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 Purchase Article
Right arrow View Shopping Cart
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 Moralez, A.
Right arrow Articles by Clemmons, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moralez, A.
Right arrow Articles by Clemmons, D.
Endocrinology Vol. 144, No. 6 2489-2495
Copyright © 2003 by The Endocrine Society

Control of Insulin-Like Growth Factor Binding Protein-5 Protease Synthesis and Secretion by Human Fibroblasts and Porcine Aortic Smooth Muscle Cells

Anna Moralez, Walker H. Busby, Jr. and David Clemmons

Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, M.D., Department of Medicine, Division of Endocrinology, CB# 7170, 6111 Thurston-Bowles, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: endo{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding protein-5 (IGFBP-5) is an important trophic factor for controlling the actions of IGF-I in human dermal fibroblasts and porcine aortic smooth muscle cells. When IGFBP-5 is associated with extracellular matrix, it acts to enhance the cell growth response to IGF-I. The amount of IGFBP-5 within the extracellular matrix is related in part to the amount that is present in conditioned medium, which is related to its rate of synthesis and degradation. A serine protease that degrades IGFBP-5 is present in the conditioned medium of both of these cell types. Because the IGFBP-5 protease activity that is secreted by fibroblasts has been shown to be due to the complement components C1r and C1s, these studies were undertaken to determine whether smooth muscle cells also secreted these proteases and to identify some of the factors that regulate their secretion by both cell types.

Both smooth muscle cells and human fibroblasts were shown to release C1r and C1s into conditioned medium. Both C1r and C1s were detected as activated forms, as determined by SDS-PAGE using reducing conditions. The addition of increasing concentrations of either IL-1ß or TNF{alpha} resulted in increased synthesis of C1s by fibroblasts and smooth muscle cells, and they each increased C1r release. TNF{alpha} (50 ng/ml) and IL-1ß (20 ng/ml) resulted in maximum stimulation of release of both proteases. In contrast dexamethasone (10-7 M) had no effect on C1s release and stimulated C1r release only by smooth muscle cells.

To determine the physiological significance of this increase in C1r and C1s, the amount of IGFBP-5 protease activity that was present in conditioned medium was determined before and after exposure to TNF{alpha}, IL-ß, and dexamethasone. All three compounds resulted in an increase in the amount of IGFBP-5 proteolytic activity. Dexamethasone inhibited the release of C1 inhibitor from fibroblasts, and this contributed to the net increase in proteolytic activity. TNF{alpha} inhibited the smooth muscle cell DNA synthesis response to IGF-I, but the effect of IGF-I was partially restored by the addition of C1 inhibitor. In conclusion, both C1r and C1s are released by cultured fibroblasts, and the release of each into fibroblast or porcine smooth muscle cells medium is stimulated by TNF{alpha} and IL-1ß. This increase results in a net increase in IGFBP-5 proteolysis, which has the potential to modify IGF-I and IGFBP-5 actions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABUNDANCE of the IGFs in extracellular fluids and their capacity to interact with cell surface receptors is controlled by IGF binding proteins (IGFBPs) (1). Human fibroblasts have been shown to synthesize three forms of IGFBPs, IGFBP-3, -4, and -5 (2). Unlike IGFBP-3 or -4, intact IGFBP-5 can be found in abundance in extracellular matrix (3); however, when conditioned medium is analyzed, there is much less intact IGFBP-5 compared with IGFBP-3 (2). After its synthesis, IGFBP-5 is rapidly proteolytically degraded (2). The physiological consequences of IGFBP-5 proteolysis are complex. Low concentrations of IGFBP-5 in conditioned medium (e.g. 10–200 ng/ml) do not result in inhibition of the cellular response to IGF-I, but are associated with accumulation of IGFBP-5 in the extracellular matrix and an enhancement of IGF-I actions (3, 4). However, increasing the amount of intact IGFBP-5 in conditioned medium to concentrations that range from 500-2000 ng/ml is associated with a progressive increase in inhibition of IGF-I-stimulated cell growth (5). If 500 ng/ml of the mutant form of IGFBP-5 that is resistant to proteolysis is added to porcine smooth muscle cells (pSMC), this results in inhibition of the ability of IGF-I (20 ng/ml; 6:1 molar ratio) to stimulate DNA synthesis and IGF-I receptor activation (6). In contrast, if wild-type IGFBP-5 and IGF-I are added at equimolar concentrations, they enhance the actions of IGF-I (3, 7). Therefore, the net effect of increasing IGFBP-5 proteolysis can be to either enhance or inhibit cell responsiveness to IGF-I depending upon the molar ratio of intact IGFBP-5 to IGF-I and the extent of IGFBP-5 proteolysis.

In previous studies we have demonstrated that IGFBP-5 is rapidly proteolytically cleaved after its secretion into fibroblast or pSMC medium (2, 3, 7). The molecular sizes of the fragments that are generated and the protease inhibitors that inhibit IGFBP-5 cleavage are similar for both cell types (2, 7). Purification and characterization of the IGFBP-5 protease activity from human fibroblasts have shown that it is attributable to the serine proteases C1r and C1s (8). Therefore, these studies were undertaken to determine whether pSMC also released C1r and C1s into conditioned medium, to identify the factors that stimulate C1r and C1s release by fibroblasts and pSMC, and to determine whether the factors that alter their release also result in a change in the amount of IGFBP-5 protease activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DMEM was purchased from Hazelton (Denver, PA). Calf serum was purchased from Colorado Laboratories, Inc. (Logan, VT). BSA and dexamethasone were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Eagle’s MEM and fetal bovine serum were purchased from Life Technologies, Inc. (Grand Island, NY). Anti-C1r, -C1s, and -C1 inhibitor antisera were purchased from Calbiochem (La Jolla, CA). Recombinant IGF-I was a gift from Genentech, Inc. (South San Francisco, CA). Human dermal fibroblasts (GM-10) were purchased from the Coriell Institute (Camden, NJ). These cells were cultured in 10-cm dishes (model 3003, Falcon Labware, BD Biosciences, Rutherford, NJ) using Eagle’s MEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% calf serum. The cells were passaged weakly using 0.1% trypsin/0.2 M EDTA and were replated at a density of 1 x 104 cells/cm2. pSMC were obtained from aortas from young (age 2–3 months) pigs as previously described (9). Stock cultures were plated and maintained in 10-cm dishes in DMEM supplemented with 10% FBS and passaged weekly as described previously (10). To quantify the amount of C1r or C1s in conditioned medium, the cells were plated at 2 x 104 cells/cm2 in six-well plates (Falcon 3046) and grown to confluence, usually for 7 d. To prepare conditioned medium, confluent cultures were washed three times with PBS, then 1.0 cc of serum-free DMEM (pSMC) or Eagle’s MEM (fibroblasts) was added. After 24 h, the medium samples were removed and frozen at -80 C until they were analyzed.

