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(Received for publication, May 15, 1995; and in revised form, August
7, 1995) From the
Previous in vivo and in vitro studies suggest
that insulin-like growth factor (IGF-I) could be a regulator of the
renal production of 1,25-(OH)
Kidney is the main site of 25-hydroxyvitamin D A
major question which remains is that of the mechanism of the IGF-I
action on the renal metabolism of vitamin D. Two main intracellular
pathways appear to be involved for the regulation of the 1-OHase
system. The first one, via the activation of cAMP-dependent kinases, is
thought to be the main route of the PTH-induced stimulation of the
1,25-(OH) Involvement of the PKA
pathway in the IGF-I action on the renal vitamin D metabolism is
unlikely as IGF-I is not known to influence cAMP synthesis in other
cell systems(15) . In the opposite, protein kinase C could be a
reasonable candidate to mediate the effect of IGF-I for the following
reasons: 1) IGF-I stimulates the PKC activity in several cell
systems(26, 27) ; 2) IGF-I increases
1,2-diacylglycerol production and calcium entry into the cell, two
potential activators of
PKC(26, 28, 29, 30) ; 3) H7 or
down-regulation of PKC inhibits some of the IGF-I effects, like the
activation of the cell replication cycle in the rat astrocyte
cells(27) . Alternatively, a third pathway could be at work
to mediate the IGF-I-induced stimulation of the
1,25-(OH) We have now developed a new in vitro model of mouse
proximal tubular cells in primary culture that produces
1,25-(OH)
Fractions coeluting with synthetic
1,25-(OH)
Figure 1:
Capacity of the
1,25-(OH)
The kidney
cells produced detectable amounts of 1,25-(OH)
Figure 2:
Effect of IGF-I on the kinetics of 1-OHase
in mouse kidney cells. A, cells pretreated for 18 h with 50
ng/ml IGF-I (open squares) or its vehicle (closed
squares) were incubated with increasing concentrations of
[
Figure 3:
IGF-I specific binding to intact cultured
mouse kidney cells. Cells, approximately 50 µg of protein per well,
were preincubated in hormone and phosphate-free medium for 18 h and
incubated for 2 h at room temperature with 50,000 dpm of
Figure 4:
A, effect of IGF-I on sodium-dependent
phosphate transport by mouse kidney cells. Cells were incubated for 18
h with either IGF-I at the indicated concentrations or its vehicle.
Values are means ± S.E. of 3-5 experiments, each done in
triplicate. B, dose-response effect of IGF-I on
1,25-(OH)
This IGF-I effect
required incubation of the cells for 18 h to be significant (Fig. 5). It was not observed when cycloheximide (1
µM) had been added to the cells 1 h prior to incubation
with IGF-I (Fig. 6). This stimulation of the 1-OHase does not
appear to result from a nonspecific increase in protein synthesis
linked to the mitogenic action of IGF-I, as total protein contents were
similar in IGF-I-treated and untreated cells, 45 ± 2.6 and 51
± 1.9 mg/well, respectively.
Figure 5:
Time course effect of IGF-I on the
1,25-(OH)
Figure 6:
Effect of cycloheximide (Cyclo)
on IGF-I-stimulated 1,25-(OH)
In contrast, incubation of the
cells for 18 h with bovine insulin did not stimulate
1,25-(OH)
Figure 7:
Effect of PKC activity on 1-OHase
regulation. A, cells were incubated for 18 h with IGF-I or its
vehicle. PMA was added for 18 h or for the last hour of incubation. B, cells were pretreated for 18 h without (Control)
or with calphostin C (1 nM) or GF109203X (1 µM).
IGF-I (50 ng/ml) or its vehicle was added 30 min later to the cultures.
5 nM [
We then tested
the possible influence of extracellular calcium on the observed IGF-I
action and found that external calcium is required for this action.
