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Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells
Kidney Internationa4 VoL 49 (1996), pp. 1071—1078
Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells PASCALE GRELLIER, MICHAEL 5ABBAH, BRUNO FOUOUERAY, KATHLEEN WOODRUFF, DOUGLAS YEE, HANNA E. ABBOUD, and 5HERRY L. ABBOUD Departments of Medicine and Pathology, University of Texas Health Science Center and Audie Murphy Veterans Hospital, San Antonio, Texas, USA
Characterization of insulin-like growth factor hinding proteins and regulation of IGFBP3 in human mesangial cells. IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-l and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays
using total RNA demonstrated that MC express all of the IGFBPs. E5251]IGFJ Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-3 arc implicated in glomerular
hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng./ml), TGF-f3 (2 ng/ml) and forskolin (10-i
at) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-j3, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-l-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-f3 is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs.
Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-p and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.
TGF-/3 to enhance extracellular matrix deposition within the glomerulus. The biologic action of IGF-I/II in the kidney may be modulated
by a family of six IGFBPs, designated IGFBP-1 to -6. These IGFBPs bind with high affinity to lOP-I and -II, but not to insulin [11]. They serve as a reservoir and prolong the biologic half-life of TOPs in the circulation. Depending on the IGFBP species, target cell and culture conditions, IGPBPs may either inhibit IGF action by forming inactive complexes with IGFs or enhance TOP action by facilitating IGF interaction with surface receptors [12]. Certain TGFBPs provide a storage site for IGFs in the extraeellular matrix [13]. In addition, IGFBP-3 and -5 have intrinsic biologic activity independent of IGFs [14, 15].
IGPBPs are abundant in the human kidney and demonstrate
distinct cellular distribution. By in situ hybridization, IGFBP-1 to -5 mRNAs arc expressed in glomeruli; however, the precise
IGFBPs produced by the different cell types that comprise the glomerulus have not been determined [16]. We have previously demonstrated that human mesangial cells express lOP-I mRNA, lOP-I receptors and release IGF-I and IGF-binding activity [17]. These cells provide a useful model for determining the spectrum of IGFBPs released by mesangial cells, the factors that regulate IGFBP gene expression, and the role of each IGFBP in modulating lOP action in the glomerulus. Previous studies have demon-
strated that adenylate cyclase agonists such as forskolin, and Insulin-like growth factors-I and -II (IGF-I and -II) play an important role in kidney embryogenesis and function [1, 2]. Glomeruli and segments of the distal nephron are targets for circulating IGFs and provide a source of local IGF-I production
[1]. The systemic administration of IGF-I has been shown to increase renal plasma flow, glomerular filtration rate and kidney volume in rats and humans [3, 4]. Increased circulating or locally produced IGF-I have also been implicated in the pathogenesis of renal hypertrophy associated with unilateral nephrectomy, diabe-
tes, hypersomatotropism, and a high protein diet [5—10]. In chronic diabetic nephropathy, lOP-I may act in concert with
cytokines including IGF-I and TOF-J3, regulate IOFBPs in fibroblasts and other cell types [18—20].
In this study, we determined the molecular identity of each IGFBP released by human mesangial cells. The effect of IGF-I, TOP-f3 and the cAMP agonist, forskolin, on IGFBPs released by mesangial cells was determined. Since IGFBP3 was markedly induced by lOP-I and TGF-3, recombinant IOFBP3 was tested for its effect on IGF-I-induced DNA synthesis in mesangial cells. Whether IGFBPs are involved in mediating the inhibitory effect of TGF-13 on TOP-I-stimulated DNA synthesis was also explored. Methods Materials
Received for publication August 24, 1995 and in revised form November 20, 1995 Accepted for publication November 20, 1995
© 1996 by the International Society of Nephrology
Porskolin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant human TGP-1 was from R&D Systems, Inc. (Minneapolis, MN, USA) and recombinant human 1071
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Gre/her et a!: IGF binding proteins and kidney
IGF-I and R3-IGF-I from GroPep (Adelaide, Australia). Recombinant human IGFBP3 was purchased from Upstate Biotechnol-
Western ligand blot
Samples were concentrated 40-fold by ultrafiltration in Centricon 10 microconcentrators (10 kDa molecular wt cutoff). Ligand blotting with [1251]IGF-I was performed by the method of Hossenlopp et a! [24] as previously described [25]. Briefly, samples were electrophoresed through a 10% SDS-polyacryiamide gel. Separated proteins were electroblotted onto nitrocellulose filters and then incubated with [1251]IGF-I overnight at 4°C and visualized by autoradiography.
ogy, Inc. (Lake Placid, NY, USA). Rainbow molecular mass markers and radiolabeled IGF-I was obtained from Amersham (Arlington Heights, IL, USA). Centricon 10 concentrators were from Amicon (Danvers, MA, USA). RNAzol B was purchased from Cinna Biotex (Houston, TX, USA) and the Riboprobe Kit from Promega (Madison, WI, USA). Antisense RNA probes. The probes for human IGFBP-2, -4, -5 and -6 were provided by Dr. Shimasaki (Whittier Institute, La Jolla, CA, USA) [11]. Each probe was derived from a eDNA [3H/thymidine ([3H]TdR) assay fragment corresponding to part of the protein-coding region and Confluent cells were made quiescent by incubation in SFIF cloned into pBluescript SKI. The IGFBP2 and IGFBP4 probes medium for 72 hours. They were then washed once with SFIF were synthesized from a StyI-EcoRV 382-bp and an EcoRI- medium and incubated in same medium with IGF-I (200 ng/ml) Hindlil 505-bp fragment, respectively. The IGFBP5 and IGFBP6 probes were synthesized from a SacII-Saci 317-bp and a PstI-PstI 267-bp fragment, respectively. IGFBP-1 and -3 probes (provided
by Dr. D. Powell, Baylor University, Waco, TX, USA) were synthesized from a PstI-PstI 369-bp and a PstI-EcoRI 535-bp fragment, respectively. The human 36B4 eDNA probe detects a ubiquitously expressed ribosomal phosphoprotein and was provided by Dr. P. Chambon (INSERM, Institut de la Sante et de Ia Recherche Medicale) [21]. Cell culture
and R3-IGF-I (200 ng/ml) alone and in combination with TGF-13 (2 ng/ml). In some experiments, IGF-I and R3-IGF-I were incu-
bated in the presence and absence of various concentrations of IGFBP3. Cells were pulsed with [3H]TdR (1 iCi/ml) for four hours prior to harvesting at 28 hours. The amount of [3H]TdR incorporated into TCA-precipitable material was determined by liquid scintillation counting. Statistical analysis Analysis of the difference of the means among treatment groups
was determined utilizing Tukey's Studentized Range Test. The results in Figure 4 represent the mean of at least three separate Human mesangial cells were cultured from glomeruli isolated experiments SEM. by differential sieving as previously described [22]. Portions of normal renal cortex were obtained from kidneys judged unsuitResults able for transplantation. Epithelial cells were removed by diIGFBP gene expression in human mesangial cells gestion with collagenase. Glomerular cores were cultured in IGFBP mRNAs are expressed in the glomerulus. We examined Waymouth's medium (GIBCO, Grand Island, NY, USA) supplemented with 15 mi Hepes, 0.6 U/mi insulin, 2 mrvi glutamine, 0.1 the specific IGFBP mRNAs expressed by mesangial cells. Total m nonessential amino acids, 1 m sodium pyruvate, antibiotic/ RNA isolated from mesangial cells was hybridized with human antifungal solution, and 17% fetal calf serum (FCS). Cells were IGFBP-i to -6 antisense RNA probes and analyzed using a sensitive RNase protection assay. Figure 1 demonstrates that used between passages three to ten. mesangial cells constitutively express abundant levels of IGFBP-2 to -6 mRNAs, and to a lesser extent IGFBP1 mRNA. For each RNase protection assay IGFBP analyzed, human breast carcinoma cell lines, MDA-MB To determine constitutive IGFBP-1 to -6 mRNA expression, total 231 or MCF-7, were used as controls since they are known to RNA was isolated from confluent cultures of mesangial cells express certain IGFBP mRNAs [26]. Autoradiograms show the maintained in Waymouth's medium with serum. Total RNA was expected protected fragment in both the control and mesangial isolated using RNAzoI B method. Concentrations were deter- cell total RNAs: 369-bp for IGFBP1; 325-bp for IGFBP2; 535-bp mined by spectrophotometry; integrity and quantitation was con- for IGFBP3; 505-bp for IGFBP4; 317-bp for IGFBP5 and 267-bp firmed by visualizing ethidium bromide-stained 28S and 18S for IGFBP6. These fragments are specific and not observed in the ribosomal bands on 1% agarose gels. For experiments designed to tRNA lane. determine the effect of forskolin, IGF-I and TGF-/3 on IGFBP3 mRNA and protein, confluent MC were washed three times with Characterization of IGFBPs in mesangial cell conditioned medium serum-free, insulin-free (SFIF) medium and incubated 24 hours in To determine if IGFBP mRNA expression is associated with the same medium. Fresh SFIF medium with or without forskolin, the release of the corresponding IGFBPs, conditioned medium IGF-I, or TGF-J3 was added. Cell-free supernatants were col- from mesangial cells was analyzed by ligand blotting with lected at 8 and 24 hours and used for Western ligand blot analysis. ['251]IGF-I. In Figure 2, the first lane shows conditioned medium Cells were also harvested for total RNA at these time points and obtained from ovarian carcinoma cells (OVCAR-3) that contain at 72 hours. RNase protection assay was carried out as we have IGFBPs of 24, 29 (not visualized), 32 kDa and a doublet at 46 kDa previously described [23]. 32P-labeled single-stranded RNA that correlate with IGFBP-4, -5, -2 and -3, respectively, as we have probes were synthesized in an antisense orientation using SP6, T7 previously shown [25]. The constitutive release of IGFBPs from or T3 RNA polymerase. After hybridization, samples were di- confluent mesangial cell cultures incubated under unstimulated, gested sequentially with RNases A and Ti and proteinase K. The serum-free, insulin-free conditions is shown in lanes labeled products were analyzed by electrophoresis through a 7 M urea/6% control (8 and 24 hr). At eight hours, the 24 kDa species predominates with less abundant species of 29, 32, and 46 kDa. At acrylamide gel, followed by autoradiography.