Human IGFBP-5 was purified to homogeneity from conditioned medium that was obtained from CHO cells (American Type Culture Collection, Manassas, VA) that had been stably transfected with a full-length human IGFBP-5 cDNA that had been inserted into the expression plasmid p-NUT obtained from Richard Palmiter, as previously described (3). The protein was purified as previously described (2). The protein was determined to be pure by amino acid sequence analysis.

Preparation of IGFBP-5 mutant
A protease-resistant IGFBP-5 mutant was prepared by using in vitro mutagenesis to alter Lys139 and Lys140 to alanines as previously described (6). This mutant was purified using a protocol that was similar to that for purifying native IGFBP-5 (6). The mutant was expressed in CHO cells, and CHO cell-conditioned medium was used as a starting material for purification. Purity was established by silver staining for IGFBP-5. The mutant was shown to be stable and was resistant to proteolytic cleavage by the IGFBP-5 protease that was present in conditioned medium after incubation for periods as long as 48 h (6).

RNA isolation and preparation of a cDNA probe
RNA was isolated from cell cultures using Tri-Reagent following the manufacturer’s instructions (Molecular Research Center, Inc., Cincinnati, OH). The total RNA content was quantified by spectrophotometry (OD, >260 nm). A specific 628-bp human IGFBP-5 cDNA was labeled to a specific activity of 200 µCi/ng by random printing as previously described (2).

RNA samples were fractionated on a 1.2% agarose/formaldehyde gel. To confirm that similar amounts of RNA were loaded, the gels were stained with ethidium bromide, and ribosomal 28S and 18S RNA bands were visualized. RNA was transferred to nylon membranes (Biotrans, ICN Biomedicals, Inc., Irvine, CA) using 20x standard saline citrate. The filters were baked in a vacuum for 2 h at 80 C, then hybridized for 14 h. The filters were washed with 2x standard saline citrate/0.1% sodium dodecyl sulfate. Autoradiography was performed at -80 C. Changes in band intensity were analyzed by scanning densitometry.

Gel electrophoresis and immunoblotting
To determine the abundance of C1r/C1s in conditioned medium, the medium was concentrated 10-fold using an Ultrafree-15 centrifugal filter device (Millipore Corp., Bedford, MA) containing a Biomax-10K NMWL membrane. The total protein concentration of the medium was determined by the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) using the manufacturer’s instructions. The concentrated medium samples (20–40 µl containing 0.5 mg protein) were mixed with 10 µl 4x Laemmli sample buffer, and 50 µl of the mixture were electrophoresed through a 9% sodium dodecyl sulfate-polyacrylamide gel. In some experiments dithiothreitol (0.1 M) was added to the sample before heating to 95 C for 10 min. The separated proteins were transferred to Immobilon filters (Millipore Corp.), then exposed to a 1:500 dilution of anti-C1r or anti-C1s antiserum or a 1:500 dilution of anti-C1 inhibitor antiserum as previously described (8). The filters were rinsed three times in Tris-buffered saline, then incubated for 2 h with a 1:500 dilution of antirabbit immunoglobulin G-alkaline phosphatase conjugate, followed by two rinses with Tris-buffered saline containing 0.01% Tween 20. The bands were visualized using Protoblot immunoblotting reagents following a technique recommended by the manufacturer (Promega Corp., Madison WI). Molecular weight standards (Life Technologies, Inc.) were run in a parallel lane. For some experiments the bands were visualized by chemiluminescence. Bands were visualized using a peroxidase-labeled secondary antibody and enhanced chemiluminescence following the manufacturer’s instructions (Pierce Chemical Co.). The immune complexes were detected by autoradiography.

IGFBP-5 protease assay
To determine the amount of proteolytic activity in conditioned medium, 30 µl conditioned medium was mixed with a 25 µl buffer containing 100 mM Tris (50 mM sodium chloride) and 8 mM calcium chloride (pH 7.2). One hundred nanograms of intact human IGFBP-5 were added, and the samples were incubated at 37 C for 14 h. The reaction products were then analyzed by SDS-PAGE (12.5% gel) with immunoblotting, using a 1:1000 dilution of guinea pig antihuman IGFBP-5 antiserum that had been prepared as previously described (2).

[3H]Thymidine incorporation
pSMC were plated in 96-well microtest plates (Falcon 3004) using a plating density of 5000 cells/well in DMEM supplemented with 10% FCS. After 5 d the medium was aspirated, and 0.2 cc serum-free DMEM containing 0.2% platelet-poor plasma, 0.5 µCi [3H]thymidine (specific activity, 32 µCi/µmol; Amersham Pharmacia Biotech, Arlington Heights, IL), and increasing concentrations of IGF-I (2–100 ng/ml) were added. Additional cultures received these concentrations of IGF-I plus TNF{alpha} (50 ng/ml) or C1 inhibitor (10-7 M). After 36 h the cultures were washed twice with serum free medium, and the amount of [3H]thymidine incorporated into DNA was determined as described previously (10).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of human fibroblast- or pSMC-conditioned medium obtained from cultures after 24 h showed easily detectable C1r and C1s bands (Fig. 1Go). In fibroblast-conditioned medium, C1r is present as two bands (e.g. 86 and 82 kDa), whereas C1s is detected only as a single band (82 kDa). In pSMC medium both the C1r and C1s are represented by single bands. To determine the effects of cytokines on C1r and C1s release into medium, the SMC and fibroblast cultures were exposed to TNF{alpha}, IL-1ß, or dexamethasone, and conditioned medium was analyzed by immunoblotting. Both TNF{alpha} and IL-1ß stimulated the release of C1r and C1s into the medium by fibroblasts and pSMC (Fig. 1Go). In the fibroblast medium, both of the C1r bands were stimulated by IL-1ß and TNF{alpha}. The lower molecular weight forms noted in lanes 3 and 7 presumably represent cleavage fragments of C1r and C1s. Dexamethasone (10-7 M) stimulated an increase in C1r release by SMC, but had no effect on C1s. It also had no effect on the release of either C1r or C1s by fibroblasts.



