| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Departments of Research and Medicine, Saint Francis Hospital and Medical Center (N.F., D.D., E.C.), Hartford, Connecticut 06105; and The University of Connecticut School of Medicine (E.C.), Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: Ernesto Canalis, Department of Research, Saint Francis Hospital and Medical Center, 114 Woodland Street, Hartford, Connecticut 06105-1299.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bone remodeling is affected by hormones and local growth factors. Insulin-like growth factors (IGFs) are autocrine factors that enhance the differentiated function of the osteoblast and have modest mitogenic activity for cells of the osteoblastic lineage (11). Recently, we found that IL-6, in the presence of sIL-6R, stimulates IGF I expression in cultures of osteoblast-enriched cells from fetal rat parietal bone (Ob cells), suggesting that IL-6 could influence bone cell function through the IGF axis (12). Osteoblasts not only express the two IGF genes, but also express six IGF binding proteins (IGFBPs), which can modify the half-life and activity of IGF-I and IGF-II (13, 14, 15, 16, 17). Some IGFBPs have specific functions, and IGFBP-5 is unique because it induces cell growth and enhances the actions of IGF I in bone cell cultures (18). Therefore, changes in IGFBP-5 synthesis could be responsible for changes in bone formation.
The synthesis of IGFBP-5 in osteoblasts is induced by prostaglandin E2 (PGE2), retinoic acid, and IGF-I, and is inhibited by transforming growth factor-ß-1, platelet-derived growth factor-BB, fibroblast growth factor-2, and cortisol (17, 19, 20, 21). Agents that modify IGFBP-5 synthesis tend to have a similar effect on IGF-I expression, so that in osteoblasts the synthesis of IGF-I and IGFBP-5 is to an extent coordinated (13, 22). Although there is information about the regulation of IGF-I, IGF-II, and IGFBP-5 by the more classic growth factors and by other agents that modify bone formation, less is known about their regulation by bone resorbing cytokines of the interleukin family, such as IL-6. Both IL-6 and IGFBP-5 have mitogenic properties for cells of the osteoblastic lineage. Furthermore, IL-6 modifies the differentiation of these cells, and the expression of IGFBP-5 depends on the stage of osteoblastic cell differentiation (23). Consequently, we postulated that IL-6 might regulate IGFBP-5 expression in osteoblast cultures.
In the present study, we examined the effects of IL-6 on IGFBP-5 expression in Ob cells, and determined possible mechanisms involved in this regulation. Because sIL-6R is known to enhance the actions of IL-6 in skeletal and nonskeletal cells, we also tested the effects of IL-6 in the presence of its soluble receptor (24, 25).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
50,000 cells/cm2) and
transferred to serum-free medium for 20–24 h, and then exposed to test
agents for 2–24 h as indicated in the text and figure legends. For the nuclear run-on experiment, subconfluent cultures of Ob cells were trypsinized, subcultured at a 1:10 dilution, and grown to confluence in DMEM supplemented with 10% FBS. Cells were serum-deprived for 24 h, and treated for 2–24 h in serum-free DMEM. Recombinant human IL-6 and sIL-6R (R&D Systems, Minneapolis, MN) were dissolved in PBS containing 0.1% BSA. Cycloheximide (Sigma Chemical Co., St. Louis, MO) was added directly to the culture medium, and 5,6-dichlorobenzimidazole riboside (DRB) and indomethacin (both from Sigma) were dissolved in absolute ethanol and diluted 1:200 and 1:1000, respectively, in DMEM. Control and treated cultures contained equal amounts of vehicle. At the completion of the treatment period, the cell layer was extracted with guanidine thiocyanate for RNA analysis or nuclei were isolated by Dounce homogenization for the nuclear run-on assay.
For protein analysis of IGFBP-5, culture medium was collected in the presence of 0.2% polyoxyethylene sorbitan monolaurate (Pierce, Rockford, IL), and the extracellular matrix was extracted as described (27, 28). Both were stored at -70 C. To assay for IGFBP-5 proteolytic activity, the culture medium was collected and tested at the completion of the cell culture period.
