Endocrinology Vol. 140, No. 11 5036-5044
Copyright © 1999 by The Endocrine Society
Response of Bipotential Human Marrow Stromal Cells to Insulin-Like Growth Factors: Effect on Binding Protein Production, Proliferation, and Commitment to Osteoblasts and Adipocytes1
Thierry Thomas,
Francesca Gori,
Thomas C. Spelsberg,
Sundeep Khosla,
B. Lawrence Riggs and
Cheryl A. Conover
Endocrine Research Unit, Division of Endocrinology, Metabolism, and
Nutrition, Department of Internal Medicine (T.T., F.G., S.K., B.L.R.,
C.A.C.), and the Department of Biochemistry and Molecular Biology
(T.C.S.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota
55905
Address all correspondence and requests for reprints to: Cheryl A. Conover, Ph.D., Mayo Clinic, 200 First Street SW, 5194 Joseph, Rochester, Minnesota 55905. E-mail: conover.cheryl{at}mayo.edu
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Abstract
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Insulin-like growth factors (IGFs) are important regulators of
the activity of mature osteoblasts, but their effects on
osteoprogenitor cells in human bone marrow stroma are unclear. In this
study, we assessed the effects of IGFs on a conditionally immortalized
human marrow stromal cell line, hMS(34), which has the ability to
differentiate to either mature osteoblasts or adipocytes. hMS(34)
cells expressed functional receptors for IGFs as well as specific
IGF-binding proteins (IGFBP-3, -4, -5, and -6). IGF treatment of
hMS(34) cells did not alter IGFBP expression, but resulted in
distinct posttranslational modifications of secreted IGFBP-3 and
IGFBP-4 proteins. IGF-I, IGF-II, and their receptor-activating analogs
significantly increased by 2-fold the proliferation rate of the
hMS(34) cells, but had a more complex effect on hMS(34) cell
differentiation. Treatment with IGFs did not affect gene expression of
Cbfa1 or peroxisome proliferator-activated receptor
2 (transcription factors involved in commitment to
osteoblast and adipocyte pathways, respectively), alkaline phosphatase,
type I collagen, and osteocalcin (markers of the osteoblast lineage),
or lipoprotein lipase and adipsin (markers of the adipocyte lineage)
and did not change alkaline phosphatase activity or type I collagen and
osteocalcin protein relative to total protein production. In contrast,
IGFs significantly increased type I collagen expression in
differentiated hMS(34) cells as well as mature osteoblasts and
promoted lipid accumulation in differentiated adipocytes. In summary,
hMS(34) cells express essential components of the IGF system and
respond to IGF treatment with increased proliferation. There was no
evidence for IGFs directly modulating the commitment of hMS(34) cells
to either osteoblast or adipocyte pathways, and their effects on
differentiation within these lineages were dependent on the stage of
cell maturation.
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Introduction
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THE INSULIN-LIKE growth factors (IGFs),
IGF-I and IGF-II, play key roles in bone formation (reviewed in Ref.
1). The IGFs are secreted by mature osteoblasts (OB) and can be stored
locally in the bone matrix until they are released through resorption
(2). Indeed, IGF-II is the most abundant of all growth factors stored
in human bone matrix (3). The IGFs have been shown to stimulate OB
proliferation and differentiated function in vitro and
in vivo by acting through specific membrane receptors
(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).
Compared with the wealth of data on mature OB, much less is known about
the effect of IGFs on marrow stromal cells, the multipotential
precursor cells that can differentiate into OB or into other
mesenchymal types, including adipocytes (15, 16, 17, 18). This is important to
assess because a decrease in the number and the differentiation
potential of bone marrow precursor cells (19) and/or alteration of the
shunt of these precursor cells between OB and adipocyte lineages (20)
have been related to postmenopausal osteoporosis and aging-induced bone
loss. In a rodent model of stromal cells, IGF-I was effective in
stimulating DNA synthesis, whereas its effects on differentiation
were limited to stimulating type I collagen (Col I) expression (21). On
the other hand, a recent study in primary cultures of human marrow
stromal cells concluded that both IGF-I and IGF-II exerted
proliferative effects, but inhibited collagen production (22). These
discrepancies underline the limitations that species differences,
heterogeneity of primary cultures, and serum-induced differentiation
before the experiments introduce in the interpretation of IGF effects.
Furthermore, neither study took into account IGF-binding proteins
(IGFBPs) that have the potential to modulate IGF action, as previously
shown in OB cells (1, 23), nor did they assess the role the IGFs may
play in the shunting between OB and adipocyte differentiation.
We have recently developed and characterized human marrow stromal (hMS)
cell lines (24, 25). These cell lines are conditionally immortalized
with a mutant temperature-sensitive simian virus 40 large T antigen
(SV40LTA) (26, 27, 28). When cultured at the restrictive temperature for
SV40LTA, these cell lines regain a normal phenotype and provide a
clonal human cell model that can be induced to differentiate either
into osteoblasts that produce matrix or into adipocytes that accumulate
neutral lipids droplets in their cytoplasm (24, 29). In this study, we
identify these bipotential hMS cells as important targets of IGF action
and define specific regulation of IGFBP production, cell proliferation,
commitment, and differentiation in response to exogenous IGFs.
