British Journal ofHaernatologg. 1995, 90, 249-257

Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells

PASCALI! GKELLIEK, DOUGLAS YEE, MYRNA GONZAJM A N D SHERRY L. ABBOIJD Departments of Medicine, Oncology and Pathology, University of Texas Health Science Center and Audie Murphy Veterans Hospital, San Antonio, Texas, U.S.A.

Received 27 September 1994; accepted for publication I3 February 199 5

Summary. Bone marrow stromal cells synthesize and secrete insulin-like growth factor (1GF)-I and IGF-binding proteins (IGFBP). IGFBPs may modulate the action of IGF-I or IGF-I1 on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP-1 to -6 mRNA expression. [1251]IGF-I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kl) species, consistent with IGFBP4 and IGFBPS. respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP-2 to -6 mRNAs. with IGFBP4, IGFBP5 and IGFBPh mRNAs predominating. Since agents that modulate cAMP

levels may influence haemopoiesis via the release of stromal- derived cytokines. we determined the effect of forskolin, a CAMP agonist, on IGFBP4 expression in TG1 cells. Forskolin (lops M) up-regulated IGFBP4 mRNA and protein secretion in a time-dependent manner. These findings suggest that IGFBP-4, -5 and -6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment,

Keywords: insulin-like growth factor, binding protein, bone marrow, stromal cells, haemopoiesis.

Insulin-like growth factors I and I1 (IGF-I, 11) enhance the growth and differentiation of a wide variety of cell types including haemopoietic stem cells, erythroid and granulo- cytic progenitors (Huang I% Terstappen, 1992: Merchav et aZ, 1992, 1993). Our previous studies demonstrated that murine TC-1 stromal cells constitutively secrete IGF-I and IGF binding proteins (IGFBP) (Abboud et al, 1991). This suggests that local release of IGF-I may act in a paracrine fashion to stimulate haemopoiesis and that this effect could be modulated by IGFBPs. The spectrum of IGFBPs released by the different stromal cell types that comprise the marrow microenvironment, however, has not been determined.

A family of IGF-binding proteins, designated IGFBP-1-6, have been isolated and molecularly cloned in human and rat (Shimasaki & Ling, 1991). IGFBPs are present in serum and other biological fluids as well as in the conditioned medium of cultured cells (Cohick & Clemmons, 1993; Rosenfeld et al,

Correspondence: Dr Sherry L. Abboud, Department of Medicine, Ilniversity of Texas Health Science Center. 7703 Floyd Curl Drive, San Antonio, TX 78284,lJ.S.A.

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1990). They bind to IGF-I and IGF-I1 with high affinity, but not to insulin. Although the physiological functions of the various IGFBPs are unknown, it is clear that they modulate IGF action by influencing the interaction between IGFs and their receptors and by regulating the distribution of IGFs between plasma and tissue compartments (Bar et al, 1990; Ritvos et al, 1988). IGFBPs may either enhance or inhibit cellular responses to IGFs in vitro depending on the IGFBP species, target cell, and culture conditions (Conover, 1992; DeMellow & Baxter, 7988; Ritvos et al, 1988). Certain IGFBPs have also been reported to have intrinsic biological activity. independent to IGF (Andress et al, 1993; Oh et al, 1993). However, they are not soluble receptor-type molecules, because there is no evidence that they partici- pate in signal transduction pathways.

Distinct patterns of IGFBP basal expression are observed in different cell types. Under serum-free culture conditions. normal human fibroblasts predominantly secrete IGFRPs with MW of 43/39, and, to a lesser extent, IFGBPs of 24 and 31 kD (Camacho-Hubner et al. 1992). By immunoblot and Northern blot analysis, these IGFBPs correspond to two

249

250 PascaZe GreZZier et al glycosylated forms of IGFBP3 (43 and 39 kD), IGFBP4 and IGFBP 5 , respectively. Microvascular endothelial cells pre- dominantly express IGFBP4 mRNA and secrete IGFBP4 protein (Yang et al, 1993). However, depending on the species and tissue of origin, endothelial cells have also been reported to express mRNAs and secrete protein for all IGFBP forms except IGFBPl (Moser et d, 1992). The IGFBPs released by macrophage-like cells have not been elucidated.

