From the Departments of Biochemistry and
¶ Medicine, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-0146
Received for publication, January 19, 2001
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ABSTRACT |
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To explore the functional role of
phospholipase C- Receptor-tyrosine kinases
(RTKs)1 transduce
extracellular signals to the nucleus provoking gene expression and
thereby orchestrating cell responses, such as mitogenesis. Growth
factor receptor signaling rapidly results in the transcription of IEGs,
which in many cases direct the synthesis of proteins that participate
in signaling pathways (1, 2). RTKs mediate these processes by
activating, following ligand binding, a variety of intracellular
signaling pathways through their intrinsic kinase activity (3).
PLC- PLC- The biological significance of PLC- Materials--
Dulbecco's modified Eagle's medium (DMEM)
containing L-glutamine and high glucose was purchased from
Life Technologies, Inc. Recombinant rat PDGF-BB was obtained from R&D
Systems, Inc, whereas [ Cell Culture and PDGF Treatment--
Spontaneously immortalized
TV-1 Plcg1-null fibroblasts were established from
Plcg1 knockout embryos (8), and PLC- Western Blotting--
After treatment without or with PDGF,
cells were lysed in TGH buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.2) supplemented with 100 mM
NaCl, protease inhibitor mixture tablets at 1 tablet/10 ml (Roche) and
1 mM Na3VO4. After centrifugation, lysate aliquots (100 µg protein) were subjected to gel
electrophoresis (10% SDS-polyacrylamide gel electrophoresis),
transferred to nitrocellulose membranes (Osmonics), and probed with the
indicated antibody. Bound antibody was detected by enhanced
chemiluminescence (ECL).
Northern Blotting--
Total RNA was isolated using a
TRIzol® Reagent according to the manufacturer's
instructions (Life Technologies, Inc). For each data point, an aliquot
(5 µg) of total RNA was electrophoresed, transferred to
Duralon-UVTM membranes (Stratagene, Inc), and probed with a
labeled cDNA fragment of each IEG according to standard procedures.
A probe for GAPDH or cyclophilin was used as
internal control. The probes were labeled with
[ PDGF-dependent Signaling--
To examine the potential
significance of PLC-
To characterize these cells for PDGF-dependent signaling,
we first established that Null and Null+ cells express equivalent levels of PDGF PDGF-dependent Induction of IEGs--
To investigate
the induction of IEGs in PDGF-stimulated Null and Null+ cells, we
assembled a collection of probes for 14 previously characterized IEGs.
Whereas the total number of IEGs expressed in growth factor-stimulated
fibroblasts exceeds 50 (15), this subset was selected for analysis by
the availability of probes and was not based on previously published
data related to their signaling requirements or promoter elements. At
various times after the addition of PDGF to Null or Null+ cells, the
mRNA level of each IEG was determined by Northern blotting. The
results for all 14 IEGs are presented in Table
I. The influence of PLC-
The data in Table I are derived from one set of Null and Null+ cell
lines, termed TV-1 (8). To substantiate that the observed differences
do represent the absence of PLC-
Those genes whose mRNA expression is compromised most by the
absence of PLC-
Lastly, we have assessed the individual capacities of PMA and ionomycin
to induce the accumulation of these five IEG mRNAs in Null cells.
The results are presented in Table II. In
the case of JE mRNA, PMA or ionomycin separately
produced significant levels of mRNA accumulation and the combined
treatment with both agents was approximately the sum of the individual
treatments. In the remaining cases, PMA and ionomycin gave a
synergistic response compared with mRNA accumulation provoked by
PMA or ionomycin alone. For JE, COX-2, and
KC mRNAs, PMA induction was significantly greater than
achieved by ionomycin, whereas c-fos and FIC
mRNA accumulation was small, but approximately equivalent for
either agent alone.
The role of PLC- Mutagenesis of Tyr-1021 in the PDGF- There are limitations to interpretations in the approaches described
above, which mainly revolve around the issue of specificity for the
inhibition of PLC- Therefore, we have used cell lines in which the Plcg1 gene
has been selectively disrupted and have then re-expressed PLC- In antigen-activated T cells, PLC- In regard to the expression of c-fos mRNA, we have
previously reported that EGF induces the mRNA for this IEG in
Plcg1+/+ and Plcg11 (PLC-
1) in the induction of immediate early
genes (IEGs), we have examined the influence of Plcg1 gene
disruption on the expression of 14 IEG mRNAs induced by
platelet-derived growth factor (PDGF). Plcg1-null embryos
were used to produce immortalized fibroblasts genetically deficient in
PLC-
1 (Null cells), and retroviral infection of those cells was used
to derive PLC-
1 re-expressing cells (Null+ cells). In terms of PDGF
activation of PDGF receptor tyrosine phosphorylation as well as the
mitogen-activated protein kinases Erk1 and Erk2, Null and Null+ cells
responded equivalently. However, the PDGF-dependent
expression of all IEG mRNAs was diminished in cells lacking
PLC-
1. The expression of FIC, COX-2, KC,
JE, and c-fos mRNAs were most strongly
compromised, as the stimulation of these genes was reduced by
more than 90% in cells lacking PLC-
1. The combination of PMA and
ionomycin, downstream analogs of PLC activation, did provoke expression
of mRNAs for these IEGs in the Null cells. We conclude that
PLC-
1 is necessary for the maximal expression of many PDGF-induced
IEGs and is essential for significant induction of at least five IEGs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is a direct tyrosine kinase substrate for most RTKs (4, 5). Following ligand binding, RTKs dimerize and autophosphorylate tyrosine
residues, which then serve as association sites for PLC-
1, facilitating its tyrosine phosphorylation and activation (3-5). Growth
factors are not known to activate PLC-
or PLC-
isoforms.