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Materials and Methods
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Reagents
Recombinant human IGF-I and IGF-II were purchased from
Amgen, Inc. (Thousand Oaks, CA) and R & D Systems
(Minneapolis, MN), respectively. The IGF analogs des(1, 2, 3)IGF-I,
des(1, 2, 3, 4, 5, 6)IGF-II, and Long-R3 (LR3) IGF-I were purchased from
GroPep Pty. Ltd. (North Adelaide, Australia). Tissue
culture media and reagents were purchased from either Sigma Chemical Co. (St. Louis, MO) or Life Technologies, Inc. (Grand Island, NY). Tissue culture plasticware was
purchased from Corning, Inc. (Corning, Inc.
NY). Molecular biology reagents and enzymes were purchased from
Roche Molecular Biochemicals (Indianapolis, IN). The RNA
Stat-60 kit was purchased from Tel-Test, Inc.
(Friendswood, TX). The Wizard PCR Preps DNA purification system was
obtained from Promega Corp. (Madison, WI).
1
,25-Dihydroxyvitamin D3, [3H]thymidine,
and [
-32P]deoxy (d)-CTP were obtained from NEN Life Science Products (Boston, MA). L-Ascorbic acid
phosphate was purchased from WAKO Chemicals USA, Inc. (Richmond, VA).
Kits for the measurement of osteocalcin (OC) and procollagen protein
were generous gifts from Metra Biosystem (Mountain View, CA). IGFBP
antibodies were provided by Diagnostics Systems Laboratories, Inc. (Webster, TX; IGFBP-3 antiserum) and Chiron Corp. (Emeryville, CA; IGFBP-4, IGFBP-5, and IGFBP-6 antisera).
IGFBP-2 antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The complementary DNA (cDNA) clones used
and their providers were: human IGFBP-1 and IGFBP-3, Dr. D. R.
Powell (Baylor College of Medicine, Houston, TX); and human IGFBP-2,
-4, -5, and -6, Dr. S. Shimasaki (Whittier Institute, La Jolla,
CA).
Cell culture
The cell line hMS(3, 4) was established in our laboratory from
human marrow stromal cells that were isolated by negative
immunoselection (25) and conditional immortalization with a mutant
temperature-sensitive SV40LTA (26, 27, 28, 29). At 34 C, the permissive
temperature for SV40LTA (24, 26, 27, 28), the large T antigen is active,
and the cells rapidly proliferate. At 39.5 C, the restrictive
temperature for SV40LTA, the large T antigen is inactive, the cells
regain a normal phenotype, proliferation is inhibited, and hMS(3, 4)
cells differentiate into either osteoblasts or adipocytes depending on
the appropriate conditions (24, 29).
The hMS(3, 4) cells were routinely cultured at 34 C in
MEM
containing 10% (vol/vol) heat-inactivated FBS (HI-FBS), 0.2 µg/ml
geneticin, and 1% stock penicillin (10,000 U/ml)-streptomycin (10,000
µg/ml), as described previously (24). Medium was changed twice a
week. For experiments, cells were plated at a density of 2 x
104 cells/cm2. After 48 h at 34 C, cells
were washed twice in PBS and incubated at 39.5 C for a further 24
h in serum-free medium plus 0.1% (wt/vol) BSA to inactivate the
SV40LTA.
Normal human osteoblast-like (hOB) cells were cultured from trabecular
bone explants obtained at the time of orthopedic procedures from
patients who had no evidence of metabolic bone disease, as previously
reported (30, 31).
Affinity cross-linking
[125I]IGF-I monolayer binding and chemical
cross-linking were performed as previously described (4). Briefly,
hMS(3, 4) cells were washed and incubated with
[125I]IGF-I (1 x 106 cpm/well) in the
presence or absence of unlabeled IGF-I (125 ng/ml) or insulin (100
µg/ml) for 2.5 h at 15 C. Cells were then washed and incubated
with 10 mM disuccinimydil suberate for 15 min at 15 C. The
reaction was stopped by the addition of Tris-EDTA quench buffer. Cells
were then solubilized, and cross-linked complexes were analyzed by 10%
SDS-PAGE under reducing conditions.