Mechanisms that govern the synthesis and secretion of each IGFBP are complex. Agents that increase intracellular cAMP levels may either stimulate or inhibit the release of cytokines from marrow stromal cells (Derigs et al, 1994). In human bone cells and fibroblasts, CAMP is a potent inducer of IGFBP4 mRNA and protein (LaTour et al, 1990; Camacho- Hubner et al, 1992).

In this study we determined the precise molecular identities of each IGFBP secreted by TC-1 and other stromal cell types. Overall, stromal cells express IGFBP-2-6 mRNAs, with a predominance of IGFBP-4, -5 and -6. Forskolin, a cAMP agonist, upregulated IGFBP4 mRNA and protein in TG1 cells.

MATERIALS AND METHODS

Materials. The culture media (Dulbecco modified Eagle medium (DMEM), Fischer's medium, RPMI 1640, L-

glutamine, penicillin/streptomycin were purchased from GIBCO (Grand Island, N.Y.). Fetal calf serum (FCS) is a product of Hyclone Co. (Logan. Utah). Hepes, hydrocorti- sone, insulin, trisma base, SDS, glycerol, Nonidet P-40, Tween-20, BSA, formamide, PIPES, EDTA and acrylamide are manufactured by Sigma Chemical Co. (St Louis, Mo.). Yeast tRNA, proteinase K, RNase A and RNase T1 are products of Boehringer Mannheim (Indianapolis, Ind.). Genescreen and oligo(dT)-cellulose were obtained from Collaborative Research (Bedford, Mass.) and New England Nuclear (Boston, Mass.), respectively. ['251]IGF-I (2000 Ci/ mmol), nick translation kit and Rainbow molecular mass markers (14.3-200 kD) were purchased from Amersham (Arlington Heights, Ill.).

Cell lines and cell culture. The stromal cell lines used were derived from adherent layers of murine long-term marrow cultures. Their phenotypic characteristics have been described previously. The TG1 stromal cells (kindly provided by Dr P. Quesenberry, University of Massachu- setts, Worcester, Mass.) were maintained in Fischer's medium, supplemented with l o r n Hepes, 2 m ~ gluta- mine, 1 m~ sodium pyruvate, penicillin 100 U/ml, strepto- mycin 100pg/ml, and 17% fetal calf serum (FCS) and incubated at 37C in 5% C02 (Song et al, 1985). Four cloned stromal cell lines (kindly provided by Dr D. Zipori, Weizmann Institute of Science, Rehovot. Israel) including MBA-1.1 (fibroblast), MBA-2 (endothelial-lie), MBA-13.2 (fibro- endothelial) and 14M1.4 (macrophage) were cultured in DMEM supplemented with 10% FCS (Zipori & Lee, 1988). GBL/6 (fibroendothelial) (kindly provided by Dr J. S. Greenberger, University of Pittsburgh Medical Center, Pittsburgh, Pa.) were cultured in DMEM supplemented with 10% FCS and M hydrocortisone (Anklesaria et al,

1987). A human ovarian carcinoma cell line (OVCAR-3), obtained from ATCC (Rockville, Md.), was grown in RPMI 1640 supplemented with 10% FCS and 10pg/ml insulin.