IGF-I stimulated 1-OHase when added to the cells in 1 or 0.5 mM calcium (Table 2). But no IGF-I effect was observed anymore
when using a calcium-depleted medium (0.05 mM). Of interest,
the calcium-depleted medium did not alter the morphology of the cells
or their protein content. It also did not influence the effect of human
1-34 PTH (Calbiochem), 5
Figure 8:
Comparison of the PTH and IGF-I effects in
relation to extracellular calcium. Cells were preincubated for 18 h in
1 mM (A) or 0.05 mM (B) calcium
before the 1-OHase assay. In order to obtain a maximal response, human
IGF-I (&cjs2100;) (50 ng/ml) was added at the beginning of this
preincubation, i.e. 18 h before the assay, and human
1-34 PTH (&cjs2112;) (5
Finally, we tested
the effect of verapamil, a specific calcium channel blocker. Addition
of 100 µM verapamil to the cells 1 h before that of IGF-I
abolished the 1-OHase response to IGF-I (Fig. 9A).
Figure 9:
A, effect of a specific calcium channel
blocker on the effect of IGF-I on 1-OHase activity. Cells were
incubated in 1 mM calcium in the presence or absence of IGF-I
(50 ng/ml) and/or verapamil, 100 µM, for 18 h. 5 nM of [
Kidney proximal tubular cells are known to be target cells
for IGF-I. This growth factor enhances renal
neoglucogenesis(40) , and it stimulates sodium-dependent
phosphate transport in isolated proximal tubules (PT) and in
established PT cell lines such as those obtained from opossum
kidney(14, 15) . The present studies on mouse PT cells
in primary culture indicate that exogenous IGF-I is also a crucial
physiological regulator of the 1,25-(OH) Our findings that low concentrations of
human recombinant IGF-I stimulate 25-OH-D IGF-I and
insulin can produce the same biological effects, due to their high
molecular structure homology and their possibility to cross-interact
with their own receptors(15, 37) . Comparison of the
dose-response curves obtained with IGF-I and insulin clearly shows that
insulin partially mimics the IGF-I effect but at doses 500-fold higher
than the minimal active IGF-I concentration (10 ng/ml). This suggests
that IGF-I exerts its effects through its own specific receptor. Understanding of the mechanism of the IGF-I action first required
the analysis of the kinetic parameters of the 1-OHase reaction. Time
course and cycloheximide experiments indicate that the IGF-I-dependent
stimulation of 1,25-(OH) Whatever the
nature of the protein(s) influenced by IGF-I, the second question we
asked was that of the intracellular pathway of the IGF-I signal. IGF-I could have indirectly influenced 1,25-(OH) IGF-I could have stimulated the 1-OHase
enzyme system via the protein kinase C pathway, as it stimulates PKC
activity in several cell systems (26, 27) and since
some of the IGF-I effects are inhibited by H7 or down-regulation of
PKC(27) . But, in the present study, PMA did not mimic the
stimulatory effect of IGF-I on the production of
1,25-(OH) Another possible pathway was therefore explored,
which has been proposed to mediate the genomic effects of IGF-I on cell
replication (26, 27, 28, 29) . IGF-I
activates a calcium-permeable cation channel in plasma membranes and
elicits continuous stimulation of calcium entry in at least two cell
types, IGF-I-responsive primed competent Balb/c/3T3 cells (26) and thyroid-stimulating hormone-primed thyroid
cells(29) . Most importantly, this calcium entry, independently
of PKC or inositol 1,4,5-trisphosphate messengers, may be the signal
for the IGF-I-dependent cell replication(26) . Our data
strongly suggest that IGF-I stimulates the 1-OHase via a similar route:
1) IGF-I-stimulated calcium entry in the mouse PT cells within the
first 10 min of incubation; 2) verapamil, the most effective calcium
channel blocker in proximal tubular cells(51) , totally blocked
both the IGF-I-induced calcium uptake and the IGF-I-induced stimulation
of the 1-OHase; 3) calcium depletion blunted the IGF-I effect on the
1-OHase. Interestingly, human 1-34 PTH also stimulated the
production of 1,25-(OH) In conclusion, this study
using primary culture of mouse kidney cells suggests that IGF-I is a
physiological regulator of the renal vitamin D metabolism. A calcium
pathway, not requiring PKC activation, appears to be involved in the
IGF-I-activated post-tyrosine kinase events leading to the stimulation
of the 1,25-(OH)
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25461-25467
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Production
STUDIES IN CULTURED MOUSE KIDNEY CELLS (*)
D
. In the present
work, the local effect of low nanomolar concentrations of IGF-I on the
25-OH-D-1
-hydroxylase activity and the mechanism of
its action have been investigated. To do so, an in vitro model
of mouse proximal tubular cells in primary culture has been developed.
