrophoresed, and transferred onto nylon membrane. Cerebellum RNA
was run as a positive control. The probe for the type I
IP3R was obtained by running the PCR product (see above)
from MMC on a low melt agarose gel and cutting out the band
corresponding to the type I IP3R. The probe was purified
and labeled via the random prime method (Boehringer Mannheim).
Hybridization and washing conditions were performed as described
previously (2). To standardize for loading, membranes were stripped and
reprobed with a
-actin cDNA probe (kindly provided by Dr. P. Norton). Densitometry was performed as described above, and mRNA
levels were calculated relative to those of
-actin.
-32-P]ATP (3000 Ci/mmol). The
immunoprecipitates were phosphorylated for 15 min at 30 °C, and the
reaction was terminated by washing the protein A-Sepharose beads three
times with phosphorylation buffer containing 1 mM unlabeled
MgATP. The phosphorylated proteins in the immunoprecipitates were
separated on 5% SDS-PAGE gels and transferred to nitrocellulose, which
was then autoradiographed. The membrane was later immunoblotted to
locate the receptor. In all experiments the IP3R was the
only phosphorylated band above the prestained myosin molecular
mass marker (200 kDa).
-mercaptoethanol, 1 µg/ml
leupeptin, 1 µg/ml aprotinin, 1 mM PMSF), and homogenized
with a Dounce homogenizer. The lysate was centrifuged for 5 min at
4 °C at 14,000 × g, and the supernatant was saved.
The protein concentrations of the supernatants were quantitated, and
equal amounts of protein were added to a reaction mixture containing 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2,
0.1 mg/ml bovine serum albumin, 200 µM IP3R
peptide substrate (RPSGRRESLTSFGNP or ARRDSVLAAS), and 3000 Ci/mmol
[
-32P]ATP, and 0.5 mM ATP per reaction.
Experiments were performed in parallel with the addition of a PKA
inhibitor peptide, TTYADFIASGRTGRRNAIHD (1 µM) (Promega).
The reaction was allowed to proceed for 5 min at 30 °C and then
terminated with the addition of 2.5 M guanidine hydrochloride. 10 µl of sample was spotted onto phosphocellulose filter paper (1 × 1 cm) and washed repeatedly with 1 M NaCl and subsequently with 1 M NaCl in 1%
H3PO4. The papers were then dried in an oven
and placed in scintillation vials for radioactive counting. The
stoichiometry of phosphorylation was assessed from the specific activity of [32P]ATP and from the amount of peptide used.
The concentration of the peptide substrate and the duration of the
in vitro kinase reaction were varied to define the
concentration and time dependence of peptide phosphorylation.
Down-regulation of Type I IP3R Protein by
TGF-1
1 (10 ng/ml) down-regulated
the expression of the IP3R if administered for 2-8 h (Fig.
1A). Fig. 1B shows the cumulative data assembled
from densitometric scans of several experiments. TGF-
1 reduced
IP3R protein expression to 42% by 2 h and 25% at 4 h, with respect to control values. The maximal effect was seen at 8 h, at which point IP3R protein expression was
reduced to 3% of control values. A similar pattern was noted with
early passage nontransformed rat glomerular mesangial cells. Type I
IP3R immunoreactivity was reduced to 57% of control at
2 h of TGF-
1 treatment and to 49% of control at 4 h of
TGF-
1 treatment (Fig. 2, A and
B).
Fig. 1.
Time course of TGF-1 inhibition of type I
IP3R immunoreactivity in MMC. Samples (20 µg of
protein/lane) of control MMC or MMC treated with TGF-
1
(10 ng/ml) for the indicated time periods were resolved on 7%
SDS-PAGE, transferred to nitrocellulose, and probed with type I
IP3R antibody. The upper band of the doublet migrated to the same position on the gel as the type I IP3R
from mouse cerebellum (data not shown) (A). Panel
B shows the densitometric quantitation of immunoreactive protein
expressed relative to the control. Data shown are the mean ± S.E.
of band intensities from three separate experiments. *,
p < 0.05 versus control.