Preparation of conditioned medium. Confluent cells were washed three times with serum-free medium and incubated in the same medium for 8 h. This medium was discarded. Cells were washed two more times and fresh serum-free medium was added. After 24-48 h, conditioned media were collected, centrifuged and cell-free supernatants stored at -20°C. MBA-1.1 and MBA-13.2 tolerated serum-free conditions for 24 h, whereas incubation of the other stromal cells in serum-free medium could be carried out for 48 h. As a control, medium conditioned by OVCAR-3. known to secrete IGFBP-2-5, was also collected. Prior to ligand blot analysis, samples were concentrated 25-50-fold by ultra- filtration in Centricon 10 microconcentrators (10 kD m.w. cutoff, Amicon, Beverly, Mass.).

Western ligand blot analysis. Ligand blotting with ['251]IGF-I was performed by the method of Hossenlopp et a1 (1986). Briefly, aliquots of conditioned media were resuspended in sample buffer [DO75 M Tris (pH 6.8), 2.5% sodium dodecyl sulphate (SDS), 0*003% bromophenol blue, and 40% glycerol), 1 0 0 ~ 1 final volume]. After boiling for 5 min, the samples were cooled and applied to a discontin- uous SDS-polyacrylamide gel and electrophoresed through a 4% stacking gel and a 10% separating gel under non- reducing conditions. The proteins were transferred (Hoefer Transphor Unit, Schleicher & Schuell, Keene. N.H.) over- night to 0.45 pm pore size nitrocellulose membranes at a 250mA constant current. Membranes were washed consecutively in buffer consisting of l o r n Tris (pH 7.4), 15Orn NaCI, and 0*5mg/ml Na azide containing 3% Nonidet P-40, 1% BSA or 0.1% Tween-20. They were then incubated overnight at 4°C with 400 000 cpm [12sI]IGF-I. washed, and visualized by autoradiography. The m.w. of the IGFBPs was estimated by comparison with prestained size markers.

RNA preparation and RNase protection assay. Confluent cultures of each stromal cell type maintained in complete medium with serum were used for total and poly(A+) RNA. Total RNA was isolated after centrifugation on CsCl gradients (Maniatis et al, 1982). Concentrations were determined by spectrophotometry; integrity and quantita- tion was confinned by visualizing ethidium bromide-stained 28s and 18s ribosomal bands on 1% agarose gels. Poly(A+) was prepared by oligo (dT)-cellulose affinity chromato- graphy. RNase protection assays were performed as previously described (Abboud et al, 1991). Briefly, "P- labelled antisense RNA probes were synthesized from linearized DNA templates using Promega Riboprobe Kit (Madison, Wis.). 30pg of total RNA, 5-1Opg of poly(A+) RNA. or 30pg of yeast tRNA as a negative control were hybridized with 5 x lo4 to 1 x 105cpm probe in 80% formamide, 40 mM Pipes, 400 rn NaCI, 1 mu EDTA for 18- 24 h at 5 5°C. The RNA was digested with 50 pg/ml RNase A and 2 pg/ml T1 for 1 h and then treated with proteinase K. The protected fragments were analysed by electrophoresis through a 7~ urea/6% acrylamide gel, followed by autoradiography.

0 1995 Blackwell Science Ltd, British JournaI of HaematoIogg 90: 249-257

IGF-binding Proteins and Marrow Stroma 251

Fig 1. Western ligand blot of IGFBPs in media conditioned by different murine stromal cell types cultured under serum-free, unstimulated conditions. Conditioned medium was collected after 2 4 h (MBA-1.1. MBA-13.2) or 48 h (14M1.4, GBL/6. TC-1) of incubation. Samples were electrophoresed through a nonreducing 10% SDS-polyacrylamide gel and transferred to nitrocellulose. After incubation with ['251]I(;F-I. filter- immobilized IGFBPs were visualized by autoradiography after 2d (Fig l a ) or 10d (Fig Ib) exposure. Lane C shows OVCAR control. The migration positions of molecular size markers (in kD) are indicated on the left.