These cells bear specific high affinity IGF-I binding sites (apparent K = 1.95 ± 0.46
nM) and express the ability to convert
[
H]25-(OH)D into
[H]1,25-(OH)D (K = 139 ± 15.7
nM). Human recombinant IGF-I (10-100 ng/ml) stimulated
both sodium-dependent phosphate uptake and 1,25-(OH)
D synthesis by these cells, in a time- and dose-dependent manner.
IGF-I did not alter the apparent Michaelis constant but increased the
maximum velocity of the 25-OH-D-1-hydroxylase
activity. This effect required protein synthesis. It was not affected
by calphostin or GF109203X, two protein kinase C inhibitors, and was
not mimicked by phorbol 12-myristate 13-acetate. In contrast, it was
blocked by verapamil, a calcium channel blocker. Calcium depletion of
the medium blunted the IGF-I effect but not that of human 1-34
parathyroid hormone 5 
10M. IGF-I
thus appears to be the first example of a physiological
calcium-dependent regulator of the renal metabolism of vitamin D.
(25-OH-D) (
)hydroxylation to
1,25-dihydroxyvitamin D
(1,25-(OH)D). This reaction is catalyzed by a
mitochondrial cytochrome P450 enzyme, the
25-OH-D-1-hydroxylase (1-OHase), whose activity is
closely regulated by several endocrine and ionic factors, including
PTH, 1,25-(OH)D itself, as well as dietary
and/or circulating
phosphate(1, 2, 3, 4, 5, 6) .
Insulin-like growth factor-I (IGF-I) could be one of these regulators,
or at least a mediator of the stimulatory effect of hypophosphatemia (7, 8, 9) . Evidence for this role has been
gathered mostly from in vivo(4, 5, 9) or in vivo/in
vitro experiments using very high concentrations of IGF-I, about
100 times higher than the K value(10, 11, 12) . But other
findings strengthen the hypothesis that IGF-I locally regulates the
production of 1,25-(OH)
D by the proximal
tubular cells of the mammalian kidney. First, these cells bear high
affinity specific binding sites for IGF-I(13) , and their
phosphate transport capacity responds to this growth factor at
concentrations in the range of the K value(14, 15) . Second, low concentrations
of IGF-I affect the activity of other cytochrome P450 enzymes involved
in the metabolism of steroids (16, 17, 18, 19, 20) .
Third, a nanomolar concentration of IGF-I has been shown recently to
stimulate the 1-OHase in a pig kidney cell line(21) .
D production(1, 22) .
The second pathway involves activation of PKC. Activators of the PKC
pathway, phorbol 12-myristate 13-acetate (PMA), for example, influence
the 1,25-(OH)D production (23, 24, 25) . Inversely the down-regulation
of PKC or its inhibition, by H7 and staurosporine, blocks the effects
of classical regulators of the 1,25-(OH)D production, like PTH(23) .D production. It involves changes in
the calcium entry through calcium channels but would not require the
PKC and inositol 1,4,5-trisphosphate messengers (26) . Such a
route has been proposed to explain the IGF-I effect on the cell
replication cycle of thyroid (29) and Balb/c/3T3
cells(26) . It has not yet been reported to be involved in the
regulation of the renal production of 1,25-(OH)DD and responds to IGF-I. The model has
first been used to investigate the kinetics of the IGF-I effect as a
local physiological regulator of the 1,25-(OH)D production in the kidney. We then tested the possibility that
IGF-I regulates the 1-OHase system via the PKC pathway or via a
calcium-dependent pathway.
