Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
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ABSTRACT |
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We have investigated the role of protein
kinase C (PKC) signal transduction pathways in parathyroid hormone
(PTH) regulation of insulin-like growth factor-binding protein-5
(IGFBP-5) gene expression in the rat osteoblast-like cell line
UMR-106-01. Involvement of the PKC pathway was determined by the
findings that bisindolylmaleimide I inhibited 40% of the PTH effect,
and 1 µM bovine PTH-(3-34) stimulated a 10-fold induction of
IGFBP-5 mRNA. PTH-(1-34) and PTH-(3-34) (100 nM) both
stimulated PKC- translocation from the membrane to the nuclear
fraction. Rottlerin, a PKC-
-specific inhibitor, and a dominant
negative mutant of PKC-
were both able to significantly inhibit
PTH-(1-34) and PTH-(3-34) induction of IGFBP-5 mRNA,
suggesting a stimulatory role for PKC-
in the effects of PTH.
Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-
translocation
from the cytosol to the membrane and inhibited ~50% of the
PTH-(1-34), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-
negatively regulates protein kinase A (PKA)-mediated induction of
IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by
PTH is both PKA and PKC dependent and PKC-
is the primary mediator
of the effects of PTH via the PKC pathway.
insulin-like growth factor-binding protein-5; osteoblast; protein kinase C; messenger RNA; protein kinase A
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INTRODUCTION |
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PARATHYROID HORMONE
(PTH) is an essential mediator of extracellular calcium
homeostasis. Its effects in vivo influence bone turnover to favor bone
resorption (11). However, PTH can also have anabolic
effects on bone if administered intermittently at low doses and is
presently in clinical testing for the treatment of osteoporosis
(24). The effects of PTH in vivo are also observed in
vitro with osteoblasts in culture and are thought to result from the
PTH/PTH-related peptide receptor's ability both to couple to
Gs and stimulate protein kinase A (PKA) and to couple to
Gq/11
and stimulate protein kinase C (PKC) (12,
18, 27). The concomitant stimulation of the PKA and PKC pathway
upon PTH-receptor activation by intact PTH mediates its cellular effects.
PTH has been shown to stimulate the production of insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) (4, 20). IGFs are important regulators of osteoblast metabolism, and their effects include stimulation of collagen production and mitogenesis (9, 16, 21). IGFBPs are thought to regulate the effects of IGFs, and IGFBP-5 specifically has been shown to potentiate the anabolic effects of IGFs, possibly by sequestering IGFs in proximity to their membrane receptors (16). IGFBP-5 has also been shown to act as an osteoblast mitogen independently of IGF, albeit with less potency (1). The effects of IGFs and IGFBP-5 may therefore contribute to the anabolic actions of PTH on bone.
Previous studies have demonstrated that pharmacological agents that stimulate the PKA pathway, such as forskolin and (Bu)2AMP, are able to induce IGFBP-5 transcript levels in both skin fibroblasts and osteoblastic cells (2, 4). Further studies in fibroblasts have indicated a role for activating protein (AP)-2 transactivation in cAMP responsiveness of the IGFBP-5 gene (5), although AP-2 does not appear to mediate PGE2 stimulation of IGFBP-5 transcription in primary osteoblasts (14). The PKC pathway has also been implicated in the stimulation of IGFBP-5 in human fibroblasts (6), and one study utilizing PTH analogs suggested that a pathway other than adenylyl cyclase mediated part of the PTH effect on IGFBP-5 mRNA in osteoblasts (25). It is not clear which PKC isozyme(s) is responsible for the regulation of IGFBP-5 in any of these cell types.
The PKC gene family consists of three subgroups based on sequence
similarity and cofactor dependence: classical PKCs (,
I,
II,
and
), novel PKCs (
,
,
, and
) and atypical PKCs (
and
). Once activated, PKC isozymes translocate to specific cellular compartments, a process that is both isozyme and cell-type specific. Each isozyme is then capable of interacting with its own specific substrates and subsequently mediate distinct cellular events (23, 35). Thus it is important to identify the specific PKC isozymes that mediate signal transduction events, because their effects in many
instances are quite distinct.
To further characterize a role for PKC isozymes in the regulation of
IGFBP-5, in the present study, we have used the well characterized
osteoblastic UMR-106-01 cell line that we and others have used
extensively to study osteoblast regulation (22, 33, 34).