Antisense RNA probes. Plasmids for rat IGFBP-1-6 were kindly provided by Dr Shunichi Shimasaki (The Whittier Institute, La Jolla, Calif.). Each of the IGFBP probes was derived from cDNA fragment (corresponding to part of the protein-coding region) cloned into pBluescript SKf (Shi- masaki &Ling, 1991). The IGFBPI (pRBP1-501) and IGFBP2 (pRBP2-501) probes were synthesized from EcoRI-Hind 111 407 bp and 397 bp fragments, respectively. IGFBP3 (pRBP3- AR) probe was synthesized from an AccI-RsaI 551 bp fragment and IGFBP4 (pRBP4-SH) probe was derived from a SrnaI-Hind111 444 bp fragment. The IGFBPS (pRBP5SH) and IGFBP6 (pRBP6-PP) probes were synthesized from an XmnI-Hind111 183 bp and a PstI-PstI 246bp fragment, respectively. In vitro transcription with the use of either T3 or T7 RNA polymerase was performed in the presence of [32P]uridine triphosphate to produce the appropriate labelled antisense probe.

Northern blot analysis. Total RNA (20 pg) and poly(Af) RNA ( 5 pg) were size-fractionated on 1% agarose-formal- dehyde gels and transferred to Genescreen. The rBP4-SH cDNA was labelled with [32P]dCTP using a nick translation kit. The membrane was prehybridized for 1 h at 42°C in 50% formamide, 0.5% SDS, 2 x Pipes-NaC1-EDTA buffer, and 0.1 mg/ml salmon sperm DNA. Hybridization was carried Out in the Same buffer containing lo6 cPm/d probe for Fig 2. Ijgand blot of IGFBPs in xrum-free medium conditioned by

at 420c. were washed sequentially in sscv MBA-2 endothelial-like cells. Conditioned medium was collected after 48 h of incubation and analyzed by Western ligand blot using [1251]IGF-I as described in Fig 1. The autoradiograph was exposed for 5 d.

o*l'k SDS for 30min at 55"c and o'2 ssc* o'lyo SDS for 3 0 min at 55°C. Membranes were exposed to AR films at -70°C with two intensifying screens.

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252 Pascale Grellier et a1

Fig 3. RNase protection assay for IGFBP4 mRNA expression by murine stromal cells. A 32P-labelled rat IGFBP4 antisense RNA probe was hybridized with total RNA (30pg) and poly(Af) RNA (5 pg) from each stromal cell type. Total RNA from normal rat and murine kidney were used as controls. The autoradiogram shows an approximately 444 bp protected fragment in rat kidney and three smaller fragments in murine kidney and stromal cells. Probe hybridized with tRNA was completely digested by RNases. Lane 14 shows undigested IGFBP4 probe. The migration of 32P-labelled DNA markers is shown to the left (in basepairs).

RESULTS ligand blot from media conditioned by murine stromal cells and human control cells may not represent the same IGFBP (Shimasaki & Ling, 1991). Identification of IGFBPs in stromal cell conditioned media

The constitutive release of IGFBPs from the various stromal cell types, measured by ligand blotting with [1251]IGF-I, is shown in Fig l(a). Lane C shows conditioned medium from ovarian carcinoma cells that contains IGFBPs of 24, 29 and 32 kD and a doublet at 46 kD. The 24,29 and 32 kD IGFBPs correlate with IGFBP-4, -5 and -2, respectively. The 46 kD doublet represents IGFBP3 (McGuire et ul, 1992). Serum-free medium from unstimulated stromal macrophages (14M1.4) contained IGFBPs with apparent m.w. of 24 and 29kD. Medium conditioned by TC-1 showed similar species as well as a doublet at 46kD. The 31-32kD band previously reported for TC-1 was also observed on a separate autoradiogram (data not shown). Our previous data showing a slightly lower m.w. doublet and no detectable 24kD species in TC-1 conditioned medium may be due to differences in ligand blot technique (Abboud et al, 1991).