1 activity catalyzes the hydrolysis of phosphatidylinositol
4,5-bisphosphate to produce the second messenger molecules inositol
1,4,5-trisphosphate and diacylglycerol (6, 7). The former provokes a
transient increase in intracellular free Ca2+
([Ca2+]i), whereas the latter
serves as a direct activator of protein kinase C. However, the
mechanism by which PLC-
1 activity participates in the mitogenic
response of cells to growth factors is not understood (4, 5). Based on
a variety of experimental approaches, some reports have concluded that
in cell culture systems, PLC-
1 activation is not essential for
growth factor induced mitogenesis, whereas other approaches have
produced the opposite conclusion (5). In the mouse knockout of
Plcg1, the null phenotype is embryonic lethal (E9.0),
indicative of an essential role in embryonic cell proliferation (8).
PLC-
1 is expressed in many tissues and cell lines, whereas the
PLC-
2 isoform is specifically expressed in hematopoietic tissues
(9). The Plcg2 mouse knockout does not have a lethal
phenotype (10), indicating that these two PLC-
isoforms do not have
a redundant function in vivo. A Drosophila PLC-
has been identified, and mutational analysis indicates that it
regulates wing vein development and may function as a negative regulator of RAS (11).
1 in T cells activated by antigen
is much clearer. In these cells PLC-
1 function is essential for the
induction of IL-2 plus other IEGs and
antigen-dependent T cell proliferation (12, 13). To explore
the functional role of PLC-
1 in the transcription of IEGs in growth
factor-dependent fibroblast proliferation, we have examined
the influence of Plcg1 disruption on the expression of
multiple IEGs induced by PDGF in mouse embryo fibroblasts.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP was purchased from
New England Life Science Products. Antibodies to PDGF-
receptor and
dual phosphorylated Erk1 and 2 were from Santa Cruz Biotechnology, Inc.
Antibody to phosphotyrosine was from Zymed Laboratories
Inc. Phorbol-12-Myristate-13-Acetate (PMA) and ionomycin were
purchased from Sigma. KC and JE cDNAs were purchased from the American Type Culture Collection.
COX-2/TIS 10 cDNA was a gift from Dr. Harvey
Herschman, University of California at Los Angeles. FIC
cDNA was a gift from Dr. Rolf-Peter Ryseck, Bristol-Myers Squibb
Pharmaceutical Research Institute. Gly96, Nup475, mTF, MKP-1, Cyr61,
JunB, Pip92, Nur77, and
Zif268 cDNAs were gifts from Dr. Lester F. Lau,
University of Illinois at Chicago College of Medicine.
cDNAs for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were gifts from Dr. Ronald Wisdom,
Vanderbilt University, whereas cyclophilin cDNA was a
gift from Dr. Chuanming Hao, Vanderbilt University.
1 was subsequently
re-expressed in these cells by retroviral infection as previously
described (14) to derive Plcg1 Null+ cells. Null+ and Null
cells were cultured in DMEM containing 10% fetal calf serum.
Subconfluent cells were incubated for 36 h in DMEM plus 0.5%
fetal calf serum prior to stimulation by PDGF-BB (25 ng/ml) or other
agonists for the indicated times.
-32P]dATP using Prime-It II random primer labeling
kit (Stratagene, Inc). The intensities of bands in Northern blots were
quantified using an imaging densitometer model GS-670 (Bio-Rad) and
standardized with GAPDH or cyclophilin. All
probes were mouse cDNAs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in the stimulation of IEG mRNA levels, we
have employed spontaneously immortalized Plcg1
/
fibroblasts that were derived from
targeted disruption of the Plcg1 gene in mice (8). These
cells do not increase
[Ca2+]i in response to
growth factors (8, 14) and are referred to herein as Null cells.