IGFBP-5 mRNA levels are extremely low in this cell line in the
basal state and are increased by stimulation with PTH. We report that
PTH selectively regulates the cellular distribution of PKC- and that
its activation, along with PKA, mediates PTH induction of
IGFBP-5 mRNA.
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MATERIALS AND METHODS |
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Cell culture. UMR-106-01 cells (a generous gift from Dr. N. Partridge, St. Louis University, St. Louis, MO) were grown in 50% Dulbecco's modified Eagle's medium and 50% F-12 medium containing 1 U/ml penicillin, 1 µg/ml streptomycin, and 0.25 µg/ml amphotericin B and supplemented with 5% fetal calf serum (Life Technologies, Burlington, ON, Canada).
Materials. Rat PTH-(1-34), bovine PTH-(1-84), human PTH-(1-31), and bovine PTH-(3-34) were purchased from Bachem Bioscience (King of Prussia, PA). Bisindolylmaleimide I, rottlerin, phorbol 12-myristate 13-acetate (PMA), H-89, forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) were purchased from Biomol (Plymouth Meeting, PA). Isozyme-specific PKC antibodies were purchased from BD Transduction Laboratories (Mississauga, ON, Canada).
Cell treatment and cellular fractionation. After 20-24 h of serum starvation, cells were treated with PTH analogs or PMA (in DMSO) in serum-free media containing 0.1% BSA for 30 min. Cytosol, membrane, and nuclear fractions of cells were prepared by previously described procedures with minor modifications (10). Briefly, cells were washed once with cold PBS and scraped from the plate on ice in 750 µl of buffer A [50 mM Tris · HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 1 mM NaF, and 1 mM sodium orthovanadate]. Cells were allowed to swell on ice for 1 h and were homogenized with 20-60 strokes of a Dounce homogenizer (pestle A) until >80% of the cells were lysed. Homogenates were centrifuged at 600 g for 20 min at 4°C to pellet a crude nuclear fraction. The supernatant was recentrifuged at 100,000 g for 30 min at 4°C to pellet membranes and the supernatant containing the cytosol. The membrane pellet was solubilized in buffer A containing 1% Triton X-100. The crude nuclear pellet was resuspended in buffer A containing 0.1% Triton X-100, layered over 30% sucrose (wt/vol in buffer A), and centrifuged at 5,000 g for 30 min at 4°C. The pellet was resuspended in buffer A containing 1% Triton X-100 and sonicated on ice for 15 s. In one experiment, total cell homogenate was prepared in the following way. Cells were transfected with respective constructs and, 24 h later, were washed once with cold PBS and scraped from the plate on ice with a rubber scraper in 750 µl of buffer B [50 mM Tris · HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, 500 µM AEBSF, 1 mM NaF, 1 mM sodium orthovanadate, 1% (vol/vol) 2-mercaptoethanol, 1% (vol/vol) NP-40, and 0.3% sodium deoxycholate]. Lysates were then sonicated on ice for 15 s. Protein concentrations were determined by the Amido Black method (30).
Western blotting of subcellular fractions. Equal amounts of protein (25-50 µg) of each fraction were separated on 11% SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes overnight at 4°C. Membranes were blocked for 1 h in PBS-0.2% Triton X-100 (PBST) containing 3% BSA. Membranes were then immunoblotted with monoclonal anti-isozyme-specific PKC antibodies in PBST and then probed with horseradish peroxidase-conjugated rabbit anti-mouse IgG antiserum, washed, and detected using an enhanced chemiluminescence detection reagent (Amersham Pharmacia, Baie d'Urfe, QC, Canada) on Kodak X-100 ARS film.
Northern and slot blot analysis.
