All-trans-retinoic Acid Increases Transforming Growth Factor-beta 2 and Insulin-like Growth Factor Binding Protein-3 Expression through a Retinoic Acid Receptor-alpha -dependent Signaling Pathway*

(Received for publication, December 24, 1996, and in revised form, February 19, 1997)

Gil-Ro Han Dagger , David F. Dohi Dagger , Ho-Young Lee Dagger , Roopmathy Rajah §, Garrett L. Walsh , Waun Ki Hong Dagger , Pinchas Cohen § and Jonathan M. Kurie Dagger par

From the Departments of Dagger  Thoracic/Head and Neck Medical Oncology and  Thoracic and Cardiovascular Surgery, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and the § Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Retinoids, including retinol and retinoic acid derivatives, maintain the normal growth and differentiation of human bronchial epithelial cells. The signaling pathways through which retinoids mediate these effects have not been defined. Insulin-like growth factor binding protein-3 (IGFBP-3) and the transforming growth factor-beta (TGF-beta ) gene family (beta 1-3) were examined as potential components of the retinoid signaling pathway in normal human bronchial epithelial cells. All-trans-retinoic acid (t-RA) increased the levels of TGF-beta 2 and IGFBP-3 mRNA and of secreted TGF-beta and IGFBP-3 proteins. An antagonist of retinoic acid receptor-alpha , LG100629, abrogated the increase in TGF-beta 2 and IGFBP-3 mRNA levels induced by t-RA. t-RA increased IGFBP-3 mRNA levels transiently from 1 to 6 h, and subsequently a sustained increase began at 72 h, which coincided with the appearance of active TGF-beta in the media. Treatment with TGF-beta 2 increased IGFBP-3 mRNA levels, but treatment with latency-associated peptide, which inactivates secreted TGF-beta , did not abrogate the effect of t-RA on IGFBP-3 expression. These findings provide evidence that t-RA increased TGF-beta 2 and IGFBP-3 expression through an retinoic acid receptor-alpha -dependent pathway, and the increase in IGFBP-3 expression by t-RA did not require activation of the TGF-beta pathway by autocrine or paracrine mechanisms.


INTRODUCTION

Retinoids control normal tracheobronchial epithelial growth and differentiation. Rodents that are deprived of vitamin A develop squamous metaplasia in the tracheobronchial epithelium, and normal epithelial differentiation is restored by vitamin A supplementation (1, 2). In tissue culture, human bronchial epithelial (HBE)1 cells undergo squamous differentiation with a variety of agents, and all-trans-retinoic acid (t-RA) inhibits this process (3-7). HBE cells treated with t-RA develop mucociliary features in collagen gels (3, 8). Grown in monolayer cultures, retinol-treated HBE cells undergo growth arrest with no evidence of morphologic differentiation (9).

Retinoids are ligands for the retinoic acid receptors (RAR-alpha , -beta , and -gamma ) and retinoid X receptors (RXR-alpha , -beta , and -gamma ), which form RAR-RXR heterodimers and RXR homodimers and are transcriptionally activated by ligand binding (reviewed in Ref. 10). In bronchial epithelial cells, RAR-alpha is expressed at high levels and has been shown to activate growth inhibitory pathways (11, 12). The signaling pathways activated by RAR-alpha that mediate growth inhibition in normal HBE cells have not been defined. Retinoids increase the expression of transforming growth factor-beta (TGF-beta ) family members (13-22). TGF-beta is secreted as a latent complex and converted to an active form, and it signals through a heteromeric complex of the type I and type II receptors (23). Activation of the TGF-beta pathway has been implicated in the effects of retinoids on cellular growth and differentiation (13-22). In addition, the secretion of insulin-like growth factor binding protein-3 (IGFBP-3) is enhanced by retinoid treatment (24-33, 35). IGFBP-3 is one of a family of seven IGFBPs (36-39). IGFBP-3 has been proposed to inhibit cell growth by reducing IGF bioavailability, by altering the responsiveness of the IGF-I receptor to IGF-I, and by mechanisms independent of the IGF-I receptor (40, 41). IGFBP-3 expression is also increased by TGF-beta 2 treatment in breast cancer cells and has been implicated in the growth inhibitory effects of TGF-beta 2 (42). These findings support the notion that TGF-beta and IGFBP-3 actions are connected through a common retinoid signal transduction pathway.