Preparation of Proximal
Tubules
3-4-week-old male mice, obtained from IFFA CREDO,
were anesthetized by pentobarbital. Their kidneys were removed, rinsed
in ice-cold buffer A (Hank's balanced solution containing 15
mM Hepes and 5 mMD-glucose), and
decapsulated, and the cortices were separated from the medulla. The
cortices were cut into 0.5-1-mm-thick sections, washed three
times in buffer A, and then incubated in buffer B (5 ml of culture
medium (see below) plus 5 ml of Hank's balanced solution)
containing 0.125% collagenase and 0.25% fatty acid-free bovine serum
albumin BSA, at 37 °C in a 5% CO, 95% air atmosphere
for 30 min. The digest was washed 2-3 times in ice-cold buffer A
and suspended in the same buffer containing 0.5% fatty acid-free BSA
for 10 min at 4 °C. Tubular fragments were collected by
centrifugation for 4 min at 1800 rpm and suspended in gradient buffer
(42% of Percoll in Krebs-Henseleit buffer solution made isotonic).
Proximal tubules were separated from the other segments by
centrifugation at 17,000 rpm for 30 min. The layer containing the
proximal tubules was collected, washed, and suspended in culture
medium.Cell Culture
Proximal tubules were placed in
24-well plastic plates containing 0.5 ml/well of culture medium (50:50
Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12
(Techgen International, France), 7.7 mM glucose, 25 mM Hepes, 21.5 mM HCO, 1 mM sodium
pyruvate, 10 ml/liter of a 100 nonessential amino acid mixture,
4 mM glutamine, 50 IU/ml penicillin, 50 µg/ml
streptomycin, 50 nM sodium selenite, 35 µg/ml transferrin,
5 nM T, 25 ng/ml PGE, 0.5 µg/ml
bovine insulin, 100 nM dexamethasone and 500 nM
retinoic acid, 1% fetal calf serum for 24 h. The medium was replaced
with medium lacking fetal calf serum and was subsequently changed every
2 days. When the cells reached confluence (5-6 days), the insulin
concentration was increased to 5 µg/ml for 24 h and returned to 0.5
µg/ml for an additional 24 h. The culture medium was changed to
hormone-free, phosphate-depleted DMEM containing various calcium
concentrations (0.05-1 mM) for 18 h. Tubular fragments
from 2 kidneys were generally used per 24-well plates. Cells were used
after 8-10 days in culture.IGF-I Binding Studies
The confluent cells,
incubated for 18 h in phosphate-free 1 mM calcium DMEM, were
washed twice with the binding buffer (phosphate-buffered saline
containing 0.2% IGF-I-free BSA, pH 7.4) and incubated in the culture
wells for 2 h at room temperature with 20,000 cpm of I-IGF-I (specific activity 80-140 µCi/µg)
with or without various concentrations of unlabeled IGF-I (0.1 to 500
ng/ml) in a total volume of 400 µl of binding buffer. The
incubation was stopped by aspirating off the medium, and the cells were
washed three times with the ice-cold binding buffer. 500 µl of 1 N NaOH was added to each well, and an aliquot of the
solubilized cell pellet was taken from each well to determine its
radioactivity. Some wells of each plate were kept for protein
determination, and their protein content was 40-60 µg.
Nonspecific binding was defined as the binding occurring in the
presence of an excess (500 ng/ml) of unlabeled IGF-I. Nonspecific
binding was subtracted from total binding to determine specific binding
and expressed as picomoles of
I-IGF-I bound/mg of
protein.
The 1-OHase Activity Studies
Confluent cells were
incubated for 18 h in 0.5 ml of hormone-free, phosphate-depleted and 1
mM calcium DMEM. IGF-I, 5-100 ng/ml (Ciba-Geigy, Basel,
Switzerland), or its vehicle (0.1% BSA in 0.1 N acetic acid)
was added to this medium 18, 12, 6, or 1 h before the 1-OHase studies.