View larger version (93K):
[in this window]
[in a new window]
  		Figure 1. Secretion of C1r and C1s by cultured fibroblasts and SMCs. Fibroblasts (A) and pSMC (B) were cultured, and serum-free conditioned media were collected as described in Materials and Methods, concentrated, and analyzed by SDS-PAGE (9% gel) with immunoblotting for C1r (lanes 1–4) or C1s (lanes 5–8). Lane 1, No treatment; lane 2, IL-1ß (20 ng/ml); lane 3, TNF{alpha} (50 ng/ml); lane 4, dexamethasone (10-7 M). For fibroblast medium in A, the two arrows denote the positions of zymogen precursor form of C1r (lanes 1–4), and the single arrow denotes the C1s precursor (lanes 5–8). For pSMC medium in B, the single arrow denotes the position of the zymogen precursor form of each protein. The results show a representative experiment that was repeated three times with similar results. Scanning densitometry of the intact bands in A gave the following values expressed as arbitrary units: lane 1, 3,552; lane 2, 6,787; lane 3, 10,004; lane 4, 4,122; lane 5, 3,410; lane 6, 9,223; lane 7, 10,998; lane 8, 3,212. B: Lane 1, 1,105; lane 2, 3,467; lane 3, 4,110; lane 4, 3,221; lane 5, 2,293; lane 6, 6,919; lane 7, 7,552; lane 8, 1,918.

 
To determine whether the increase in the amount of C1s present in medium was due to the release of presynthesized peptide that was released from cell surfaces or intracellular storage compartments or whether it was due to increased synthesis, Northern blotting was undertaken. As shown in Fig. 2Go, analysis of human fibroblast (two upper panels) and pSMC (two lower panels) mRNA showed that a single 4.2-kb mRNA transcript was present. The abundance of the C1s steady state mRNA levels was increased 3.0 ± 0.3- and 2.1 ± 0.2-fold (n = 3) in fibroblasts after incubation with TNF{alpha} and IL-1ß, respectively. For SMCs, the increases were 2.1 ± 0.4- and 1.9 ± 0.3-fold, respectively. Lesser changes (e.g. 1.4 ± 0.3- and 1.2 ± 0.2-fold; n = 3) were detected after dexamethasone. To further confirm that these changes in mRNA abundance also resulted in increases in secreted protein, increasing concentrations of each compound were incubated with fibroblast cultures. As shown in Fig. 3Go, both IL-1ß and TNF{alpha} caused a concentration-dependent increase in the amount of C1r (A) and C1s (B) present in fibroblast medium. The maximum increases in C1r were 2.2 ± 0.2-fold with TNF{alpha} and 2.1 ± 0.2-fold with IL-1ß (n = 3). The maximum increases in C1s were 3.3 ± 0.4- and 4.4 ± 0.5-fold (n = 3) for TNF{alpha} and IL-1ß, respectively. Dexamethasone had no effect on the release of either protein. For pSMC, there was also a concentration-dependent increase in C1r (Fig. 4AGo) and C1s (Fig. 4BGo) noted in response to TNF{alpha}. The maximal responses were 3.8 ± 0.4- and 4.0 ± 0.4-fold (n = 3) for C1r and C1s, respectively. When increasing concentrations of IL-1ß were added, a similar increase was noted, but dexamethasone had no effect (data not shown). After its release from cells, C1r can autoactivate, and activated C1r is necessary to cleave and activate C1s. However, after cleavage, both forms migrate with the same molecular weight estimates as the precursor forms because they contain an intact interchain disulfide bond (11). To determine the degree of activation of the forms of C1r and C1s present in the conditioned medium, fibroblast medium obtained after 24 h of exposure to each of these cytokines was analyzed by SDS-PAGE using reducing conditions. As shown in Fig. 5Go, the amounts of a 62-kDa C1r band and a 52-kDa C1s band were increased after exposure to TNF{alpha} or IL-1ß. Because both C1r and C1s contain an interchain disulfide bond, after cleavage and reduction, they are detected as lower molecular weight forms. Of note, only the cleaved forms of C1r and C1s were detected.



View larger version (107K):
[in this window]
[in a new window]
  		Figure 2. Induction of C1s mRNA. Fibroblast cultures (lanes 1–4) and pSMC cultures (lanes 5–8) were exposed to the three treatments for 14 h, then total RNA was harvested as described in Materials and Methods. The samples were analyzed using Northern blotting as described in Materials and Methods. The results of hybridization using a radiolabeled C1s probe are shown in the two upper panels. The integrity of 18S and 28S RNA is shown in the two lower panels. Lanes 1 and 6, IL-1ß (20 ng/ml); lanes 2 and 8, TNF{alpha} (50 ng/ml); lanes 3 and 5, serum-free medium; lanes 4 and 7 dexamethasone (10-7 M). Scanning densitometry units were as follows: lane 1, 11,801; lane 2, 15,906; lane 3, 5,104; lane 4, 7,509; lane 5, 6,112; lane 6, 12,091; lane 7, 7,015; lane 8, 14,998. The experiment was repeated three times with similar results.