Northern blot analysis
Total cellular RNA was extracted using the RNA STAT-60 kit,
following the manufacturers instructions. Total RNA (20 µg) was
separated on a 1.5% agarose gel containing 2.2 mol/liter formaldehyde
and transferred to nylon membranes (Nytran, Schleicher & Schuell, Inc., Keene, NH). Relative loading and integrity of the RNA were
assessed by UV shadowing. The membranes were UV autocross-linked,
prehybridized at 43 C for 6 h, and hybridized at 43 C overnight
with 106 cpm/ml 32P-labeled cDNA probe (31, 32). Human IGFBP cDNA probes were labeled with [32P]dCTP
using a random primed DNA labeling kit (New England Nuclear Corp.,
Boston, MA). Filters were washed twice in 6 x SSPE (0.15
M NaCl, 0.01 M NaH2PO4,
1 mM EDTA, pH 7.4)-0.1% SDS at room temperature,
twice in 2 x SSPE-0.1% SDS at 42 C, and once in 0.1 x
SSPE-0.1% SDS at 60 C. Membranes were stripped according to the
manufacturers instructions for sequential probing for the six
IGFBPs.
Cell-conditioned medium
hMS(3, 4) cells were washed twice and incubated at 39.5 C in
MEM containing 0.1% (vol/vol) HI-FBS and 0.1% (wt/vol) of BSA with
the indicated experimental additions for 48 h. The conditioned
media were collected, centrifuged to eliminate cell debris, aliquoted,
and stored at -80 C.
Western ligand blot analysis
Western ligand blotting was performed as described previously
(31, 32, 33, 34). Aliquots of hMS(3, 4)-conditioned media (50 µl) were
analyzed by SDS-PAGE using a 7.515% gradient under nonreducing
conditions. The separated proteins were electroblotted onto
nitrocellulose filters, and the IGFBPs were identified by incubation
with [125I]IGF-I and -II at 4 C overnight and
visualization by autoradiography.
Immunoprecipitation analysis
hMS(3, 4) cell-conditioned media (1 ml) were incubated with
primary antibody (1:100 final dilution) and Protein G Plus/Protein
A-agarose (Oncogene Science, Inc., Uniondale, NY)
overnight at 4 C. Immunoprecipitated proteins were washed and analyzed
by Western ligand blotting.
Cell-free IGFBP-4 protease assay
IGFBP-4 protease activity was measured as previously described
(31, 33, 34). Aliquots of hMS(3, 4)-cell-conditioned media (50 µl)
were incubated at 37 C with [125I]IGFBP-4 without or with
5 nM IGF-II in a microfuge tube. Reaction products were
analyzed by SDS-PAGE and autoradiography.
Cell proliferation
[3H]Thymidine incorporation was assessed as
described previously (24, 31, 35), with minor modifications.
Preliminary experiments in hMS(3, 4) cells indicated that maximum
[3H]thymidine incorporation occurred 4872 h after a
mitogenic stimulus (data not shown). Subconfluent cells were incubated
in 0.1% HI-FBS plus experimental additions for 72 h at 39.5 C
(the restrictive temperature for the SV40LTA), and 1 µCi
[3H]thymidine was added for the last 24 h.
To assess changes in cell numbers, cells were harvested by
trypsinization and counted using an electronic cell counter (Coulter
Electronics Ltd., Hialeah, FL).
Apoptosis
hMS(3, 4) cells were treated as described for the cell
proliferation studies. At the end of the 72-h incubation with and
without IGFs, cells were stained with Hoechst 33342 (36). Total and
apoptotic cells were identified and captured by digital camera and
quantitated using UTHSCSA Image Tool (http://ddsdx.uthscsa.edu). Three
fields for each of quadruplicate measurements for each condition were
analyzed in two separate experiments.
Cell differentiation
Differentiation of hMS cells was studied in a medium containing
10% HI-FBS, 10-8 M dexamethasone,
10-8 M 1,25-dihydroxyvitamin D3,
10 mM ß-glycerol phosphate, and 100 µM
L-ascorbate phosphate. We previously demonstrated that the
hMS cell lines have n equal opportunity to differentiate along either
the OB or adipocyte pathway in this differentiation medium (29). Cells
were refed every 3 days with differentiation medium and fresh IGFs
throughout the treatment interval unless otherwise specified.
Semiquantitative RT-PCR
Total cellular RNA was extracted using the RNA STAT-60 kit
following the manufacturers instructions. cDNA were synthesized from
2 µg total RNA in a 20-µl reaction mix containing 4 µl 5 x
incubation buffer for AMV reverse transcriptase; 50 pmol
poly(deoxythymidine)15 primer; 20 nmol each of dATP, dCTP,
dGTP, and dTTP; 20 U ribonuclease inhibitor; and 20 U AMV reverse
transcriptase. The reaction time was 1 h at 42 C.