Following immortalization, retroviral infection was employed to
re-express PLC-
1 in the Null cells (14). The resulting cells, which
do mobilize Ca2+ in response to growth factors, are termed
Null+ cells. The level of PLC-
1 expressed in the Null and Null+
cells is shown in Fig. 1A.
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Fig. 1.
Characterization of signaling components in
Null and Null+ cells. A, aliquots (100 µg) of lysates from
Null and Null+ cells Western blotted with antibody to PLC- 1. B, quiescent cells were incubated without or with PDGF (25 ng/ml) for 5 min, and cell lysates (1 mg) were precipitated with
anti-PDGF-
receptor. Subsequently, the samples were blotted with
anti-phosphotyrosine, stripped, and reprobed for PDGF-
receptor.
C, quiescent cells were stimulated for the indicated times
with PDGF, and lysate aliquots (100 µg) were blotted with antibody to
dual phosphorylated Erk1 and 2, stripped, and then reprobed for Erk1
and 2.
-receptors and that they respond in a comparable manner to the addition of PDGF-BB. The data in Fig. 1B show
that both cell lines do express approximately the same amount of PDGF
-receptor and that these receptors are activated to similar levels by the addition of PDGF. Also, we have compared the
PDGF-dependent activation of Erk1 and 2 in the Null and
Null+ cell lines. As shown in Fig. 1C, these MAP kinase
isoforms are activated to similar levels with comparable kinetics
regardless of the presence or absence of PLC-
1. Hence, these studies
indicate that PDGF signal transduction is not compromised in unexpected
ways by the absence of PLC-
1.
1 on the level
of each mRNA was established by comparing the amount of mRNA at
the peak induction times for both cell lines. In no case did the
absence of PLC-
1 alter the time of peak mRNA expression following the addition of PDGF. The results show that for each mRNA, the absence of PLC-
1 had an attenuating influence that ranged from ~50% (Zif268/egr-1,
Nur77, Pip92) to over 90% (FIC, COX-2, JE, KC, c-fos).
These data indicate that PLC-
1 has a role of varying magnitude in
the PDGF-dependent induction of many IEGs.
Immediate early genes induction by PDGF-BB stimulation of Null+ and
Null cells
1, a second independent set of Null
and Null+ cells, known as TV-II (8), were assayed for the
PDGF-dependent induction of five IEGs. These genes and the
level of induction in the absence of PLC-
1 are as follows: COX-2 (1%), KC (6%), c-fos (4%),
JunB (35%), and zif268 (47%). Comparison
of these results to those reported in Table I for TV-I-derived cells
indicates quantitatively similar data were obtained for both TV-I and
TV-II Null and Null+ cell lines.
1 were further studied by determining whether the
addition of PMA and ionomycin, which represent downstream pharmacologic
mimetics of PLC function (6, 7), to Null cells could induce the
accumulation of these IEG mRNAs. The data in Fig.
2 show Northern blot analyses of the
mRNA induction time course for FIC, COX-2,
c-fos, KC, and JE in PDGF-treated Null and Null+ cells, as well as PMA plus ionomycin-treated Null cells. In
each case, the addition of PMA and ionomycin restored mRNA accumulation in the Null cells, indicating that these genes are fully
inducible in the Null cells by pharmacologic replacement of PLC
function. In most cases, the time course of mRNA accumulation in
the presence of PMA and ionomycin was slightly delayed relative to that
produced by PDGF. In the case of COX-2, mRNA levels
accumulated to a greater extent following the addition of PMA and
ionomycin compared with PDGF. In contrast, KC mRNA
accumulation following treatment with PMA and ionomycin was less than
that achieved by PDGF. Hence, both the time course and quantity of
mRNA accumulation were somewhat different for PDGF compared with
PMA plus ionomycin induction of these genes.
View larger version (53K):
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Fig. 2.
Induction of FIC,
COX-2, c-fos, KC,
and JE mRNAs by PDGF or PMA plus
ionomycin. Quiescent cells were prepared as previously described.
After the addition of PDGF (25 ng/ml) to Null+ and Null cells, or PMA
(100 ng/ml) plus ionomycin (2 µM) addition to Null cells
for the indicated times, equal aliquots (5 µg) of total RNA were
electrophoresed, transferred to membranes, and probed with a labeled
cDNA fragment for each IEG. A probe for cyclophilin
(cycl) or GAPDH (GA) was used as internal
control.
Influence of PMA and/or ionomycin on the induction of immediate early
genes in Null cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in the induction of one IEG,
c-fos, by PDGF or epidermal growth factor (EGF)
receptor-tyrosine kinases has previously been examined in other
laboratories using strategies that involve either a dominant-negative
fusion protein/antibody inhibition approach or an approach that
exploits the specificity of Tyr-1021 of the PDGF
-receptor for
PLC-
1 association and activation. Using microinjection of either a
fusion protein of glutathione S-transferase with one SH2
domain from PLC-
1 or antibody to PLC-
1, Roche et al.
