help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, H.-L.
Right arrow Articles by Feldman, E. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, H.-L.
Right arrow Articles by Feldman, E. L.
Endocrinology Vol. 140, No. 10 4478-4485
Copyright © 1999 by The Endocrine Society

ARTICLES

Regulation of Insulin-Like Growth Factor-Binding Protein-5 Expression during Schwann Cell Differentiation1

Hsin-Lin Cheng, Michael Shy and Eva L. Feldman

Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109; and the Department of Neurology, Wayne State University (M.S.), Detroit Michigan 48201

Address all correspondence and requests for reprints to: Eva L. Feldman, M.D., Ph.D., Department of Neurology, University of Michigan, 200 Zina Pitcher Place, 4414 Kresge III, Ann Arbor, Michigan 48109-0588. E-mail: efeldman{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have reported that immortalized Schwann cells (SC) express the insulin-like growth factor I receptor and IGF-binding protein-5 (IGFBP-5). IGF-I promotes SC survival and protects IGFBP-5 in SC-conditioned medium from proteolysis. In the current study we examined the roles of IGF-I and IGFBP-5 in primary SC. IGF-I enhances primary SC differentiation and gene and protein expression of IGFBP-5 and the myelinating protein, P0. SC that stably overexpress human IGFBP-5 also have higher levels of P0 gene expression. The phosphatidylinositol-3 kinase inhibitor (LY294002), but not the mitogen-activated protein kinase kinase inhibitor (PD98059), blocks IGF-I enhancement of IGFBP-5 gene and protein expression. Collectively, these results suggest that IGF-I promotes SC differentiation, and this may occur in part by enhancing IGFBP-5 expression via phosphatidylinositol-3 kinase activation. These data support a link between enhanced IGFBP-5 expression and cellular differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) promotes cell proliferation and differentiation (1, 2). In the peripheral nervous system, IGF-I plays an important role in the development of Schwann cells (SC). In vivo, IGF-I immunoreactivity is detected in SC (3). In vitro, IGF-I promotes SC proliferation and DNA synthesis (3). In addition, IGF-I enhances SC differentiation by increasing expression of myelin protein, P0 (4, 5). In SC/dorsal root ganglion neuron cocultures, IGF-I enhances SC motility, which results in SC attachment on dorsal root ganglion axons and leads to myelin sheath formation (6).

The primary actions of IGF-I are mediated by activation of the IGF-I receptor (IGF-IR) (2). IGF-I binding induces tyrosine phosphorylation of the IGF-IR and activates subsequent downstream signaling molecules, which are divided into two major pathways, the Ras-Raf mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3 kinase (PI-3K) pathways (7, 8). In neurons, the MAPK pathway is important for IGF-I-induced gene expression and differentiation (9, 10), whereas IGF-I activation of the PI-3K pathway is responsible for neuronal survival, process extension, and cytoskeleton rearrangement (11, 12, 13, 14). The physiological effects of IGF-I signaling in SC are less well known.

The binding of IGF-I to IGF-IR is modulated by IGF-binding proteins (IGFBPs). There are six IGFBPs (IGFBP-1 to IGFBP-6) with high affinity for IGF-I (2, 15). The IGFBPs both promote and inhibit the actions of IGF-I (15). IGFBP-1, -3, and -4 sequester IGF-I in extracellular fluid reservoirs, thus preventing IGF-I binding to cell surface IGF-IR (15). In contrast, IGFBP-5 associates with the extracellular matrix and provides IGF-I to nearby cell surface receptors, thus enhancing the actions of IGF-I (16).

Previously, we reported that IGF-I promotes the mitogenesis of undifferentiated SC (3). We also characterized the expression of IGF-IR and IGFBP-5 in a transfected, immortalized SC line (3) and demonstrated up-regulation of IGFBP-5 associated with the expression of P0 during cAMP-induced SC differentiation (4). In the current study, using primary SC, we found that IGF-I promotes both P0 and IGFBP-5 gene and protein expression during differentiation. P0 gene expression is also increased in SC that overexpress human IGFBP-5. Studies with MAPK (PD98059) and PI-3K (LY294002) inhibitors indicate the PI-3K pathway is responsible for IGF-I enhancement of IGFBP-5 expression. In contrast, blocking neither MAPK nor PI-3K pathways has an effect on IGF-I enhancement of P0 expression, suggesting that IGF-I regulates IGFBP-5 and P0 expression via distinct signaling mechanisms. The current results reveal an association between IGFBP-5 expression and SC differentiation and suggest that elevated IGFBP-5 levels in differentiating SC might facilitate the actions of IGF-I.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DMEM was purchased from Life Technologies, Inc. (Gaithersburg, MD), and FBS was obtained from HyClone Laboratories, Inc. (Logan, UT). Other tissue culture supplies were obtained from Corning Glass Works (Corning, NY) and Costar (Cambridge, MA). Restriction enzymes were obtained from Life Technologies, Inc., and New England Biolabs, Inc. (Beverly, MA). [32P]Deoxy-CTP was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Recombinant human IGF-I was a generous gift of Cephalon (West Chester, PA) and was stored in 100 mM acetic acid in -80 C until use. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Antibodies
The anti-IGFBP-5 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) used in this study is a polyclonal antisera raised against immunoaffinity-purified IGFBP-5 from the conditioned media of transfected CHO cells. This antiserum recognizes IGFBP-5 from human and rat. The cross-reactivities with other binding proteins are as follows: IGFBP-1, less than 0.5%; IGFBP-2, less than 0.1%; IGFBP-3, less than 0.1%; and IGFBP-4, less than 0.5%. The anti-P0 antiserum (P07) was a gift from Dr. Juan J. Archelos, Max Planck Institute for Psychiatry (Munich, Germany) (17). The anti-IGF-IR ß-subunit antibody is purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Complementary DNA (cDNA) probes
The rat IGFBP-5 cDNA was received from Dr. Shunichi Shimasaki, The Whittier Institute (La Jolla, CA). A 300-bp rat IGFBP-5 cDNA, which encodes portions of the mature peptide, in pBluescript SK+ plasmid was generated by SacII and HindIII digestion. A 1.9-kb P0 cDNA probe was isolated from pBluescript plasmid by EcoRI digestion. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was a gift from Dr. Ellen Zimmermann (University of Michigan, Ann Arbor, MI). The 780-bp cDNA probe was isolated by PstI/XbaI digestion from a pBR322 vector. The cDNA probes were purified with a Magic PCR Prep DNA Purification Kit from Promega Corp. (Madison, WI).