In this study, we examined the regulation of IGFBP-3 and TGF-beta gene family expression by t-RA in normal HBE cells. We demonstrated that t-RA increased the levels of TGF-beta 2 and IGFBP-3 mRNA and of secreted TGF-beta and IGFBP-3 proteins. These events were inhibited by a retinoid that functions as an RAR-alpha antagonist. Treatment with TGF-beta 2 increased IGFBP-3 mRNA levels, demonstrating a linkage of IGFBP-3 with TGF-beta 2 signaling pathways. However, the addition of latency-associated peptide (LAP), which inactivates secreted TGF-beta , did not abrogate the effect of t-RA on IGFBP-3 expression. These findings provide evidence that t-RA increased TGF-beta 2 and IGFBP-3 expression through an RAR-alpha -dependent pathway, and the increase in IGFBP-3 expression by t-RA did not require activation of the TGF-beta pathway by autocrine or paracrine mechanisms.


EXPERIMENTAL PROCEDURES

Cells and Culture Conditions

Normal HBE cells were cultured from bronchial mucosal biopsy samples taken from fresh surgical specimens as monolayer cultures on standard plasticware in keratinocyte serum-free medium (Life Technologies, Inc.) containing bovine pituitary extract (BPE) and epidermal growth factor as described previously (11). Mink lung epithelial cells (MLECs) stably transfected with a luciferase reporter plasmid containing a truncated plasminogen activator inhibitor type I promoter (16) were a gift from Drs. Irene Nunes and Daniel Rifkin (Department of Cell Biology and Kaplan Cancer Center, New York University Medical Center, New York, NY). MLECs were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum as described previously (16). t-RA was purchased from Sigma. The RAR-alpha antagonist LG100629 (43) was obtained from Ligand Pharmaceuticals, Inc. (La Jolla, CA), and LAP was provided by R & D Systems, Inc. (Minneapolis, MN). Recombinant TGF-beta 2 was purchased from Genzyme, Inc. (Cambridge, MA). Actinomycin D and cycloheximide were purchased from Sigma.

Northern Blot Analysis

Total cellular RNA was prepared from normal HBE cells, electrophoresed (20 µg/lane) on a 1% agarose gel containing 2% formaldehyde, transferred to a nylon membrane (Zetaprobe, Bio-Rad), hybridized to an [alpha -32P]dCTP-labeled cDNA probe, washed, and autoradiographed as described previously (11). cDNAs for TGF-beta 1, -beta 2, and -beta 3 (44-46) were obtained from Dr. Rik Derynck (University of California, San Francisco, CA), and the IGFBP-3 cDNA (47) was obtained from Dr. William Wood (Genentech, Inc., San Francisco, CA).

MLEC Assay for TGF-beta Activity in Conditioned Media

Normal HBE cells were seeded on 10-cm plates (105 cells/plate), treated with t-RA for different time periods or with medium alone, and conditioned medium samples were collected simultaneously 144 h after cell seeding. For the medium sample that represents the 0-24-h time point, treatment with 10-6 M t-RA in BPE-free medium (to eliminate exogenous TGF-beta ) was begun 120 h after cell seeding, and an aliquot was collected at 144 h. For the sample that represents 24-48 h, treatment was begun 96 h after cell seeding, replaced at 120 h with BPE-free medium containing t-RA, and an aliquot was collected at 144 h. For the sample that represents 48-72 h, treatment was begun 72 h after cell seeding and replaced at 120 h with BPE-free medium containing t-RA, and an aliquot was collected at 144 h. For the sample that represents 72-96 h, treatment was begun 48 h after cell seeding and replaced at 120 h with BPE-free medium containing t-RA, and an aliquot was collected at 144 h. For the sample that represents 96-120 h, treatment was begun 24 h after cell seeding and replaced at 120 h with BPE-free medium containing t-RA, and an aliquot was collected at 144 h. For the control (untreated) medium sample, no t-RA was added, the medium was changed to BPE-free medium at 120 h, and an aliquot was collected at 144 h. Conditioned medium samples were transferred to three separate wells on a six-well plate seeded with MLECs that were stably transfected with the TGF-beta -responsive luciferase vector (105 cells/well). MLECs were treated for 16 h, and then cytosolic luciferase activity was measured as described previously (11). Luciferase values reflect TGF-beta bioactivity in the conditioned medium. To examine bioactivity that reflects total (active plus latent) TGF-beta levels, MLECs were treated with conditioned medium previously heated to 80 °C for 5 min, which activates latent TGF-beta (21). The results represent the means and standard deviations of luciferase activities from three identical wells, and luciferase activities were corrected for normal HBE cell numbers determined at the end of t-RA treatment. The presence of t-RA in the conditioned medium did not appreciably alter luciferase activity in MLECs (data not shown).