[View Larger Version of this Image (27K GIF file)]
Fig. 2.
Time course of TGF-1 inhibition of type I
IP3R immunoreactivity in rat mesangial cells. Shown is
a study similar to that in Fig. 1 using rat mesangial cells treated
with TGF-
1 (10 ng/ml) for 2 and 4 h. The upper band
of the doublet migrated to the same position on the gel as the type I
IP3R from rat cerebellum (data not shown) (A).
Panel B shows the densitometric quantitation of
immunoreactive protein expressed relative to the control. Data shown
are mean ± S.E. of band intensities from three separate experiments. *, p < 0.05 versus
control.
[View Larger Version of this Image (14K GIF file)]
1 Inhibits the Expression of the IP3R
mRNA
1 on inhibition of the IP3R protein expression may
be due to an effect on synthesis of new protein, we evaluated possible
regulation by TGF-
1 at the messenger RNA level of type I
IP3R. Northern analysis of poly(A) mRNA from MMC
revealed a single >10-kilobase mRNA band (Fig.
3A). A band of identical size was noted with
mouse and rat cerebellum RNA (data not shown). MMC were treated with TGF-
1 for 1 and 4 h, and the results from a representative
Northern analysis are shown in Fig. 3A. Quantitative
analysis demonstrates a decrease to 41% of control type 1 IP3R mRNA expression as early as 1 h of exposure
to TGF-
1, and it remains suppressed at 4 h of exposure to
TGF-
1 (31%) (Fig. 3B). Thus, the reduction in mRNA
for the type I IP3R precedes the reduction in protein.
Fig. 3.
Effects of TGF-1 on type I
IP3R mRNA expression in MMC. Shown is a
representative Northern blot of poly(A) mRNA from MMC (3 µg)
treated with TGF-
1 (10 ng/ml) for 1 and 4 h and hybridized with
a radioactive 580-bp probe for the type I IP3R
(A). The blot was stripped and reprobed with
-actin
cDNA to standardize the amount of RNA loaded. Panel B
shows the densitometric quantitation of IP3R
mRNA/
-actin mRNA expressed relative to the control. Data shown are the mean ± S.E. of band intensities from three separate experiments. *, p < 0.01 versus
control.
[View Larger Version of this Image (20K GIF file)]
Form of the
IP3R
) or both the
SII
and SII+ of the type I IP3R (13, 14). To evaluate the
length of the IP3R in mesangial cells and mouse kidney, we
chose primers that surround the SII domain of the IP3R
cDNA (13). Fig. 4 shows that mesangial cells and
kidneys from mice only express a 580-bp DNA segment, whereas cerebellum
cDNA expresses a 700-bp segment. As described previously (13), the
cerebellum fragment corresponds to the SII+ of the IP3R,
and the 580-bp fragment corresponds to the SII
of the
IP3R that has the SII segment deleted. This was confirmed
by sequencing of the 580-bp DNA segment.
Fig. 4.
PCR products from mouse cerebellum,
kidney, and mesangial cell cDNAs. RNA isolated from
mouse tissues and MMC was reverse transcribed to cDNA, and PCR was
performed using primers flanking the SII insertion site. The location
of the molecular size markers for 700 and 500 base pairs are
indicated.
[View Larger Version of this Image (36K GIF file)]
1
1 could affect the phosphorylation status of the type I
IP3R. MMC labeled with [32P]orthophosphate
were treated with TGF-
1 for variable times, and the IP3R
was immunoprecipitated and resolved by SDS-PAGE. A time course
demonstrating the peak effect of TGF-
1 on phosphorylation of the
IP3R is shown in Fig. 5A.