Cell culture
SC were isolated from sciatic nerves of 3-day-old Sprague Dawley rats (Harlan-Sprague Dawley, Indianapolis, IN) as described previously (18). Cells were maintained in culture medium [DMEM and 10% FBS with 2 µM forskolin and 10 µg/ml bovine pituitary extract (Life Technologies, Inc.)] on poly-L-lysine-coated plates. Cells were passaged upon reaching confluence and used for four passages. For serum-free conditions, cells were washed twice and cultured in defined medium [DMEM/F12 medium supplemented with transferrin (10 mg/liter), putresine (10 µM), progesterone (20 nM), and sodium selenite (30 nM)].

Northern analysis
SC were plated (6 x 106 cells/150 mm plate), and upon reaching confluence, media were removed, and cells were rinsed twice in DMEM then placed in defined medium with 10 nM IGF-I with or without 2 µM forskolin. After 24 h, the culture medium was collected, and the cells were harvested for RNA using the guanidine isothiocyanate method (19). Total RNA (10 µg) was separated by size in formaldehyde-agarose gels and transferred to nylon Nytran membranes (Amersham Pharmacia Biotech, Arlington Heights, IL). Membranes were hybridized with 32P-deoxy-CTP-labeled (0.1–10 x 108 cpm/mg) cDNA probes for rat IGFBP-5, P0, and GAPDH and exposed to X-Omat films (Eastman Kodak Co., Rochester, NY) after extensive rinsing. Individual Nytran membranes were hybridized by stripping and reprobing, as described in the manufacturer’s instructions. The relative OD for each hybridization was determined densitometrically by averaging several exposures in the linear range of the film. The densitometric values of IGFBP-5 or P0 were divided by values of GAPDH hybridization from the same conditions and expressed as a percentage of the serum free control value (20).

Immunoblotting
Fifty micrograms of protein from concentrated conditioned medium (for IGFBP-5 immunoblots) or whole cell lysates (for P0 immunoblots) were mixed at a 5:1 ratio with 10 x SDS sample buffer [100 mM Tris (pH 8), 10 mM EDTA, 10% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue, and 20% glycerol] and boiled for 3–5 min. Samples were separated by SDS-PAGE (12.5%) and electrophoretically transferred to nitrocellulose. Nitrocellulose membranes were blocked with 5% nonfat dried milk in TBST (20 mM Tris, 0.16 M NaCl, and 0.10% Tween-20, pH 7.4). The membranes were then incubated for 2 h with IGFBP-5 antiserum (1:1000), IGF-IR ß-subunit (1:1000), or anti-P0 (1:5000), in TBST plus 5% milk, washed extensively over a period of 30 min with TBST, and then incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit antibody (1:7500; Santa Cruz) for IGFBP-5 and IGF-IR ß-subunit or horse antimouse (1:1000, Santa Cruz) for P0 in TBST plus 5% milk. After extensive washing over a period of 40 min, nitrocellulose membranes were incubated with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). Bound protein-antibody complexes were visualized by autoluminography on enhance chemiluminescence films (Amersham Pharmacia Biotech).

[125I]IGF-I ligand blotting
Ligand blotting was performed as previously described (4). Fifty micrograms of protein from concentrated SC-conditioned medium were separated by SDS-PAGE in nonreducing conditions before being transferred to nitrocellulose membranes. The membranes were rinsed with TBST and incubated with TBST and 1% BSA with 25 x 104 cpm [125I]IGF-I overnight. After extensive rinsing with TBST, the membranes were dried and exposed to X-Omat films.