Immunoblotting and Western Ligand Blotting (WLB) of IGFBP-3

Conditioned medium samples were collected from t-RA-treated normal HBE cells as described for the TGF-beta bioactivity assay. Aliquots determined on the basis of equal cell number were concentrated 1:10 with Centriprep 10 filters (Amicon, Inc., Beverly, MA), loaded on a 1% SDS, 12% polyacrylamide gel, blotted to a nitrocellulose membrane (BAS-83, Schleicher & Schuell, Inc., Burlingame, VT), incubated for 1 h at room temperature with an anti-IGFBP-3 affinity purified monoclonal antibody (Diagnostic Systems Laboratories, Webster, TX), and detected with the enzyme-linked chemiluminesence assay (ECL kit, Amersham Corp.). For WLB, the membrane was incubated with a 2 × 106 cpm mixture of 125I-labeled IGF-I and IGF-II and exposed to film as described previously (48).

Effect of LAP on Cell Growth

Normal HBE cells were seeded at a density of 105 cells/10-cm plate and treated for 120 h with media alone, 10-6 M t-RA alone, and 10-6 M t-RA in combination with LAP at concentrations of 1, 10, 100, 200, and 500 ng/ml. At 120 h, the cells were trypsinized and stained with trypan blue, and the total viable cell number was counted by using an hemocytometer.


RESULTS

We examined the regulation of IGFBP-3 and TGF-beta 1, -beta 2, and -beta 3 mRNA levels in normal HBE cells during 10-6 M t-RA treatment by Northern analysis (Fig. 1). An increase in TGF-beta 2 mRNA levels was detected between 6 and 12 h; this increase continued through 120 h. TGF-beta 1 was expressed constitutively with no change during treatment. TGF-beta 3 mRNA was not detected (data not shown). IGFBP-3 mRNA was expressed in a bimodal pattern. A transient increase was observed at 6 h, and a sustained increase appeared at 72 h of treatment. Examination of earlier time points revealed that increased IGFBP-3 mRNA was detectable at 1 h of 10-6 M t-RA treatment (Fig. 2A). The increase in IGFBP-3 mRNA at 6 h was inhibited by treatment with actinomycin D or cycloheximide (Fig. 2B), demonstrating the necessity of both gene transcription and protein synthesis for activation of this retinoid signaling event.


Fig. 1. Northern analysis of the indicated genes was performed on total cellular RNA prepared from normal HBE cells. Expression was examined in cells treated with 10-6 M t-RA for different time periods (6, 12, 24, 72, and 120 h) or with media alone (t = 0) (A). Expression was examined in cells treated for 120 h with media alone (lane C) or with 10-6, 10-8, or 10-10 M t-RA (B). Photographs of representative ethidium bromide-stained gels illustrate the relative amounts of RNA loaded per lane.
[View Larger Version of this Image (25K GIF file)]


Fig. 2. Northern analysis of IGFBP-3 expression was performed on total cellular RNA prepared from normal HBE cells. Expression was examined in cells treated with 10-6 M t-RA for different time periods (0.5, 1, 2, 3, 4, 5, or 6 h) (A). The effect of 10-6 M t-RA treatment on IGFBP-3 expression was examined in the presence of 1 µg/ml actinomycin D (Act D) or 10 µg/ml cycloheximide (CHX) (B). Photographs of ethidium bromide-stained gels illustrate the relative amount of RNA loaded per lane. The absence (-) and the presence (+) of an agent are indicated.
[View Larger Version of this Image (20K GIF file)]