Aliquots of 1 µl cDNA were amplified in a 25-µl PCR mixture that
contained 2.5 µl 10 x Expand high fidelity PCR buffer
(Roche Molecular Biochemicals, Indianapolis, IN) with
MgCl2 15 mM; 5 pmol 5'- and 3'-oligoprimers;
2.5 nmol each of dATP, dCTP, dGTP, and dTTP; 0.25 µl
[
-32P]dCTP (10 µCi/µl); and 0.35 U Expand high
fidelity Taq DNA polymerase. Each cDNA sample was amplified
in duplicate PCRs for each gene. Amplifications were performed in a
GeneAmp 9600 thermal cycler (Perkin-Elmer Corp., Norwalk,
CT), for the following cDNAs: Cbfa1, bone/liver/kidney alkaline
phosphatase (AP), OC, Col I, peroxisome proliferator-activated receptor
2 (PPAR
2), plain lipoprotein lipase
(LPL), and adipsin. The housekeeping gene glyceraldehyde phosphate
dehydrogenase (GAPDH) was amplified as a control for RNA loading of RT
and variation in cDNA synthesis efficiency. Primer sequences and
amplification profiles used for all of these genes were reported
previously (25, 27, 29). Amplification of LPL and Col I was performed
for 35 and 30 cycles, respectively, with the following temperature
profile: denaturation at 94 C (30 sec), annealing at 55 C (60 sec), and
extension at 72 C (120 sec). Amplifications of AP and OC were performed
for 30 cycles, and those of PPAR
2 and adipsin were
performed for 35 cycles, with denaturation at 94 C (30 sec), annealing
at 55 C (30 sec), and extension at 72 C (30 sec). All PCR ended in a
7-min incubation at 72 C.
PCR products were analyzed as described previously (25). Briefly,
9-µl samples were electrophoresed on a 1.5% (wt/vol) agarose gel
containing 0.01% (wt/vol) ethidium bromide. Visualized PCR product
bands were sliced from gel, and radioactivity within gel slices was
quantitated using an LS600 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Quantification of PCR product was
normalized to GAPDH PCR product. The different gene products were
purified using a Wizard PCR Preps DNA kit (Promega Corp.).
For sequence analysis, approximately 150 ng of each purified cDNA
fragment were added to 3.2 pmol of either 5'- or 3'-primer and analyzed
in both directions in an automated DNA sequence analyzer.
Bone-related proteins
The AP enzyme activity was quantitated in cell lysates by
spectrophotometric measurement of p-nitrophenol 1-h release
at 37 C (37). Total cellular protein values were measured from cell
lysate by the Bradford method (Bio-Rad Laboratories, Inc.,
Hercules, CA). Col I (Prolagen-C, Metra Biosystem) and OC (Novocalcin,
Metra Biosystem, Mountain View, CA) proteins were measured in
hMS(3, 4)-conditioned medium samples using an enzyme-linked
immunosorbent assay.
Cytoplasmic lipid droplet formation
Cytoplasmic inclusions of neutral lipids were assessed by Oil
Red O staining, as described previously (24, 29). Cells were observed
using a Nikon Diaphot inverted microscope and a
Nikon 35-mm camera (Nikon, Melville, NY). The
percentage of Oil Red O-positive cells was determined by counting cells
in 30 contiguous fields/well after a random start. Results were
normalized to cell number per well.
Statistical analysis
All values are expressed as the mean ± SEM.
ANOVA was used for dose- and time-dependent differences and when
comparing multiple groups with a single control.
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Results
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Expression of IGF receptors in hMS cells
As shown in Fig. 1
, cross-linking of
[125I]IGF-I to hMS(3, 4) cells and SDS-PAGE of the cell
lysate under reducing conditions identified one major band of
approximately 130 kDa. Labeling of this protein band was displaceable
by unlabeled IGF-I or insulin at high concentrations, consistent with
the
-subunit of the type I IGF receptor (4). In addition, we
observed a faint 40- to 50-kDa band displaceable by unlabeled IGF-I but
not by insulin, indicating very low levels of cell-associated IGFBPs
(4). Cross-linking of [125I]IGF-II to hMS(3, 4) cells
also demonstrated low expression of the type II IGF receptor (data not
shown). However, it is generally accepted that both IGF-I and -II exert
their anabolic effects through the type I receptor (1, 38, 39). These
data indicate that hMS(3, 4) cells, with documented IGF receptors, are
potential targets for IGF stimulation.

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Figure 1. Affinity cross-linking of
[125I]IGF-I to hMS(3 4 ) cells in the absence (Total) or
presence of unlabeled IGF-I or insulin (Ins). Cross-linked complexes
were analyzed by 10% SDS-PAGE under reducing conditions. The migration
of molecular size markers (in kilodaltons) is indicated on the
left. The arrows indicate a 130-kDa band
displaceable by unlabeled IGF-I and insulin ( -subunit of the type I
IGF receptor) and 40- to 50-kDa bands displaceable by unlabeled IGF-I,
but not by insulin (cell-associated IGFBPs).
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Expression and regulation of IGFBPs in hMS cells
Cell-associated and soluble IGFBPs are capable of modulating
IGF/receptor interaction as well as having IGF-independent function
(40, 41). Furthermore, in a number of cell systems IGFs can regulate
IGFBP bioavailability through both receptor-mediated and
nonreceptor-mediated processes (32, 42). Northern blot analyses (Fig. 2
) showed that hMS(3, 4) cells
constitutively express IGFBP-3, -4, -5, and -6. No expression of
IGFBP-1 or IGFBP-2 was evidenced under these conditions (data not
shown). In addition, treatment of hMS(3, 4) cells for 48 h with
IGF-I or an IGF-I analog (LR3-IGF-I) that has similar affinity for the
type I IGF receptor (43) had no apparent effect on IGFBP expression.