Quantitative analysis demonstrates that phosphorylation was increased
by 2-fold at 15 min, 5-fold at 30 min, and 4-fold at 60 min of TGF-
1
treatment (Fig. 5B). As shown in Fig. 6,
TGF-
1 and forskolin treatment for 15 min enhanced phosphorylation of
the IP3R to a similar degree.
Fig. 5.
Time course of phosphorylation of type I
IP3R by TGF-1 in MMC. MMC labeled with
[32P]orthophosphate were incubated with TGF-
1 (10 ng/ml) for the designated time periods (lanes 3-8). After
washing, the cells were solubilized with Triton X-100, and the
IP3R was immunoprecipitated as described under
"Experimental Procedures." The immunoprecipitates were analyzed on
5% SDS-PAGE, and the polypeptides were transferred to nitrocellulose.
Panel A shows the autoradiogram of a representative experiment. Panel B shows the histogram of mean ± S.E.
of data derived from three separate experiments. *, p < 0.01 versus control.
[View Larger Version of this Image (23K GIF file)]
Fig. 6.
Phosphorylation of the type I
IP3R by TGF-1 and forskolin. MMC labeled with
[32P]orthophosphate were incubated with TGF-
1 (10 ng/ml, 15 min) or forskolin (10 µM, 15 min).
Immunoprecipitated IP3R was analyzed on 5% SDS-PAGE,
transferred to nitrocellulose, and exposed to autoradiography.
[View Larger Version of this Image (37K GIF file)]
1 inhibition of [Ca2+]i mobilization (10),
we asked if PKA might be playing a role in TGF-
1 phosphorylation of
the IP3R. Triton X-100 extracts of unlabeled control or
TGF-
1-treated mesangial cells were immunoprecipitated, and the
immunoprecipitates were phosphorylated in vitro after incubation with [32P]ATP and the catalytic subunit of PKA
(Fig. 7A, upper panel). The major
polypeptide phosphorylated in the immunoprecipitates could be shown to
be IP3R by immunoblotting (Fig. 7A, lower
panel). The enhanced phosphorylation of the protein in
TGF-
1-treated mesangial cells in vivo markedly lowered
the incorporation of 32P in the in vitro
phosphorylation assay. Quantitative analysis demonstrates that the
degree of in vitro phosphorylation of the IP3R
by PKA was decreased to 34% of control with 15 min of TGF-
1 treatment (Fig. 7B). This experiment suggests that TGF-
1
stimulates phosphorylation of the IP3R at sites that are
phosphorylated by protein kinase A.
Fig. 7.
Back phosphorylation of IP3R by
PKA in immunoprecipitates prepared from control and TGF-1-treated
cells. Triton X-100 extracts prepared from control and
TGF-
1-treated (10 ng/ml, 15 min) MMC were immunoprecipitated and
back phosphorylated in vitro with the catalytic subunit of
PKA and [
-32P]ATP as described under "Experimental
Procedures." The phosphorylated immunoprecipitates were run out on
5% SDS-PAGE, transferred to nitrocellulose, and autoradiographed
(A, top). The nitrocellulose membrane was then
immunoblotted with IP3R antibody (A,
bottom). A histogram of back phosphorylation results is
shown in B. Data are the mean ± S.E. from four
separate experiments. *, p < 0.01 versus
control.
[View Larger Version of this Image (12K GIF file)]
1 Phosphorylates the Type I IP3R Peptides
RPSGRRESLTSFGNP and ARRDSVLAAS via PKA
1-treated MMC.
Increasing the concentration of the peptide RPSGRRESLTSFGNP from 25 to
400 µM in the in vitro kinase reaction (Fig.
8A) led to an increasing amount of
phosphorylation of the peptide, with a maximal effect at 200 µM. Increasing the time of the kinase reaction from 2 to 15 min enhanced the amount of phosphorylated peptide (Fig.