We investigated the contribution of RAR-alpha -dependent signaling pathways to the increased TGF-beta 2 and IGFBP-3 mRNA levels induced by t-RA. Normal HBE cells were treated for 120 h with 10-8 M t-RA and different doses of LG100629 (43), which is a synthetic retinoid that functions as an RAR-alpha antagonist, and total cellular RNA was prepared for Northern analysis. In combination with 10-6 M t-RA, LG100629, which must be in molar excess to inhibit t-RA-induced activation of RAR-alpha , was toxic to normal HBE cells (data not shown). LG100629 abrogated the increase in TGF-beta 2 and IGFBP-3 expression induced by 10-8 M t-RA (Fig. 3). TGF-beta 1 mRNA levels, which did not change with t-RA treatment, were not altered by LG100629. LG100629 also inhibited the increase in IGFBP-3 expression observed at 6 h of t-RA treatment (data not shown).


Fig. 3. Northern analysis of the indicated genes was performed on total cellular RNA prepared from normal HBE cells treated for 120 h with 10-8 M t-RA alone or in the presence of different doses of the RAR-alpha antagonist LG100629. A photograph of a representative ethidium bromide-stained gel illustrates the relative amount of RNA loaded per lane.
[View Larger Version of this Image (44K GIF file)]

The effect of t-RA on TGF-beta bioactivity in conditioned medium was investigated. Normal HBE cells were treated with 10-6 M t-RA, and conditioned medium samples that reflect defined 24-h periods (0-24, 24-48, 48-72 h, etc.) of t-RA treatment were collected as described under "Experimental Procedures." MLECs that are stably transfected with a reporter plasmid containing a truncated plasminogen activator inhibitor type I promoter (16) were treated with conditioned medium samples for 16 h and subjected to luciferase assays to determine relative levels of TGF-beta bioactivity in the conditioned media. Total TGF-beta (active plus latent) bioactivity was measured by heating the conditioned medium samples to convert latent TGF-beta into its active form. Luciferase activities increased during t-RA treatment (Fig. 4), demonstrating increases in total and active TGF-beta . The increase in active TGF-beta occurred between 48 and 72 h of treatment.


Fig. 4. Luciferase assays were performed on MLECs stably transfected with a reporter plasmid containing a TGF-beta response element (as described under "Experimental Procedures"). For these studies, medium samples were collected from normal HBE cells treated with media alone (control) and at 24-h intervals (0-24, 24-48 h, etc.) during 10-6 M t-RA treatment. At the beginning of each collection period, the medium was changed to BPE-free medium to remove TGF-beta from medium constituents. MLECs seeded onto six-well plates were treated with conditioned media for 16 h and subjected to luciferase assays. The luciferase values represent levels of TGF-beta bioactivity in the conditioned media. Total TGF-beta (active plus latent) bioactivity was measured by heating the conditioned medium samples to convert latent TGF-beta into its active form. The results represent the means and standard deviations of luciferase activities from three identical wells, and luciferase activities were corrected for normal HBE cell numbers determined at the end of t-RA treatment.
[View Larger Version of this Image (15K GIF file)]

The effect of t-RA on IGFBP-3 protein secretion was examined in conditioned medium samples collected at 24-h intervals (as described for the MLEC luciferase assay) by performing immunoblot and WLB (Fig. 5). IGFBP-3 was detected in the media of untreated cells (t = 0) by WLB, and increased IGFBP-3 levels, first detected by immunoblot at 24 h, occurred with t-RA treatment.


Fig. 5. Immunoblot and WLB analyses of IGFBP-3 expression were performed on conditioned medium samples collected from normal HBE cells at defined 24-h periods of 10-6 M t-RA treatment (0-24, 24-48, 48-72 h, etc.) and from cells treated with media alone (control). Sample aliquots for loading were determined on the basis of equal cell number. The position of a molecular mass size marker is indicated. IMB, immunoblot.
[View Larger Version of this Image (18K GIF file)]