This experiment was repeated with similar results.

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Figure 2. Northern blot analysis of steady state mRNA levels
for IGFBPs from hMS(3 4 ) cells treated for 48 h with vehicle
(lane a), 10 nM IGF-I (lane b), or 10 nM
LR3-IGF-I (lane c). A, Total RNA was electrophoresed, transferred to a
nylon membrane, and hybridized with 32P-labeled cDNA probes
for the indicated IGFBPs. B, Assessment of 18S and 28S RNA by UV.
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Western ligand blot analysis of the conditioned medium indicated that
hMS(3, 4) secrete 40-, 24-, and 30-kDa IGFBPs consistent with the sizes
of IGFBP-3, -4, and -6, respectively (Fig. 3
). The identities of these hMS IGFBPs
were confirmed by immunoprecipitation with specific IGFBP antibodies
(data not shown). No IGFBP-5 protein was detected, and a 34-kDa IGFBP
appeared to be contributed by 0.1% HI-FBS in the medium. In contrast
to negligible effects on messenger RNA (mRNA) levels, IGF treatment had
marked effects on IGFBP protein levels in the medium. Treatment of
hMS(3, 4) cells with IGF-I or IGF-II resulted in elevated IGFBP-3 and
loss of detectable IGFBP-4 in the conditioned medium. On the other
hand, high concentrations of insulin, which bind type I IGF receptors
but do not bind IGFBPs, had no effect on IGFBP levels. Treatment of
cells with LR3-IGF-I likewise had no effect on IGFBP-3 levels, but
resulted in a loss of IGFBP-4. As treatments had no effect on IGFBP
mRNA expression that could account for these changes in IGFBP protein
levels (see Fig. 2
), the data suggest specific posttranslational
regulation of IGFBP-3 and -4 by IGFs in these cells, similar to what
has been reported for human OB cells and normal human fibroblasts (31, 33, 42). Indeed, the loss of IGFBP-4 induced by IGF-I and IGF-II was
demonstrated by cell-free assay to be due to IGF-dependent proteolysis
of 24-kDa IGFBP-4 into 18- and 14-kDa fragments (Fig. 4
). Although treatment of hMS(3, 4) cells
with LR3-IGF-I resulted in a decrease in medium IGFBP-4, LR3-IGF-I did
not directly induce IGFBP-4 proteolysis in a cell-free assay.
Conditioned medium from hMS(3, 4) cells also exhibited constitutively
active IGFBP-5 proteolysis (data not shown), which probably accounts
for the failure to detect intact IGFBP-5 protein by Western ligand
blot. Thus, hMS(3, 4) cells express specific IGFBPs, which, in turn,
can be modulated by IGFs.

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Figure 3. Western ligand blot of IGFBPs in unconditioned
medium (0.1% HI-FBS; lane a) and in conditioned medium from hMS(3 4 )
cells after 48-h treatment with vehicle (lane b), 10 nM
IGF-I (lane c), 10 nM IGF-II (lane d), 1 µM
insulin (lane e), or 10 nM LR3-IGF-I (lane f). The
migration of molecular size markers (in kilodaltons) is indicated on
the left.
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Figure 4. Cell-free IGFBP-4 protease assay.
hMS(3 4 )-conditioned medium and [125I]IGFBP-4 were
incubated under cell-free conditions without (lane b) and with 10
nM IGF-I (lane c), 10 nM IGF-II (lane d), 1
µM insulin (lane e), or 10 nM LR3-IGF-I (lane
f) for 6 h at 37 C. Lane a is an unincubated sample,
i.e. intact [125I]IGFBP-4. Reactions
products were analyzed by SDS-PAGE and autoradiography.
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Effect of IGFs on hMS cell proliferation
Acknowledging the potential confounding effects of IGFBPs
produced by hMS cells and present in FBS, the effects of IGF-I, IGF-II,
and IGF analogs were compared in biological response experiments. These
analogs [des(1, 2, 3)IGF-I, des(1, 2, 3, 4, 5, 6)IGF-II, and LR3-IGF-I] have normal
affinity for IGF receptors, but markedly decreased affinity for IGFBPs
(43, 44, 45). Both [3H]thymidine incorporation and cell
number were significantly increased (1.5- to 2-fold) in hMS(3, 4) cells
treated with 10 nM IGF-I, des(1, 2, 3)IGF-I, IGF-II,
des(1, 2, 3, 4, 5, 6)IGF-II, and LR3-IGF-I (Fig. 5
).