A longer exposure time of the same blot in Fig l(a) was required to visualize IGFBPs secreted by MBA-13.2, MBA- 1.1 and GBL/6. Fig l(b) shows that fibroendothelial MBA- 13.2 cell conditioned medium contained 24kD and 29kD IGFBPs as well as a single band migrating at 46 kD. MBA-1.1 and GBL/6 secreted 24 and 29 kD IGFBPs. The ligand blot in Fig 2 demonstrates the presence of 24 and 46 kD IGFBPs in medium conditioned by MBA-2 endothelial cells. Each cell type secreted a similar pattern of IGFBPs when media was tested at 24 or 48 h, although IGFBP abundance varied at these time points. These results demonstrate that although conditioned medium from each stromal cell type shows a unique pattern of IGFBPs. they all have in common the release of 24 kD and 29 kD IGFBPs.

Ligand blot analysis is a rapid method to screen for the presence of IGFBPs: however, further confirmation of the specific IGFBPs is necessary. The various glycosylated forms of certain IGFBPs may overlap in the same m.w. range of other IGFBPs. In some cases it may be difficult to distinguish IGFBP-1, -2 and -5 on the basis of ligand blot alone (Conover et al, 1993a). Furthermore, IGFBPs of the same m.w. on

IGFBP gene expression in stromal cells To determine whether the IGFBPs secreted by stromal cells correlate with the predicted IGFBP mRNA expression, total and poly(A+) RNA was isolated from each stromal cell type and analysed using a sensitive RNase protection assay. Rat antisense RNA probes were utilized to detect murine IGFBP- 1-6 mRNAs. For certain IGFBPs the protected fragment(s) in murine samples did not correlate with the predicted fragment observed in rat control tissues. It is likely that

Fig 4. Northern blot analysis of IGFBP4 mRNA in TG1 cells. Total RNA (15pg) and poly(A+) RNA ( 5 p g ) from TC-1 cells were electrophoresed in 1% agarose-formaldehyde gel and transferred to Genescreen. The blot was hybridized with a nick-translated "P- labelled rat IGFBP4 cDNA probe as described in Materials and Methods. Total RNA (15 pg) from rat kidney, murine liver and murine kidney were used as controls. IGFBP4 probe detects a 2.6 kb transcript in TG1. similar to that observed in murine and rat tissues. The migration of the 28s and 185 ribosomal RNA bands is shown to the left.

0 1995 Blackwell Science Ltd, British Journal of Haematologg 9 0 249-257

IGF-binding Proteins and Marrow Strorna 2 5 3

these findings are due to differences in the nucleotide sequences between rat and murine species, resulting in alternate sites in RNA-RNA hybrids sensitive to RNase digestion. Northern blot analyses performed for IGFBP4 mRNA (shown below) support this conclusion, because a single transcript of the same size is observed for both rat and murine samples.

Secretion of 24 and 29kD IGPBPs by all stromal cells suggest that they synthesize IGFBP4 and IGFBP5 mRNA, respectively. RNase protection assay shown in Fig 3 of total and poly(A+) RNA isolated from each cell type confirmed the presence of IGFBP4 mRNA in all cells. On a separate autoradiogram, MBA-1.1 also expressed IGFBP4 mRNA (data not shown). Lane 1 shows rat kidney control with the expected 444 bp protected fragment. The mouse kidney control and stromal cells show three prominent lower M.W. protected fragments of 290,242 and 190 bp that are specific and not observed in the tRNA lane. There is differential expression of IGFBP4 mRNA among the stromal cells, with the highest steady-state levels observed for TC-1 and 14M1.4.