Because the increase in active TGF-beta protein in the media coincided with the increase in IGFBP-3 mRNA levels at 72 h, we examined whether IGFBP-3 expression is responsive to activation of TGF-beta 2 signaling pathways. Normal HBE cells were treated with 5 ng/ml TGF-beta 2, which has been shown to increase IGFBP-3 expression in human breast cancer cells (42), and Northern analysis was performed at 72 h, revealing increased IGFBP-3 mRNA (Fig. 6). The contribution of TGF-beta signaling pathways to the t-RA-induced increase in IGFBP-3 mRNA was explored by treatment with 10-6 M t-RA combined with varying doses of LAP, which noncovalently associates with active TGF-beta in the extracellular space, converting TGF-beta into its inactive form. Examination of conditioned medium samples collected on day 5 revealed that LAP abrogated the increase in TGF-beta activity induced by 10-6 M t-RA (Fig. 7), confirming its inhibitory effect on extracellular TGF-beta activity. However, LAP did not measurably alter the effects of t-RA on IGFBP-3 mRNA or secreted protein levels (Fig. 8).


Fig. 6. Northern analysis of IGFBP-3 expression was performed using total cellular RNA prepared from normal HBE cells treated for 72 h with media alone, 10-6 M t-RA, or 5 ng/ml TGF-beta 2. A photograph of the ethidium bromide-stained gel illustrates the relative amount of RNA loaded per lane.
[View Larger Version of this Image (30K GIF file)]


Fig. 7. Luciferase assays were performed on MLECs stably transfected with a reporter plasmid containing a TGF-beta response element (as described under "Experimental Procedures"). For these studies, normal HBE cells were treated for 120 h with media alone, 10-6 M t-RA alone, or 10-6 M t-RA combined with different doses of LAP. Fresh medium (containing BPE) was added at 72 h, and conditioned medium samples were collected at 120 h. MLECs seeded onto six-well plates were treated with the conditioned medium samples for 16 h and subjected to luciferase assays. The results represent the means and standard deviations of luciferase activities from three identical wells, and luciferase activities were corrected for normal HBE cell numbers determined at the end of t-RA treatment. RA, retinoic acid.
[View Larger Version of this Image (10K GIF file)]


Fig. 8. Northern (A) and immunoblot analysis (B) were performed using total cellular RNA and conditioned medium samples, respectively, prepared from normal HBE cells treated for 120 h with media alone, 10-6 M t-RA, or 10-6 M t-RA combined with different doses of LAP. A photograph of the ethidium bromide-stained gel illustrates the relative amounts of RNA loaded per lane. Media were replaced at 72 h, and conditioned medium samples were collected at 120 h. Sample aliquots for loading were determined on the basis of equal cell number. The positions of molecular size markers are indicated for the immunoblot.
[View Larger Version of this Image (17K GIF file)]


DISCUSSION

In this study, we demonstrated that t-RA increased IGFBP-3 and TGF-beta 2 mRNA and protein levels in normal HBE cells. t-RA increases the expression of IGFBP-3 and TGF-beta family members in a variety of transformed and nontransformed cell lines (13-35). In contrast to our findings in normal HBE cells, the levels of TGF-beta 1 and -beta 2 both increase in t-RA-treated hamster tracheal epithelial cells grown in collagen gels (14), providing evidence that retinoid signaling in bronchial epithelial cells depends upon the culture conditions and is species-specific. Further, we found that t-RA stimulated the conversion of latent TGF-beta into its active form, suggesting that t-RA activated a protease that cleaves latent TGF-beta in normal HBE cells. This TGF-beta activity accounted for a relatively small fraction of total TGF-beta , suggesting that protease activity is tightly regulated. In bovine epithelial cells, t-RA activates latent TGF-beta through enhanced cell-associated plasmin activity (21), whereas in HL-60 cells, activation of TGF-beta by t-RA occurs through plasmin-independent mechanisms (16).

We examined the role of RAR-alpha , which is expressed constitutively and activates growth inhibitory pathways in normal HBE cells (11), in these retinoid signaling events. An RAR-alpha antagonist inhibited the effects of t-RA on TGF-beta 2 and IGFBP-3 expression. Normal HBE cells exhibited a bimodal pattern of IGFBP-3 mRNA expression, and the antagonist inhibited the increase at both time points. Treatment with an RAR-alpha antagonist also blocked the increase in transglutaminase type II expression induced by t-RA in rat tracheobronchial epithelial cells (12). Further, RAR activation increases lGFBP-3 expression in human ectocervical epithelial cell lines (32). These studies support a role for RARs in specific retinoid signaling events. Retinoid receptors regulate gene expression directly by binding to gene promoters or indirectly by protein-protein interactions with other transcription factors. We found that transcriptional mechanisms contribute to the increase in IGFBP-3 expression, and t-RA induced this effect rapidly (1 h). Based on these findings, we will investigate direct binding of retinoid receptors to the IGFBP-3 gene promoter and its role in the activation of IGFBP-3 expression by t-RA.