Northern blot analysis
Total cellular RNA was isolated with guanidine thiocyanate, at
acid pH, followed by a phenol-chloroform (Sigma) extraction or by
RNeasy kit in accordance with manufacturer’s instructions (Qiagen,
Chatsworth, CA) (29). The RNA recovered was quantitated by
spectrometry, and equal amounts of RNA from control or test samples
were loaded on a formaldehyde agarose gel following denaturation. The
gel was stained with ethidium bromide to visualize ribosomal RNA
(rRNA), documenting equal RNA loading of the various experimental
samples. RNA was then blotted onto Gene Screen Plus charged nylon
(DuPont, Wilmington, DE), and the uniformity of transfer documented by
revisualization of rRNA. A 300 bp HindIII restriction
fragment of a rat IGFBP-5 complementary DNA (cDNA) (kindly provided by
Dr. S. Shimasaki, La Jolla, CA) and a 700-bp
BamHI-SphI fragment of a mouse 18S rRNA cDNA
clone (ATCC, Rockville, MD) were purified by agarose gel
electrophoresis (30). IGFBP-5 and 18S rRNA cDNAs were labeled with
[
-32P] deoxyadenosine triphosphate (dATP) and
[-32P] deoxycytosine triphosphate (dCTP) (50 µCi
each at a specific activity of 3,000 Ci/mmol; DuPont) using the random
hexanucleotide primed second strand synthesis method (31).
Hybridizations were carried out at 42 C for 16–48 h, and posthybridization washes were performed at 65 C in 1x SSC for IGFBP-5 and 0.1x SSC for 18S. The bound radioactive material was visualized by autoradiography on DuPont Reflection Film (DuPont), employing Cronex Lightning Plus intensifying screens (DuPont) (32). Relative hybridization levels were determined by densitometry. Northern analyses are representative of three or more cultures.
Nuclear run-on assay
To examine changes in the rate of transcription, nuclei were
isolated by Dounce homogenization in a Tris buffer, pH 7.4, containing
0.5% Nonidet P-40 (32). Nascent transcripts were labeled by incubation
of nuclei in a reaction buffer containing 500 µM each
adenosine, cytidine, and guanosine triphosphates, 150 U RNasin
(Promega, Madison, WI) and 250 µCi [-32P]uridine
triphosphate (UTP) (3000 Ci/mmol, DuPont) (33). RNA was isolated by
treatment with DNase I and proteinase K, followed by phenol-chloroform
extraction and ethanol precipitation. Linearized plasmid DNA containing
approximately 1 µg cDNA was immobilized onto GeneScreen Plus by slot
blotting according to manufacturer’s directions (DuPont). The plasmid
vector pGL2-Basic (Promega) was used as a control for nonspecific
hybridization, and a mouse 18S rRNA cDNA clone was used to estimate
uniformity of the loading. Equal cpm of [32P]RNA from
each sample were hybridized to cDNAs at 42 C for 72 h and washed
in 1x SSC at 65 C for 20 min. Hybridized cDNAs were visualized by
autoradiography, and IGFBP-5 hybridization levels were determined by
densitometry. Nuclear run-on assay was performed in two separate
experiments.
Western immunoblot analysis
Extracellular matrix was prepared as described (27, 28).
Briefly, Ob cells were rinsed in PBS, and cell membranes were removed
with 0.5% Triton X-100 (Sigma), pH 7.4, and nuclei and cytoskeleton
were removed by incubation with 2 mM ammonium acetate, pH
9.0. The extracellular matrix was rinsed with PBS and scraped from the
culture plates. Conditioned medium was concentrated approximately three
times by speed vac centrifugation. Aliquots of extracellular matrix
extracts or conditioned medium were mixed with Laemmli sample buffer to
give a final concentration of 2% SDS. Samples were fractionated by
electrophoresis on a 12% polyacrylamide gel (34). Proteins were
transferred to Immobilon P membranes (Millipore, Bedford, MA), blocked
with 2% BSA, and exposed to a 1:500 dilution of rabbit antiserum
raised against native human IGFBP-5 (UBI, Lake Placid, NY) in 1% BSA
overnight. Blots were exposed to goat antirabbit IgG antiserum
conjugated to horseradish peroxidase, washed, and developed with a
horseradish peroxidase chemiluminescence detection reagent (DuPont).