8B). Prolonged incubation (>15 min) decreased kinase
activity, possibly due to endogenous phophatase activity present in the
lysate. TGF-
1 treatment for 15 min demonstrated enhanced kinase
activity at all concentrations of the peptide and at all durations of
the in vitro kinase reaction. Varying the concentration and
time of the kinase reaction of the peptide ARRDSVLAAS gave similar
relationships (data not shown) as noted for the peptide
RPSGRRESLTSFGNP. For the subsequent experiments, the concentration of
the peptide was 200 µM, and the duration of the kinase
reaction was 5 min. TGF-
1 treatment of MMC for 5 and 15 min
stimulated phosphorylation of the peptide RPSGRRESLTSFGNP by 150 and
158% of control values, respectively (Table I and Fig.
9). Forskolin treatment resulted in slightly greater
phosphorylation (201%) at 5 min but phosphorylation similar to that of
TGF-
1 at 15 min (157%). The addition of the peptide inhibitor of
PKA (PKI) completely prevented the enhanced phosphorylation of this
peptide by both forskolin and TGF-
1 (Fig. 9). Based on the specific
activity of [32P]ATP and the amount of IP3R
peptide added to the reaction mixture, under control conditions
0.038 ± 0.003 mol of phosphate was incorporated per mol of the
IP3R peptide RPSGRRESLTSFGNP. TGF-
1 treatment (10 ng/ml
for 15 min) increased the phosphorylation to 0.059 ± 0.001 mol of
phosphate/mol of the IP3R peptide RPSGRRESLTSFGNP. Although
dilute, crude cell lysates do not appear to contain enough kinase
activity to demonstrate stoichiometric phosphorylation on the
IP3R peptides, this method does allow for comparison of relative kinase activities from treated and untreated cells.
Fig. 8.
Concentration and time course of
phosphorylation of IP3R peptide substrate RPSGRRESLTSFGNP.
In vitro kinase assays were performed as described under
"Experimental Procedures." Panel A shows kinase activity
in pmol of ATP/min with cell lysate from control or TGF-1-treated
(10 ng/ml, 15 min) samples with varying concentrations of the peptide
RPSGRRESLTSFGNP. Panel B shows kinase activity in pmol of
ATP/µg of cell lysate from control or TGF-
1-treated (10 ng/ml, 15 min) samples with 200 µM concentration of the peptide RPSGRRESLTSFGNP and varying the time of the in vitro kinase
reaction. Experiments were repeated twice with essentially the same
results.
[View Larger Version of this Image (13K GIF file)]
1 and
forskolin
1 (10 ng/ml) for 5 and 15 min or forskolin
(10 µM) for 5 and 15 min. Cells were lysed with SDS buffer, and the cell lysate (16 µg) from each sample was added to an
in vitro reaction mixture that included
[
-32P]ATP and IP3R peptide substrate (200 µM). The in vitro reaction time was 5 min.
Data are expressed as the mean ± S.E. of pmol of ATP/min/µg of
protein lysate from three separate experiments.
Condition
RPSGRRESLTSFGNP
ARRDSVLAAS
pmol/min/µg protein
pmol/min/µg protein
Control
0.94 ± 0.06
0.77
± 0.03
TGF-
1 (5 min)
1.40 ± 0.13a
0.93
± 0.03a
TGF-
1 (15 min)
1.47
± 0.01a
0.94 ± 0.06a
Forskolin (5 min)
1.86 ± 0.44a
1.17 ± 0.19a
Forskolin (15 min)
1.48 ± 0.22a
1.02
± 0.09a
a
p < 0.05 versus control
kinase activity with the corresponding IP3R peptide
substrate.
Fig. 9.
In vitro kinase assays with
IP3R peptide substrate RPSGRRESLTSFGNP. MMC were
treated with TGF-1 (10 ng/ml) for 5 and 15 min or forskolin (10 µM) for 5 and 15 min. In vitro kinase assays
were performed as described under "Experimental Procedures" and as
noted in Table I. Cell lysate was added to an in vitro reaction mixture that included [
32P]ATP and
IP3R peptide substrate RPSGRRESLTSFGNP (200 µM) with or without PKI (1 µM). Data are
calculated as percentage increase over control, with the control value
assigned as 100%, and are presented as mean ± S.E. from three
separate experiments. *, p < 0.05 versus
control.