1073
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Gre/tier et at: IGF binding proteins and kidney
Fig. 1. RNase protection assays for IGFBP mRNA expression by mesangial cells. 32P-labeled LGFBP-1 to -6 antisense RNA probes were hybridized with
total RNA (30 sg) isolated from mesangial cells. Total RNA from human breast cancer cell lines (MDA-MB-23 1 or MCF-7) were used as controls. Autoradiograms show the predicted protected fragment in both controls and mesangial cells. Probes hybridized with tRNA were completely digested by RNases. Last lane of each panel shows undigested IGFBP probes. The migration of 32P-labeled DNA markers is shown to the left (in basepairs).
24 hours, there is a predominance of the 46 kDa doublet and the levels were similar in control and forskolin-treated samples, and a 32 kD IGFBP. Less abundant IGFBP species are seen at 24 kDa decrease in 32 kDa IGFBP2 was observed. IGF-I primarily and at approximately 26 and 34 kDa; the 29 kDa IGFBP present induced IGFBP-2 and -5 and slightly increased 46 kDa IGFBP3 at eight hours is no longer observed at 24 hours. The precise doublet at eight hours. By 24 hours, accumulation of IGFBP-2 identity of the 26 and 34 kDa species is unknown, but the 26 kDa and -3 markedly increased in response to IGF-I, an effect also
band may represent either a glycosylated form of IGFBP4 or
observed on the 26 kDa species. TGF-f3 primarily induced
IGFBP1; the 34 kDa band may be a glycosylated IGFBP. The 46
IGFBP-4 and -2 and slightly increased IGFBP3 at eight hours. Similar to IGF-I, TGF-f3 markedly increased the accumulation of IGFBP-2 and -3 at 24 hours. This effect persisted in cultures exposed to TGF-J3 for 48 hours (data not shown).
kDa doublet present in the medium was more abundant at 48 hours compared to 24 hours (data not shown). These findings
indicate that the relative abundance of IGFBPs in mesangial cell conditioned medium varies in a time-dependent fashion. Release Regulation of JGFBP3 mRNA by forskolin, IGF-J and TGF-13 of IGFBP-2, -3, -4, -5 and possibly -1 and -6 by mesangial cells correlates with expression of these IGFBP mRNAs. IGFBP6 may Since IGFBP3 showed the strongest response to cytokines, not be detected by [1251]IGF-I ligand blot since IGFBP6 protein experiments were carried out to determine if IGFBP3 is regulated has a higher affinity for IGF-II than IGF-I. at the level of mRNA abundance (Fig. 3). RNA was isolated from the same experiment described in Figure 2 and from an additional Regulation of IGFBPs in mesangial cell conditioned medium by 48 hours time point. In unstimulated cells, IGFBP3 mRNA forskolin, JGF-I and TGF-/3 increased in a time-dependent manner (control 8, 24 and 48 hr), cAMP agonists and various cytokines have been shown to similar to the protein levels observed on the ligand blot. Forskolin regulate IGFBP proteins in other systems. We performed time increased IGFBP3 mRNA expression at eight hours with a peak course experiments to determine the effect of forskolin, IGF-I and effect observed at 24 hours, and a decline to basal levels by 48 TGF-13 on IGFBPs released by mesangial cells. Figure 2 shows hours. Although a corresponding increase in IGFBP3 protein was that IGFBPs are differentially regulated by these agents. Forsko- not observed in this experiment, other experiments showed induc-
lin induced 24 kDa IGFBP4 and, to a lesser extent, 29 kDa tion of IGFBP3 protein by forskolin. While IGF-l appeared to IGFBP5 at eight hours compared to control. At 24 hours, IGFBP4 slightly decrease IGFBP3 mRNA at each time point tested,
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Grellier et al: JGF binding proteins and kidney
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21.5— Fig. 2. Time course for the effect of forskolin, IGF-I and TGF-P on IGFBPs by Western ligand blot analysis. Confluent cells were made quiescent by placing
them in SFIF medium for 24 hours. Forskolin (10 M), IGF-l (100 ng/ml) or TGF-/3 (2 ng/ml) were then added and cells incubated for thc indicated time points. Samples were electrophoresed through a nonreducing 10% SDS-polyacrylamide gel and transferred to nitrocellulose. After incubation with [1251j1GF-l, filter-immobilized IGFBPs were visualized by autoradiography after two days exposure. The migration positions of molecular size markers (in kDa) are indicated on the left.
TGF-/3 was a potent inducer of IGFBP3 mRNA at 24 and 48 hours compared to control. Thus, the effect of TGF-/3, but not IGF-1, on IGFBP3 mRNA expression correlated with IGFBP3 protein abundance as measured by ligand blotting. Control 36B4 mRNA did not vary in response to the test agents, demonstrating that the response in IGFBP3 mRNA does not simply reflect a generalized nonspecific change in total RNA expression (Fig. 3, lower panel). Similar results were observed in two separate
DNA synthesis, suggesting that any endogenous production of IGFs by cells, under these experimental conditions, did not result in autocrine regulation. Perhaps released IGF-I is immediately sequestered by endogenously released IGFBPs.
experiments.
inhibits DNA synthesis in mesangial cells and blocks the mitogenic effect of certain cytokines {27. The marked induction of IGFBP3 protein by TGF-13 suggests that TGF-f3 may inhibit the mitogenic effect of IGF-I via the release of IGFBP3. Figure 5 shows that IGF-I and R3-IGF-I stimulated DNA synthesis in mesangial cells compared to control. TGF-/3 alone inhibited basal DNA synthesis. When co-incubated with TGF-13, the mitogenic effects of both IGF-I and R3-IGF-I were markedly inhibited. These findings suggest that the inhibitory effect of TGF-J3 is not mediated via an IGF-binding protein.