IGFBP-5 was identified by comigration with purified human IGFBP-5
(kindly provided by Dr. D. Clemmons, Chapel Hill, NC). Western blot
analyses of the conditioned medium and extracellular matrix are
representative of two and eight different cultures, respectively.
To detect IGFBP-5 proteolytic activity in the culture medium of Ob cells, 100 ng of a purified human IGFBP-5 standard were incubated with 100 µl conditioned medium from control or treated cultures in the presence and absence of 1 mM 1,10-phenanthrolene, 10 mM EDTA, or 10 mM phenylmethylsulfonyl fluoride (PMSF). Samples were incubated at 37 C for 0–16 h in a shaking water bath, and proteolytic degradation was terminated by adding 2x Laemmli sample buffer and heating at 100 C for 3 min. Samples were fractionated by electrophoresis and processed for Western immunoblot analysis. Western blot analysis to detect IGFBP-5 proteolytic activity was performed three times in the absence and twice in the presence of protease inhibitors.
Statistical methods
Data are expressed as means ± SEM. Slopes of
messenger RNA (mRNA) decay were examined by linear regression, and
differences in decay were determined by the method of Sokal and Rolf
(35).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). This effect was
magnified when Ob cells were treated with IL-6 in the presence of
sIL-6R. IL-6 at 100 ng/ml in the presence of sIL-6R at 125 ng/ml
induced a 2- to 3-fold increase in IGFBP-5 mRNA levels after 6 h
and a 5- to 7-fold increase after 24 h, as determined by
densitometry (Fig. 1). IL-6, when tested by itself, was effective at
doses of 100 and 300 ng/ml, which increased IGFBP-5 mRNA levels by 1.5-
to 2-fold after 24 h (Fig. 2). When tested in the
presence of sIL-6R at 125 ng/ml, IL-6 was modestly active at a dose of
1 ng/ml, and the effect was maximal at 10–300 ng/ml; at these doses
IL-6 increased IGFBP-5 transcripts by 6- to 8-fold after 24 h
(Fig. 2). In the absence of IL-6, sIL-6R caused a small increase in
IGFBP-5 mRNA levels, and at 125–250 ng/ml it increased IGFBP-5
transcripts by approximately 1.5-fold (Fig. 3). In the
same experiment, when sIL-6R was tested in the presence of IL-6 at 100
ng/ml, it was effective at 31–250 ng/ml, increasing IGFBP-5 by 3.5- to
4.5-fold after 24 h. In other experiments, sIL-6R at 125 ng/ml
with IL-6 at 100 ng/ml for 24 h increased IGFBP-5 mRNA by 5- to
7-fold (Figs. 1 and 2).
|
|
|
). To determine whether the effect of IL-6 and
sIL-6R was protein synthesis dependent, Ob cells were treated with IL-6
at 100 ng/ml and sIL-6R at 50 ng/ml in the presence or absence of
cycloheximide at 3.6 µM. Doses of cycloheximide of 2
µM and higher were shown previously to inhibit protein
synthesis in Ob cell cultures by 80–85% (36). Cycloheximide caused a
marked decrease in IGFBP-5 mRNA levels, and the effects of IL-6 and
sIL-6R on IGFBP-5 transcripts were virtually undetectable in its
presence (Fig. 5).