[26,27-H]25-OH-D (5
nM-1 µM) (Amersham) dissolved in ethanol
(10 µl) was added to measure the activity of the 1-OHase. The
reaction was stopped 30 to 120 min later by removing the medium which
was saved in glass tubes. The cells were washed with 0.5 ml of methanol
which was added to the incubation medium. 1 N NaOH (200
µl) was added to each well, and their protein content was
determined. The [H]25-OH-D and other
labeled steroids were extracted from the medium with an additional 0.5
ml of methanol plus 1 ml of chloroform. The labeled compounds were
separated by two-step straight phase HPLC. The chloroform extracts were
cochromatographed with 100 ng of synthetic 1,25-(OH)D on an ultrasphere Si, 5 µm, 4.6 mm 25 cm column
(Beckman) with 92:8 n-hexane/isopropyl alcohol solvent, and a
flow rate of 2 ml/min. The absorbance at 254 nm was recorded
continuously to determine the elution position of synthetic
1,25-(OH)D. 1-min fractions of the eluting
solvent were collected, and an aliquot of each fraction was removed for
radioactivity measurement.D were pooled and rechromatographed on
a second straight phase HPLC system using the same column eluted with a
95:5 methylene chloride:isopropyl alcohol solvent at a flow rate of 1
ml/min. Fractions of eluate were collected every minute, and their
radioactivity was determined. The percent conversion of
[H]25-OH-D to
[H]1,25-(OH)D was
calculated as follows: 1-OHase activity = radioactivity in the
1,25-(OH)D region after the first
chromatography (expressed as percent of the total radioactivity
recovered from the column) radioactivity in the
1,25-(OH)D region after the second
chromatography (expressed as percent of the total radioactivity
recovered from the column).Protein Determination
The protein concentration
was determined by the method of Bradford using BSA as
standard(31) .Characterization of the
1,25-(OH)
Cells have been incubated as described above
for the 1-OHase studies with one exception: unlabeled 25-OH-DDProduced (1 µM) was added to the incubation medium instead of
[H]25-OH-D. The chloroform extract of
the incubation medium was cochromatographed with 5 nCi of
[H]1,25-(OH)D as a marker
of the elution position of 1,25-(OH)D. After
two successive purifications on HPLC using first the 92:8 n-hexane:isopropyl alcohol system and then the 95:5 methylene
chloride:isopropyl alcohol, the kidney-produced
1,25-(OH)D was tested for its ability to
compete with
[H]1,25-(OH)D for the
binding to chick intestinal cytosol(32) .Phosphate Transport Studies
Cells were incubated
with 6-50 ng/ml IGF-I for 18 h in the hormone-free medium
described for the 1-OHase studies. P
uptake was measured,
in the presence or absence of sodium, as described
previously(33) .Determination of Calcium Influx Rate
Calcium
influx rate was measured by the initial uptake of Ca as
was described(34) . Briefly, cells grown in the 24-well plates
were incubated in DMEM hormone-free medium in the presence or absence
of IGF-I and/or verapamil.
Ca uptake was determined by the
addition of 3-4 µCi/ml
[
Ca]CaCl
. The reaction was stopped
by aspirating the medium and washing the cells four times with ice-cold
Tris/HCl buffer (pH 7.4) containing 144 mM NaCl and 5
mM CaCl. The radioactivity and protein content
were determined after the addition of 1 N NaOH.Determination of PKC Activities
Membrane-bound PKC
activity was measured, using the PKC enzyme assay system RPN 77
(Amersham), as described(35) . Cells were preincubated for 30
min with 1 µM GF109203X (Roussel-Uclaf, France) and then
incubated or not with 200 ng/ml PMA (Sigma) for 5 min.
1,25-(OH)
Confluent
mouse kidney cells, cultured for 18 h in a phosphate-free, protein and
hormone-free medium containing 1 mM calcium, metabolized
25-OH-DDProduction by Cultured Mouse Kidney Cells to a more polar compound similar to synthetic
1,25-(OH)D. It coeluted with
1,25-(OH)D on two HPLC systems, 92:8
hexane:isopropyl alcohol and 95:5 methylene chloride:isopropyl alcohol.