 


View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. Concentration-dependent increase in C1r and C1s in fibroblast medium. Increasing concentrations of dexamethasone (left panel), TNF{alpha} (middle panel), and IL-1ß (right panel) were added for 24 h. The conditioned media were collected as described in Materials and Methods, then analyzed by immunoblotting for C1r (A) or C1s (B). The 86- to 88-kDa band in A represents intact C1r. The high molecular weight doublet may represent a polymerized form. In B, the 86-kDa band represents intact C1s, and the 58-kDa band represents a C1s fragment. Control serum-free medium is shown in lane 1 for each treatment. The treatments shown are a follows: dexamethasone: lane 2, 10-9 M; lane 3, 10-8 M; lane 4, 10-7 M; lane 5, 10-6 M. For IL-1ß, they were: lane 2, 1 ng/ml; lane 3, 2 ng/ml; lane 4, 5 ng/ml; lane 5, 10 ng/ml; lane 6, 20 ng/ml. For TNF{alpha}, they were: lane 2, 2 ng/ml; lane 3, 5 ng/ml; lane 4, 10 ng/ml; lane 5, 20 ng/ml; lane 6, 50 ng/ml. Scanning densitometry units were as follows: A: Left panel, lane 1, 2481; lane 2, 2702; lane 3, 2689; lane 4, 2416; lane 5, 2764. Middle panel, lane 1, 2804; lane 2, 3502; lane 3, 3699; lane 4, 4587; lane 5, 5969; lane 6, 6452. Right panel, lane, 1, 2402; lane 2, 3886; lane 3, 4425; lane 4, 4653; lane 5, 4874; lane 6, 5112. B. Left panel, lane 1, 1107; lane 2, 1495; lane 3, 1322; lane 4, 1006; lane 5, 1154. Middle panel, lane 1, 1398; lane 2, 2889; lane 3, 3467; lane 4, 4228; lane 5, 4569; lane 6, 4732. Left panel, lane 1, 976; lane 2, 1807; lane 3, 2147; lane 4, 2506; lane 5, 3629; lane 6, 4516.

 


View larger version (84K):
[in this window]
[in a new window]
  		Figure 4. Concentration-dependent increase in C1r and C1s in pSMC medium. SMC cultures were exposed to increasing concentrations of TNF{alpha}, and conditioned medium was collected, then immunoblotted for C1r (A) or C1s (B) as described in Materials and Methods. Control cultures receiving serum-free medium are shown in lane 1. Additional pSMC cultures were exposed to TNF{alpha}: lane 2, 2 ng/ml; lane 3, 5 ng/ml; lane 4, 10 ng/ml; lane 5, 20 ng/ml; lane 6, 50 ng/ml. Scanning densitometry units for A were as follows: lane 1, 1911; lane 2, 2706; lane 3, 4862; lane 4, 5136; lane 5, 7987; lane 6, 7552. For B, they were: lane 1, 1015; lane 2, 1544; lane 3, 1809; lane 4, 2844; lane 5, 3489; lane 6, 4156. The experiment was repeated three times with similar results.

 


View larger version (69K):
[in this window]
[in a new window]
  		Figure 5. Change in activated C1r and C1s concentrations after exposure to TNF{alpha}, dexamethasone, or IL-1ß. All of the test compounds were incubated with fibroblast cultures for 24 h, and conditioned media were collected as described previously. Before electrophoresis, the samples were heated to 95 C with dithiothreitol (50 mM) for 5 min. The proteins were then analyzed by SDS-PAGE and transferred to Immobilon filters that were probed for C1r (A) or C1s (B). The 62-kDa band in A corresponds to the molecular mass estimate of the largest fragment of C1r after cleavage, and the 52-kDa band in B corresponds to the largest fragment of cleaved C1s. Treatments were as follows: serum-free medium (lanes 1 and 2), 10 ng/ml IL-1ß (lane 3), 20 ng/ml IL-1ß (lane 5), 20 ng/ml TNF{alpha} (lane 4), and 50 ng/ml TNF{alpha} (lane 6). Scanning densitometry units for A were: lane 1, 923; lane 2, 1214; lane 3, 3604; lane 4, 3827; lane 5, 4612; lane 6, 5437. For B, they were: lane 1, 125; lane 2, 568; lane 3, 996; lane 4, 2218; lane 5, 3987; lane 6, 4574. The experiment was repeated three times with similar results.

 
To determine whether the increases in activated C1r and C1s that are occurring in fibroblast medium result in increased IGFBP-5 proteolysis, fibroblast-conditioned medium obtained after 24 h of exposure to these compounds was incubated overnight with IGFBP-5, and the degree of proteolysis was assessed. As shown in Fig. 6Go, medium conditioned by exposure to IL-1ß, TNF{alpha}, or dexamethasone resulted in substantially more degradation of IGFBP-5. This experiment was repeated using pSMC cultures, and the medium obtained after each of the three treatments showed enhanced degradation of IGFBP-5 compared with control medium that contained no additives.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 6. IGFBP-5 proteolysis during exposure to conditioned media. Conditioned media were obtained from fibroblast (lanes 2–5) or pSMC cultures (lanes 6–9) after a 24-h exposure to each of the three test compounds. IGFBP-5 was added to each sample, then they were analyzed for IGFBP-5 protease activity as described in Materials and Methods. The treatments were as follows: lane 1, IGFBP-5 standard, no protease added; lanes 2 and 7, dexamethasone (10-7 M); lanes 3 and 8, TNF{alpha} (50 ng/ml); lanes 4 and 6, IL-1ß (20 ng/ml); lanes 5 and 9, serum-free medium. The arrows denote the positions of intact IGFBP-5 and its principle 22-kDa fragment. The results show significant cleavage of IGFBP-5 under each of the conditions with the greatest cleavage occurring with TNF{alpha} exposure. The scanning densitometry units for the intact IGFBP-5 band were: lane 1, 33,443; lane 2, 9,196; lane 3, 3,483; lane 4, 5,126; lane 5, 14,575; lane 6, 5,304; lane 7, 6,688; lane 8, 3,391; lane 9, 14,041. For the major fragment band, they were: lane 1, 0; lane 2, 8,113; lane 3, 9,275; lane 4, 8,066; lane 5, 2,268; lane 6, 7,877; lane 7, 7,924; lane 8, 7,369; lane 9, 4,554. The experiment was repeated three times with similar results.

