Insulin-like growth factor I, a unique calcium-dependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells.

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)2D3. In the present work, the local effect of low nanomolar concentrations of IGF-I on the 25-OH-D3-1α-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 Kd = 1.95 ± 0.46 nM) and express the ability to convert [3H]25-(OH)D3 into [3H]1,25-(OH)2D3 (Km = 139 ± 15.7 nM). Human recombinant IGF-I (10-100 ng/ml) stimulated both sodium-dependent phosphate uptake and 1,25-(OH)2D3 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-D3-1α-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 × 10−8M. IGF-I thus appears to be the first example of a physiological calcium-dependent regulator of the renal metabolism of vitamin D.

using very high concentrations of IGF-I, about 100 times higher than the K d value (10 -12). But other findings strengthen the hypothesis that IGF-I locally regulates the production of 1,25-(OH) 2 D 3 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 d value (14,15). Second, low concentrations of IGF-I affect the activity of other cytochrome P450 enzymes involved in the metabolism of steroids (16 -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) 2 D 3 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) 2 D 3 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) 2 D 3 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 -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) 2 D 3 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) 2 D 3 We have now developed a new in vitro model of mouse proximal tubular cells in primary culture that produces 1,25-(OH) 2 D 3 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) 2 D 3 production in the kidney. We then tested the possibility that IGF-I regulates the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Preparation of Proximal
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 3 , 1 mM sodium pyruvate, 10 ml/liter of a 100 ϫ nonessential amino acid mixture, 4 mM glutamine, 50 IU/ml penicillin, 50 g/ml streptomycin, 50 nM sodium selenite, 35 g/ml transferrin, 5 nM T 3 , 25 ng/ml PGE 1 , 0.5 g/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 g/ml for 24 h and returned to 0.5 g/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 125 I-IGF-I (specific activity 80 -140 Ci/g) with or without various concentrations of unlabeled IGF-I (0.1 to 500 ng/ml) in a total volume of 400 l 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 l 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 g. 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 125 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, H]25-OH-D 3 (5 nM-1 M) (Amersham) dissolved in ethanol (10 l) 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 l) was added to each well, and their protein content was determined. The [ 3 H]25-OH-D 3 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) 2 D 3 on an ultrasphere Si, 5 m, 4.6 mm ϫ 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) 2 D 3 . 1-min fractions of the eluting solvent were collected, and an aliquot of each fraction was removed for radioactivity measurement.
Fractions coeluting with synthetic 1,25-(OH) 2 D 3 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 [ 3 H]25-OH-D 3 to [ 3 H]1,25-(OH) 2 D 3 was calculated as follows: 1-OHase activity ϭ radioactivity in the 1,25-(OH) 2 D 3 region after the first chromatography (expressed as percent of the total radioactivity recovered from the column) ϫ radioactivity in the 1,25-(OH) 2 D 3 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) 2 D 3 Produced-Cells have been incubated as described above for the 1-OHase studies with one exception: unlabeled 25-OH-D 3 (1 M) was added to the incubation medium instead of [ 3 H]25-OH-D 3 . The chloroform extract of the incubation medium was cochromatographed with 5 nCi of [ 3 H]1,25-(OH) 2 D 3 as a marker of the elution position of 1,25-(OH) 2 D 3 . 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) 2 D 3 was tested for its ability to compete with [ 3 H]1,25-(OH) 2 D 3 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 i 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 45 Ca as was described (34). Briefly, cells grown in the 24-well plates were incubated in DMEM hormonefree medium in the presence or absence of IGF-I and/or verapamil. 45 Ca uptake was determined by the addition of 3-4 Ci/ml [ 45 Ca]CaCl 2 . 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 2 . 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 M GF109203X (Roussel-Uclaf, France) and then incubated or not with 200 ng/ml PMA (Sigma) for 5 min. RESULTS
The kidney cells produced detectable amounts of 1,25-(OH) 2 D 3 30 min after the addition of 5 nM [ 3 H]25-OH-D 3 and production increased to a maximum after 60 -120 min. As shown in Fig. 2, the apparent Michaelis constant (K m ) (139 Ϯ 15.7 nM) and maximum velocity (V max ) (115 Ϯ 7 fmol/mg of protein/min) were similar to those in chick kidneys cells (37) and isolated rat kidney mitochondria (38).
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 125 I-IGF-I bound/50 g of protein (Fig. 3). Half-maximal displacement of 125 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 Ϯ 0.46 nM (mean Ϯ S.E., n ϭ 5), and the specific binding capacities were 0.72 Ϯ 0.16 pmol of IGF-I/mg of protein (n ϭ 5).
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 ala- 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) 2 D 3 production was obtained with 17.5 ng/ml IGF-I. Kinetic parameter analysis showed that IGF-I did not alter the apparent K m of the enzymatic conversion of 25-OH-D 3 (139 Ϯ 15.7 nM and 146 Ϯ 8.5 nM, n ϭ 4), but it significantly increased (p Ͻ 0.01) the apparent V max of the reaction from 115 Ϯ 7 to 166 Ϯ 9 fmol/mg of protein/min (Fig. 2).
This IGF-I effect required incubation of the cells for 18 h to be significant (Fig. 5). It was not observed when cycloheximide (1 M) 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 Ϯ 2.6 and 51 Ϯ 1.9 mg/well, respectively.
In contrast, incubation of the cells for 18 h with bovine insulin did not stimulate 1,25-(OH) 2 D 3 production at concentrations between 25 and 1000 ng/ml. Stimulation only occurred with a very high, 5 g/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 calphostin C (1 nM) or GF109203X (1 M) had been added 30 min prior to IGF-I (Fig. 6). The tested GF109203X concentration was 50 times higher than the IC 50 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 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 II). But no IGF-I effect was observed anymore when using a calciumdepleted 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 ϫ 10 Ϫ8 M, on the 1-OHase (Fig. 8).
Finally, we tested the effect of verapamil, a specific calcium channel blocker. Addition of 100 M verapamil to the cells 1 h before that of IGF-I abolished the 1-OHase response to IGF-I (Fig. 9A).
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 M verapamil, for 1 h, blunted the IGF-I effect on calcium accumulation (Fig. 9B). DISCUSSION 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) 2 D 3 production (7-9). 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 3 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 i ) transport in the present mouse PT cell model and in other PT cells (14,15). They are in the range of the apparent K d 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) 2 D 3 production requires protein synthesis, as its effect was only detected   after incubations for 18 h and was blocked by preincubation with 1 M cycloheximide. IGF-I did not alter the apparent K m of the enzymatic system, but it increased its apparent V max , from 115 Ϯ 7.4 to 166 Ϯ 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 -20). But, this hypothesis cannot be tested at the present time because the P450 involved in the 1,25-(OH) 2 D 3 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) 2 D 3 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) 2 D 3 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) 2 D 3 . 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) 2 D 3 synthesis.
Another possible pathway was therefore explored, which has been proposed to mediate the genomic effects of IGF-I on cell replication (26 -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 thyroidstimulating 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-Iinduced 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) 2 D 3 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-Iactivated post-tyrosine kinase events leading to the stimulation of the 1,25-(OH) 2 D 3 production. IGF-I would thus be the first example of a calcium-dependent regulator of the renal metabolism of vitamin D.