|
|
). To test whether IL-6 and sIL-6R modified the
rate of transcription of the IGFBP-5 gene, nuclear run-on assays were
performed on nuclei from Ob cells exposed to control medium or to IL-6
at 100 ng/ml and sIL-6R at 50 ng/ml for 2 and 6 h (Fig. 7, Exp A) or 24 h (Fig. 7, Exp B). After 2–24 h,
IL-6 in the presence of sIL-6R increased IGFBP-5 transcription rates by
2.5- to 3-fold, demonstrating a transcriptional effect.
|
|
, left). Immunoreactive bands of 45–50 kDa were also
observed, but these are not detected by Western ligand blot analysis
using 125I-IGF-II as a probe indicating that they are not
IGFBPs (20). IL-6 at 100 ng/ml in the presence of sIL-6R at 50–125
ng/ml did not change the levels of the 31-kDa form of IGFBP-5 after 24
or 48 h in the extracellular matrix (Fig. 8, left)
(n = 8). The 31-kDa form of IGFBP-5, as well as an immunoreactive
protein migrating with a molecular mass of approximately 24 kDa, which
is the known molecular mass of one of the IGFBP-5 proteolytic
fragments, were detected in the culture medium (Fig. 8, right) (38, 39). IL-6 in the presence of its soluble
receptor decreased the 31-kDa form of IGFBP-5 in the medium and
increased the 24-kDa fragment after 24 and 48 h (n = 2). To
test for the presence of IGFBP-5 proteolytic activity, purified human
IGFBP-5 was incubated with conditioned medium from control and
IL-6/sIL-6R-treated cultures for 0–16 h. There was a decrease in the
31-kDa form of IGFBP-5 and the generation of immunoreactive proteins of
approximately 17, 20, and 24 kDa (Figs. 9 and 10) (n = 3). The appearance of the proteolytic
fragments and the decay of the IGFBP-5 standard were accelerated and
enhanced when recombinant IGFBP-5 was incubated in the presence of
conditioned medium from IL-6/sIL-6R-treated cultures (Figs. 9 and 10).
The generation of proteolytic fragments was virtually prevented by the
metalloprotease inhibitors 1,10 phenanthrolene and EDTA, and only
partially prevented by the serine protease inhibitor PMSF (Fig. 10)
(40).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we demonstrated that IL-6 alone caused a modest increase in IGFBP-5 mRNA levels in Ob cells, but when tested in the presence of its soluble receptor, the effect was amplified and observed at lower doses of IL-6. This is not surprising because sIL-6R binds IL-6 with similar affinity as the membrane bound receptor, and the sIL-6R/IL-6 complex mediates IL-6 signaling by binding and activating the signal transducing gp 130 (24). By these mechanisms, IL-6R may confer IL-6 responsiveness to cells expressing low levels of IL-6 transmembrane receptor (24). Furthermore, the effect of IL-6 on osteoclast formation also is observed in the presence of its soluble receptor (25). The effect of IL-6 and sIL-6R on IGFBP-5 mRNA was observed at concentrations detected in mouse and human serum, suggesting that it is physiologically relevant (45, 46). sIL-6R alone had a small effect, and its activity is probably due to the presence of significant levels of IL-6 in the bone cell microenvironment (47). Although IL-6 stimulates PGE2 synthesis in osteoblastic cells, and PGE2 stimulates the transcription of IGFBP-5 in osteoblasts, the stimulatory effect of IL-6 and sIL-6R on IGFBP-5 is not prostaglandin synthesis dependent, suggesting that IL-6 influences bone cell growth through various mechanisms (8, 48). IL-6 and its soluble receptor also increase IGF-I transcripts and polypeptide levels, and IGF-I increases IGFBP-5 transcription (12, 19). However, IGF-I probably does not mediate the acute effects observed with IL-6 and its soluble receptor on IGFBP-5 synthesis. This is because the effect on IGFBP-5 transcription was detected after 2 h, whereas 24 h of exposure to IL-6 and sIL-6R are needed to detect a stimulation of IGF-I mRNA in Ob cells (N. Franchimont and E. Canalis, unpublished observations). Furthermore, indomethacin decreases the effect of IL-6 and sIL-6R on IGF-I expression without modifying the expression of IGFBP-5. Nevertheless, the increase in IGF-I synthesis by IL-6 and sIL-6R may have an important secondary delayed effect not only on the stimulation of IGFBP-5 transcription in osteoblasts, but also on the stabilization of the protein (19, 49).