 
To further confirm that the increase in proteolytic IGFBP-5 activity was due to an increase in C1r/C1s in conditioned medium, conditioned medium that had been collected after exposure to TNF{alpha} or IL-1ß was incubated with native IGFBP-5, a protease-resistant mutant form of IGFBP-5 or C1 inhibitor. As shown in Fig. 7Go, there was no significant change in the amount of intact IGFBP-5 when the mutant form was incubated with the conditioned medium. Exposure to either TNF{alpha} or IL-1ß resulted in increased proteolysis of native IGFBP-5. When C1 inhibitor was added, the amount of the 22-kDa fragment detected in medium from cells exposed to either TNF{alpha} or IL-1ß was decreased, and the amount of intact IGFBP-5 was increased.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 7. Specificity of IGFBP-5 protease degradation. To confirm that the increase in protease activity in the conditioned medium was due to an increase in C1r and C1s, conditioned media were collected from fibroblast cultures after exposure to medium alone (lanes 4 and 8), TNF{alpha} (lanes 1–3), or IL-ß (lanes 5–7) for 24 h. The media were incubated in vitro with 80 ng native IGFBP-5 (lanes 2–5, 7, and 8) as described in Materials and Methods. The protease-resistant mutant form of IGFBP-5 (80 ng) was used in lanes 1 and 6. Purified C1 inhibitor (10-7 M) was also included in lanes 2 and 5. The extent of IGFBP-5 cleavage was determined by immunoblotting. The arrows denote the positions of intact IGFBP-5 and the 22-kDa fragment. The scanning densitometry units for intact IGFBP-5 were: lane 1, 26,422; lane 2, 29,300; lane 3, 9,195; lane 4, 15,366; lane 5, 12,908; lane 6, 10,856; lane 7, 1,854; lane 8, 2,933. For the 22-kDa fragment, they were: lane 1, 1,121; lane 2, 1,440; lane 3, 20,060; lane 4, 14,877; lane 5, 1,114; lane 6, 706; lane 7, 4,278; lane 8, 3,091. The experiment was reported three times with similar results.

 
Fibroblasts have been shown to secrete C1 inhibitor, a protein that inhibits C1r autoactivation (12, 13). Therefore, a possible explanation for the discrepancy between C1s release and IGFBP-5 protease activation that was noted with dexamethasone might be due to a decrease in the release of C1 inhibitor, thus allowing more C1s activation. As shown in Fig. 8Go, both fibroblasts and pSMC secreted C1 inhibitor. After exposure of either cell type to dexamethasone, the abundance of C1 inhibitor in the medium was decreased.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 8. Secretion of C1 inhibitor. Conditioned media were collected from fibroblasts (lanes 1–3) and SMCs (lanes 4–6), concentrated, then analyzed for the presence of C1 inhibitor by immunoblotting. The arrows in lanes 1–3 denote the positions of intact C1 inhibitor and a fragment. The single band detected in lanes 4–6 represents intact C1 inhibitor. As shown, the addition of increasing concentrations of dexamethasone (10-7 M; lanes 1 and 6) resulted in a decrease in the release of the inhibitor compared with 10-9 M dexamethasone (lanes 2 and 5) or serum-free medium (lanes 3 and 4). The arrow denotes the position of the C1 inhibitor. Values (mean ± SD) obtained using scanning densitometry for three separate experiments were as follows: lane 1, 105 ± 102; lane 2, 5449 ± 743; lane 3, 5386 ± 802; lane 4, 3436 ± 595; lane 5, 2874 ± 422; lane 6, 1239 ± 266.

 
To determine the effect of increasing IGFBP-5 proteolysis on the pSMC DNA synthesis response to IGF-I, SMC cultures were exposed to increasing concentrations of IGF-I in the presence or absence of TNF{alpha}, and [3H]thymidine incorporation into DNA was quantified (Fig. 9Go). IGF-I alone (100 ng/ml) stimulated a 7.2-fold increase. Concomitant exposure of TNF{alpha} resulted in inhibition of this response. Coincubation with C1 inhibitor in the present of TNF{alpha} resulted in a partial restoration of the cellular response to IGF-I.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 9. Effects of IGFBP-5 proteolysis on the SMC DNA synthesis response to IGF-I. Quiescent SMC cultures were exposed to increasing concentrations of IGF-I ({bullet}) as described in Materials and Methods. Additional cultures received the same concentrations of IGF-I in the presence of TNF{alpha} (50 ng/ml; {circ}) or TNF{alpha} (50 ng/ml) plus C1 inhibitor (10-7 M; X). After 36 h, [3H]thymidine incorporation into DNA was determined as described in Materials and Methods. The results shown are the mean of triplicate determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These findings show that both cultured aortic SMCs and dermal fibroblasts secrete C1r and C1s. We have previously demonstrated that C1r and C1s account for much of the IGFBP-5 proteolytic activity produced by cultured human fibroblasts (8). These studies extend that finding to SMCs showing that the same proteins are also released into conditioned medium by this cell type. Both C1r and C1s are synthesized and secreted as a precursor forms (11). C1r autoactivates and cleaves itself into the active serine protease moiety; it then cleaves C1s. However, after cleavage, both C1r and C1s remain detectable only as higher molecular weight forms, because they retain an interchain disulfide bond. Therefore, reduction is usually required to detect the mature active protease. Our studies show that after their secretion, analysis by reducing SDS-PAGE shows that C1r and C1s are present in the conditioned medium of both cell types as activated forms.