At this concentration, the mitogenic activities of the IGFs and their
analogs were not significantly different. Similarly, dose-response
curves did not reveal statistically significant differences between
IGFs and their analogs (data not shown). Although IGFs have been shown
to have antiapoptotic effects in a number of cell systems (46),
treatment of hMS(3, 4) cells with 10 nM IGF-I or -II had no
suppressive effect on apoptosis in these cells (data not shown). Thus,
hMS(3, 4) cells respond to mitogenic stimulation by IGFs.

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Figure 5. Effects of IGFs on proliferation of hMS(3 4 )
cells. Subconfluent cells were treated at 39.5 C for 72 h with 10
nM of the indicated IGFs. A, [3H]Thymidine
incorporation; B, cell number. Results are the mean ±
SEM of quadruplicate determinations, expressed as a
percentage of the control value. *, P < 0.05; **,
P < 0.01; ***, P < 0.001
(compared with the corresponding control values).
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Effect of IGFs on hMS cell differentiation
Expression of phenotype marker genes. A representative RT-PCR
experiment is shown in Fig. 6
. Gene
expression of Cbfa1, a transcription factor involved in commitment to
the osteoblast lineage (29, 47, 48, 49), was not affected by treatment with
10 nM des(1, 2, 3)IGF-I or des(1, 2, 3, 4, 5, 6)IGF-II. This was true when
measured over a period of 30 min to 6 days (data not shown). Expression
of osteoblast marker genes, Col I, AP, and OC, was not altered by
treatment with 10 nM des(1, 2, 3)IGF or des(1, 2, 3, 4, 5, 6)IGF-II. Gene
expression of PPAR
2, the transcription factor involved
in commitment to the adipocyte lineage (29, 50), also was not affected
by treatment with 10 nM IGF. Furthermore, expression of
adipocyte markers LPL and adipsin up to 6 days was not altered. Similar
results were obtained with 10 nM IGF-I or IGF-II and
increasing doses of LR3-IGF-I (data not shown). These experiments were
repeated, and the results were confirmed under serum-free conditions
(data not shown).

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Figure 6. Effect of IGFs on phenotype marker gene expression
in hMS cells. hMS(3 4 ) cells were treated at 39.5 C in differentiation
medium without (Control) or with 10 nM des(1 2 3 )-IGF-I and
des(1 2 3 4 5 6 )-IGF-II for 4 days. For each condition, total RNA was
collected from three individual wells, and cDNA was run in duplicate
PCR. Aliquots of cDNA synthesized from 2 µg total RNA were amplified
in a 25-µl PCR reaction mixture with 0.25 µl
[ -32P]dCTP (10 µCi/µl) and corrected for GAPDH
expression. Base pairs are indicated on the right.
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Interestingly, when hMS(3, 4) cells were first induced to differentiate
for 15 days and then treated with 10 nM IGFs, a significant
increase in type I procollagen mRNA expression was observed; a similar
response was obtained in mature hOB cells (Fig. 7
). Furthermore, there was a differential
response to IGF-I and LR3-IGF-I in hOB cells, indicating an influence
of IGFBPs.
Production of phenotype marker proteins. As shown in
Fig. 8
, treatment with 10 nM
des(1, 2, 3)IGF-I or des(1, 2, 3, 4, 5, 6)IGF-II for 39 days did not affect the AP
activity of hMS(3, 4) cells. Similar data were obtained with IGF-I and
-II (data not shown). Type I procollagen and OC production normalized
to total protein varied during culture, but was not affected by 10
nM des(1, 2, 3)IGF-I or des(1, 2, 3, 4, 5, 6)IGF-II for up to 15 days of
treatment (Fig. 9
). Equivalent results
were obtained when cells were treated with IGF-I, IGF-II, or LR3-IGF-I
(data not shown).

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Figure 8. Effects of IGFs on AP activity. hMS(3 4 ) cells
were treated at 39.5 C in differentiation medium for 3, 6, and 9 days
with 10 nM des(1 2 3 )-IGF-I (circles) and
des(1 2 3 4 5 6 )-IGF-II (squares). The AP activity was
quantified as nanomoles of p-nitrophenylphosphate
released per h/µg total cellular proteins. Results are the mean
± SEM expressed as a percentage of the control value. The
data are representative of three separate experiments performed in
quadruplicate.
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Figure 9. Effect of IGFs on Col I (A) and OC (B) secretion
in hMS(3 4 ) cells. The cells were treated at 39.5 C for 315 days in
differentiation medium with 10 nM des(1 2 3 )-IGF-I
(solid circles), des(1 2 3 4 5 6 )-IGF-II (solid
squares), or vehicle (open circles). Col I and
OC values were normalized to micrograms of total cellular proteins.
Results are expressed as the mean ± SEM. The data
shown are representative of three separate experiments performed in
quadruplicate.