[View Larger Version of this Image (36K GIF file)]
1 treatment of MMC for 5 and 15 min also stimulated
phosphorylation of the peptide ARRDSVLAAS by 121 and 123% of control values (Table I and Fig. 10). Forskolin treatment of
MMC resulted in a greater degree of phosphorylation of this peptide
(153 and 133% at 5 and 15 min, respectively). The enhanced
phosphorylation was reversed if PKI was added to the reaction mix (Fig.
10). These results suggest that TGF-
1-stimulated phosphorylation of
the IP3R peptides is mediated via PKA activation in murine
mesangial cells.
Fig. 10.
In vitro kinase assays with
IP3R peptide substrate ARRDSVLAAS. MMC were treated
with TGF-1 (10 ng/ml) for 5 and 15 min or forskolin (10 µM) for 5 and 15 min. In vitro kinase assays were performed as described under "Experimental Procedures" and as
noted in Table I. Cell lysate was added to an in vitro
reaction mixture that included [
32P]ATP and
IP3R peptide substrate ARRDSVLAAS (200 µM)
with or without PKI (1 µM). Data are calculated as
percentage increase over control, with the control value assigned as
100%, and are presented as mean ± S.E. from three separate
experiments. *, p < 0.05 versus control.
[View Larger Version of this Image (44K GIF file)]
1. TGF-
1 has a potent ability to inhibit the protein expression of the type I
IP3R after 2-4 h of TGF-
1 exposure in glomerular
mesangial cells. The decreased protein expression of the type I
IP3R may be partly due to diminished synthesis of the
IP3R as we found a decrease in mRNA levels prior to
observing a decrease in protein levels. Our finding that TGF-
1
decreases steady-state IP3R mRNA levels can be
explained either by an effect on transcription of the type I
IP3R gene or by enhancement of the degradation rate of the
mRNA and will require further studies.
-untranslated region of the
type I IP3R cDNA in both the mouse and rat (28, 29)
suggests that mRNA stability and/or translation may be subject to
regulation (30, 31). Apart from regulation by TGF-
1 of the mRNA
for the type I IP3R, it is likely that degradation of the
protein is also enhanced by TGF-
1, since we have previously found
the half-life of type I IP3R protein to be 8-11 h
(32).
1 inhibits type I IP3R
expression also demonstrates that factors that do not directly induce a
[Ca2+]i flux may affect IP3R
expression. Carbachol-induced IP3R degradation has been
linked to regulation of functional [Ca2+]i stores
(19), although the mechanism underlying this linkage has not been
demonstrated. TGF-
1 has not been found to directly affect
[Ca2+]i in many cell types evaluated (33, 34),
including mesangial cells (10). Therefore, it is likely that TGF-
regulation of the IP3R differs from the down-regulation
observed with [Ca2+]i-mobilizing agonists.
1-treated cell should be less sensitive to mobilize
[Ca2+]i when exposed to any agonist that
stimulates IP3-mediated [Ca2+]i
mobilization via the IP3R. Thus, our findings may explain
the previous observations that long term exposure (>1 h) to TGF-
1
would inhibit [Ca2+]i mobilization by
isoproterenol (8) and PDGF.2
1 in mesangial and smooth
muscle cells are also sufficient to block agonist-induced [Ca2+]i mobilization (9, 10). This effect would
not be explained by decreased expression of type I IP3R,
since it requires at least 1 h of exposure to TGF-
. The short
term effect may be mediated via TGF-
1-induced phosphorylation of the
IP3R. It has been demonstrated that phosphorylation of the
IP3R may affect its ability to release
[Ca2+]i upon exposure to IP3 (17).