Effect of recombinant JGFBP3 on JGF-J-induced DNA synthesis in mesangial cells
Since IGFBP3 may modulate IGF-I action, the effect of recom-
binant human IGFBP3 on IGF-I-stimulated DNA synthesis in mesangial cells was determined. Figure 4 shows that IGFBP3 inhibited IGF-I-induced mitogenesis in a dose-dependent manner with approximately 35% inhibition at 25 nM and 50% inhibition at
50 mM IGFBP3. Addition of 50 nM IGFBP3, a concentration twofold higher than IGF-I, completely abrogated the mitogenic effect of IGF-I. Although there was some variation in IGF-
Effect of TGF-13 on IGF-I and R3-IGF-I-induced DNA synthesis in mesangial cells
We and others have previously demonstrated that TGF-p
Discussion The present study demonstrates that human mesangial cells in induced DNA synthesis from 1.5- to 2.5-fold compared to control, the addition of 25 and 50 tiM IGFBP3 consistently inhibited the culture constitutively express IGFBP-1 to -6 mRNAs and release effect of IGF-I. To determine if the effect of IGFBP3 was specific IGFBPs. Recent work has shown that human glomeruli express for IGF-I-stimulated DNA synthesis, and required the interaction IGFBP-1 to -5 mRNAs. IGFBP1 mRNA was localized to parietal between the binding protein and ligand, we tested the effect of epithelial cells along the glomerular capsule and IGFBP2 mRNA
IGFBP3 on R3-IGF-I-induced DNA synthesis. R3-IGF-1 is an
was present in glomerular cells with a distribution pattern of
analogue of IGF-l that retains affinity for IGF-1 receptors but fails to complex with IGFBPs. As shown in Figure 4, 50 nM IGFBP3 did not influence DNA synthesis induced by R3-IGF-1. Incubation of cells with IGFBP3 alone showed no significant effect on basal
epithelial cells {16. However, the precise molecular identities of each IGFBP expressed by the individual cell types that comprise the glomerulus are unknown. By RNase protection assay, human mesangial cells expressed abundant IGFBP-2 to -6 mRNAs and,
1075
Gre/tier et at: JGF binding proteins and kidney
24hr
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Fig. 3. Time course for the effect of forskolin, IGF-I and TGF-13 on IGFBP-3 mRNA expression. RNase protection assay of total RNA (30 xg/1ane) isolated from the same experiment described in Figure 2. 32P-labeled human antisense IGFBP-3 and 36B4 RNA probes were simultaneously hybridized with
mesangial cell total RNA. Autoradiogram shows the expected 535-hp protected fragment for IGFBP-3.
200 -
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IGFBP3, flM Fig. 4. Inhibitoiy effect of IGFBP-3 on IGF-I-induced DNA synthesis. Cells
were plated in 24-well dishes at 3 to 5
x io cells/well in Waymouth's
medium with serum. At confluence, cells were made quiescent by incubation in SFIF medium for 72 hours and then placed in the same medium
with or without IGF-I and R3-IGF-I (200 ng/ml) in the presence of increasing concentrations of IGFBP-3. Control wells were incubated with SFIF medium alone. Cells were pulsed with [3H]TdR (1 rCi/ml) for four hours prior to harvesting at 28 hours. Data represent the mean SEM of
three to four separate experiments each performed in triplicate wells. Symbols are: (LI) control; (U) IGF-I; () R3-IGF-I. Asterisks indicate significant difference from cells treated with IGF-I in the absence of IGFBP-3 or cells treated with R3-IGF-l in the presence and absence of IGFBP-3 (*P < 0.01; < 0.001).
IGF-l R3-IGF-l
—
TGF-
—
—
+
—
+
—
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+
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Fig. 5. Inhibitoiy ejfrct of TGF-/3 on IGF-I and R-IGF-I-induced DNA synthesis. Confluent quiescent mesangial cells were incubated with IGF-l
and R3-IGF-I (200 ng/ml) in the presence and absence of TGF-/3 (2 nglml). Cells were pulsed and harvested as described in Figure 4. Data represent the mean SEM of triplicate determinations from one of two similar experiments. Asterisks indicate significant difference from cells treated with IGF-I (*P < 0.001) or R3-IGF-l (**P < 0.001) alone.
require further confirmation by immunoblot analysis. These findings also demonstrate that IGFBP6 is expressed by glomerular
cells, in contrast to previous studies that have failed to detect IGFBP6 mRNA in the human kidney [161.
to a lesser extent, IGFBP1 mRNA. IGFBP gene expression The expression of multiple IGFBPs by mesangial cells suggests correlated with the release of the corresponding IGFBP into that the ratio of one IGFBP to another may be an important conditioned medium, although the release of IGFBP-1 and -6 will
factor in determining autocrine and paracrine actions of IGF-I in
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Grellier et al: IGF binding proteins and kidney
the glomerulus [28]. The overall net effect of IGF-I would depend upon the relative abundance of each IGFBP that may interact in either a synergistic or opposing manner to modulate IGF action.
IGFBP-1, -2, -3 and -5 may either enhance or inhibit IGFmediated effects under varying incubation conditions, while
IGF-I receptors, but near-normal affinity for IGFBP-3, decreased
surface bound IGFBP-3 and increased IGFBP-3 in the conditioned medium. IGFBP-3 was regulated in a similar manner by heparin, an agent that releases proteins attached to cell surface proteoglycans. These findings suggest that IGF-I regulates IGFBP-3 post-transcriptionally by increasing the release of IGFBP-3 from the cell surface or matrix proteoglycans. Perhaps in mesangial cells, IGF-I also releases cell-surface associated IGFBP-3, although this remains to be determined. Alternatively, IGF-I may influence IGFBP levels by other post-transcriptional
IGFBP4 demonstrates only inhibitory activity. For IGFBPI, the biologic effect depends on the prevalence of phosphorylated and nonphosphorylated forms [29]. Although the effect of these IGFBPs in modulating IGF actions in renal disease are unknown, recent studies suggest that they play an important role. Induction of diabetes in rats by streptozotocin mechanisms. In certain systems, IGF-I induces certain IGFBPs by causes rapid renal hypertrophy that is preceded by a rise in kidney directly binding to them, thereby protecting against proteolysis tissue IGF-I levels 24 to 48 hours after the onset of diabetes [7]. [28]. It is also possible that IGF-I, like other agents, increases During this time, there is a coordinant increase in IGFBP3 and a IGFBPs by inducing a specific protease inhibitor [42]. Whether
30 kDa IGFBP species in whole kidney extracts and an increase in one or a combination of these mechanisms play a role in IGFBP1 mRNA expression [30, 31]. By in situ hybridization, there IGF-I-mediated increase of IGFBP-3 protein released by mesanis a predominant increase in IGFBP-l and -5 mRNA levels in the gial cells requires further study. cortex and medulla, respectively [32]. The observation that treatRelease of endogenous IGFBP-3 may influence the mitogenic ment of rats with insulin, a potent inhibitor of IGFBP1, prevents effect of IGF-I on mesangial cells. In fibroblast cultures, simultathe induction of IGFBPs and inhibits renal growth, suggests that neous addition of IGFBP-3 with IGF-I inhibits the mitogenic IGFBP1 may be important for the initiation of renal hypertrophy action, while preincubation of the cells with IGFBP-3 prior to the [33, 34]. addition of IGF-I potentiates IGF action [12]. Studies suggest that The relative abundance of IGFBPs in the kidney is dependent,
inhibition of IGF action is through interaction with soluble
in part, on the regulation of each IGFBP by growth factors and IGFBP-3, while potentiation occurs via cell surface-associated hormones. In other systems, IGFBPs are regulated by agents that IGFBP-3 [43]. Co-incubation of IGFBP-3 with IGF-I or R3-IGF-I increase cAMP, IGF-I and TGF-/3, factors also implicated in the completely abrogated IGF-I, but not R3-IGF-I-induced mitogendevelopment of renal pathology [35—38]. Mechanisms that govern the synthesis and secretion of each IGFBP are complex. IGFBP esis in confluent mesangial cultures. These findings suggest that levels in mesangial cell conditioned medium may be influenced by inhibition of IGF-I bioactivity is due to IGFBP-3 sequestering several factors including differential transcription rate, post-translational modifications, alterations in membrane-associated fractions of certain IGFBPs and specific IGFBP proteases [39]. Under
IGF-I, thereby preventing the interaction of IGF-I with its receptor. This is also supported by the observation that IGFBP-3 alone had no effect on mesangial cell DNA synthesis. In other
basal conditions, the relative ratio of IGFBPs in mesangial systems, however, IGFBP-3 has been shown to have a direct inhibitory action on cell growth in the absence of IGF-I or -II [14]. Induction of IGFBP-3 by TGF-f3 suggests that IGFBP-3 may
conditioned medium changed with time. IGFBP-2 and -3 protein levels increased while IGFBP-4 and -5 decreased between 8 to 24 hours. In the case of IGFBP-3, increased protein levels correlated with a time-dependent increase in IGFBP-3 mRNA abundance. In mesangial conditioned medium forskolin, IGF-I and TGF-13 induced distinct patterns of IGFBPs, with a prominent increase in IGFBP-3. Forskolin, a direct activator of adenylate cyclase, was used as a tool to determine the effect of increased cAMP levels on
mediate the inhibitory effect of TGF-f3 on IGF-I-induced DNA synthesis. Inhibition of both IGF-I and R3-IGF-I-induced DNA synthesis by TGF-13, however, indicates that the inhibitory effect of TGF-/3 is not mediated via an IGFBP. These results are in contrast to those reported by Yateman et al [20], where TGF-13 blocked the mitogenic response of fibroblasts to IGF-I via release
manner [40]. In MDBK epithelial cells and in fibroblasts, forskolin
IGF-I signaling pathways by TGF-13 [44].