To verify that the RNase protection assay was specific for IGFBP4 mRNA expression in stromal cells, Northern blot analysis was carried out to determine the size of the IGFBP4 mRNA transcript. Fig 4 shows a single mRNA transcript of 2.6 kb in TC-1, murine and rat control tissues, consistent with the reported size of IGFBP4 mRNA (Shimasaki et al,

Fig 5 demonstrates that all stromal cells, to a variable degree, express IGFBP5 mRNA, with the strongest signal observed in TC-1. The rat and murine tissue controls and the stromal cell lines show the expected 183bp protected fragment. Since IGFBPS protein may migrate in close proximity to IGFBPl and IGFBP2 on ligand blot, stromal cells were also examined for mRNA expression of these IGFBPs. None of cell types expressed IGFBPl mRNA and only MBA-13.2 and TG1 cells expressed IGFBP2 mRNA (data not shown).

By ligand blot analysis, TG1 MBA-13.2 and MBA-2 appear to secrete IGFBP3 protein. Fig 6 confirms the presence of IGFBPS mRNA in TC-1 and MBA-2; other cell

1990).

Fig 5. RNase protection assay for IGFBP5 mRNA expression by murine stromal cells. A 32P-labelled rat IGEBPS antisense RNA probe was hybridized with total RNA (30 pg) and/or poly(A+) RNA (10 pg) from each stromal cell type. Total RNA (20pg) from normal rat and mouse kidney were used as controls. The autoradiogram shows the predicted 183 bp protected fragment in both control tissues and stromal cells.

types did not show evidence of IGFBP3 mRNA production (data not shown). Rat tissue shows the single predicted 551 bp species. In contrast, the mouse liver and stromal cells exhibit a distinct doublet of protected fragments of approximately 309 bp. It is unlikely that the 46kD IGFBP in MBA-13.2-conditioned media represents IGFBP3. and additional studies are required to identify this IGFBP.

Since ['251]IGF-I ligand blot analysis is unlikely to detect IGFBP6 protein due its higher affinity for IGF-11, cells were screened for the presence of IGFBP6 mRNA. All stromal cells, except GBL/6, constitutively express IGFBP6 mRNA (Fig 7), with the strongest signals observed for 14M1.4 and MBA- 1.1. Longer exposure of the blot in Fig 7 showed IGFBPG

Fig 6. RNase protection assay for IGPBP3 mRNA expression by murine stromal cells. Hybridization of total RNA (1Opg) from rat liver control with 32P-labelled rat IGFBP3 antisense RNA probe shows the predicted 551 bp fragment. Total RNA (30pg) and/or poly(A+) RNA (5 pg) from stromal cells show two smaller protected fragments similar in size to IGFBP3 mRNA detected in murine liver controI.

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254 Pascale Grellier et a1

Table I. IGFBP mRNA expression by stromal cells.

Fig 7. RNase protection assay for IGFBPG mRNA expression by murine stromal cells. Total RNA (30pg) and poly(Af) RNA (5 pg) from stromal cells show the same 246 bp protected fragment observed with total RNA (30 Gg) from control rat tissue.

mRNA expression in MBA-2 cells. Table I summarizes the relative IGFBP exmession bv stroma.

BP1 BP2 BP3 BP4 BP5 BP6

TC-1 - + ++ +++ ++ +R MBA-1.1 - -

MBA-2 -

MBA-13.2 - +R ? + + +R 14M1.4 - - - +++ + +++R GBL/G - - - + + -

- + + ++R - ++ + + +R

R = detected by RNase protection only. + to f i- + is weakest to strongest mRNA expression: - represents no detectable mRNA signal.

Induction of IGFBW mRNA and protein by forskolin in TC-1 In some systems IGFBP4 mRNA is stimulated by agents that increase intracellular cAMP (Yang et al, 1993). Time course experiments for the effect of forskolin, a cAMP agonist, on IGFBP4 mRNA expression in TC-1 were performed. Fig 8(a) shows that lo-' M forskolin induces IFGBP4 mKNA in a time-dependent manner, with increased levels by 4 h and a peak effect observed at 16 h. mRNA levels remained elevated up to 24 h. To confirm these findings, a second experiment shown in Fig 8(b) was carried out using controls for each time point tested. At 16. 24 and 48 h, forskolin stimulated IGFBP4 mRNA above control levels. The relative abundance