IL-6 induces the transcription of the IGFBP-5 gene in osteoblasts, and does not stabilize IGFBP-5 mRNA in transcriptionally arrested Ob cells. The gene sequences responsible for the effect have not been determined. In other cells, IL-6 activates the synthesis of the activating protein-1 family of transcription factors, induces nuclear factor-IL-6, a member of the CCAAT enhancer binding protein family, acute-phase response factor, and the octamer binding proteins (50, 51, 52, 53). Examination of the IGFBP-5 promoter region reveals potential binding sites for nuclear factor-IL-6 and activating protein-1 in the bp -2700 to +1 region of the gene (54). It is possible that IL-6 acts by inducing or activating transcription factors that bind to one or more of these sequences.
Whereas most of the IGFBPs appear to have inhibitory activities on bone formation, IGFBP-5 has been shown to increase bone cell growth and enhance the actions of IGF-I on this process (14, 18, 55, 56). Although IL-6 and sIL-6R increased IGFBP-5 mRNA, they did not cause a detectable increase in polypeptide levels, suggesting additional effects at the translational or posttranslational level. Our studies demonstrate that IL-6 and sIL-6R enhance the production of IGFBP-5 fragments due to increased proteolytic activity, and this is probably the cause for the lack of a substantial accumulation of intact IGFBP-5 in Ob cells. The osteoblastic MC3T3 cells and fibroblasts secrete serine proteases and matrix metalloproteinases that degrade IGFBP-5 (38, 39, 57, 58). Ob cells seem to secrete similar proteases, because the IGFBP-5 proteolytic activity was inhibited both by the serine protease inhibitor PMSF, and by the metalloproteinase inhibitors EDTA and 1,10 phenanthrolene. Recent studies from our laboratory demonstrated that IL-6 and sIL-6R have stimulatory activity on the synthesis of collagenase 3 or matrix metalloproteinases-13 and of the 72-kDa gelatinase in Ob cell cultures (59), but it is not known whether or not IL-6 also induces serine proteases in these cells. Therefore, the induction of collagenase or other proteases may be responsible for the proteolysis of IGFBP-5 by IL-6 and sIL-6R in Ob cells. It is not clear why IL-6 and sIL-6R induce IGFBP-5 synthesis as well as proteolytic activity for this binding protein. This may be a local mechanism to prevent excessive accumulation of intact IGFBP-5 and binding of IGF-I. The function of the IGFBP-5 fragments is not well established, and it is not known whether they have effects distinct from those of the intact protein. The significance of IGFBP-5 fragmentation is not clear, but it is possible that the fragments have specific actions on bone cell function.
In conclusion, IL-6 in the presence of sIL-6R enhances IGFBP-5 mRNA expression by transcriptional mechanisms in osteoblast cultures, and stimulates the production of IGFBP-5 proteolytic activity and the formation of IGFBP-5 fragments. The effects of IL-6 and sIL-6R on the IGF-IGFBP axis could constitute a pathway for IL-6 effects on bone formation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Recipient of a Fellowship Award from the Catherine and Weldon
Donaghue Foundation.