Cell transfection
Cells were cultured until 80% confluence, then rinsed and transfected with human IGFBP-5 in a mammalian expression vector (pRc/RSV, Invitrogen, Carlsbad, CA; gift from Dr. Cunming Duan, University of Michigan) using the lipofectamine method (21). Briefly, 1 µg IGFBP-5 containing expression vector or control vector was mixed with 10 µl lipofectamine in 200 µl DMEM. After 45 min at room temperature, liposomes were formed and introduced to cells cultured in serum-free medium. After 6 h of transfection, cells were rinsed and cultured for 3 days, then selected in medium containing 400 µg/ml G418. In all experiments using IGFBP-5 transfectants, at least two pools of transfected SC, representing separate transfections, were used in each of three experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I enhances morphological differentiation and P0 expression in SC
In the current studies, we used forskolin-treated primary SC to examine the effects of IGF-I on differentiation. Forskolin treatment blocks SC mitogenesis, allowing us to examine the effects of IGF-I on a more differentiated SC phenotype (4). We monitored both SC morphology and myelin protein expression to ascertain the degree of cellular differentiation. To evaluate SC morphology, cells were serum deprived overnight and then treated with serum-free medium or serum-free medium plus 10 nM IGF-I with or without 2 µM forskolin. After 24 h, SC morphology was examined using a phase contrast microscope (Fig. 1Go). Under serum-free conditions, SC exhibited an undifferentiated phenotype with multiple, small scattered processes (Fig. 1AGo). With IGF-I treatment alone, SC processes became longer, and their number increased (Fig. 1CGo). As we have previously reported (4), 2 µM forskolin conferred a more differentiated SC phenotype. SC became bipolar spindle cells with a tendency to orient in the same direction. Coaddition of IGF-I with forskolin resulted in a much more differentiated phenotype. Spindle-shaped SC become densely packed together with very long cell processes aligned in the same orientation (Fig. 1D).



View larger version (131K):
[in this window]
[in a new window]
  		Figure 1. IGF-I enhances SC differentiation. SC morphology after treatment with serum-free medium (A), 2 µM forskolin (B), 10 nM IGF-I (C), and 2 µM forskolin and 10 nM IGF-I (D) for 24 h. Scale bar, 10 µm. Pictures are representative of three independent experiments.

 
Our previous studies with neurons suggested that IGF-I-mediated process formation reflects enhanced cellular differentiation. In neurons, phenotypic markers of differentiation parallel IGF-I-induced changes in gene and protein expression (9, 22). We reasoned that a similar sequence of events occurred in SC and examined the effects of IGF-I on P0 expression under the same experimental paradigm that produced changes in SC morphology. P0 immunoblots are presented in Fig. 2Go. With either serum-free or serum-free plus 10 nM IGF-I treatment, P0 protein levels were minimal (Fig. 2Go). Forskolin treatment induced P0 expression, and IGF-I coadministration further enhanced the level of P0 (Fig. 2Go). These results in conjunction with the morphological studies suggest that IGF-I enhances forskolin-induced SC differentiation.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. IGF-I enhances P0 expression in forskolin-treated SC. SC were treated with serum-free medium (C), 2 µM forskolin (F), 2 µM forskolin plus 10 nM IGF-I (F+I), or 10 nM IGF-I (I) for 24 h. P0 and IGF-IR ß-subunit immunoblots were performed as described in Materials and Methods. Data are from one of three representative experiments.

 
In some cell types, IGF-IR expression increases with cellular differentiation (23). To rule out the possibility that the observed effects of IGF-I on SC differentiation are secondary to changes in IGF-IR expression, we examined IGF-IR ß-subunit expression by immunoblot analysis. Forskolin treatment had no effect on IGF-IR expression (Fig. 2Go), whereas IGF-I treatment down-regulated the level of IGF-IR. These results suggest that changes in IGF-IR are not responsible for IGF-I enhancement of SC differentiation in the presence of forskolin.

IGF-I enhances IGFBP-5 expression in SC
IGFBP-5 expression correlated with the differentiation of several cell types. In the current study, we first asked which IGFBPs are expressed by differentiating SC. Using [125I]IGF-I ligand blotting, a single 30-kDa IGFBP was present in conditioned medium from SC. Maximal signaling was seen in conditioned medium from SC treated with both forskolin and IGF-I (Fig. 3AGo), and less signal was detected in medium from cells treated with IGF-I alone. No signal was present in medium from SC grown under serum-free conditions or with the addition of forskolin alone, in agreement with our previous reports that IGF-I protects IGFBP-5 from proteolysis (4).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 3. SC IGFBP expression is regulated by forskolin and IGF-I treatment. SC were treated with serum-free medium (C), 2 µM forskolin (F), 2 µM forskolin plus 10 nM IGF-I (F+I), or 10 nM IGF-I (I) for 24 h. Conditioned media were concentrated, and 50 µg protein were used for Western ligand blots using [125I]IGF-I as a ligand (A) and IGFBP-5 immunoblots (B). A, Ligand blots detected a 30-kDa IGFBP secreted by SC. B, IGFBP-5 immunoblots not only confirmed that the 30-kDa IGFBP is IGFBP-5, but also detected a 23-kDa fragment. Data are from one of three representative experiments.

 
We predicted that the 30-kDa IGFBP would be IGFBP-5. Immunoblots confirmed the presence of IGFBP-5 in SC-conditioned medium with the same expression pattern as that seen with [125I]IGF-I ligand blots (Fig. 3BGo). The 23-kDa degradation product of IGFBP-5, not visualized with ligand blotting, was present with immunoblot analysis (Fig. 3BGo). Forskolin treatment enhanced the levels of the 30-kDa intact and the 23-kDa degraded IGFBP-5 fragments, suggesting that forskolin increases IGFBP-5 expression. In comparison, IGF-I not only promoted IGFBP-5 expression, but also preserved the 30-kDa IGFBP-5 from degradation in either the presence or absence of forskolin.