These studies analyzed the regulatory control of C1s synthesis and C1r and C1s release by fibroblasts and SMCs. Exposure of SMCs to TNF{alpha} or IL-1ß resulted in increased steady state mRNA levels of C1s, thus showing that they stimulate its synthesis. This led to increased amounts of C1s in the medium of both cell types. Analysis of the secreted C1r and C1s using reducing conditions showed that the forms present in medium had been activated, suggesting either that C1r was being autoactivated or that the active form of this protease was released after exposure to these two stimuli. We did not detect evidence of TNF{alpha} or IL-ß stimulating increased conversion from the C1r or C1s precursors to the active forms. In contrast, after exposure to dexamethasone, we did not detect an increase in the release of either C1r or C1s by fibroblasts, but we did detect an increase in C1r in SMCs, and for both cell types there was an increase in IGFBP-5 proteolytic activity. Further analysis showed that the secretion of C1 inhibitor by both cell types was suppressed by dexamethasone, which would be predicted to lead to an increase in net proteolytic activity. When we analyzed protease activity in the conditioned medium that was obtained after exposure to both TNF{alpha} and IL-1ß, the increase detected in IGFBP-5 protease activity was inhibitable with the C1 inhibitor. Therefore, multiple lines of evidence suggest that stimulation of secretion of C1r and C1s by these cell types results in increased cleavage of IGFBP-5.

Our prior studies have shown that C1r in fibroblast medium is capable of autoactivation, and after autoactivation it cleaves the C1s precursor that is also synthesized by this cell type (8). Our prior study showed that incubation of a mutant form of IGFBP-5 containing substitutions for two basic residues (K138N and K139N) with fibroblast medium or purified C1r/C1s showed no cleavage. This suggests that the mutations had altered the cleavage site, making it resistant to proteolysis (5, 8). These studies show that the IGFBP-5 proteolytic activity that is increased by exposure to TNF{alpha} or IL-1ß is accounted for by a protease that does not cleave this mutant, and its activity is inhibited by C1 inhibitor. Therefore, we conclude that the increase in proteolytic activity is also due to an increase in C1r and C1s.

Both the C1r and C1s have been shown to be synthesized by multiple cell types; however, identification of the factors that regulate their synthesis has received limited analysis (11, 12, 13, 14, 15, 16, 17, 18, 19). Given their important role in tissue repair processes and in the catabolic response to injury, it is not surprising that IL-1ß and TNF{alpha} are important stimuli of the synthesis of these proteases. IL-1ß has been shown previously to stimulate a 12-fold increase in C1r/C1s synthesis in amnion cells (15). Similarly, cytokines associated with amyloid plaques such as a IL-1 and TNF{alpha} were shown to stimulate C1r and C1s secretion by glial cells (20). It has also been generally acknowledged that these enzymes are important in terms of cartilage repair (14, 16). Recently, we reported that an inhibitor of C1r/C1s activation that inhibited their activation in joint fluid during the development of osteoarthritis resulted in inhibition of degradation of IGFBP-5 and an improved anabolic response to IGF-I (21). That in vivo study suggested that these proteases may have a significant role in controlling cell and tissue responsiveness to this growth factor. In addition, other investigators reported that IGFBP-5 has IGF-I-independent effects to enhance cell growth, and therefore, its cleavage by these proteases might represent a means of inhibiting those actions (22). Because increasing the amount of intact IGFBP-5 deposited in extracellular matrix enhances the cellular responsiveness to IGF-I, the enhanced secretion of two proteases that cleave IGFBP-5 could result in attenuation of cellular responsiveness to IGF-I (3). This is consistent with our finding in this study that the addition of TNF{alpha} with IGF-I inhibited the DNA synthesis response to IGF-I, and concomitant inhibition of IGFBP-5 proteolysis resulted in a partial restoration of IGF-I’s stimulatory effect. Other interpretations of this result must be considered, because TNF{alpha} could also alter the rate of IGFBP-5 synthesis and could have direct effects on cellular replication not mediated through changes in components of the IGF system. However, the balance between the effects of IGF-I and TNF{alpha} could be of significance in catabolic states where cytokines often have actions that oppose the anabolic effects of IGF-I (23). Our findings suggest that accelerated degradation of IGFBP-5 is one mechanism by which these catabolic cytokines could counteract the effect of this anabolic growth factor.

The interactions between cytokines, such as IL-1ß or TNF{alpha}, and IGF-I has been analyzed in multiple different tissues, and several different mechanisms have been identified by which they antagonize each other’s effects. The most extensive analysis has been undertaken in neural tissue, in which astrocytes, oligodendrocytes, and glial cells as well as neurons have been analyzed (20, 24, 25, 26). A similar extensive analysis has occurred in chondrocytes (27, 28, 29, 30, 31). Multiple mechanisms of antagonism have been proposed. Specifically, it has been shown in neural cell types that TNF{alpha} can inhibit the ability of IGF-I to stimulate insulin receptor substrate-2 phosphorylation and concomitant activation of phosphoinositol 3-kinase is markedly inhibited by prior exposure to TNF{alpha} (25). IGF-I can reverse the effects of TNF{alpha} on inhibitor of nuclear factor-{kappa}B (I{kappa}B) phosphorylation and that inhibiting TNF{alpha} resulted in stimulation of nuclear factor-{kappa}B translocation (26). In this manner, IGF-I appears to inhibit proapoptotic effects of TNF{alpha}. Similarly, in chondrocytes IGF-I has been shown to antagonize the effects of both TNF{alpha} and IL-1ß on inhibition of collagen mRNA synthesis (28, 29). IGF-I also increases proteoglycan synthesis in cartilage, an effect antagonized by IL-1ß (32). Conversely, TNF{alpha} inhibits the synthesis of IGF-I by cartilage and osteoblasts (33). Furthermore, both of these cytokines have been shown to be potent inducers of several matrix metalloproteases (MMPs), including MMP-2, -9, and -13, by chondrocytes (27). IGF-I treatment inhibits the ability of these cytokines to activate these MMPs. This may be directly relevant to the mechanisms proposed in this paper, where these cytokines have been shown to activate C1s, which cleaves IGFBP-5, a known potentiator of IGF-I action. These cytokines may also attenuate the effects of IGF-I by the induction of proteolytic activities such as C1r/C1s in SMCs. Anwar et al. (34) recently demonstrated that rat arterial wall SMC responded to these cytokines with an increase in the secretion of IGFBP-3, a binding protein known to inhibit the effect of IGF-I on vascular smooth muscle, thus leading to a reduction in IGF-I bioactivity. Similarly, TNF{alpha} was shown to suppress IGF-I synthesis (34). Therefore, TNF{alpha} could use all three mechanisms to attenuate SMC responsiveness to IGF-I.

