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Endocrinology Vol. 140, No. 11 5036-5044
Copyright © 1999 by The Endocrine Society

ARTICLES

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, 5–194 Joseph, Rochester, Minnesota 55905. E-mail: conover.cheryl{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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(3–4), which has the ability to differentiate to either mature osteoblasts or adipocytes. hMS(3–4) cells expressed functional receptors for IGFs as well as specific IGF-binding proteins (IGFBP-3, -4, -5, and -6). IGF treatment of hMS(3–4) 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(3–4) cells, but had a more complex effect on hMS(3–4) cell differentiation. Treatment with IGFs did not affect gene expression of Cbfa1 or peroxisome proliferator-activated receptor {gamma}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(3–4) cells as well as mature osteoblasts and promoted lipid accumulation in differentiated adipocytes. In summary, hMS(3–4) 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(3–4) cells to either osteoblast or adipocyte pathways, and their effects on differentiation within these lineages were dependent on the stage of cell maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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{alpha},25-Dihydroxyvitamin D3, [3H]thymidine, and [{alpha}-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 {alpha}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 manufacturer’s 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 manufacturer’s 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 {alpha}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 7.5–15% 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 48–72 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 manufacturer’s 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 [{alpha}-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 {gamma}2 (PPAR{gamma}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{gamma}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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IGF receptors in hMS cells
As shown in Fig. 1Go, 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 {alpha}-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 ({alpha}-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).

 
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. 2Go) 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.

 
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. 3Go). 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. 2Go), 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. 4Go). 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.

 
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. 5Go). 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).

 
Effect of IGFs on hMS cell differentiation
Expression of phenotype marker genes. A representative RT-PCR experiment is shown in Fig. 6Go. 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{gamma}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 [{alpha}-32P]dCTP (10 µCi/µl) and corrected for GAPDH expression. Base pairs are indicated on the right.

 
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. 7Go). Furthermore, there was a differential response to IGF-I and LR3-IGF-I in hOB cells, indicating an influence of IGFBPs.



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  		Figure 7. Differential effect of IGFs on Col I mRNA expression in hMS and hOB cells. Early hMS(3 4 ) cells, late hMS(3 4 ) cells (15 days before incubation in differentiation medium at 39.5 C), and mature hOB cells were treated at 39.5 C in differentiation medium with vehicle (Control; solid bars), 10 nM IGF-I (open bars) or 10 nM LR3-IGF-I (hatched bars) for 4 days. Col I mRNA was determined as described in Fig. 6Go. Results are the mean ± SEM of quadruplicate determinations expressed as the treatment/control ratio. *, P < 0.05; **, P < 0.01 (compared with control values). {ddagger}, P < 0.05 (significant difference between IGF-I and LR3-IGF-I).

 
Production of phenotype marker proteins. As shown in Fig. 8Go, treatment with 10 nM des(1, 2, 3)IGF-I or des(1, 2, 3, 4, 5, 6)IGF-II for 3–9 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. 9Go). 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 3–15 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.

 
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. 10Go). 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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{gamma}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{gamma}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. Back

Received March 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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