During muscle cell differentiation, increased IGFBP-5 protein levels reflect increased IGFBP-5 gene expression (24). Our data suggest similar changes in SC. IGF-I treatment alone enhanced IGFBP-5 gene expression by less than 2-fold, whereas forskolin treatment induced a 3-fold increase in IGFBP-5 messenger RNA (mRNA) expression (Fig. 4Go, A and B). Under conditions of maximal SC differentiation (forskolin plus IGF-I), IGFBP-5 gene expression was enhanced 9-fold (Fig. 4Go, A and B). Northern analysis for P0 of the same blots demonstrated similar patterns of P0 up-regulation by both forskolin and forskolin plus IGF-I treatment (Fig. 4Go, A and C). IGF-I enhancement of IGFBP-5 and P0 gene expression by forskolin-treated SC was dose-dependent (Fig. 4DGo). Our results suggest that there is a positive correlation between SC differentiation and IGFBP-5 expression.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 4. SC P0 and IGFBP-5 gene expression is regulated by forskolin and IGF-I treatment. A, SC were treated with serum-free medium (C), 2 µM forskolin (F), 2 µM forskolin plus 10 nM IGF-I (F+I), or 10 nM IGF-I (I) for 24 h. SC mRNA was isolated, and Northern analysis was performed and probed for IGFBP-5, P0, and GAPDH (as a loading control). Data are from one of three representative experiments. Densitometric analysis demonstrated a significant increase in IGFBP-5 (B) and P0 (C) gene expression in response to forskolin and/or IGF-I treatments. *, P < 0.05 compared with control, by unpaired two-tailed t test. Data are from three separate experiments. D, In the presence of 2 µM forskolin, IGF-I enhances IGFBP-5 and P0 gene expression in a dose-dependent manner. Data are from one of three representative experiments.

 
IGF-I enhances IGFBP-5 expression in SC via PI-3K signaling
IGF-I binding to IGF-IR activates two major downstream signaling pathways: the MAPK and PI-3K pathways. In the current study, we examined the relative roles of these two IGF-IR signaling cascades in IGF-I enhancement of IGFBP-5 expression and P0 expression. For these studies, we applied 10 µM PD98059 or 10 µM LY294002 to serum-starved SC 30 min before the addition of 2 µM forskolin with or without 10 nM IGF-I. Cells were incubated for 24 h, and mRNA was collected for Northern analysis. LY294002 (10 µM) blocked IGF-I induced IGFBP-5 gene expression, whereas PD98059 had no effect (Fig. 5Go, A and B). IGFBP-5 immunoblots of proteins from conditioned medium demonstrated a similar pattern of regulation, i.e. the PI-3K inhibitor blocked and the MAPK inhibitor had no effect on IGF-I enhancement of IGFBP-5 protein expression (Fig. 5DGo). In contrast, densitometric analysis of P0 mRNA demonstrated that neither inhibitor had an effect on IGF-I enhancement of P0 gene expression (Fig. 5Go, A and C). Our findings suggest that there are distinct IGF-IR signaling pathways that govern IGFBP-5 and P0 expression during SC differentiation. Our data also imply that multiple signaling cascades are involved in SC differentiation.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 5. IGF-I enhancement of IGFBP-5 expression is blocked by LY294002. SC were treated with 2 µM forskolin (F), 2 µM forskolin and 10 nM IGF-I (F+I), or 2 µM forskolin and 10 nM IGF-I plus 10 µM PD98059 or 10 µM LY294002 for 24 h. Northern analysis was performed for IGFBP-5, P0, and GAPDH (as a loading control). Data are from one of three representative experiments. Densitometric analysis demonstrated that IGF-I enhancement of IGFBP-5 gene expression was blocked by LY294002, but not PD98059 (B). Neither inhibitor had an effect on IGF-I enhancement of P0 gene expression (C). *, P < 0.05; **, P > 0.05 (compared with F+I, by unpaired two-tailed t test). Data are from three separate experiments. D, IGFBP-5 immunoblots demonstrated that LY294002, but not PD98059, blocked IGF-I enhancement of IGFBP-5 protein expression. Data are from one of three representative experiments.

 
Overexpression of IGFBP-5 by SC leads to differentiation
To better evaluate the effects of IGFBP-5 on IGF enhancement of SC differentiation, SC were transfected with a control vector (pRc/RSV) or a vector containing human IGFBP-5 cDNA. To evaluate IGFBP-5 expression, vector control and IGFBP-5-transfected cells were serum deprived and then treated with forskolin with or without 10 nM IGF-I for 24 h. IGFBP-5 immunoblots using concentrated conditioned medium revealed that more IGFBP-5 was secreted by IGFBP-5 transfectants (Fig. 6AGo). Northern analysis revealed increased IGFBP-5 gene expression in IGFBP-5 transfectants (Fig. 6BGo).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 6. SC are transfected to overexpress human IGFBP-5. SC transfected with a control vector or human IGFBP-5 were treated with serum-free medium (C), 2 µM forskolin (F), or 2 µM forskolin plus 10 nM IGF-I (F+I) for 24 h. A, IGFBP-5 immunoblots demonstrated a higher level of IGFBP-5 secretion from SC that overexpress IGFBP-5. B, IGFBP-5-transfected SC also express more IGFBP-5 mRNA than cells transfected with the control vector. Data are from one of three representative experiments.