After 20-24 h of serum starvation in media containing 0.1% BSA,
UMR-106-01 cells were treated for 6 h with PTH or other
agents, and total cellular RNA was isolated with TRIzol reagent (Life Technologies, Burlington, ON, Canada). Having verified by Northern blot
that both the IGFBP-5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes recognized single transcripts, size 6.0 and 1.3 kb,
respectively, we subsequently employed slot blots to assess transcript
levels. Samples (3 µg) of total RNA were directly blotted onto a
nylon membrane by means of a slot blot apparatus and then ultraviolet
cross-linked. Blots were prehybridized for 1 h in 0.1 M sodium
phosphate buffer containing 0.1% BSA, 9 mg/ml salmon sperm DNA, and
7% SDS at 65°C. The 300-bp IGFBP-5 cDNA probe was obtained by
digesting IGFBP-5 cDNA (kindly provided by Dr. S. Shimasaki, The
Whittier Institute for Diabetes and Endocrinology, La Jolla, CA) with
HindIII and SacII (31). The probe
was labeled with [
-32P]dCTP by use of the random
hexanucleotide-primed method (7). Hybridizations were
carried out in the prehybridization solution overnight at 65°C, and
washes were performed at 65°C in 30 mM sodium phosphate buffer
containing 0.1% SDS. As an internal control, parallel blots were
prepared and hybridized under the same conditions with a 950-bp GAPDH
cDNA probe, obtained by digesting GAPDH cDNA with
SflI and BstEII, as previously
described (8). Bound RNA was visualized by autoradiography
on Kodak X-AR5 film. Signals were quantitated from a phosphorimager
using ImageQuant.
Transient transfections.
The mouse dominant negative PKC- K376R (DNPKC-
), and PKC-
K376R (DNPKC-
) constructs were kindly provided by Dr. I. B. Weinstein (Columbia University, New York, NY) (32). Cells
were grown to 60-70% confluence in 6-well plates over 48 h
and transfected with DNPKC-
, DNPKC-
, or vector alone (pcDNA
3.1+) with Lipofectamine reagent (Life Technologies,
Burlington, ON, Canada). Two micrograms of DNA and 8 µl of
transfection reagent were used for each well in serum-free media. After
a 24-h incubation, cells were treated with PTH or other agents for an
additional 6 h, and then RNA was isolated and assessed as
outlined. Transfection rates of ~30% were found using this protocol
and were assessed as follows. Cells were transfected with the plasmid
pSV-
-gal (Promega, Madison, WI), which encodes the reporter gene
-galactosidase. Twenty-four hours after transfection, cells were
fixed in 3% formaldehyde (in PBS) for 15 min at room temperature,
washed three times with PBS, and then stained with X-gal-containing
solution (in PBS: 4 mM K+ ferrocyanate, 4 mM K+
ferricyanate, 4 mM MgCl2, and 0.4 mg/ml X-gal in dimethyl
fluoride) for 2 h at 37°C. Transfection rates were determined as
the percentage of blue-stained cells compared with the total number of cells.
Presentation of data and statistical analysis. The slot blots presented are representative of at least three experiments. The quantitated values of IGFBP-5 mRNA obtained from individual samples were corrected for GAPDH. Statistical analysis was performed by Student's t-test. The results are expressed as means ± SD.
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RESULTS |
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Mechanisms of PTH-mediated induction of IGFBP-5 mRNA.
Previous studies suggested that part of the induction by PTH of IGFBP-5
mRNA in osteoblasts occurs through pathways other than PKA; however,
the involvement of the PKC pathway has not been investigated. We
utilized PTH fragments that selectively stimulate the PKC or
PKA-plus-PKC pathways to determine their relative
contribution to PTH effects on IGFBP-5 transcript. As demonstrated in
Fig. 1A, under basal
conditions, there were very low levels of IGFBP-5 transcript as
assessed by phosphorimaging. PTH-(1-34), PTH-(1-84), and
PTH-(1-31), each of which can stimulate PKA and PKC, all
stimulated a 30- to 45-fold induction of IGFBP-5 mRNA levels.
PTH-(3-34), an analog that has been shown to stimulate PKC but not
PKA, stimulated a 5- to 20-fold induction of IGFBP-5, suggesting that
PKC activation mediates part of PTH stimulation of IGFBP-5 transcript
levels.
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Effects of PTH-(1-34), PTH-(3-34), and PMA on PKC isozyme cellular distribution. Our initial findings suggested that IGFBP-5 mRNA is stimulated by PTH partially via the PKC pathway. However, direct activation of PKC by PMA had no effect. Because there are 11 members of the PKC family and only a subset of these is stimulated by PMA, we wished to determine which isozymes were activated by PTH or PMA.