IGFBPs. RNase protection assay demonstrated that forskolin of an endogenous IGFBP, probably IGFBP-3. Perhaps other increased IGFBP-3 mRNA levels. Previous studies have shown factors are involved in mediating the effect of TGF-f3 in mesangial that IGFBP-3 is regulated in a tissue- and cell type-specific cells such as down-regulation of IGF-I receptors or alteration of
also increased IGFBP-3 mRNA and protein levels, whereas in osteoblasts, forskolin inhibited IGFBP-3 [18, 28, 41]. IGF-I and TGF-f3 were potent inducers of IGFBP-3 protein
release in mesangial cells; however, TGF-p, but not IGF-I, markedly increased IGFBP-3 mRNA levels. These findings suggest that IGFBP-3 is regulated by distinct mechanisms: forskolin and TGF-13 act by increasing de novo synthesis of IGFBP-3, while
IGF-I acts by post-transcriptional mechanism(s). Similar results have been obtained in human fibroblasts [19]. In these cells, the effect of TGF-p on IGFBP-3 was at the level of mRNA abun-
The physiological significance of IGFBPs in renal disease remains to be elucidated. In diabetic nephropathy, one may speculate that IGF-I and insulin regulate IGFBPs. The transient increase in IGFBP-1, IGFBP-3 and perhaps other IGFBPs and the relative ratio of stimulatoiy versus inhibitory IGFBPs in the glomerular microenvironment may play a key role in determining IGF action. Both IGF-I and TGF-J3 may act synergistically to enhance matrix formation, as well as modulate IGFBP levels that, in turn, influence IGF-I-mediated effects. Further understanding of the mechanisms involved in regulating IGFBP expression and
dance, while IGF-I altered the amount of surface bound their interaction with IGFs in the glomerulus is required to IGFBP-3. IGFBPs synthesized by fibroblasts may remain cell understand the complex role of this system in glomerular patholsurface-associated. IGF-I analogues with reduced affinity for ogy.
Grellier et al: IGF binding proteins and kidney
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Acknowledgments This work was supported in part by the Veterans Administration Medical Research Service (S.A, HA), National Institutes of Health Grants AR42306 (S.A), DK33665 (HA.), CA52952 (DY.) and by a
t8. COHICK WS, CLEMMONS DR: Regulation of IGFBP secretion and
Cancer Center Support Grant P30-CA54174 to the Cancer Therapy and Research Center. D.Y. is a Pew Scholar in the Biomedical Sciences.
131:1703—1710, 1992 20. YATEMAN ME, CLAFFEY DC, CWYFAN HUGhES SC, FROST VJ, WASS
Portions of this work were published in abstract form at the annual meeting of the American Society of Nephrology, Orlando, Florida, October 26—29, 1994.
Reprint requests to Sheriy L. Abboud, M.D., Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA.
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and IGF-binding proteins in the physiological and pathological pro-
cesses of the kidney. Virchows Archiv B Cell Pathol 62:207—220, 1992 3. TIIRSCHBERG R, KOI'PLE JD: Evidence that insulin-like growth factor
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KOV H: Kidney IGF-l mRNA in initial renal hypertrophy in experimental diabetes in rats. Diabetologia 33:334—338, 1990 8. CATANESE VM, SclAvouNo PJ, LANGO MN: Discordant, organspecific regulation of insulin-like growth factor-I messenger ribonucleic acid in insulin-deficient diabetes in rats. Endocrinology 132:496— 503, 1993 9. O'SHEA MH, LAYISH DT: Growth hormone and the kidney: A case presentation and review of the literature. JAm Soc Nephrol 3:157—161, 1992 10. CHIN E, B0NDY CA: Dietaly protein-induced renal growth: correla-
tion between renal IGF-t synthesis and hyperplasia. Am J Physiol 266:C1037—C1045, 1994 11. SHIMASAKI 5, LING N: Identification and molecular characterization of
insulin-like growth factor binding proteins (IGFBP-1,-2,-3,-4,-5 and
-6), Prog Growth Factor Res 3:243—266, 1991 12. DEMELLOW JSM, BAxtER RC: Growth hormone-dependent insulinlike growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts. Biochem Biophys Res Commun 156:199—204, 1988 13. JONES JI, GOCKERMAN A, BUSBY WIT JR, CAMACHO-HURNER C,
CLEMMONS DR: Extracellular matrix contains insulin-like growth factor binding protein-5: Potcntiation of the effects of IGF-I. J Cell Biol 121:679—687, 1993 14. OH Y, MULLER HL, LAMSON U, ROSENFELD RG: Insulin-like growth
factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. J Biol Chem 268:14964—14971, 1993 15. ANDRESS DL, Loon SM, ZAPF J, KIEFER MC: Carboxy-truncated insulin-like growth factor binding protein-S stimulates mitogenesis in osteoblast-like cells. Biochem Biophys Res Commun 195:25—30, 1993 16. CHIN E, MICHELS K, BONDY CA: Partition of insulin-like growth
factor (IGF)-binding sites between the IGF-I and IGF-lI receptors and IGF-binding proteins in the human kidney. J Clin Endocrinol Metab 78:156—164, 1994 17. AR0N DC, ROSENZWEIG JL, ABBOUD HE: Synthesis and binding of insulin-like growth factor by human glomerular mesangial cells. J COn Endocrinol Metab 68:585—59 1, 1989
modulation of cell growth in MDBK cells. Growth Regul 3:20—23, 1993 19. MARTIN JL, BALLESTEROS M, BAXTER RC: Insulin-like growth factor-I
(IGF-I) and transforming growth factor-pI release IGF-binding protein-3 from human fibroblasts by different mechanisms. Endocrinology JAH, Ho[.[,Y iMP: Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production. J Endocrinol 137:151—159, 1993 21. BASSET P, BELLOCQ JP, W0I.F C, STOLL 1, HUTIN P, LIMACHER JM,
PODHAJCER OL, CHENARD MP, RIO MC, CHAMBON P: A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348:699—704, 1990 22. SHULTZ PJ, DECORLETO PE, SILVER BJ, ABBOUD HE: Mesangial cells
express PDGF mRNAs and proliferate in response to PDGF. Am J Physiol 255:F674—F684, 1988
23. Aisnouu SL, BETHEL CR, ARON DC: Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine hone marrow stromal cells. J Clin Invest 88:470—475, 1991 24. HOSSENLOPP P, SEURIN D, SEGOVIA-QUINSON B, HARDOVIN S, BIN-
oux M: Analysis of serum insulin-like growth factor binding proteins using Western blotting: Use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138—143, 1986 25. MCGUIRE WL JR, JACKSON JG, FIGUEROA JA, SHIMASAKI S, POWELL
DR, YEE D: Regulation of insulin-like growth factor-binding protein
(LGFBP) expression by breast cancer cells: Use of IGFBP-l as an inhibitor of insulin-like growth factor action. J Nail Cancer Inst 84:1336—1341, 1992 26. FIGUEROA JA, JACKSON JG, MCGUIRE WL, KRYWICKI RF, YEE D:
Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status. J Cell Biochem 52:196—205, 1993 27. JAFFER F, SAUNDERS C, SIIuI:I'z P, THROCKMORTON D, WEINSHELL E,
ABBOUD HE: Regulation of mesangial cell growth by polypeptide mitogens. Inhibitory role of transforming growth factor beta. Am J Pathol 135:261—269, 1989 28. CAMACHO-IIUBNER C, BUSBY WH, MCCUSKER RH, WRIGHT G,