This issue is complex, since there are different lengths of the type I
IP3R in neuronal and nonneuronal tissues that may affect
the site and consequence of phosphorylation (14). Cerebellar type I
IP3R is noted to be in the long form with insertion of an
SII domain, which is situated between the two major serines (serines
1589 and 1755) that are phosphorylated by PKA. Using cerebellar derived
IP3R in liposomes, Cameron et al. (16) recently
demonstrated that PKA-induced phosphorylation impairs
IP3-mediated [Ca2+]i, whereas protein
kinase C-induced phosphorylation of the cerebellar IP3R
enhances IP3-mediated [Ca2+]i
release. PKA-induced phosphorylation of the purified nonneuronal short
form of the type I IP3R primarily occurs on serine 1589 and
on serine 1755 (14). Our studies by PCR determined that, similar to
other peripheral tissues previously examined (13, 14), mesangial cells
and kidney tissue only contain the SII
form of the receptor. The
functional result of nonneuronal PKA-induced IP3R
phosphorylation remains unclear. Our in vitro kinase assays
demonstrate that forskolin treatment of mesangial cells induces
PKA-mediated phosphorylation of both IP3R peptides that
contain serine 1589 and serine 1755. Thus, both sites are potential
phosphorylation sites on the nonneuronal IP3R by PKA. TGF-
1 treatment also resulted in significantly increased
phosphorylation of both peptides, although the degree of
phosphorylation on the peptide containing serine 1755 was twice as much
as the peptide containing serine 1589. It should be noted that prior
studies evaluating the site of IP3R phosphorylation
employed purified kinases and purified IP3R. Our findings
employed mesangial cell-derived kinase preparations that were activated
by agonists in the intact cell. Our studies do not exclude the
possibility that other sites on the type I IP3R may also be
phosphorylated by TGF-
1, which may affect its function.
1 with the type I IP3R probably
leads to important modulatory influences on glomerular mesangial
cells in vivo. Chronic glomerular overexpression of TGF-
has been demonstrated in experimental diabetic kidney disease,
experimental glomerulonephritis, and puromycin-induced nephrosis (1).
In experimental diabetes, other factors including PDGF-BB, fibroblast
growth factor, endothelin, and the renin-angiotensin II system are also
found to be up-regulated (6, 35). The diabetic kidney demonstrates
chronic vasodilation of the afferent arteriole, mesangial cell
stretching, and glomerular hypertrophy without a great degree of
mesangial cell proliferation. Theoretically, TGF-
-induced
IP3R down-regulation may impair angiotensin- and
endothelin-induced smooth muscle cell and mesangial cell contraction and impair PDGF- and fibroblast growth factor-induced mesangial cell
proliferation. Whether this property of TGF-
would be beneficial or
deleterious in contributing to chronic renal disease progression remains to be investigated.
*
This work was supported by National Institutes of Health
Grant KO8 DK02308 (to K. S.), a National Kidney Foundation Young Investigator Award (to K. S.), and by National Institutes of Health Post-doctoral Training Grant T32-AA07463 (to S. B.) and Grant R01-AA10971 (to S. K. J.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Div. of Nephrology,
Dept. of Medicine, Thomas Jefferson University, Suite 353, JAH, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-503-6950; Fax:
215-923-7212; E-mail: sharma1{at}jeflin.tju.edu.
1
The abbreviations used are: TGF, transforming
growth factor; IP3, inositol 1,4,5-trisphosphate;
IP3R, IP3 receptor; PDGF, platelet-derived
growth factor; MMC, murine mesangial cells; PBS, phosphate-buffered
saline; DMEM, Dulbecco's modified Eagle's medium; PKA, protein kinase
A; PKI, protein kinase A inhibitor; PMSF, phenylmethylsulfonyl
fluoride; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase
chain reaction; bp, base pair.
2
K. Sharma, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.