This latter system separates 1,25-(OH)D from
the two isomers of 19-nor-10-oxo-25-OH-D(36) . The
kidney-produced and synthetic 1,25-(OH)D both
absorbed UV light at 254 nm (data not shown) and competed with
[H]1,25-(OH)D for its
binding to chick intestinal cytosol (Fig. 1).
D produced by primary cultures of
mouse kidney cells to compete with
[H]1,25-(OH)D for binding
to its specific receptor in chick intestinal cytosol. The capacity of
the cell-produced material (closed circles and squares) was compared to that of known amounts of synthetic
1,25-(OH)D (open circles). Two
different preparations of kidney-produced metabolites were tested. See
details for their production and purification under ``Materials
and Methods.''
D 30 min after the addition of 5 nM
[H]25-OH-D and production increased
to a maximum after 60-120 min. As shown in Fig. 2, the
apparent Michaelis constant (K
) (139 ± 15.7
nM) and maximum velocity (V
) (115
± 7 fmol/mg of protein/min) were similar to those in chick
kidneys cells (37) and isolated rat kidney
mitochondria(38) .
H]25-OH-D for 45 min. Values are
means ± S.E. of 4 separate incubations. B,
Lineweaver-Burk plots of 1-OHase kinetics.
IGF-I Binding Studies
Intact confluent kidney
cells bound IGF-I when incubated with increasing concentrations of
recombinant human IGF-I and a fixed amount of radiolabeled IGF-I. The
binding was saturable and reached a plateau at 10 pmol of I-IGF-I bound/50 µg of protein (Fig. 3).
Half-maximal displacement of
I-IGF-I was obtained with 15
ng/ml IGF-I. Scatchard transformation of the data gave linear plots
compatible with two classes of binding sites. The interexperimental
dissociation constants for the high affinity class of receptors
averaged 1.95 ± 0.46 nM (mean ± S.E., n = 5), and the specific binding capacities were 0.72
± 0.16 pmol of IGF-I/mg of protein (n = 5).
I- IGF-I with or without the indicated concentrations of
unlabeled IGF-I. Values are means ± S.E. of 5 separate
experiments, each done in duplicate. Scatchard analysis of the data was
obtained in one experiment. Comparable results were obtained in the
five experiments.
Effect of IGF-I on Sodium-dependent Phosphate
Uptake
Human IGF-I significantly stimulated the sodium-dependent
uptake of phosphate by mouse kidney cells (Fig. 4A)
over the concentration range (6-50 ng/ml). These IGF-I doses had
no effect on other sodium-dependent transports, such as the alanine and
glucose transports (data not shown).
D production by mouse kidney cells.
Cells were incubated for 18 h with either IGF-I at the indicated
concentrations or its vehicle. 5 nM
[H]25-OH-D was then added to
determine the 1-OHase activity.
Effect of IGF-I and Insulin on
1,25-(OH)
Exogenous human IGF-I increased the amount
of [DProductionH]1,25-(OH)D detected
in the incubation medium 45-120 min after the addition of
[H]25-OH-D. This stimulation occurred
with an IGF-I concentration as low as 10 ng/ml and was close to maximum
with 100 ng/ml (Fig. 4B). Estimated half-maximal
stimulation of the 1,25-(OH)D production was
obtained with 17.5 ng/ml IGF-I. Kinetic parameter analysis showed that
IGF-I did not alter the apparent K of the
enzymatic conversion of 25-OH-D (139 ± 15.7 nM and 146 ± 8.5 nM, n = 4), but it
significantly increased (p < 0.01) the apparent V of the reaction from 115 ± 7 to 166
± 9 fmol/mg of protein/min (Fig. 2).
D production by mouse kidney cells.
Cells were incubated with IGF-I (50 ng/ml) for 1, 6, 12, or 18 h or
with its vehicle (C). 5 nM
[H]25-OH-D were then added for 1 h.