Systemic administration of TNF{alpha} or IL-1ß has also been shown to inhibit IGF-I actions. IL-1ß decreases IGF-I expression in skeletal muscle and increases IGFBP-1 and -2 in serum, leading to attenuation of IGF-I action (35). Administration of TNF{alpha} to animals results in decreased concentrations of IGF-I in serum and an increase in the amount of inhibitory forms of IGFBPs, such as IGFBP-1 (32, 35). IL-1ß can inhibit the induction of IGF-I mRNA in hepatocytes by GH, thereby directly blocking IGF-I synthesis and secretion into the blood (36). Thus, there appear to be multiple mechanisms by which TNF{alpha} and IL-1ß interact to inhibit IGF-I actions, suggesting that all of these mechanisms, including that described in this study, contribute to the ability of these two cytokines to antagonize the anabolic effects IGF-I.


   Acknowledgments
 
We thank Ms. Laura Lindsey for her help in preparing the manuscript.


   Footnotes
 
This work was supported by NIH Grant AG-02331.

Abbreviations: IGFBP, IGF binding protein; MMP, matrix metalloprotease; pSMC, porcine smooth muscle cells.

Received August 26, 2002.

Accepted for publication February 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jones JI, Clemmons DR 1995 Insulin like growth factor and their binding proteins: biologic actions. Endocr Rev 16:3–34[CrossRef][Medline]
  2. 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]
  3. Jones JI, Gockerman A, Busby WH, 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]
  4. Parker A, Busby WH, Clemmons DR 1996 Identification of the extracellular matrix binding site for insulin like growth factor binding protein-5. J Biol Chem 271:13523–13529[Abstract/Free Full Text]
  5. Imai Y, Moralez A, Andag U, Clarke JB, Busby WH, Clemmons DR 2000 Substitutions for hydrophobic amino acids in the N terminal domain of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions. J Biol Chem 275:18188–18194[Abstract/Free Full Text]
  6. Imai Y, Busby WH, Smith CE, Clark JB, Horvitz GD, Rees C, Clemmons DR 1997 Protease resistant form of insulin-like growth factor binding protein-5 is an inhibitor of insulin-like growth factor-I actions in porcine smooth muscle cells in culture. J Clin Invest 100:2596–25605[Medline]
  7. Parker A, Rees C, Clarke JB, Busby WH, Clemmons DR 1998 Binding of insulin-like growth factor binding protein-5 to smooth muscle cell extracellular matrix is a major determinant of the cellular response to IGF-I. Mol Cell Biol 9:2383–2392
  8. Busby WH, Nam TJ, Moralez A, Smith C, Jennings M, Clemmons DR 2000 The complement component C1s is the protease that accounts for cleavage of insulin-like growth factor binding protein-5 in fibroblast medium. J Biol Chem 275:37638–37644[Abstract/Free Full Text]
  9. Clemmons DR, Shaw DS 1983 Variables controlling somatomedin production by cultured human fibroblasts. J Cell Physiol 115:137–143[CrossRef][Medline]
  10. Clemmons DR, Gardner LI 1990 A factor contained in plasma is required for IGF binding protein-1 to potentiate the effect of IGF-I on smooth muscle cell DNA synthesis. J Cell Physiol 145:129–135[CrossRef][Medline]
  11. Spycher SE, Nick H, Rickli EE 1986 Human complement component C1s: partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation. Eur J Biochem 156:49–57[Medline]
  12. Katz Y, Strunk RC 1998 Synovial fibroblast-like cells synthesize seven proteins of the complement system. Arthritis Rheum 31:1365–1370
  13. Katz Y, Strunk RC 1989 Synthesis and regulation of C1 inhibitor by human skin fibroblasts. J Immunol 42:2041–2045
  14. Bradley K, North J, Saunders D, Schwaeble W, Jeziorska M, Woolley DE, Whaley K 1996 Synthesis of classical pathway complement components by chondrocytes. Immunology 88:648–656[Medline]
  15. Katz Y, Gur S, Aladjem M, Strunk RC 1995 Synthesis of complement proteins in amnion. J Clin Endocrinol Metab 80:2027–2032[Abstract]
  16. Breitner S, Storkel S, Reichel W, Loos M 1995 Complement components Cq C1r/C1s, and C1INH in rheumatoid arthritis. Correlation of in situ hybridization and Northern blot results with function and protein concentration in synovium and primary cell cultures. Arthritis Rheum 38:492–498[Medline]
  17. Ramadori G, Heinz HP, Martin H, Meyer sum Buschenfelde KH, Loos M 1986 Biosynthesis of the subcomponents of C1q, C1r and C1s of the first component of complement (C1) by guinea pig hepatocyte primary cultures. Eur J Immunol 16:1137–1141[Medline]
  18. Sakai H, Nakashima S, Yoshimura S, Nishimura Y, Sakai N, Nozawa Y 1998 Molecular cloning of a cDNA encoding a serine protease homologous to complement C1s precursor from rat C6 glial cells and its expression during glial differentiation. Gene 209:87–94[CrossRef][Medline]
  19. Legoedec J, Gasque P, Jeanne JF, Scotte M, Fontaine M 1997 Complement classical pathway expression by human skeletal myoblasts in vitro. Mol Immunol 34:734–741
  20. Veerhuis R, Janssen I, De Groot CJ, Van Muiswinkel FL, Hack CE, Eikelenboom P 1999 Cytokines associated with amyloid plaques in Alzheimer’s disease brain stimulate human glial and neuronal cell cultures to secrete early complement proteins, but not C1-inhibitor. Exp Neurol 160:289–299[CrossRef][Medline]
  21. Clemmons DR, Busby WH, Garmong A, Schultz DR, Howell DS, Carr R 2002 Inhibition of IGFBP-5 proteolysis in articular cartilage and joint fluid results in enhanced concentrations of IGF-I and improved osteoarthritis. Arthritis Rheum 46:694–703[CrossRef][Medline]