We found increased TGF-beta bioactivity in the media at 72 h of t-RA treatment, which was coincident with the increase in IGFBP-3 mRNA levels, and TGF-beta 2 treatment increased IGFBP-3 expression. Similarly, TGF-beta 2 treatment increases IGFBP-3 expression in breast cancer cells (42). Based on these findings, we investigated whether t-RA-induced IGFBP-3 expression was TGF-beta -dependent. Supporting this possibility, retinoid signaling events such as increased expression of thrombospondin and fibronectin are TGF-beta -dependent in rat prostatic epithelial cells (17). We found that LAP did not abrogate the increase in IGFBP-3 expression induced by t-RA, suggesting that the increase in IGFBP-3 mRNA levels by t-RA did not require activation of the TGF-beta 2 signaling pathway through autocrine or paracrine mechanisms. It is possible that multiple retinoid signaling pathways, including TGF-beta -dependent ones, activate IGFBP-3 expression, and blocking one retinoid signaling pathway is not sufficient to alter the effects of t-RA on IGFBP-3 expression. Further, TGF-beta may activate IGFBP-3 expression through intracrine mechanisms, which LAP cannot inhibit. Supporting this possibility, intracrine signaling has been reported to be a mechanism of TGF-beta 1 actions in mammary epithelial differentiation (34).

Prior work in a variety of cell types has demonstrated the importance of the TGF-beta and IGFBP-3 signaling pathways in the biologic effects of t-RA. Blocking antibodies to TGF-beta inhibit the mucous differentiation of hamster tracheal epithelial cells by t-RA and partially abrogate the growth inhibitory effects of t-RA in lymphoma cells, rat prostatic epithelial cells, and keratinocytes (13, 14, 17, 18). In breast cancer cells, t-RA increases IGFBP-3 expression, and antisense IGFBP-3 oligonucleotides abrogate the growth inhibitory effects of t-RA (24). In contrast, LAP did not alter the growth inhibitory effects of t-RA on normal HBE cells (data not shown). These findings suggest that the role of the TGF-beta pathway in cell growth and differentiation depends on the tissue of origin, culture conditions, and transformation state of the cells examined. Additional investigations into the roles of the TGF-beta and IGFBP-3 signaling pathways in the biologic effects of retinoid treatment are warranted.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant R29 CA 67353 and by grants from the University Cancer Foundation and the Physicians Referral Service of the M. D. Anderson Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Dept. of Thoracic/Head and Neck Medical Oncology, M. D. Anderson Cancer Center, Box 80, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-6363; Fax: 713-796-8655.
1   The abbreviations used are: HBE, human bronchial epithelial; t-RA, all-trans-retinoic acid; TGF-beta , transforming growth factor-beta ; MLEC, mink lung epithelial cells; IGFBP-3, insulin-like growth factor binding protein-3; BPE, bovine pituitary extract; LAP, latency-associated peptide; WLB, Western ligand blot; RAR, retinoic acid receptor; RXR, retinoid X receptors.