 
To determine the effects of IGFBP-5 overexpression on SC differentiation, both vector and IGFBP-5 transfectants were treated with 2 µM forskolin with or without 10 nM IGF-I for 24 h. In comparison with the vector-transfected cells, IGFBP-5 transfectants expressed more P0 in the absence of IGF-I or forskolin, suggesting that they were more differentiated (Fig. 7Go). Forskolin treatment enhanced P0 expression in both vector and IGFBP-5 transfectants; the addition of 10 nM IGF-I further enhanced P0 expression.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 7. SC that overexpress IGFBP-5 express more P0. SC transfected with a control vector and human IGFBP-5 were treated with serum-free medium (C), 2 µM forskolin (F), 2 µM forskolin plus 10 nM IGF-I (F+I), or 10 nM IGF-I (I) for 24 h. A, Northern analysis for P0 and GAPDH were performed. SC that overexpress IGFBP-5 have a higher level of P0 expression than the vector-transfected control. Data are from one of three representative experiments. B, Densitometric analysis confirms the different levels of expression. *, P < 0.05 compared with control vector in the same experimental condition, by unpaired two-tailed t test. Data are from three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported that IGFBP-5 expression is increased in a simian virus 40 (SV40) large T antigen-transfected SC line (MT4H1) after treatment with cAMP or Matrigel matrix, a paradigm that promotes cellular differentiation (4). IGF-I protects IGFBP-5 from proteolytic degradation in conditioned medium from these same cells, but has no effect on IGFBP-5 gene expression (3). Our previous work was confounded by the fact the SC had been immortalized with SV40, which decreases IGFBP-5 expression in fibroblasts (25). To avoid the potential interference of SV40 T antigen on IGFBP-5 expression, untransfected primary SC were used in the current study.

Our current results demonstrate that primary SC express IGFBP-5 and IGF-I protects IGFBP-5 from proteolytic degradation. Gene expression of IGFBP-5 is increased by forskolin, a stimulator of adenylate cyclase, which induces SC differentiation. Increased expression of IGFBP-5 is associated with up-regulation of the myelin protein P0, a cell differentiation marker, suggesting that IGFBP-5 expression augments SC differentiation.

In agreement with our current findings, cAMP activates IGFBP-5 expression in muscle cells (26), osteosarcoma cells (27, 28), and human fibroblasts (29, 30). In osteosarcoma cells, PTH increases IGFBP-5 expression in a cAMP-dependent manner (27, 28). In human fibroblasts, Duan and colleagues located an activating protein-2-binding site on the IGFBP-5 promoter. These data strongly suggest a direct induction of IGFBP-5 expression by cAMP-responsive transcriptional factors (29).

In our previous study using the SV40 large T antigen-transfected SC, IGF-I enhanced IGFBP-5 expression only if the cells were pretreated with forskolin. In the current study, IGF-I promotes IGFBP-5 expression in primary SC regardless of forskolin treatment. These data agree with previous reports that cellular transfection with SV40 T antigen has negative effects on IGF-I enhancement of IGFBP-5 expression (25). Our present data also agree with previous in vivo and in vitro studies showing that IGF-I enhances IGFBP-5 expression. In vivo, transgenic mice that overexpress IGF-I have larger brains, which contain higher percentages of IGFBP-5 mRNA, suggesting that IGF-I regulates IGFBP-5 expression (31). In vitro, numerous studies report that IGF-I enhances IGFBP-5 gene expression in a variety of cell types (32).

The mechanism underlying IGF-I regulation of IGFBP-5 expression is cell type specific. In SC, both forskolin and IGF-I enhance IGFBP-5 expression. Using L6 muscle cells, McCusker and colleagues report that IGF-I up-regulation of IGFBP-5 gene expression is mediated by IGF-IR in a cAMP-independent manner (26). In SC, the effects of IGF-I and forskolin on IGFBP-5 expression are more than additive, in contrast to reports in L6 muscle cells, where a similar phenomenon is not present (26). Taken together, these data suggest that different signaling pathways are involved in IGF-I regulation of IGFBP-5 expression. Our results also indicate that IGF-I mediates changes in IGFBP-5 SC expression via PI-3K signaling. The effects of IGF-I on SC IGFBP-5 expression are blocked by LY294002, a PI-3K inhibitor. In contrast to our data, Rousse and colleagues report that IGF-I up-regulation of IGFBP-5 expression in myoblasts can be blocked by rapamysin, an inhibitor of p70 ribosomal protein-S6 kinase (p70S6k), but not by the PI-3K inhibitors, LY294002 and wortmannin (33). Collectively, these findings suggest IGFBP-5 gene expression is regulated in a cell-specific manner involving multiple signaling pathways.

In the current study, P0 serves as a marker of SC differentiation. Although IGF-I treatment alone changes SC morphology, pretreatment with forskolin is required for IGF-I addition to produce a differentiated SC phenotype. Similarly, IGF-I has negligible effects on P0 expression unless SC are also treated with forskolin. Although IGF-I enhances IGFBP-5 expression via the PI-3 kinase pathway, IGF-I enhancement of P0 expression in the presence of forskolin is independent of either PI-3K or MAP kinase signaling. Our data suggest that forskolin treatment primes SC to respond to IGF-I treatment by signaling pathways other than PI-3K and MAP kinase, including protein kinase C (34, 35, 36) and G proteins (37, 38, 39, 40).