Fig 8. Regulation of IGFBP4 mRNA by M forskolin. RNase protection assay of total RNA (15 pg/lane) from TG1. Confluent TC-1 cells were made quiescent by placing them in serum-free medium with 0.2% BSA for 48 h. Forskolin was then added and cells incubated for the indicated time points. Cytoplasmic RNA was isolated from the cells and RNase protection assay was performed as described in the legend to Fig 3. Control in Fig 8(a) refers to RNA isolated from cells incubated 48 h in serum-free medium alone. Controls in Fig 8(b) were from cells incubated in medium alone for the indicated time points.

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of the three protected Gagments did not change after induction. Ligand blot analysis of conditioned media from the 16 and 24 h time points demonstrated increased IGFBP4 protein in forskolin-treated cells as compared to controls (data not shown).

DISCUSSION

The action of IGF-I and IGF-I1 on haemopoiesis may be influenced by their interaction with IGFBPs: however, the specific IGFBPs released by stromal cells in the microenvironment is unknown. Our findings indicate that: (1) each stromal cell type has a unique pattern of IGFBP gene expression: (2) as a whole, stromal cells preferentially express IGFBP-4, -5 and -6 mRNAs and secrete detectable levels of IGFBP-4 and -5: (3) IGFBP4 gene expression in TC-1 is induced by agents that increase intracellular CAMP.

IGFBPs detected by Western ligand blot may be distin- guished by ligand immunoblot or immunoprecipitation using specific antibodies. Since murine IGFBP antibodies are not available, the identity of secreted IGFBP forms may be inferred from expression of IGFBP mRNAs. IGFBP mRNA expression in stromal cells as measured by RNase protection assay correlated well with secretion of the corresponding protein. By ligand blot, 24, 29, 32 and 46kD IGFBPs correlated with IGFBP-4, -5, -2 and -3 mRNA expression, respectively. Although IGFBP6 secretion was not apparent by [1251]IGF-I ligand blotting, IGFBP6 mRNA expression was readily detected by RNase protection. The various stromal cells exhibited different steady-state levels of IGFBP expres- sion. The strongest mRNA signals were observed for IGFBP4 (TC-1, 14M1.4) and IGFBPG (14M1.4). It is curious that the two fibroendothelial cell lines, MBA-13.2 and GBL/6, demonstrate different patterns of IGFBP expression. This may be due to their original derivation from different strains of mice.

The distinct patterns of IGFBP expression and protein secretion by various stromal cell types suggests that the ratio of one IGFBP to another may be an important factor in determining IGFs action (Camacho-Hubner et al, 1992). The overall net effect of IGF on haemopoiesis would depend upon the relative abundance of each IGFBP that may interact in either a synergistic or opposing manner to modulate IGF action. Thus, a highly integrated network of IGFBPs would serve to tightly regulate IGF bioavailability and bioactivity in the marrow.

Predominant expression of IGFBP-4, -5 and -6 by stromal cells suggest that these IGFBPs play a key role in regulating IGF-mediated haemopoietic effects. Their effect on IGF action has been examined in other systems. IGPBP4 and IGFBP6 are potent inhibitors of IGF-induced mitogenesis in bone- derived cells and other cell types (Kiefer et d, 1992). IGFBPS is mainly localized in extracellular matrix where it potentiates IGF action: however, soluble IGFBP5 is an inhibitor of IGF action (Jones et al. 1993; Kiefer et al, 1992). The presence of IGFBP3 in some stromal cells is consistent with its wide distribution in vivo. IGFBP3 may either stimulate or inhibit IGF action depending upon its equilibrium between the ce€l membrane and extracellular

IGF-binding Proteins and Marrow Stroma 2 55 fluid (Conover, 1992; &Mellow & Baxter. 1988). The absence of IGFBPl and the low prevalance of IGFBP2 expression in stromal cells is not unexpected, because their expression is tissue and developmentally restricted (Shima- saki & Ling, 1991).