Received February 26, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. Giustina, G. Mazziotti, and E. Canalis Growth Hormone, Insulin-Like Growth Factors, and the Skeleton Endocr. Rev., August 1, 2008; 29(5): 535 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. Lang, B. J. Krawiec, D. Huber, J. M. McCoy, and R. A. Frost Sepsis and inflammatory insults downregulate IGFBP-5, but not IGFBP-4, in skeletal muscle via a TNF-dependent mechanism Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2006; 290(4): R963 - R972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamagami, S. Yokoo, T. Mimura, and S. Amano Effects of TGF-{beta}2 on Immune Response-Related Gene Expression Profiles in the Human Corneal Endothelium Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 515 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ueland, K. Brixen, L. Mosekilde, L. Mosekilde, A. Flyvbjerg, and J. Bollerslev Age-Related Changes in Cortical Bone Content of Insulin-Like Growth Factor Binding Protein (IGFBP)-3, IGFBP-5, Osteoprotegerin, and Calcium in Postmenopausal Osteoporosis: A Cross-Sectional Study J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1014 - 1018. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Devlin, Z. Du, V. Buccilli, V. Jorgetti, and E. Canalis Transgenic Mice Overexpressing Insulin-Like Growth Factor Binding Protein-5 Display Transiently Decreased Osteoblastic Function and Osteopenia Endocrinology, October 1, 2002; 143(10): 3955 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Tanno, A. Negroni, R. Vitali, M. C. Pirozzoli, V. Cesi, C. Mancini, B. Calabretta, and G. Raschella Expression of Insulin-like Growth Factor-binding Protein 5 in Neuroblastoma Cells Is Regulated at the Transcriptional Level by c-Myb and B-Myb via Direct and Indirect Mechanisms J. Biol. Chem., June 21, 2002; 277(26): 23172 - 23180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-C. C. Yeh and J. C. Lee Identification of an Osteogenic Protein-1 (Bone Morphogenetic Protein-7)-Responsive Element in the Promoter of the Rat Insulin-Like Growth Factor-Binding Protein-5 Gene Endocrinology, September 1, 2000; 141(9): 3278 - 3286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Varghese, S. Rydziel, and E. Canalis Basic Fibroblast Growth Factor Stimulates Collagenase-3 Promoter Activity in Osteoblasts through an Activator Protein-1-Binding Site Endocrinology, June 1, 2000; 141(6): 2185 - 2191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Boonyaratanakornkit, D. D. Strong, S. Mohan, D. J. Baylink, C. A. Beck, and T. A. Linkhart Progesterone Stimulation of Human Insulin-like Growth Factor-binding Protein-5 Gene Transcription in Human Osteoblasts Is Mediated by a CACCC Sequence in the Proximal Promoter J. Biol. Chem., September 10, 1999; 274(37): 26431 - 26438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Pereira, V. Jorgetti, and E. Canalis Triiodothyronine induces collagenase-3 and gelatinase B expression in murine osteoblasts Am J Physiol Endocrinol Metab, September 1, 1999; 277(3): E496 - E504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Varghese, K. Yu, and E. Canalis Leukemia inhibitory factor and oncostatin M stimulate collagenase-3 expression in osteoblasts Am J Physiol Endocrinol Metab, March 1, 1999; 276(3): E465 - E471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gazzerro, S. Rydziel, and E. Canalis Skeletal Bone Morphogenetic Proteins Suppress the Expression of Collagenase-3 by Rat Osteoblasts Endocrinology, February 1, 1999; 140(2): 562 - 567. [Abstract] [Full Text]
|
|
|
|
|
|
N. Franchimont, V. Gangji, D. Durant, and E. Canalis Interleukin-6 with Its Soluble Receptor Enhances the Expression of Insulin-Like Growth Factor-I in Osteoblasts Endocrinology, December 1, 1997; 138(12): 5248 - 5255. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
) or
without sIL-6R (•) relative to untreated controls was quantitated by
densitometry. Symbols represent means ± SEM for three
cultures. Inset, Representative Northern blot.
) was
subjected to Northern blot analysis and hybridized with a
32P-labeled rat IGFBP-5 cDNA. IGFBP-5 mRNA was visualized
by autoradiography, and changes in mRNA levels quantitated by
densitometry. Values are means ± SEM for four to six
cultures obtained from two experiments. Slopes from control and
experimental cultures were not different.