The current data demonstrate that IGFBP-5 overexpression promotes the expression of P0, implying an association between IGFBP-5 and SC differentiation. Several reports suggest that this association is also dependent on cell type (32). For example, IGFBP-5 is not detected in proliferating myoblasts, but is present upon myoblast differentiation (24). In contrast, James and colleagues report that expression of murine IGFBP-5 in C2 myoblasts inhibits cell differentiation (41). The inhibitory effect is associated with IGFBP-5 deposition in the extracellular matrix and is reversed by exogenous IGF-I, suggesting that extracellular matrix plays a critical role in modulating IGFBP-5 function. In SC, we postulate that transcription factors that regulate SC differentiation, including Oct-6 (SCIP or Tst-1), Krox, and Oct-1 (42, 43), are activated by IGF-I and forskolin treatment and interact with the IGFBP-5 promoter to enhance IGFBP-5 expression. In support of this idea, Rotwein and colleagues report a 156-nucleotide region of the IGFBP-5 promoter that contains several binding sites for muscle-specific transcriptional factors that may mediate the up-regulation of IGFBP-5 associated with muscle differentiation (44).

Recent studies suggest that IGFBP-5 may also act independently of IGF-I (45). In the absence of IGF-I, IGFBP-5 promotes osteoblast proliferation (45) and migration (46). Andress reports a 420-kDa IGFBP-5 receptor (47), and along with IGFBP-1 and -3, IGFBP-5 can be serine phosphorylated (48). These data suggest that IGFBP-5 has a function other than to modulate the actions of IGF-I. In our studies, the elevated levels of IGFBP-5 expression might reflect in part an IGF-I-independent mechanism. We are currently addressing this question in our laboratory using primary SC.

In summary, although the function of IGF-I and IGFBP-5 remains unknown in SC, accumulating data suggest a role for both factors in differentiation. IGF-I enhances primary SC differentiation and IGFBP-5 gene and protein expression, whereas SC, which stably overexpress human IGFBP-5, have a more differentiated phenotype. Collectively, these results suggest that IGF-I promotes SC differentiation in part by enhancing IGFBP-5 expression. Further studies aimed at understanding IGF-I-independent roles of IGFBP-5 will help clarify the role of IGFBP-5 in SC differentiation and a potential role for this binding protein in the treatment of dysmyelinating disorders.


   Acknowledgments
 
The authors thank Dr. Cunming Duan for helpful discussions, Mr. Michael Peacock for technical assistance, and Ms. Judy Boldt for secretarial assistance.


   Footnotes
 
1 This work was supported by NIH Grants NS-36778 and NS-38849 and grants from the Juvenile Diabetes Foundation and American Diabetes Association. Back