CLEMMONS DR: Identification of the forms of insulin-like growth factor-binding proteins produced by human fibroblasts and the mechanisms that regulate their secretion. J Biol Chem 267:11949—11956, 1992 29. JONES JI, D'ERCOLE AJ, CAMACIIO-HUBNER C, CLEMMONS DR:
Phosphorylation of insulin-like growth factor (IGF)-binding protein I in cell culture and in vivo: Effects on affinity for IGF-l. Proc NatlAcad Sci USA 88:7481—7485, 1991 30. FLYVBJERG A, KESSLER U, DoRIc B, FUNK B, ORSKOV H, KIESS W:
Transient increase in renal insulin-like growth factor binding proteins
during inital kidney hypertrophy in experimental diabetes in rats. Diabetologia 35:589—593, 1992
31. LEROITH D, WERNER H, PHILLIP M, ROBERTS CT JE: Role of insulin-like growth factors in diabetic kidney disease. Am J Kidney Dis 22:722—726, 1993 32. LANDAU D, CHIN E, BONDY C, DOMENE H, ROBERTS CT JR, GRONBAEK H, FLYVBJERG A, LEROFrH D: Expression of insulin-like growth
factor binding proteins in the rat kidney: Effects of long-term diabetes. Endocrinology 136:1835—1842, 1995 33. BRISMAR K, FERNOVIST-FORBES E, WARREN J, HALL K: Effect of
insulin on the hepatic production of insulin-like growth factor-binding protein-i (IGFBP-i), IGFBP-3, and IGF-I in insulin-dependent diabetes. J Clin Endocrinol Metab 79:872—878, 1994 34. QUIN JD, FtSHER BM, MACCUISH AC, BEASTALL GH, SMALL M, HOLLY JMP, COTFERILL AM: Insulin-like growth factor binding protein 1 response to acute insulin induced hypoglycaemia in Type I diabetes. Clin Endocrinol 41:225—229, 1994 35. SAVIC V, BLANCHARD A, VLAHOVIC P, STEFANOVIC V, ARDAILI.OU N,
ARDAILLOU R: Cyclic adenosine monophosphate-stimulating agents induce ecto-5'-nucleotidase activity and inhibit DNA synthesis in rat cultured mesangial cells. Arch Biochem Biophys 290:202—206, 1991
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36. YAMAMOTO T, NAKAMURA T, NOBLE NA, RUOSLAHTI E, BORDER
WA: Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy. Proc NatI Acad Sci USA 90:1814—1818, 1993
37. BAR RS, DAKE BL, STUECK S: Stimulation of proteoglycans by IGF-I
and IGF-II in microvessel and large vessel endothelial cells. Am J Physiol 253:E21—E27, 1987 38. NAKAMURA T, EBIHARA I, Fuiui M, OSADA S, NAGAOKA I, HORIKO-
Sm S, Toimo Y, K0IDE H: Messenger RNA expression for growth factors in glomeruli from focal glomerular sclerosis. Clin Immunol Immunopathol 66:33—42, 1993
39. CHEN TL, CHANG LY, DIGREGORLO DA, PERLMAN AJ, HUANG Y-F:
Growth factor modulation of insulin-like growth factor binding proteins in rat osteoblast-like cells. Endocrinology 133:1382—1389, 1993 40. ALBISTON AL, HERINGTON AC: Tissuc distribution and regulation of
insulin-like growth factor (IGF)-binding protein-3 messenger ribonu-
cleic acid (mRNA) in the rat: Comparison with IGF-I mRNA expression. Endocrinology 130:497—502, 1992
41. MOHAN 5: Insulin-like growth factor binding proteins in bone cell regulation. Growth Regul 3:67—70, 1993 42. CONOVER CA, CLARKSON JT, BALE LK: Phorbol ester tumor promot-
ers regulate insulin-like growth factor-binding protein-4 proteolysis. Endocrinology 133:1347—1351, 1993
43. Cortovp. CA, RaNK M, LOMBANA F, POWELL DR: Structural and biological characterization of bovine insulin-like growth factor binding protein-3. Endocrinology 127:2795—2803, 1990 44. BESSET V, COLLETTE J, CHAIJVIN MA, FRANCI-TIMONT P, BENAHMED
M: Effect of transforming growth factor-l on the insulin-like growth factor system in cultured porcine Leydig cells. Mol Cell Endocrinol 99:251—257, 1994
Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells PASCALE GRELLIER, MICHAEL 5ABBAH, BRUNO FOUOUERAY, KATHLEEN WOODRUFF, DOUGLAS YEE, HANNA E. ABBOUD, and 5HERRY L. ABBOUD Departments of Medicine and Pathology, University of Texas Health Science Center and Audie Murphy Veterans Hospital, San Antonio, Texas, USA
Characterization of insulin-like growth factor hinding proteins and regulation of IGFBP3 in human mesangial cells. IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-l and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays
using total RNA demonstrated that MC express all of the IGFBPs. E5251]IGFJ Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-3 arc implicated in glomerular
hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng./ml), TGF-f3 (2 ng/ml) and forskolin (10-i
at) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-j3, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-l-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-f3 is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs.
Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-p and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.
TGF-/3 to enhance extracellular matrix deposition within the glomerulus. The biologic action of IGF-I/II in the kidney may be modulated
by a family of six IGFBPs, designated IGFBP-1 to -6. These IGFBPs bind with high affinity to lOP-I and -II, but not to insulin [11]. They serve as a reservoir and prolong the biologic half-life of TOPs in the circulation. Depending on the IGFBP species, target cell and culture conditions, IGPBPs may either inhibit IGF action by forming inactive complexes with IGFs or enhance TOP action by facilitating IGF interaction with surface receptors [12]. Certain TGFBPs provide a storage site for IGFs in the extraeellular matrix [13]. In addition, IGFBP-3 and -5 have intrinsic biologic activity independent of IGFs [14, 15].
IGPBPs are abundant in the human kidney and demonstrate
distinct cellular distribution. By in situ hybridization, IGFBP-1 to -5 mRNAs arc expressed in glomeruli; however, the precise
IGFBPs produced by the different cell types that comprise the glomerulus have not been determined [16]. We have previously demonstrated that human mesangial cells express lOP-I mRNA, lOP-I receptors and release IGF-I and IGF-binding activity [17]. These cells provide a useful model for determining the spectrum of IGFBPs released by mesangial cells, the factors that regulate IGFBP gene expression, and the role of each IGFBP in modulating lOP action in the glomerulus. Previous studies have demon-
strated that adenylate cyclase agonists such as forskolin, and Insulin-like growth factors-I and -II (IGF-I and -II) play an important role in kidney embryogenesis and function [1, 2]. Glomeruli and segments of the distal nephron are targets for circulating IGFs and provide a source of local IGF-I production
[1]. The systemic administration of IGF-I has been shown to increase renal plasma flow, glomerular filtration rate and kidney volume in rats and humans [3, 4]. Increased circulating or locally produced IGF-I have also been implicated in the pathogenesis of renal hypertrophy associated with unilateral nephrectomy, diabe-
tes, hypersomatotropism, and a high protein diet [5—10]. In chronic diabetic nephropathy, lOP-I may act in concert with
cytokines including IGF-I and TOF-J3, regulate IOFBPs in fibroblasts and other cell types [18—20].