Activated PKC isozymes have been shown to translocate from one intracellular location to another (26). Therefore, translocation can be used to identify which isozyme(s) could mediate the PTH downstream effects. Cytosol, membrane, and nuclear fractions of UMR-106-01 cells that were untreated (control) or incubated with PTH-(1-34), PTH-(3-34), or PMA were prepared, and the quantity of PKC isozymes present in each fraction was assessed by immunoblotting with isozyme-specific antibodies. Figure 2 demonstrates the distribution of representatives of each of the PKC subfamilies (PKC-
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Involvement of PKC- in PTH-mediated stimulation of IGFBP-5 mRNA.
Having established the ability of PTH to regulate the cellular
distribution of PKC-
, we next investigated whether this isozyme mediated part of the PTH induction of IGFBP-5 transcript. Cells were
treated with PTH-(1-34) or PTH-(3-34) alone or in the
presence of the PKC-
-specific inhibitor rottlerin. When administered
alone, rottlerin had no effect on IGFBP-5 mRNA levels (data not shown). Increasing concentrations of rottlerin progressively suppressed PTH-(1-34) induction of IGFBP-5, such that 5 µM rottlerin
inhibited ~45% of the PTH signal (Fig.
4A). Rottlerin was similarly
able to inhibit PTH-(3-34) induction of IGFBP-5 mRNA (Fig.
4B).
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Effect of PMA on PKA-induced stimulation of IGFBP-5 mRNA.
Our PKC isozyme translocation studies indicated that PMA stimulated
the translocation of PKC- and downregulated PKC-
. The lack of
effect of PMA alone on IGFBP-5 mRNA levels suggested that PKC-
does
not stimulate IGFBP-5 transcription (Fig. 1). We also investigated
whether PMA could affect IGFBP-5 gene expression stimulated by PKA.
UMR-106-01 cells were treated with 5 µM forskolin, 1 mM
8-BrcAMP, or 10 nM PTH-(1-34) for 6 h in the presence or absence of 1 µM PMA. PMA blocked 50% of PTH-, forskolin-, and 8-BrcAMP-induced stimulation of IGFBP-5 transcript (Fig.
6).
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DISCUSSION |
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In the present report, we have demonstrated that both the PKA and PKC pathways mediate PTH stimulation of IGFBP-5 mRNA levels in UMR-106-01 osteoblastic cells. The functional involvement of PKA in PTH induction of IGFBP-5 is demonstrated in the present study by partial inhibition in the presence of H-89. However, this PKA inhibitor could not suppress 40% of PTH activation of IGFBP-5. Evidence that the PKC pathway mediated this additional PTH-stimulated IGFBP-5 came from several observations. First, the selective PKC inhibitor bisindolylmaleimide I partially blocked PTH stimulation of IGFBP-5. Second, PTH-(3-34), which is not able to stimulate adenylyl cyclase in UMR cells, was able to significantly stimulate IGFBP-5.
Because there was very little evidence for PKC regulation of IGFBP-5
transcription in the literature, we proceeded to investigate the
regulation of the activity of PKC isozymes in UMR-106-01 cells. It
has been reported by Sanders and Stern (29) that the
related UMR-106 cell line expresses eight PKC isoforms, including
PKC-, -
I, -
II, -
, -
, -
, -
, and-
. We were unable
to detect expression of either the
- or
-isoforms in the
106-01 cells, likely due to differences in the cell lines or the
antibodies used in the two studies. We found that PTH-(1-34) and
PTH-(3-34) had no effect on the distribution of PKC-
, -
, and
-
but were able to stimulate translocation of PKC-
to the
nucleus, with a corresponding decrease in the membrane fraction over a
10- to 40-min time course. Others have previously reported that PKC-
is translocated to the nucleus, in a number of different cell lines.
For example, Wang et al. (35) demonstrated by means of
fluorescence microscopy that bryostatin stimulated the rapid
translocation of PKC-
to the nucleus in Chinese hamster ovary cells.