REFERENCES

  1. Wolbach, S. B., and Howe, P. T. (1925) J. Exp. Med. 42, 753-778
  2. Chopra, D. P. (1982) J. Natl. Cancer Inst. 69, 895-901 [Medline] [Order article via Infotrieve]
  3. Jetten, A. M., Rearick, J. I., and Smits, H. L. (1986) Biochem. Soc. Trans. 14, 930-933 [Medline] [Order article via Infotrieve]
  4. Jetten, A. M., Shirley, J. E., and Stoner, G. (1986) Exp. Cell Res. 167, 539-549 [Medline] [Order article via Infotrieve]
  5. Masui, T., Wakefield, L. M., Lechner, J. F., LaVeck, M. A., Sporn, M. B., and Harris, C. C. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2438-2442 [Abstract]
  6. Willey, J. C., Moser, C. E., Jr., Lechner, J. F., and Harris, C. C. (1984) Cancer Res. 44, 5124-5126 [Abstract]
  7. Saunders, N. A., and Jetten, A. M. (1994) J. Biol. Chem. 269, 2016-2022 [Abstract/Free Full Text]
  8. Jetten, A. M., Brody, A. R., Deas, M. A., Hook, G. E. R., Rearick, J. I., and Thacher, S. M. (1987) Lab. Invest. 56, 654-664 [Medline] [Order article via Infotrieve]
  9. Miller, L. A., Cheng, L. Z., and Wu, R. (1993) Cancer Res. 53, 2527-2533 [Abstract]
  10. Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841-850 [Medline] [Order article via Infotrieve]
  11. Kim, Y.-H., Dohi, D. F., Han, G. R., Zou, C.-P., Oridate, N., Walsh, G. L., Nesbitt, J. C., Xu, X.-C., Hong, W. K., Lotan, R., and Kurie, J. M. (1995) Cancer Res. 55, 5603-5610 [Abstract]
  12. Zhang, L.-X., Mills, K. J., Dawson, M. I., Collins, S. J., and Jetten, A. M. (1994) J. Biol. Chem. 270, 6022-6029 [Abstract/Free Full Text]
  13. Glick, A. B., Flanders, K. C., Danielpour, D., Yuspa, S. H., and Sporn, M. B. (1989) Cell. Regul. 1, 87-97 [Medline] [Order article via Infotrieve]
  14. Niles, R. M., Thompson, N. L., and Fenton, F. (1994) In Vitro Cell. Dev. Biol. Anim. 30, 256-262
  15. Glick, A. B., McCune, B. K., Abdulkarem, N., Flanders, K. C., Lumadue, J. A., Smith, J. M., and Sporn, M. B. (1991) Development 111, 1081-1086 [Abstract]
  16. Nunes, I., Kojima, S., and Rifkin, D. B. (1996) Cancer Res. 56, 495-499 [Abstract]
  17. Danielpour, D. (1996) J. Cell. Physiol. 166, 231-239 [CrossRef][Medline] [Order article via Infotrieve]
  18. Turley, J. M., Funakoshi, S., Ruscetti, F. W., Kasper, J., Murphy, W. J., Longo, D. L., and Birchenall-Roberts, M. C. (1995) Cell Growth & Differ. 6, 655-663 [Abstract]
  19. Nugent, P., Potchinsky, M., Lafferty, C., and Greene, R. M. (1995) Exp. Cell Res. 220, 495-500 [CrossRef][Medline] [Order article via Infotrieve]
  20. Cohen, P. S., Letterio, J. J., Gaetano, C., Chan, J., Matsumoto, K., Sporn, M. B., and Thiele, C. J. (1995) Cancer Res. 55, 2830-2386
  21. Kojima, S., and Rifkin, D. B. (1993) J. Cell. Physiol. 155, 323-332 [Medline] [Order article via Infotrieve]
  22. Batova, A., Danielpour, D., Pirisi, L., and Creek, K. E. (1992) Cell Growth & Differ. 3, 763-772 [Abstract]
  23. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massagué, J. (1994) Nature 370, 341-347 [CrossRef][Medline] [Order article via Infotrieve]
  24. Gucev, Z. S., Oh, Y., Kelly, K. M., and Rosenfeld, R. G. (1996) Cancer Res. 56, 1545-1550 [Abstract]
  25. Zhou, Y., Mohan, S., Linkhart, T. A., Baylink, D. J., and Strong, D. D. (1996) Endocrinology 137, 975-983 [Abstract]
  26. Sheikh, M. S., Shao, Z.-M., Chen, J.-C., Clemmons, D. R., Roberts, C. T., Jr., Leroith, D., and Fontana, J. A. (1992) Biochem. Biophys. Res. Commun. 188, 1122-1130 [Medline] [Order article via Infotrieve]