Values are means ± S.E. of 4-12 incubations as indicated
in parentheses.***, p < 0.001 as compared to
untreated cells.
D production by
mouse kidney cells. Cells were pretreated for 1 h by 1 µM cycloheximide before the addition of IGF-I (50 ng/ml) or its
vehicle (C). After 18 h, the 1-OHase activity was determined
by adding [H]25-OH-D (5 nM)
for 60 min. Values are means ± S.E. of 3-5 incubations.**, p < 0.01 as compared to untreated cells. ***, p < 0.001 as compared to cells treated by cycloheximide plus
IGF-I.
D production at concentrations between
25 and 1000 ng/ml. Stimulation only occurred with a very high, 5
µg/ml, insulin concentration. The stimulation was 0.7-fold that
obtained with 50 ng/ml IGF-I.Studies on the Mechanism of the IGF-I Action
In an
attempt to determine the intracellular pathway of the IGF-I action,
cells were incubated either with an activator, PMA (Sigma), or two
dissimilar inhibitors, calphostin C (Calbiochem) and GF109203X
(Roussel-Uclaf, France), of PKC activities (Fig. 7). Cell
incubation with 200 ng/ml PMA for 1 h or 18 h had an effect opposite to
IGF-I as it decreased the cell's ability to convert
[H]25-OH-D into
[H]1,25-(OH)D. Moreover,
the stimulatory effect of IGF-I was not blocked when calphostin C (1
nM) or GF109203X (1 µM) had been added 30 min
prior to IGF-I (Fig. 6). The tested GF109203X concentration was
50 times higher than the IC reported for its inhibitory
effect on PKC activity(39) , and it was verified that it
prevented the PMA-induced stimulation of membrane-bound PKC activity in
the present experimental condition (Table 1).
H]25-OH-D was then
added for 60 min to determine the 1-OHase activity. Values are means
± S.E. of 3-11 separate incubations as indicated.**, p < 0.01;***, p < 0.001 versus homologous
controls (not treated with IGF-I).
10M, on the 1-OHase (Fig. 8).
10M)
was added at the 17th h of the preincubation, i.e. 1 h before
the assay. 5 nM [
H]25-OH-D were then added for 1 h to determine the 1-OHase activity.
Results are expressed as percent of the values obtained in untreated
cells incubated with the same calcium concentration (Control,
). Values are mean ± S.E. of 5-8 different
incubations. *, p < 0.03;**, p < 0.005, as
compared to paired untreated cultures incubated in the same calcium
concentration.
H]25-OH-D were then added to
determine the 1-OHase activity. Values are mean ± S.E. of
4-8 different incubations.**, p < 0.005, as compared
to untreated cells. B, effect of a specific calcium channel
blocker on the IGF-I effect on calcium influx rate. Preincubated cells,
in hormone-free medium for 18 h, were treated with 50 ng/ml IGF-I for
10 min in the presence or absence of 100 µM verapamil
added 1 h before the addition of IGF-I. Calcium uptake was measured
after the addition of 3-4 µCi of
[Ca]CaCl
. Values are means ±
S.E. of 3-4 different experiments. *, p < 0.025, as
compared to their homologous control.
Effect of IGF-I on Calcium Influx
Calcium uptake
by the cells was linear for at least 90 s. Addition of 50 ng/ml IGF-I
stimulated the initial calcium influx rate measured at 30, 60, and 90
s. A significant stimulation of the calcium influx rate was observed 10
min and 1 h after addition of IGF-I to the medium: calcium uptake was
1400, 1860, and 2150 cpm/min/well in the control, 10-min treated and
1-h treated wells, respectively. No IGF-I effect was observed in the
18-h treated cells (1400 versus 1540 cpm/min/well).
Preincubation of the cells with 100 µM verapamil, for 1 h,
blunted the IGF-I effect on calcium accumulation (Fig. 9B).
D production (7, 8, 9) . Its mechanism of
action appears to be unique among the other known regulators of the
vitamin D metabolism as it may be mediated via a calcium-dependent
pathway not requiring PKC. hydroxylation in
a dose-dependent manner (10-100 ng/ml) strongly support the
hypothesis that IGF-I physiologically regulates vitamin D metabolism.
These concentrations are similar to those that affect the
(Na-P) transport in the present mouse PT cell model and in
other PT cells(14, 15) . They are in the range of the
apparent K
value for IGF-I binding measured here
in intact kidney cells, as well as in tubular cell membranes (13) or in other cell types(41, 42) . Finally,
they are similar to the IGF-I concentrations which stimulate the
phosphorylation of its own specific receptor(43) .D production requires
protein synthesis, as its effect was only detected after incubations
for 18 h and was blocked by preincubation with 1 µM cycloheximide. IGF-I did not alter the apparent K of the enzymatic system, but it increased its apparent V, from 115 ± 7.4 to 166 ± 9.07
fmol/mg of protein/min. This increase may reflect enhancement in the
protein-enzyme expression or an acceleration of the turnover of the
substrate hydroxylation. The protein synthesized in the presence of
IGF-I could be the cytochrome P450 component of the 1-OHase enzymatic
system. Indeed, low concentrations of IGF-I have been shown to
stimulate the synthesis of several other steroid-linked cytochromes
P450, 11
-hydroxylase(16) ,
3-hydroxydehydrogenase(17) , and cholesterol side chain
cleavage enzymes(18, 19, 20) . But, this
hypothesis cannot be tested at the present time because the P450
involved in the 1,25-(OH)D synthesis has not
been isolated and no probe detecting and measuring its mRNA is
available. Other protein candidates could be ferredoxin, a component of
the 1-OHase system which can be modulated by regulators of vitamin D
metabolism at the transcriptional and post-transcriptional
levels(44, 45) , or proteins required for the
phosphorylation or dephosphorylation of these two 1-OHase components,
ferredoxin or cytochrome P450 (45, 46, 47, 48) .D production through changes in phosphate uptake by the cells after
the activation of tyrosine kinase(14) . Interactions between
IGF-I and phosphate on the production of 1,25-(OH)D have been extensively
documented(5, 7, 8, 9, 10, 11, 12) ,
and IGF-I modulates the sodium-dependent phosphate uptake of
kidney(14, 15) , bone (49) , and cartilage (50) cells. Our results, obtained with cells incubated in a
phosphate-free medium, demonstrate that stimulation of the 1-OHase by
IGF-I does not require phosphate transfer from the extracellular
compartment to the cell.D. In the opposite, it decreased this
production, a result consistent with those obtained with TPA in chick
and rat kidney cells(24, 25) . Second, the
IGF-I-induced stimulation of 1-OHase was not affected by preincubation
of the cells with calphostin C or GF109203X while this treatment
blocked PMA-stimulated PKC activity in the cell membranes. Thus, PKC
activation does not appear to be a crucial step in the IGF-I-induced
events leading to the stimulation of the 1,25-(OH)D synthesis.D in our cell system.
But, unlike that induced by IGF-I, the PTH stimulation was not affected
by the calcium concentration of the incubation medium, 1 or 0.05
mM. Thus, PTH and IGF-I appear to influence the renal
metabolism of vitamin D via different signaling routes. The PTH
signaling pathway(s) involved in the present experimental conditions
has not been tested. Yet, whatever its nature, activation of the cAMP
system (22) and/or of the PKC pathway(23) , it does not
appear to be influenced by external calcium. In contrast, that involved
for IGF-I is clearly calcium-dependent.D production. IGF-I would thus
be the first example of a calcium-dependent regulator of the renal
metabolism of vitamin D.
),
25-hydroxyvitamin D; 1,25-(OH)D,
1,25-dihydroxyvitamin D; PTH, parathyroid hormone; PKC,
protein kinase C; IGF-I, insulin-like growth factor I; BSA, bovine
serum albumin; DMEM, Dulbecco's modified Eagle's medium;
HPLC, high performance liquid chromatography; PMA, phorbol 12-myristate
13-acetate; PT, proximal tubules.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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