In this study, we determined the molecular identity of each IGFBP released by human mesangial cells. The effect of IGF-I, TOP-f3 and the cAMP agonist, forskolin, on IGFBPs released by mesangial cells was determined. Since IGFBP3 was markedly induced by lOP-I and TGF-3, recombinant IOFBP3 was tested for its effect on IGF-I-induced DNA synthesis in mesangial cells. Whether IGFBPs are involved in mediating the inhibitory effect of TGF-13 on TOP-I-stimulated DNA synthesis was also explored. Methods Materials
Received for publication August 24, 1995 and in revised form November 20, 1995 Accepted for publication November 20, 1995
© 1996 by the International Society of Nephrology
Porskolin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant human TGP-1 was from R&D Systems, Inc. (Minneapolis, MN, USA) and recombinant human 1071
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Gre/her et a!: IGF binding proteins and kidney
IGF-I and R3-IGF-I from GroPep (Adelaide, Australia). Recombinant human IGFBP3 was purchased from Upstate Biotechnol-
Western ligand blot
Samples were concentrated 40-fold by ultrafiltration in Centricon 10 microconcentrators (10 kDa molecular wt cutoff). Ligand blotting with [1251]IGF-I was performed by the method of Hossenlopp et a! [24] as previously described [25]. Briefly, samples were electrophoresed through a 10% SDS-polyacryiamide gel. Separated proteins were electroblotted onto nitrocellulose filters and then incubated with [1251]IGF-I overnight at 4°C and visualized by autoradiography.
ogy, Inc. (Lake Placid, NY, USA). Rainbow molecular mass markers and radiolabeled IGF-I was obtained from Amersham (Arlington Heights, IL, USA). Centricon 10 concentrators were from Amicon (Danvers, MA, USA). RNAzol B was purchased from Cinna Biotex (Houston, TX, USA) and the Riboprobe Kit from Promega (Madison, WI, USA). Antisense RNA probes. The probes for human IGFBP-2, -4, -5 and -6 were provided by Dr. Shimasaki (Whittier Institute, La Jolla, CA, USA) [11]. Each probe was derived from a eDNA [3H/thymidine ([3H]TdR) assay fragment corresponding to part of the protein-coding region and Confluent cells were made quiescent by incubation in SFIF cloned into pBluescript SKI. The IGFBP2 and IGFBP4 probes medium for 72 hours. They were then washed once with SFIF were synthesized from a StyI-EcoRV 382-bp and an EcoRI- medium and incubated in same medium with IGF-I (200 ng/ml) Hindlil 505-bp fragment, respectively. The IGFBP5 and IGFBP6 probes were synthesized from a SacII-Saci 317-bp and a PstI-PstI 267-bp fragment, respectively. IGFBP-1 and -3 probes (provided
by Dr. D. Powell, Baylor University, Waco, TX, USA) were synthesized from a PstI-PstI 369-bp and a PstI-EcoRI 535-bp fragment, respectively. The human 36B4 eDNA probe detects a ubiquitously expressed ribosomal phosphoprotein and was provided by Dr. P. Chambon (INSERM, Institut de la Sante et de Ia Recherche Medicale) [21]. Cell culture
and R3-IGF-I (200 ng/ml) alone and in combination with TGF-13 (2 ng/ml). In some experiments, IGF-I and R3-IGF-I were incu-
bated in the presence and absence of various concentrations of IGFBP3. Cells were pulsed with [3H]TdR (1 iCi/ml) for four hours prior to harvesting at 28 hours. The amount of [3H]TdR incorporated into TCA-precipitable material was determined by liquid scintillation counting. Statistical analysis Analysis of the difference of the means among treatment groups
was determined utilizing Tukey's Studentized Range Test. The results in Figure 4 represent the mean of at least three separate Human mesangial cells were cultured from glomeruli isolated experiments SEM. by differential sieving as previously described [22]. Portions of normal renal cortex were obtained from kidneys judged unsuitResults able for transplantation. Epithelial cells were removed by diIGFBP gene expression in human mesangial cells gestion with collagenase. Glomerular cores were cultured in IGFBP mRNAs are expressed in the glomerulus. We examined Waymouth's medium (GIBCO, Grand Island, NY, USA) supplemented with 15 mi Hepes, 0.6 U/mi insulin, 2 mrvi glutamine, 0.1 the specific IGFBP mRNAs expressed by mesangial cells. Total m nonessential amino acids, 1 m sodium pyruvate, antibiotic/ RNA isolated from mesangial cells was hybridized with human antifungal solution, and 17% fetal calf serum (FCS). Cells were IGFBP-i to -6 antisense RNA probes and analyzed using a sensitive RNase protection assay. Figure 1 demonstrates that used between passages three to ten. mesangial cells constitutively express abundant levels of IGFBP-2 to -6 mRNAs, and to a lesser extent IGFBP1 mRNA. For each RNase protection assay IGFBP analyzed, human breast carcinoma cell lines, MDA-MB To determine constitutive IGFBP-1 to -6 mRNA expression, total 231 or MCF-7, were used as controls since they are known to RNA was isolated from confluent cultures of mesangial cells express certain IGFBP mRNAs [26]. Autoradiograms show the maintained in Waymouth's medium with serum. Total RNA was expected protected fragment in both the control and mesangial isolated using RNAzoI B method. Concentrations were deter- cell total RNAs: 369-bp for IGFBP1; 325-bp for IGFBP2; 535-bp mined by spectrophotometry; integrity and quantitation was con- for IGFBP3; 505-bp for IGFBP4; 317-bp for IGFBP5 and 267-bp firmed by visualizing ethidium bromide-stained 28S and 18S for IGFBP6. These fragments are specific and not observed in the ribosomal bands on 1% agarose gels. For experiments designed to tRNA lane. determine the effect of forskolin, IGF-I and TGF-/3 on IGFBP3 mRNA and protein, confluent MC were washed three times with Characterization of IGFBPs in mesangial cell conditioned medium serum-free, insulin-free (SFIF) medium and incubated 24 hours in To determine if IGFBP mRNA expression is associated with the same medium. Fresh SFIF medium with or without forskolin, the release of the corresponding IGFBPs, conditioned medium IGF-I, or TGF-J3 was added. Cell-free supernatants were col- from mesangial cells was analyzed by ligand blotting with lected at 8 and 24 hours and used for Western ligand blot analysis. ['251]IGF-I. In Figure 2, the first lane shows conditioned medium Cells were also harvested for total RNA at these time points and obtained from ovarian carcinoma cells (OVCAR-3) that contain at 72 hours. RNase protection assay was carried out as we have IGFBPs of 24, 29 (not visualized), 32 kDa and a doublet at 46 kDa previously described [23]. 32P-labeled single-stranded RNA that correlate with IGFBP-4, -5, -2 and -3, respectively, as we have probes were synthesized in an antisense orientation using SP6, T7 previously shown [25]. The constitutive release of IGFBPs from or T3 RNA polymerase. After hybridization, samples were di- confluent mesangial cell cultures incubated under unstimulated, gested sequentially with RNases A and Ti and proteinase K. The serum-free, insulin-free conditions is shown in lanes labeled products were analyzed by electrophoresis through a 7 M urea/6% control (8 and 24 hr). At eight hours, the 24 kDa species predominates with less abundant species of 29, 32, and 46 kDa. At acrylamide gel, followed by autoradiography.
1073
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Fig. 1. RNase protection assays for IGFBP mRNA expression by mesangial cells. 32P-labeled LGFBP-1 to -6 antisense RNA probes were hybridized with
total RNA (30 sg) isolated from mesangial cells. Total RNA from human breast cancer cell lines (MDA-MB-23 1 or MCF-7) were used as controls. Autoradiograms show the predicted protected fragment in both controls and mesangial cells. Probes hybridized with tRNA were completely digested by RNases. Last lane of each panel shows undigested IGFBP probes. The migration of 32P-labeled DNA markers is shown to the left (in basepairs).