  27. Sheikh, M. S., Shao, A.-M., Hussain, A., Clemmons, D. R., Chen, J.-C., Roberts, C. T., Jr., LeRoith, D., and Fontana, J. A. (1993) J. Cell. Physiol. 155, 556-567 [Medline] [Order article via Infotrieve]
  28. Leyen, S. A.-V., Hembree, J. R., and Eckert, R. L. (1994) J. Cell. Physiol. 160, 265-274 [Medline] [Order article via Infotrieve]
  29. Adamo, M. L., Shao, Z.-M, Lanau, F., Chen, J. C., Clemmons, D. R., Roberts, C. T., Jr., LeRoith, D., and Fontana, J. A. (1992) Endocrinology 131, 1858-1866 [Abstract]
  30. Martin, J. L., Coverley, J. A., Pattison, S. T., and Baxter, R. C. (1995) Endocrinology 136, 1219-1226 [Abstract]
  31. Katz, J., Weiss, H., Goldman, B., Kanety, H., Stannard, B., LeRoith, D., and Shemer, J. (1995) J. Cell. Physiol. 165, 223-227 [Medline] [Order article via Infotrieve]
  32. Hembree, J. R., Agarwal, C., Beard, R. L., Chandraratna, R. A. S., and Eckert, R. L. (1996) Cancer Res. 56, 1794-1799 [Abstract]
  33. Sheikh, M. S., Shao, Z.-M., Hussain, A., Clemmons, D. R., Chen, J.-C., Roberts, C. T., Jr., LeRoith, D., and Fontana, J. A. (1993) J. Cell. Physiol. 155, 556-567 [Medline] [Order article via Infotrieve]
  34. Kordon, E. C., McKnight, R. A., Jhappan, C., Henninghausen, L., Merlino, G., and Smith, G. H. (1995) Dev. Biol. 168, 47-61 [CrossRef][Medline] [Order article via Infotrieve]
  35. Fontana, J. A., Burrows-Mezu, A., Clemmons, D. R., and LeRoith, D. (1991) Endocrinology 128, 1115-1122 [Abstract]
  36. Cohen, P., Fielder, P. J., Hasegawa, Y., Frisch, H., Giudice, L. C., and Rosenfeld, R. G. (1991) Acta Endocrinol. 124, 74-85 [Medline] [Order article via Infotrieve]
  37. Holly, J. M. P., and Martin, J. L. (1994) Growth Regul. 4, 20-30 [Medline] [Order article via Infotrieve]
  38. Drop, S. L. S., Schuller, A. G. P., Lindenbergh-Kortelve, D. J., Groffen, C., Brinkman, A., and Zwarthoff, E. C. (1992) Growth Regul. 2, 69-79 [Medline] [Order article via Infotrieve]
  39. Swisshelm, K., Ryan, K., Tsuchiya, K., and Sager, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4472-4476 [Abstract]
  40. Valentinis, B., Bhala, A., DeAngelis, T., Baserga, R., and Cohen, P. (1995) Mol. Endocrinol. 9, 361-367 [Abstract]
  41. Conover, C. A. (1992) Endocrinology 130, 3191-3199 [Abstract]
  42. Oh, Y., Muller, H. M., Ng, L., and Rosenfeld, R. G. (1995) J. Biol. Chem. 270, 13589-13592 [Abstract/Free Full Text]
  43. Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7129-7133 [Abstract]
  44. Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, M. B., and Goeddel, D. V. (1985) Nature 316, 701-705 [Medline] [Order article via Infotrieve]
  45. De Martin, R., Haendler, B., Hofer-Warbinek, R., Gaugitsch, H., Wrann, M., Schlusner, H., Seifert, J. M., Bodmer, S., Fontana, A., and Hofer, E. (1987) EMBO J. 6, 3673-3677 [Abstract]
  46. Derynck, R., Lindquist, P. B., Lee, A., Wen, D., Tamm, J., Graycar, J. L., Rhee, L., Mason, A. J., Miller, D. A., Coffey, R. J., Moses, H. L., and Chen, E. Y. (1988) EMBO J. 7, 3737-3743 [Abstract]
  47. Wood, W. I., Cachianes, G., Henzel, W. J., Winslow, G. A., Spencer, S. A., Hellmiss, R., Martin, J. L., and Baxter, R. C. (1988) Mol. Endocrinol. 2, 1176-1185 [Abstract]
  48. Cohen, P., Peehl, D. M., Lamson, G., and Rosenfeld, R. G. (1991) J. Clin. Endocrinol. Metab. 73, 491-407

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