(16) reported that expression of a
-galactosidase reporter
containing the c-fos promoter was decreased by ~90% in PDGF-stimulated NIH 3T3 cells. They also demonstrated that these reagents blocked PDGF-dependent induction of DNA synthesis.
Analogously, Wang et al. (17) microinjected a fusion protein
of glutathione S-transferase with both SH2 domains plus the
SH3 domain of PLC-
1 into NIH 3T3 cells and reported that expression
of a c-fos serum response element reporter construct was
blocked in PDGF-treated cells. Similar results were reported with these
reagents in EGF-treated MDCK cells. This group also reported that the
EGF-dependent or PDGF-dependent initiation of
DNA synthesis in these cells was blocked by microinjection of the
dominant-negative protein. These papers, therefore, indicate that
PLC-
1 is necessary for PDGF- or EGF-dependent expression
of the c-fos promoter and for the induction of DNA synthesis
in quiescent cells.
receptor abrogates PLC-
1
association with the receptor, and this prevents tyrosine phosphorylation and activation of this signaling protein (18-21). However, PDGF-induced mitogenesis is either not impaired (18) or is
slightly decreased (21) by this receptor mutation, leading to the
conclusion that PLC-
1 is dispensable for PDGF-dependent mitogenesis. However, this receptor mutant (Y1021F) has not been employed to assess the role of PLC-
1 in the induction of any IEG. In
a related approach, termed an "add back" strategy (22), the data
were interpreted to indicate that the PLC-
1 pathway in the absence
of several other, but not all, PDGF receptor-dependent signaling pathways can support the induction of several IEG mRNAs, including c-fos, KC, and JunB (23).
However, another study of these PDGF receptor add back mutants
concluded that the PLC-
1 specific Tyr-1021 site, could support the
expression of KC mRNA but not other IEG mRNAs
(24).
1 function and/or the extent to which apparent
PLC-
1 regulation of reporter constructs accurately reflects regulation of endogenous mRNA expression. Also, differences in cell
lines may account for some of the contradictory results obtained with
various experimental approaches. The interpretation of add back mutant
data are complex, as the strategy is meant to reveal what downstream
responses can be activated through PLC-
1 in the absence of other
potentially supportive pathways.
1 in
these cells to compare a closely related set of cells in which PLC-
1
is either absent or present and in which other
PDGF-dependent signaling pathways have not been altered. A
major finding is that the absence of PLC-
1 attenuates to varying
extents the PDGF-dependent accumulation of all IEGs
studied. This suggests a wide role for PLC-
1 in IEG signaling
downstream of the PDGF receptor. Whereas the data presented in this
manuscript argue for a significant role of PLC-
1 in the induction of
multiple IEG mRNAs, it is highly likely that other signaling
pathways independent of PLC-function, such as the Ras/MAP kinase
pathway, also participate in the maximal induction of these IEGs.
isoforms are thought to be
essential for the downstream activation of multiple IEGs, such as
IL-2 (12, 13). However, that information is somewhat
indirect as it relies on the capacity of immunosuppressive agents, such as cyclosporin, to block the Ca2+-sensitive phosphatase
calcineurin. Recently it has been reported that immunosuppressive
agents, including cyclosporin, also interfere with T cell
receptor-dependent activation of Jun kinase and p38 pathways in a calcineurin-independent manner (24). This illustrates the
difficulty of specifically interrupting signal transduction pathways
with pharmacologic inhibitors.
/
cells (25). This implies that c-fos induction by EGF is not reliant on PLC-
1. This observation has been confirmed comparing the
Null and Null+ cells employed in this
study.2 Therefore, at least
for induction of this one IEG, the EGF and PDGF receptors display a
distinctly different requirement for PLC-
1. Also, we have measured
the PDGF-dependent incorporation of
[3H]thymidine in quiescent serum-starved Null and Null+
cells. The stimulation of thymidine incorporation was approximately
equivalent (about 3-fold) for both cell lines. Hence, maximal
expression of many IEGs does not seem to be essential for
PDGF-dependent entry of these cells into S phase.
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ACKNOWLEDGEMENT |
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We thank David Coon for technical assistance.
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FOOTNOTES |
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* This work was supported by Grant CA75195 from the National Institutes of Health.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.
§ Present address: Onyx Pharmaceuticals Inc., Richmond, CA 94806.
To whom correspondence should be addressed. Tel.:
615-322-6678; Fax: 615-322-2931; E-mail:
graham.carpenter@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.C100030200
2 H-J. Liao and G. Carpenter, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: RTKs, receptor-tyrosine kinases; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IEG, immediate early gene; MAP kinase, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PLC, phospholipase C; PMA, phorbol-12-myristate-13-acetate.
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