24 hours, there is a predominance of the 46 kDa doublet and the levels were similar in control and forskolin-treated samples, and a 32 kD IGFBP. Less abundant IGFBP species are seen at 24 kDa decrease in 32 kDa IGFBP2 was observed. IGF-I primarily and at approximately 26 and 34 kDa; the 29 kDa IGFBP present induced IGFBP-2 and -5 and slightly increased 46 kDa IGFBP3 at eight hours is no longer observed at 24 hours. The precise doublet at eight hours. By 24 hours, accumulation of IGFBP-2 identity of the 26 and 34 kDa species is unknown, but the 26 kDa and -3 markedly increased in response to IGF-I, an effect also
band may represent either a glycosylated form of IGFBP4 or
observed on the 26 kDa species. TGF-f3 primarily induced
IGFBP1; the 34 kDa band may be a glycosylated IGFBP. The 46
IGFBP-4 and -2 and slightly increased IGFBP3 at eight hours. Similar to IGF-I, TGF-f3 markedly increased the accumulation of IGFBP-2 and -3 at 24 hours. This effect persisted in cultures exposed to TGF-J3 for 48 hours (data not shown).
kDa doublet present in the medium was more abundant at 48 hours compared to 24 hours (data not shown). These findings
indicate that the relative abundance of IGFBPs in mesangial cell conditioned medium varies in a time-dependent fashion. Release Regulation of JGFBP3 mRNA by forskolin, IGF-J and TGF-13 of IGFBP-2, -3, -4, -5 and possibly -1 and -6 by mesangial cells correlates with expression of these IGFBP mRNAs. IGFBP6 may Since IGFBP3 showed the strongest response to cytokines, not be detected by [1251]IGF-I ligand blot since IGFBP6 protein experiments were carried out to determine if IGFBP3 is regulated has a higher affinity for IGF-II than IGF-I. at the level of mRNA abundance (Fig. 3). RNA was isolated from the same experiment described in Figure 2 and from an additional Regulation of IGFBPs in mesangial cell conditioned medium by 48 hours time point. In unstimulated cells, IGFBP3 mRNA forskolin, JGF-I and TGF-/3 increased in a time-dependent manner (control 8, 24 and 48 hr), cAMP agonists and various cytokines have been shown to similar to the protein levels observed on the ligand blot. Forskolin regulate IGFBP proteins in other systems. We performed time increased IGFBP3 mRNA expression at eight hours with a peak course experiments to determine the effect of forskolin, IGF-I and effect observed at 24 hours, and a decline to basal levels by 48 TGF-13 on IGFBPs released by mesangial cells. Figure 2 shows hours. Although a corresponding increase in IGFBP3 protein was that IGFBPs are differentially regulated by these agents. Forsko- not observed in this experiment, other experiments showed induc-
lin induced 24 kDa IGFBP4 and, to a lesser extent, 29 kDa tion of IGFBP3 protein by forskolin. While IGF-l appeared to IGFBP5 at eight hours compared to control. At 24 hours, IGFBP4 slightly decrease IGFBP3 mRNA at each time point tested,
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Grellier et al: JGF binding proteins and kidney
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21.5— Fig. 2. Time course for the effect of forskolin, IGF-I and TGF-P on IGFBPs by Western ligand blot analysis. Confluent cells were made quiescent by placing
them in SFIF medium for 24 hours. Forskolin (10 M), IGF-l (100 ng/ml) or TGF-/3 (2 ng/ml) were then added and cells incubated for thc indicated time points. Samples were electrophoresed through a nonreducing 10% SDS-polyacrylamide gel and transferred to nitrocellulose. After incubation with [1251j1GF-l, filter-immobilized IGFBPs were visualized by autoradiography after two days exposure. The migration positions of molecular size markers (in kDa) are indicated on the left.
TGF-/3 was a potent inducer of IGFBP3 mRNA at 24 and 48 hours compared to control. Thus, the effect of TGF-/3, but not IGF-1, on IGFBP3 mRNA expression correlated with IGFBP3 protein abundance as measured by ligand blotting. Control 36B4 mRNA did not vary in response to the test agents, demonstrating that the response in IGFBP3 mRNA does not simply reflect a generalized nonspecific change in total RNA expression (Fig. 3, lower panel). Similar results were observed in two separate
DNA synthesis, suggesting that any endogenous production of IGFs by cells, under these experimental conditions, did not result in autocrine regulation. Perhaps released IGF-I is immediately sequestered by endogenously released IGFBPs.
experiments.
inhibits DNA synthesis in mesangial cells and blocks the mitogenic effect of certain cytokines {27. The marked induction of IGFBP3 protein by TGF-13 suggests that TGF-f3 may inhibit the mitogenic effect of IGF-I via the release of IGFBP3. Figure 5 shows that IGF-I and R3-IGF-I stimulated DNA synthesis in mesangial cells compared to control. TGF-/3 alone inhibited basal DNA synthesis. When co-incubated with TGF-13, the mitogenic effects of both IGF-I and R3-IGF-I were markedly inhibited. These findings suggest that the inhibitory effect of TGF-J3 is not mediated via an IGF-binding protein.
Effect of recombinant JGFBP3 on JGF-J-induced DNA synthesis in mesangial cells
Since IGFBP3 may modulate IGF-I action, the effect of recom-
binant human IGFBP3 on IGF-I-stimulated DNA synthesis in mesangial cells was determined. Figure 4 shows that IGFBP3 inhibited IGF-I-induced mitogenesis in a dose-dependent manner with approximately 35% inhibition at 25 nM and 50% inhibition at
50 mM IGFBP3. Addition of 50 nM IGFBP3, a concentration twofold higher than IGF-I, completely abrogated the mitogenic effect of IGF-I. Although there was some variation in IGF-
Effect of TGF-13 on IGF-I and R3-IGF-I-induced DNA synthesis in mesangial cells
We and others have previously demonstrated that TGF-p
Discussion The present study demonstrates that human mesangial cells in induced DNA synthesis from 1.5- to 2.5-fold compared to control, the addition of 25 and 50 tiM IGFBP3 consistently inhibited the culture constitutively express IGFBP-1 to -6 mRNAs and release effect of IGF-I. To determine if the effect of IGFBP3 was specific IGFBPs. Recent work has shown that human glomeruli express for IGF-I-stimulated DNA synthesis, and required the interaction IGFBP-1 to -5 mRNAs. IGFBP1 mRNA was localized to parietal between the binding protein and ligand, we tested the effect of epithelial cells along the glomerular capsule and IGFBP2 mRNA
IGFBP3 on R3-IGF-I-induced DNA synthesis. R3-IGF-1 is an
was present in glomerular cells with a distribution pattern of
analogue of IGF-l that retains affinity for IGF-1 receptors but fails to complex with IGFBPs. As shown in Figure 4, 50 nM IGFBP3 did not influence DNA synthesis induced by R3-IGF-1. Incubation of cells with IGFBP3 alone showed no significant effect on basal
epithelial cells {16. However, the precise molecular identities of each IGFBP expressed by the individual cell types that comprise the glomerulus are unknown. By RNase protection assay, human mesangial cells expressed abundant IGFBP-2 to -6 mRNAs and,
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Gre/tier et at: JGF binding proteins and kidney
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Fig. 3. Time course for the effect of forskolin, IGF-I and TGF-13 on IGFBP-3 mRNA expression. RNase protection assay of total RNA (30 xg/1ane) isolated from the same experiment described in Figure 2. 32P-labeled human antisense IGFBP-3 and 36B4 RNA probes were simultaneously hybridized with
mesangial cell total RNA. Autoradiogram shows the expected 535-hp protected fragment for IGFBP-3.
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. F
'5
100
-
50
-
0
0—
6CX
-LI
0
10
25
50
IGFBP3, flM Fig. 4. Inhibitoiy effect of IGFBP-3 on IGF-I-induced DNA synthesis. Cells
were plated in 24-well dishes at 3 to 5
x io cells/well in Waymouth's
medium with serum. At confluence, cells were made quiescent by incubation in SFIF medium for 72 hours and then placed in the same medium
with or without IGF-I and R3-IGF-I (200 ng/ml) in the presence of increasing concentrations of IGFBP-3. Control wells were incubated with SFIF medium alone. Cells were pulsed with [3H]TdR (1 rCi/ml) for four hours prior to harvesting at 28 hours. Data represent the mean SEM of
three to four separate experiments each performed in triplicate wells. Symbols are: (LI) control; (U) IGF-I; () R3-IGF-I. Asterisks indicate significant difference from cells treated with IGF-I in the absence of IGFBP-3 or cells treated with R3-IGF-l in the presence and absence of IGFBP-3 (*P < 0.01; < 0.001).
IGF-l R3-IGF-l
—
TGF-
—
—
+
—
+
—
+ —— + +
+
+
Fig. 5. Inhibitoiy ejfrct of TGF-/3 on IGF-I and R-IGF-I-induced DNA synthesis. Confluent quiescent mesangial cells were incubated with IGF-l
and R3-IGF-I (200 ng/ml) in the presence and absence of TGF-/3 (2 nglml). Cells were pulsed and harvested as described in Figure 4. Data represent the mean SEM of triplicate determinations from one of two similar experiments. Asterisks indicate significant difference from cells treated with IGF-I (*P < 0.001) or R3-IGF-l (**P < 0.001) alone.
require further confirmation by immunoblot analysis. These findings also demonstrate that IGFBP6 is expressed by glomerular
cells, in contrast to previous studies that have failed to detect IGFBP6 mRNA in the human kidney [161.
to a lesser extent, IGFBP1 mRNA. IGFBP gene expression The expression of multiple IGFBPs by mesangial cells suggests correlated with the release of the corresponding IGFBP into that the ratio of one IGFBP to another may be an important conditioned medium, although the release of IGFBP-1 and -6 will
factor in determining autocrine and paracrine actions of IGF-I in
1076
Grellier et al: IGF binding proteins and kidney
the glomerulus [28]. The overall net effect of IGF-I would depend upon the relative abundance of each IGFBP that may interact in either a synergistic or opposing manner to modulate IGF action.