Received December 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zhang Y, Wick DA, Seetharam B, Dahms NM 1995 Expression of IGF-II and IGF binding proteins in differentiating human intestinal caco-2 cells. Am J Physiol 269:E804–E813
  2. Sara VR, Hall K 1990 Insulin-like growth factors and their binding proteins. Physiol Rev 70:591–614[Free Full Text]
  3. Cheng H-L, Randolph A, Yee D, Delafontaine P, Tennekoon G, Feldman EL 1996 Characterization of insulin-like growth factor-I (IGF-I), IGF-I receptor and binding proteins in transected nerves and cultured Schwann cells. J Neurochem 66:525–536[Medline]
  4. Cheng H-L, Feldman EL 1997 Insulin-like growth factor-I (IGF-I) and IGF binding protein-5 in Schwann cell differentiation. J Cell Physiol 171:161–167[CrossRef][Medline]
  5. Stewart HJ, Bradke F, Tabernero A, Morrell D, Jessen KR, Mirsky R 1996 Regulation of rat Schwann cell Po expression and DNA synthesis by insulin-like growth factors in vitro. Eur J Neurosci 8:553–564[CrossRef][Medline]
  6. Russell JW, Cheng H-L, Feldman EL Insulin-like growth factor-I promotes myelination of peripheral sensory axons. Glia, in press
  7. Barbacid M 1987 ras genes. Annu Rev Biochem 56:779–827[CrossRef][Medline]
  8. Myers MG Jr, Grammer TC, Wang L-M, Sun XJ, Pierce JH, Blenis J, White MF 1994 Insulin receptor substrate-1 mediates phosphatidylinositol 3'-kinase and p70S6k signaling during insulin, insulin-like growth factor-1, and interleukin-4 stimulation. J Biol Chem 269:28783–28789[Abstract/Free Full Text]
  9. Kim B, Leventhal PS, Saltiel AR, Feldman EL 1997 Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires MAP kinase activation. J Biol Chem 272:21268–21273[Abstract/Free Full Text]
  10. Feldman EL, Sullivan KA, Kim B, Russell JW 1997 Insulin-like growth factors regulate neuronal differentiation and survival. Neurobiol Dis 4:201–214[CrossRef][Medline]
  11. Leventhal PS, Shelden EA, Kim B, Feldman EL 1997 Tyrosine phosphorylation of paxillin and focal adhesion kinase during insulin-like growth factor-I-stimulated lamellipodial advance. J Biol Chem 272:5214–5218[Abstract/Free Full Text]
  12. Leventhal PS, Feldman EL 1997 Insulin-like growth factors as regulators of cell motility: signaling mechanisms. Trends Endocrinol Metab 8:1–6
  13. Kim B, Feldman EL 1998 Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization. J Neurochem 71:1333–1336[Medline]
  14. Kim B, Cheng H-L, Margolis B, Feldman EL 1998 Insulin receptor substrate-2 and Shc play different roles in insulin-like growth factor-I signaling. J Biol Chem 273:34543–34550[Abstract/Free Full Text]
  15. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[Abstract/Free Full Text]
  16. Jones JI, Gockerman A, Busby WH Jr, Camacho-Hubner C, Clemmons DR 1993 Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I. J Cell Biol 121:679–687[Abstract/Free Full Text]
  17. Archelos JJ, Roggenbuck K, Schneider-Schaulies J, Linington C, Toyka KV, Hartung H-P 1993 Production and characterization of monoclonal antibodies to the extracellular domain of P0. J Neurosci Res 35:46–53[CrossRef][Medline]
  18. Brockes JP, Fields KL, Raff MC 1979 Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res 165:105–118[CrossRef][Medline]
  19. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  20. Martin DM, Feldman EL 1993 Regulation of insulin-like growth factor-II expression and its role in autocrine growth of human neuroblastoma cells. J Cell Physiol 155:290–300[CrossRef][Medline]
  21. Singleton JR, Randolph AE, Feldman EL 1996 Insulin-like growth factor I receptor prevents apotosis and enhances neuroblastoma tumorigenesis. Cancer Res 56:4522–4529[Abstract/Free Full Text]
  22. Sumantran VN, Feldman EL 1993 Insulin-like growth factor I regulates c-myc and GAP-43 messenger ribonucleic acid expression in SH-SY5Y human neuroblastoma cells. Endocrinology 132:2017–2023[Abstract/Free Full Text]
  23. Martin DM, Carlson RO, Feldman EL 1993 Interferon-{gamma} inhibits DNA synthesis and insulin-like growth factor-II expression in human neuroblastoma cells. J Neurosci Res 34:489–501[CrossRef][Medline]
  24. James PL, Jones SB, Busby WH Jr, Clemmons DR, Rotwein P 1993 A highly conserved insulin-like growth factor-binding protein (IGFBP-5) is expressed during myoblast differentiation. J Biol Chem 268:22305–22312[Abstract/Free Full Text]
  25. Reeve JG, Guada–o A, Xiong J, Morgan J, Bleehen NM 1995 Diminished expression of insulin-like growth factor (IGF) binding protein-5 and activation of IGF-I-mediated autocrine growth in simian virus 40-transformed human fibroblasts. J Biol Chem 270:135–142[Abstract/Free Full Text]
  26. McCusker RH, Clemmons DR 1998 Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells. J Cell Physiol 174:293–300[CrossRef][Medline]
  27. Torring O, Firek AF, Heath H III, Conover CA 1991 Parathyroid hormone and parathyroid hormone-related peptide stimulate insulin-like growth factor-binding protein secretion by rat osteoblast-like cells through a adenosine 3',5'-monophosphate-dependent mechanism. Endocrinology 128:1006–1014[Abstract/Free Full Text]
  28. Conover CA, Bale LK, Clarkson JT, Torring O 1993 Regulation of insulin-like growth factor binding protein-5 messenger ribonucleic acid expression and protein availability in rat osteoblast-like cells. Endocrinology 132:2525–2530[Abstract/Free Full Text]
  29. Duan C, Clemmons DR 1995 Transcription factor AP-2 regulates human insulin-like growth factor binding protein-5 gene expression. J Biol Chem 270:24844–24851[Abstract/Free Full Text]
  30. Camacho-Hubner C, Busby WH, McCusker RH, Wright G, Clemmons DR 1992 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[Abstract/Free Full Text]
  31. Ye P, D’Ercole J 1998 Insulin-like growth factor I (IGF-I) regulates IGF binding protein-5 gene expression in the brain. Endocrinology 139:65–71[Abstract/Free Full Text]
  32. Clemmons DR 1997 Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev 8:45–62[CrossRef][Medline]
  33. Rousse S, Montarras D, Pinset C, Dubois C 1998 Up-regulation of insulin-like growth factor binding protein-5 is independent of muscle cell differentiation, sensitive to rapamycin, but insensitive to wortmannin and LY294002. Endocrinology 139:1487–1493[Abstract/Free Full Text]