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After 15 days in culture, the number of hMS(3, 4) cells per field
containing cytoplasmic droplets stained with Oil Red O normalized to
the total number of cells per well was significantly increased with
des(1, 2, 3)IGF-I or des(1, 2, 3, 4, 5, 6)IGF-II treatment (Fig. 10
). Similar results were obtained when
cells were treated with 10 nM IGF-I (data not shown).

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|
Figure 10. Effects of IGFs on cytoplasmic lipid droplet
formation number in hMS(3 4 ) cells. The cells were treated at 39.5 C
for 15 days in differentiation medium without (Control) or with 10
nM des(1 2 3 )-IGF-I or des(1 2 3 4 5 6 )-IGF-II. To quantify the
formation of cytoplasmic inclusions of neutral lipids, Oil Red O
histochemical staining was performed. The percentage of Oil Red
O-positive cells was determined by counting cells in 30 contiguous
fields/well. Results are expressed as the mean ± SEM.
The data shown are representative of three separate experiments
performed in quadruplicate. *, P < 0.05 compared
with the corresponding control values.
|
|
 |
Discussion
|
|---|
In the present study, we confirm that human marrow stromal cells
are potential targets for IGFs by demonstrating expression of specific
receptors for the IGFs and response to exogenous IGF with increased
cell proliferation and, subsequent to commitment, enhanced
differentiated function of osteoblasts and adipocytes. There was no
evidence of a direct effect of IGFs on early genes regulating
commitment per se. We also describe, for the first time,
IGFBP expression in human marrow stromal cells and regulation by
IGFs.
The IGFs are potent mitogenic factors for a variety of cell types (1, 51). We demonstrate that they are mitogenic for human marrow stromal
cells as well, with the potential for providing an increased number of
progenitors available for new units of bone formation. These findings
are consistent with the study by Canalis (6) showing that in organ
culture of fetal rat calvaria, the increase in thymidine uptake was
observed both in the periosteum, which is rich in osteoprogenitors, and
in the central area, which is rich in mature osteoblasts.
Interestingly, treatment of hMS(3, 4) cells with IGFs or their analogs
induced equivalent increases in the proliferation rate, suggesting that
under our in vitro conditions, endogenous IGFBPs did not
modulate the effects of IGFs on cell proliferation. This is probably
due to experimental conditions, i.e. stimulating the cells
in fresh serum-free medium conditions, allowing the IGFs to bind to
their receptors before any release of IGFBPs would occur. Furthermore,
our cells have very low levels of cell-associated IGFBPs that could
potentially modulate IGF bioactivity. IGF-I has also been shown to
stimulate [3H]thymidine incorporation in murine TC-1
stromal cells and primary cultures of human stromal cells grown under
conditions that promote osteogenic differentiation (21, 22).
The effects induced by the IGFs on differentiation were more complex.
Differentiation of marrow stromal cells into mature cells is under the
control of transcriptional factors that regulate the commitment and
early differentiation of these progenitors to the different lineages.
Cbfa1 is a gene expressed early during differentiation, and its product
serves as a transcriptional activator of the commitment to the
osteoblastic lineage (47, 48, 49). Similarly, PPAR
2 is an
early response gene that is involved in commitment to the adipocyte
pathway (50). As assessed by semiquantitative RT-PCR, IGFs induced no
change in the gene expression of Cbfa1 or PPAR
2,
suggesting that the IGFs do not participate in the commitment to the
osteoblast or to the adipocyte pathway. Investigating the effects of
the IGFs on early osteoblast differentiation, we observed no changes in
AP, Col I, and OC gene expression. We could rule out a negative effect
being due to IGFBPs because similar results were obtained using
LR3-IGF-I, des(1, 2, 3)-IGF-I, or des(1, 2, 3, 4, 5, 6)-IGF-II, analogs of the IGFs
possessing a similar affinity to the IGF receptor but a very low
affinity to the IGFBPs largely present in FBS (43, 44, 45). At the protein
level, IGFs had no effect on AP activity, but increased Col I and OC
levels in the conditioned medium. However, this increase in Col I and
OC disappeared after normalization to total protein level, suggesting
that the IGFs exert a posttranscriptional effect on overall protein
synthesis (7) without specifically triggering the differentiation of
human marrow stromal cells along the osteoblast pathway.
Recently, Langdhal and colleagues reported inhibitory effects of IGF-I
and IGF-II on Col I protein levels in primary cultures of human marrow
cells, whereas IGF-I decreased and IGF-II increased AP activity (22).
Methodological differences probably explain the differing results.
These researchers used primary cultures of human marrow stromal cell
aspirates with a heterogeneous cell population within each bone marrow
sample and, consequently, variability between samples. Also, the
results were not normalized to total protein. Using primary rodent bone
marrow stromal cells, Tanaka and Liang observed an increase in Col I
gene and protein levels and no difference in AP activity with IGF-I
treatment (21). However, IGF-I was added after 10 days in culture in
the presence of 10% FBS, suggesting that part of the population may
have already differentiated. Furthermore, these researchers reported in
the same model either no effect (21) or an increase in osteopontin mRNA
levels in response to IGF-I treatment (52), suggesting variability
and/or heterogeneity of their cell population and/or confounding
effects of IGFBPs in the FBS.