Translocation occurs after PKC activation, and peptide inhibitors of
PKC isozyme translocation act as antagonists of PKC activity
(15). To directly demonstrate the involvement of PKC-
in PTH-(1-34) stimulation of IGFBP-5, we blocked the kinase
activity of PKC-
with the selective inhibitor rottlerin at
concentrations between 0.5 and 5 µM. Rottlerin is a natural toxin
that has been shown to inhibit PKC-
with an IC50 of
3-6 µM by interaction with the ATP-binding domain
(36). Other PKC isozymes and PKA are inhibited at higher
rottlerin concentrations of 30-100 µM, but the concentrations of
rottlerin used in our assays were too low to affect these kinases. The
only other kinase reported to be significantly inhibited by this
compound in the low micromolar range is calmodulin kinase III
(rottlerin IC50 of 5.3 µM). Therefore, we used a dominant
negative PKC-
mutant, DNPKC-
(K367R), to discriminate further the
role of PKC-
in PTH effects on IGFBP-5. DNPKC-
significantly
blocked the PTH regulation of IGFBP-5, whereas DNPKC-
had no effect
on PTH-(1-34) induction of IGFBP-5. PTH-(3-34) stimulation of
IGFBP-5 transcript was also partially inhibited by both rottlerin and
DNPKC-
. The failure of these agents to completely inhibit the
PTH-(3-34) effect suggests that a fraction of the PTH-(3-34)
regulation of IGFBP-5 transcript is mediated by a pathway that is both
PKA and PKC-
independent. There are a number of signaling pathways
that could be stimulated by G
subunits released after
PTH-(3-34) stimulation of Gq/11
through the PTH
receptor (3). The IGFBP-5 gene has been shown to be under
the transcriptional control of a plethora of transactivating factors
and, subsequently, a large number of signaling pathways
(28). Thus
-subunits released by PTH-(3-34) could potentially mediate the stimulation of a pathway(s) whose activity also regulates IGFBP-5.
Previous attempts to investigate whether the PKC pathway was involved
in PTH regulation of IGFBP-5 were performed by attempting to mimic the
PTH effect with PMA. Nasu et al. (25) found that PMA was
unable to mimic PTH stimulation of IGFBP-5 transcript. We also found
that PMA could not induce IGFBP-5 mRNA; furthermore, it inhibited the
stimulation of IGFBP-5 by PTH-(1-34). Because PMA selectively
activated PKC- in our cells, as evidenced by its translocation to
the cell membrane, PKC-
appears to have an inhibitory effect on
IGFBP-5 transcription. Our demonstration that PMA is also able to
partially abrogate the stimulation of IGFBP-5 mRNA induced by forskolin
or 8-BrcAMP indicates that the inhibitory effects of PMA occur through
cross-regulation of the PKA pathway downstream of adenylyl cyclase. The
inhibitory effects of PMA were also directly demonstrated in
osteoblast-enriched cultures from fetal rat calvaria, where PMA was
shown to inhibit basal expression of IGFBP-5 mRNA (28).
PMA could also be regulating IGFBP-5 through the downregulation of
PKC-
; however, this seems unlikely, given that induction of IGFBP-5
by forskolin and 8-BrcAMP likely occurs by PKA activation independently
of PKC-
regulation.
IGF-I has been shown to act through phosphatidylinositol 3-kinase to regulate IGFBP-5 gene expression in vascular smooth muscle cells (6). Osteoblasts also produce IGF-I, and PTH stimulates its synthesis through a PKA-dependent mechanism. We found that, after 6 h of treatment with exogenously added IGF-I, IGFBP-5 transcript levels were not changed in our cells (M. S. Erclik, unpublished observations), a finding consistent with previous reports by others (4). Thus it is unlikely that there is an IGF-I component to the PTH-induced accumulation of IGFBP-5 mRNA under our assay conditions.
Because there were very low amounts of the transcript in unstimulated cells, it is likely that PTH induces IGFBP-5 mRNA through transcriptional mechanisms. The IGFBP-5 promoter has been cloned and well characterized. A number of AP-2 consensus binding sites have been identified throughout the length of the promoter, and studies in human fibroblasts have demonstrated AP-2 regulation of IGFBP-5 gene expression (5). Because AP-2 has been shown to mediate transcription events in response to both PKA and PKC activation (13), it is an attractive hypothesis to suggest that PTH-stimulated PKC activation works through AP-2 to stimulate IGFBP-5 gene expression. Further studies to characterize the molecular mechanisms of regulation of the IGFBP-5 promoter by PTH are necessary to lend insight into the complex regulation by both PKA and PKC of IGFBP-5 gene expression.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Canadian Institute of Health Research of Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Mitchell, Dept. of Pharmacology, Univ. of Toronto, 1 King's College Circle, Rm. 4342, Toronto, ON, M5S 1A8 CANADA (E-mail: jane.mitchell{at}utoronto.ca).
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.
10.1152/ajpendo.00417.2001
Received 18 September 2001; accepted in final form 24 October 2001.
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