  34. Li W, Jiang YX, Zhang J, Soon L, Flechner L, Kapoor V, Pierce JH, Wang LH 1998 Protein kinase C-delta is an important signaling molecule in insulin-like growth factor I receptor-mediated cell transformation. Mol Cell Biol 18:5888–5898[Abstract/Free Full Text]
  35. Liu Q, Ning W, Dantzer R, Freund GG, Kelley KW 1998 Activation of protein kinase C-{zeta} and phosphatidylinositol 3'-kinase and promotion of macrophage differentiation by insulin-like growth factor-I. J Immunol 160:1393–1401[Abstract/Free Full Text]
  36. Heidenreich KA, Zeppelin T, Robinson LJ 1993 Insulin and insulin-like growth factor I induce c-fos expression in postmitotic neurons by a protein kinase C-dependent pathway. J Biol Chem 268:14663–14670[Abstract/Free Full Text]
  37. Pertseva MN, Plesneva SA, Kuznetsova LA, Shpakov AO, Derkach KV 1996 On the tyrosine kinase mechanism of the novel effect of insulin and insulinlike growth factor I: stimulation of the adenylyl cyclase system in muscle tissue. Biochem Pharmacol 52:1867–1874[CrossRef][Medline]
  38. Thomson FJ, Jess TJ, Moyes C, Plevin R, Gould GW 1997 Characterization of the intracellular signalling pathways that underlie growth factors-stimulated glucose transport. Biochem J 325:637–643
  39. Delafontaine P, Anwar A, Lou H, Ku L 1996 G-protein coupled and tyrosine kinase receptors: evidence that activation of the insulin-like growth factor I receptor is required for thrombin-induced mitogenesis of rat aortic smooth muscle cells. J Clin Invest 97:139–145[Medline]
  40. Begeot M, Langlois D, Saez JM 1989 Insulin-like growth factor-I and insulin increase the stimulatory guanine nucleotide binding protein (Gs) in cultured bovine adrenal cells. Mol Cell Endocrinol 66:53–57[CrossRef][Medline]
  41. James PL, Stewart CEH, Rotwein P 1996 Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism. J Cell Biol 133:683–693[Abstract/Free Full Text]
  42. Zorick TS, Syroid DE, Arroyo E, Scherer SS, Lemke G 1996 The transcription factors SCIP and Krox-20 mark distinct stages and cell fates in Schwann cell differentiation. Mol Cell Neurosci 8:129–145[CrossRef][Medline]
  43. Jaegle M, Mandemakers W, Broos L, Zwart R, Karis A, Visser P, Grosveld F, Meijer D 1996 The POU factor Oct-6 and Schwann cell differentiation. Science 273:507–510[Abstract]
  44. Rotwein P, James PL, Kou K 1995 Rapid activation of insulin-like growth factor binding protein-5 gene transcription during myoblast differentiation. Mol Endocrinol 9:913–923[Abstract/Free Full Text]
  45. Andress DL, Birnbaum RS 1992 Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action. J Biol Chem 267:22467–22472[Abstract/Free Full Text]
  46. Abrass CK, Berfield AK, Andress DL 1997 Heparin binding domain of insulin-like growth factor binding protein-5 stimulates mesangial cell migration. Am J Physiol 273:F899–F906
  47. Andress DL 1998 Insulin-like growth factor binding protein-5 (IGFBP-5) stimulates phosphorylation of the IGFBP-5 receptor. Am J Physiol 274:E744–E750
  48. Coverley JA, Baxter RC 1997 Phosphorylation of insulin-like growth factor binding proteins. Mol Cell Endocrinol 128:1–5[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
J. M Fleming, J. A Brandimarto, and W. S Cohick
The mitogen-activated protein kinase pathway tonically inhibits both basal and IGF-I-stimulated IGF-binding protein-5 production in mammary epithelial cells
J. Endocrinol., August 1, 2007; 194(2): 349 - 359.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Ning, B. Hoang, A. G. P. Schuller, T. P. Cominski, M.-S. Hsu, T. L. Wood, and J. E. Pintar
Delayed Mammary Gland Involution in Mice with Mutation of the Insulin-Like Growth Factor Binding Protein 5 Gene
Endocrinology, May 1, 2007; 148(5): 2138 - 2147.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. E. LeBlanc, S.-W. Jang, R. M. Ward, L. Wrabetz, and J. Svaren
Direct Regulation of Myelin Protein Zero Expression by the Egr2 Transactivator
J. Biol. Chem., March 3, 2006; 281(9): 5453 - 5460.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
T. L. Hagemann, S. A. Gaeta, M. A. Smith, D. A. Johnson, J. A. Johnson, and A. Messing
Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal fibers reveals a stress response followed by glial activation and neuronal dysfunction
Hum. Mol. Genet., August 15, 2005; 14(16): 2443 - 2458.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
D. Kiepe, S. Ciarmatori, A. Hoeflich, E. Wolf, and B. Tonshoff
Insulin-Like Growth Factor (IGF)-I Stimulates Cell Proliferation and Induces IGF Binding Protein (IGFBP)-3 and IGFBP-5 Gene Expression in Cultured Growth Plate Chondrocytes via Distinct Signaling Pathways
Endocrinology, July 1, 2005; 146(7): 3096 - 3104.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
L. J. Cobb, D. A. M. Salih, I. Gonzalez, G. Tripathi, E. J. Carter, F. Lovett, C. Holding, and J. M. Pell
Partitioning of IGFBP-5 actions in myogenesis: IGF-independent anti-apoptotic function
J. Cell Sci., May 1, 2004; 117(9): 1737 - 1746.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. A. M. Salih, G. Tripathi, C. Holding, T. A. M. Szestak, M. I. Gonzalez, E. J. Carter, L. J. Cobb, J. E. Eisemann, and J. M. Pell
Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice
PNAS, March 23, 2004; 101(12): 4314 - 4319.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. M. Frago, C. Paneda, S. L. Dickson, A. K. Hewson, J. Argente, and J. A. Chowen
Growth Hormone (GH) and GH-Releasing Peptide-6 Increase Brain Insulin-Like Growth Factor-I Expression and Activate Intracellular Signaling Pathways Involved in Neuroprotection
Endocrinology, October 1, 2002; 143(10): 4113 - 4122.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
E. Tonner, M. C. Barber, G. J. Allan, J. Beattie, J. Webster, C. B. A. Whitelaw, and D. J. Flint
Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice
Development, January 10, 2002; 129(19): 4547 - 4557.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, H.-L.
Right arrow Articles by Feldman, E. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, H.-L.
Right arrow Articles by Feldman, E. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals