(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)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
10
M. IGF-I
thus appears to be the first example of a physiological
calcium-dependent regulator of the renal metabolism of vitamin D.
Kidney is the main site of 25-hydroxyvitamin 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) .
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)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) .
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)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)
D
We have now developed a new in vitro model of mouse
proximal tubular cells in primary culture that produces
1,25-(OH)D
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.
Fractions coeluting with synthetic
1,25-(OH)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).
Figure 1:
Capacity of the
1,25-(OH)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.''
The kidney
cells produced detectable amounts of 1,25-(OH)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) .
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
[H]25-OH-D
for 45 min. Values are
means ± S.E. of 4 separate incubations. B,
Lineweaver-Burk plots of 1-OHase kinetics.
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 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.
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)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.
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)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.
Figure 6:
Effect of cycloheximide (Cyclo)
on IGF-I-stimulated 1,25-(OH)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.
In contrast, incubation of the
cells for 18 h with bovine insulin did not stimulate
1,25-(OH)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.
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 [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).
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 10
M, on the 1-OHase (Fig. 8).
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 10
M)
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.
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 [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.
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)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.
Our findings that low concentrations of
human recombinant IGF-I stimulate 25-OH-D 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) .
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)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) .
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)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.
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)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.
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)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.
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)D
production. IGF-I would thus
be the first example of a calcium-dependent regulator of the renal
metabolism of vitamin D.