A predominant enhancing versus inhibitory effect of IGFBPs may be dependent, in part, on regulation of IGFBPs by growth factors and hormones. Previous studies have demonstrated that increased intracellular cAMP is a potent stimulator of IGFBP4 synthesis in a variety of cells (Yang et al, 1993: Conover et aI, 1993a: LaTour et a!, 1990; Cohick & Clemmons, 1993; Camacho-Hubner et al, 1992).

Certain hormones, cytokines and drugs modulate intra- cellular cAMP levels that, in turn, may mediate haemo- poietic responses. cAMP has been shown to regulate haemopoietic cell proliferation and the release of cytokines produced by progenitors and stromal cells. For example, elevation of cAMP inhibits granulocyte-macrophage colony formation and proliferation of bone marrow macrophages induced by CSF-1, GM-CSF, IL-3 and PMA (Kurland et al, 1977; Vairo et al, 1990). Induction of CSF-1 mRNA in HI,- 60 cells by TNF is also blocked by CAMP analogues or PGEz (Sherman et al, 1990). In marrow stromal cells, cAMP differentially regulates cytokine production. Agents that increase intracellular cAMP levels inhibit GM-CSF mRNA and protein induced by IL1, whereas cAMP agents alone or in combination with cytokines enhance the synthesis of IL-6 (Derigs et d, 1994). Agents may modulate CAMP levels by either stimulating adenylate cyclase or inhibiting phospho- diesterase activity. Forskolin, a direct activator of adenylate cyclase, was used as a tool to determine the effect of increased cAMP levels on IGFBP4 expression in TC-1 cells. Our results demonstrate that forskolin induces IGFBP4 mRNA and protein in TC-1. This suggests that ambient CAME' levels may indirectly modulate IGF-mediated haemo- poietic responses, via the release of IGFBP4. It is also possible that, during inflammation. PGE2 may stimulate the release of IGFBP4 in the microenvironment via CAMP.

Increased IGFBP4 mRNA abundance and protein secre- tion by TC-1 suggests that cAMP may, in part, regulate IGFBP4 gene expression at the transcriptional level. This is supported by recent evidence demonstrating the presence of three cAMP responsive elements and three AP1 binding sites in the rat IGFBP4 gene promotor (Gao et al, 1993). Post- translational modification of IGFBP4 has also been reported. Phorbol ester increases IGFBP4 protein accumulation in medium conditioned by fibroblasts via the release of IGFBP4 protease inhibitors. whereas IGF-I and IGF-I1 decrease IGFBP4 protein through activation of specific IGFBP4 proteases (Conover et al, 1993b. c). Other IGFBPs may also be regulated post-translationally by changes in phos- phorylation and by alterations in membrane-associated fractions (Jones et a], 1991; Martin et al, 1992). Whether the effects on mRNA levels in TC-1 by forskolin represent stimulation of gene transcription, stabilization of pre-existing mRNA, or a combination of the two, awaits further study.

In conclusion, the relative abundance of IGFBPs with stimulatory versus inhibitory properties is likely to be a key determinant of the haemopoietic response to IGFs. Further

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256 Pascale Grellier et al understanding of the intracellular pathways involved in regulating the synthesis and release of stromal-derived IGFBPs will help to elucidate the biological role of IGFs in haemopoiesis.

ACKNOWLEDGMENTS

This work was supported in part by the Veterans Admini- stration Medical Research Service (S.L.A.), National Insti- tutes of Health Grants AR42306 (S.L.A.). CA52952 (D.Y.) and by a cancer center support grant P30-CA54174 to the Cancer Therapy and Research Center. D.Y. is a Pew Scholar in the Biomedical Sciences.

Portions of this work were published in abstract form at the annual meeting of the American Society of Hematology, St Louis, Missouri, 3-7 December 1993.

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