Unlike hMS(3, 4) cells, mature hOB cells significantly increased Col I
expression in response to IGF treatment, with IGF analogs being more
effective than the native IGFs. A greater abundance of cell-associated
IGFBP in hOB cells compared with hMS cells may account for this
differential response. In addition, treating hMS(3, 4) cells for 4 days
with IGF analogs after 15 days in culture conditions promoting their
differentiation provided a similar increase in Col I gene and protein
expression. Overall, these data suggest that in mature osteoblasts, the
IGFs induce an increase in collagen synthesis through both
transcriptional and translational mechanisms. These data are in
agreement with the initial observation by Canalis (6) and support the
hypothesis that the effects of IGFs on differentiation are dependent on
the maturation and origin of the cells (53).
Treatment of hMS(3, 4) cells with IGFs did not modulate gene expression
of LPL or adipsin, early differentiation markers of the adipocyte
lineage. These results are consistent with previous studies showing a
lack of IGF effect on differentiation of a preadipocyte 3T3-F442A cell
line without a prestimulation with GH triggering the early stages of
differentiation (54). In the adipocyte lineage, it is known that IGFs
promote adipocyte differentiation predominantly by acting on adipocytes
at a late stage of differentiation (54, 55, 56). Our observation that in
long term experiments with culture conditions triggering the early
stages of differentiation the IGFs were able to increase lipid droplet
accumulation provides further evidence that the stage of cell
maturation plays a critical role in the response to IGFs during
adipocyte differentiation. We previously reported that addition of
insulin with dexamethasone to hMS cell cultures did not increase the
number of Oil Red O-positive cells above that with dexamethasone alone
(24). These observations support the hypothesis that IGFs could
increase lipid accumulation by acting through their specific
receptors.
IGFBPs are important determinants of IGF action at the cellular level
(40). hMS(3, 4) cells produce a pattern of IGFBPs similar to what
our group previously described in mature hOB cells and SV40-transformed
hOB cells (23, 31, 57). Thus, hMS(3, 4) cells in culture constitutively
expressed and secreted IGFBP-3, -4, -5, and -6. Treatment with IGF-I or
LR3-IGF-I had no effect on steady state mRNA levels for these IGFBPs.
However, treatment of hMS(3, 4) cells with IGF-I or IGF-II increased
IGFBP-3 medium levels and induced the loss of detectable IGFBP-4.
LR3-IGF-I or insulin treatment could not duplicate the increase in
IGFBP-3, suggesting receptor-independent regulation as previously
reported in human fibroblasts (32, 42) and other cells (58, 59).
Whereas IGF-I and -II did not alter IGFBP-4 mRNA steady state levels,
they induced a dramatic decrease in IGFBP-4 protein levels related to
activation of a specific IGFBP-4 protease, similar to what we and
others previously described in hOB cells (31, 57, 60). Interestingly,
treatment of hMS cells with LR3-IGF-I, an IGF analog that does not bind
IGFBP-4 and does not induce proteolysis in a cell-free assay, was as
effective as IGF-I and -II in producing proteolysis of secreted
IGFBP-4. These data are in contrast to those from other cell systems,
and they suggest that LR3-IGF-I may be acting indirectly to induce the
primary activator of IGFBP-4 proteolysis. We are in the process of
determining whether LR3-IGF-I treatment up-regulates IGF expression in
these cells. Our results for IGFBP expression in hMS(3, 4) cells differ
from those reported for the murine marrow stromal cell line, TC-1
(61, 62, 63). In this mouse cell model, IGF-I induced an increase in
IGFBP-4 and IGFBP-5 at a posttranscriptional level and an increase in
IGFBP-3 at both transcriptional and posttranscriptional levels,
underlining the known species differences in the IGF system. The
significance of IGFBPs in the marrow stroma is unknown, but they have
been proven to be critical determinants of IGF action in other systems
as well as possessing intrinsic growth regulatory potential.
In summary, we demonstrated that human marrow stromal cells express
important components of the IGF system and are potential targets for
IGF regulation. Whereas the IGFs exert mitogenic effects on human
marrow stromal cells, their effects on differentiation are dependent on
the stage of maturation of the cells. The IGFs do not directly modulate
the genes determining either commitment to the osteoblast or the
adipocyte lineages or early differentiation, but they enhance the
differentiated function of mature osteoblasts and adipocytes. These
data further support an important role for IGFs in bone formation and
suggest that IGF therapy might be especially useful in bone diseases
related to an insufficiency of osteoprogenitor cells.
 |
Acknowledgments
|
|---|
We thank Ms. Laurie Bale and Ms. Marcy Schroeder for their
excellent technical assistance.
 |
Footnotes
|
|---|
1 This work was supported by NIH Research Grant AG-0487505. 
Received March 11, 1999.
 |
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