  22. Miyakoshi N, Richman C, Kasukawa Y, Linkhart TA, Baylink DJ, Mohan S 2001 Evidence that IGF-binding protein-5 functions as a growth factor. J Clinic Invest 107:7381
  23. Lang CH, Fan J, Cooney R, Vary RC 1996 IL-1 receptor antagonist attenuates sepsis induced alterations in IGF system and protein synthesis. Am J Physiol 270:430–437
  24. Ye P, D’Ercole AJ 1999 Insulin-like growth factor I protects oligodendrocytes from tumor necrosis factor-{alpha}-induced injury. Endocrinology 140:3063–3072[Abstract/Free Full Text]
  25. Venters HD, Tan Q, Lui Q, VanHoy RW, Dantzer R, Kelley KW 1999 A new mechanism of neurodegeneration: a proinflammatory cytokine inhibits receptor signaling. Proc Natl Acad Sci USA 96:9879–9884[Abstract/Free Full Text]
  26. Pons S, Torres-Aleman I 2000 Insulin-like growth factor-I stimulates dephosphorylation of I{kappa}B through the serine phosphatase calcineurin (protein phosphatase 2-B). J Biol Chem 275:38620–38625[Abstract/Free Full Text]
  27. Hui W, Rowan AD, Cawston T 2001 Insulin-like growth factor 1 blocks collagen release and down regulates matrix metalloproteinase-1, -3, -8 and -13 mRNA expression in bovine nasal cartilage stimulated with oncostatin M in combination with interleukin 1{alpha}. Ann Rheum Dis 60:254–261[Abstract/Free Full Text]
  28. Smith P, Shuler FD, Georgescu HI, Ghivizzani SC, Johnstone B, Niyibizi C, Robbins PD, Evans CH 2000 Genetic enhancement of matrix synthesis by articular chrondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1. Arthritis Rheum 43:1156–1164[CrossRef][Medline]
  29. Frisbie DD, Nixon AJ 1997 Insulin-like growth factor-1 and corticosteroid modulation of chondrocyte metabolic and mitogenic activities in interleukin 1-conditioned equine cartilage. Am J Vet Res 58:524–530[Medline]
  30. Kondo S, Cha SH, Xie WF, Sandell LJ 2001 Cytokine regulation of cartilage-derived retinoic acid-sensitive protein (CD-RAP) in primary articular chrondrocytes: suppression by IL-1, bfGF, TGFß and stimulation by IGF-1. J Orthpaed Res 19:712–719[CrossRef][Medline]
  31. Koletta E, Muir HJ, Barrett JC, Hardingham TE 2001 Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor. Rheumatology 40:1146–1156[Abstract/Free Full Text]
  32. Lang CH, Nystrom GJ, Frost RA 2001 Tissue-specific regulation of IGF-I and IGF-binding proteins in response to TNF{alpha}. Growth Horm IGF Res 11:250–260[CrossRef][Medline]
  33. Gilbert L, He X, Farmer P, Boden S, Kozlowski M, Rubin J, Nanes MS 2000 Inhibition of osteoblast differentiation by tumor necrosis factor-{alpha}. Endocrinology 141:3956–3964[Abstract/Free Full Text]
  34. Anwar A, Zahid AA, Scheidegger KJ, Brink M, Delafontaine P 2002 Tumor necrosis factor-{alpha} regulates insulin-like growth factor-1 and insulin-like growth factor binding protein-3 expression in vascular smooth muscle. Circulation 105:1220–1225[Abstract/Free Full Text]
  35. Fan J, Woinar MM, Theodorakis M, Lang CH 1996 Regulation of insulin-like growth factor (IGF)-I mRNA and peptide and IGF-binding proteins by interleukin-1. Am J Physiol 270:R621–R629
  36. Thissen JP, Verniers J 1997 Inhibition of interkeukin-1ß and tumor necrosis factor-{alpha} of the insulin-like growth factor I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture. Endocrinology 138:1078–1084[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
K. S. Kim, Y. B. Seu, S.-H. Baek, M. J. Kim, K. J. Kim, J. H. Kim, and J.-R. Kim
Induction of Cellular Senescence by Insulin-like Growth Factor Binding Protein-5 through a p53-dependent Mechanism
Mol. Biol. Cell, November 1, 2007; 18(11): 4543 - 4552.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K.-H. Chang, T. Chan-Ling, E. L. McFarland, A. Afzal, H. Pan, L. C. Baxter, L. C. Shaw, S. Caballero, N. Sengupta, S. L. Calzi, et al.
IGF binding protein-3 regulates hematopoietic stem cell and endothelial precursor cell function during vascular development
PNAS, June 19, 2007; 104(25): 10595 - 10600.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
K. R. von Schalburg, M. L. Rise, G. D. Brown, W. S. Davidson, and B. F. Koop
A Comprehensive Survey of the Genes Involved in Maturation and Development of the Rainbow Trout Ovary
Biol Reprod, March 1, 2005; 72(3): 687 - 699.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
L. J. Spicer
Proteolytic Degradation of Insulin-Like Growth Factor Binding Proteins by Ovarian Follicles: A Control Mechanism for Selection of Dominant Follicles
Biol Reprod, May 1, 2004; 70(5): 1223 - 1230.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Z. T. Resch, B.-K. Chen, L. K. Bale, C. Oxvig, M. T. Overgaard, and C. A. Conover
Pregnancy-Associated Plasma Protein A Gene Expression as a Target of Inflammatory Cytokines
Endocrinology, March 1, 2004; 145(3): 1124 - 1129.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
P. Delafontaine, Y.-H. Song, and Y. Li
Expression, Regulation, and Function of IGF-1, IGF-1R, and IGF-1 Binding Proteins in Blood Vessels
Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 435 - 444.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 Moralez, A.
Right arrow Articles by Clemmons, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moralez, A.
Right arrow Articles by Clemmons, D.

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