IGFBP-1, -2, -3 and -5 may either enhance or inhibit IGFmediated effects under varying incubation conditions, while
IGF-I receptors, but near-normal affinity for IGFBP-3, decreased
surface bound IGFBP-3 and increased IGFBP-3 in the conditioned medium. IGFBP-3 was regulated in a similar manner by heparin, an agent that releases proteins attached to cell surface proteoglycans. These findings suggest that IGF-I regulates IGFBP-3 post-transcriptionally by increasing the release of IGFBP-3 from the cell surface or matrix proteoglycans. Perhaps in mesangial cells, IGF-I also releases cell-surface associated IGFBP-3, although this remains to be determined. Alternatively, IGF-I may influence IGFBP levels by other post-transcriptional
IGFBP4 demonstrates only inhibitory activity. For IGFBPI, the biologic effect depends on the prevalence of phosphorylated and nonphosphorylated forms [29]. Although the effect of these IGFBPs in modulating IGF actions in renal disease are unknown, recent studies suggest that they play an important role. Induction of diabetes in rats by streptozotocin mechanisms. In certain systems, IGF-I induces certain IGFBPs by causes rapid renal hypertrophy that is preceded by a rise in kidney directly binding to them, thereby protecting against proteolysis tissue IGF-I levels 24 to 48 hours after the onset of diabetes [7]. [28]. It is also possible that IGF-I, like other agents, increases During this time, there is a coordinant increase in IGFBP3 and a IGFBPs by inducing a specific protease inhibitor [42]. Whether
30 kDa IGFBP species in whole kidney extracts and an increase in one or a combination of these mechanisms play a role in IGFBP1 mRNA expression [30, 31]. By in situ hybridization, there IGF-I-mediated increase of IGFBP-3 protein released by mesanis a predominant increase in IGFBP-l and -5 mRNA levels in the gial cells requires further study. cortex and medulla, respectively [32]. The observation that treatRelease of endogenous IGFBP-3 may influence the mitogenic ment of rats with insulin, a potent inhibitor of IGFBP1, prevents effect of IGF-I on mesangial cells. In fibroblast cultures, simultathe induction of IGFBPs and inhibits renal growth, suggests that neous addition of IGFBP-3 with IGF-I inhibits the mitogenic IGFBP1 may be important for the initiation of renal hypertrophy action, while preincubation of the cells with IGFBP-3 prior to the [33, 34]. addition of IGF-I potentiates IGF action [12]. Studies suggest that The relative abundance of IGFBPs in the kidney is dependent,
inhibition of IGF action is through interaction with soluble
in part, on the regulation of each IGFBP by growth factors and IGFBP-3, while potentiation occurs via cell surface-associated hormones. In other systems, IGFBPs are regulated by agents that IGFBP-3 [43]. Co-incubation of IGFBP-3 with IGF-I or R3-IGF-I increase cAMP, IGF-I and TGF-/3, factors also implicated in the completely abrogated IGF-I, but not R3-IGF-I-induced mitogendevelopment of renal pathology [35—38]. Mechanisms that govern the synthesis and secretion of each IGFBP are complex. IGFBP esis in confluent mesangial cultures. These findings suggest that levels in mesangial cell conditioned medium may be influenced by inhibition of IGF-I bioactivity is due to IGFBP-3 sequestering several factors including differential transcription rate, post-translational modifications, alterations in membrane-associated fractions of certain IGFBPs and specific IGFBP proteases [39]. Under
IGF-I, thereby preventing the interaction of IGF-I with its receptor. This is also supported by the observation that IGFBP-3 alone had no effect on mesangial cell DNA synthesis. In other
basal conditions, the relative ratio of IGFBPs in mesangial systems, however, IGFBP-3 has been shown to have a direct inhibitory action on cell growth in the absence of IGF-I or -II [14]. Induction of IGFBP-3 by TGF-f3 suggests that IGFBP-3 may
conditioned medium changed with time. IGFBP-2 and -3 protein levels increased while IGFBP-4 and -5 decreased between 8 to 24 hours. In the case of IGFBP-3, increased protein levels correlated with a time-dependent increase in IGFBP-3 mRNA abundance. In mesangial conditioned medium forskolin, IGF-I and TGF-13 induced distinct patterns of IGFBPs, with a prominent increase in IGFBP-3. Forskolin, a direct activator of adenylate cyclase, was used as a tool to determine the effect of increased cAMP levels on
mediate the inhibitory effect of TGF-f3 on IGF-I-induced DNA synthesis. Inhibition of both IGF-I and R3-IGF-I-induced DNA synthesis by TGF-13, however, indicates that the inhibitory effect of TGF-/3 is not mediated via an IGFBP. These results are in contrast to those reported by Yateman et al [20], where TGF-13 blocked the mitogenic response of fibroblasts to IGF-I via release
manner [40]. In MDBK epithelial cells and in fibroblasts, forskolin
IGF-I signaling pathways by TGF-13 [44].
IGFBPs. RNase protection assay demonstrated that forskolin of an endogenous IGFBP, probably IGFBP-3. Perhaps other increased IGFBP-3 mRNA levels. Previous studies have shown factors are involved in mediating the effect of TGF-f3 in mesangial that IGFBP-3 is regulated in a tissue- and cell type-specific cells such as down-regulation of IGF-I receptors or alteration of
also increased IGFBP-3 mRNA and protein levels, whereas in osteoblasts, forskolin inhibited IGFBP-3 [18, 28, 41]. IGF-I and TGF-f3 were potent inducers of IGFBP-3 protein
release in mesangial cells; however, TGF-p, but not IGF-I, markedly increased IGFBP-3 mRNA levels. These findings suggest that IGFBP-3 is regulated by distinct mechanisms: forskolin and TGF-13 act by increasing de novo synthesis of IGFBP-3, while
IGF-I acts by post-transcriptional mechanism(s). Similar results have been obtained in human fibroblasts [19]. In these cells, the effect of TGF-p on IGFBP-3 was at the level of mRNA abun-
The physiological significance of IGFBPs in renal disease remains to be elucidated. In diabetic nephropathy, one may speculate that IGF-I and insulin regulate IGFBPs. The transient increase in IGFBP-1, IGFBP-3 and perhaps other IGFBPs and the relative ratio of stimulatoiy versus inhibitory IGFBPs in the glomerular microenvironment may play a key role in determining IGF action. Both IGF-I and TGF-J3 may act synergistically to enhance matrix formation, as well as modulate IGFBP levels that, in turn, influence IGF-I-mediated effects. Further understanding of the mechanisms involved in regulating IGFBP expression and
dance, while IGF-I altered the amount of surface bound their interaction with IGFs in the glomerulus is required to IGFBP-3. IGFBPs synthesized by fibroblasts may remain cell understand the complex role of this system in glomerular patholsurface-associated. IGF-I analogues with reduced affinity for ogy.
Grellier et al: IGF binding proteins and kidney
1077
Acknowledgments This work was supported in part by the Veterans Administration Medical Research Service (S.A, HA), National Institutes of Health Grants AR42306 (S.A), DK33665 (HA.), CA52952 (DY.) and by a
t8. COHICK WS, CLEMMONS DR: Regulation of IGFBP secretion and
Cancer Center Support Grant P30-CA54174 to the Cancer Therapy and Research Center. D.Y. is a Pew Scholar in the Biomedical Sciences.
131:1703—1710, 1992 20. YATEMAN ME, CLAFFEY DC, CWYFAN HUGhES SC, FROST VJ, WASS
Portions of this work were published in abstract form at the annual meeting of the American Society of Nephrology, Orlando, Florida, October 26—29, 1994.
Reprint requests to Sheriy L. Abboud, M.D., Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA.
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