©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirement of Transforming Growth Factor- (TGF-) Type II Receptor for TGF--induced Proliferation and Growth Inhibition (*)

(Received for publication, October 11, 1995; and in revised form, December 12, 1995)

Yun Zhao (§) Stephen L. Young

From the Department of Medicine, Duke University Medical Center and Research Service, Durham Veterans Administration Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Growth regulation of fibroblasts is important for lung development and repair of lung injury. In this study, we investigated the role of transforming growth factor-beta (TGF-beta) type II receptor in the TGF-beta-dependent proliferative response of lung fibroblasts. TGF-beta stimulated the proliferation of adult lung fibroblasts at a low concentration (1 ng/ml), but inhibited the growth of fetal lung fibroblasts in a dose-dependent fashion (0.1-10 ng/ml). Cross-linking and Northern analysis demonstrated that the two lung fibroblast cell lines expressed the TGF-beta type I receptor (TbetaRI) and type II receptor (TbetaRII). We overexpressed in lung fibroblasts a truncated derivative of TbetaRII that lacked the cytoplasmic serine/threonine kinase domain (TbetaRIIDeltaK). TbetaRIIDeltaK was a dominant-negative inhibitor of TGF-beta signal transduction blocking not only TGF-beta-induced mitogenic action upon adult lung fibroblasts but also TGF-beta-induced growth inhibition of fetal lung fibroblasts. The results indicate that the type II receptor is indispensable for mediating both the mitogenic and antiproliferative effects of TGF-beta upon lung fibroblasts.


INTRODUCTION

Transforming growth factor-beta (TGF-beta) (^1)is a family of multifunctional cytokines that regulates cell growth, differentiation, and extracellular matrix deposition(1) . Three isoforms TGF-beta1, -2, and -3 have been identified in lung and found to act on many different lung-derived cell types and to regulate a wide variety of cellular activities(2, 3) . TGF-betas elicit their biological effects on cells through binding to cell surface transmembrane receptors. A number of different types of putative receptors for TGF-beta, including three distinct size classes termed type I (TbetaRI, 50-60 kDa), type II (TbetaRII, 75-85 kDa), and type III (TbetaRIII, a 280-kDa proteoglycan with a 120-kDa core protein), have been identified by affinity cross-linking experiments(4, 5) . Molecular cloning of cDNAs coding type I and type II receptors for the TGF-beta superfamily have shown that both types belong to a novel family of transmembrane serine/threonine kinases with a small extracellular domain, a single transmembrane segment, and an intracellular region with a serine/threonine kinase domain(6, 7, 8) . Sequence analysis of TbetaRIII revealed that it is a transmembrane proteoglycan with a short and highly conserved cytoplasmic domain that has no apparent signal motif (9, 10) . Current evidence indicates that a complex of TbetaRI and TbetaRII, but not the individual components, mediates TGF-beta signal transduction. A ligand-induced heterodimer model was proposed for TGF-beta signal transduction(11, 12) .

TGF-beta may act as either a positive or a negative regulator of cell division. TGF-beta stimulates the proliferation of mesenchyme-derived cells such as fibroblasts and osteoblasts, but acts as a powerful growth inhibitor of cells of epithelial and endothelial origin(13, 14) . TGF-beta inhibits epithelial cell proliferation by delaying or arresting progression through the late portion of G1(15) . TbetaRII was found to be essential for TGF-beta growth inhibition signal. Mv1Lu mink lung epithelial cells are highly responsive to the growth inhibition of TGF-beta. A chemically mutated Mv1Lu cell line defective in TbetaRII lacks TGF-beta-induced growth inhibition. Transfection of the human TbetaRII to this mutant cell line restored the inhibition of growth by TGF-beta(16, 17) . A similar conclusion has been drawn by expression of a kinase-defective truncation of the human TbetaRII in Mv1Lu mink lung epithelial cells(18) .

TbetaRII is required to mediate antiproliferative responses to TGF-beta, but its involvement in TGF-beta signaling that lead to growth stimulation has not been established. In the present studies, we demonstrate a bifunctional action of TGF-beta in lung fibroblast cells. An adult rat lung fibroblast cell line expressed TbetaRII and was responsive to the growth stimulation of TGF-beta, whereas a fetal rat lung fibroblast cell line expressed TbetaRII and displayed an unexpected response, growth inhibition, to TGF-beta. To further determine the role of TbetaRII in modulating the growth stimulation effects of TGF-beta, we transfected into rat lung fibroblast cells an expression plasmid containing rat TbetaRII cDNA that was lacking the kinase domain. Overexpression of the dominant-negative TbetaRII mutant blocked both the stimulating and the inhibitory effects of TGF-beta on rat lung fibroblasts. These experiments provide evidence for a functional role of TbetaRII in TGF-beta-induced proliferation and growth inhibition of lung fibroblasts.


MATERIALS AND METHODS

Cell Cultures

Fetal rat lung fibroblasts were isolated from day 16 gestational age fetal rat lung tissues as described(19) . Adult rat lung fibroblasts isolated from 9-week-old rats were kindly provided by Dr. J. Clarke McIntosh from Duke University Medical Center. These cells exhibited typical fibroblastoid morphology, and they were vimentin-positive and cytokeratin-negative. These lung fibroblasts were also characterized by expression of extracellular matrix protein tenascin and fibronectin(19) . Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. Cultures were grown in humidified 5% CO(2) and 95% air at 37 °C.

Mitogenesis Assay

[^3H]Thymidine incorporation was used to determine TGF-beta sensitivity of fibroblasts to exogenous TGF-beta treatment. Fetal rat lung fibroblasts or adult rat lung fibroblasts were plated in 24-well plates at a density of 2 times 10^4 cells/well in DMEM supplemented with 10% fetal bovine serum. After 48 h of incubation, the cells were serum-starved for 24 h in serum-free medium. Quiescent cultures were then incubated with serum-free DMEM in the presence of various concentration of TGF-beta (0.1 ng/ml-10 ng/ml), as indicated. After receiving a 6-h pulse with 2 µCi/ml of [^3H]thymidine (Amersham Corp.), cells were rinsed with phosphate-buffered saline three times, and twice with 10% trichloroacetic acid, then lysed in 0.1 M NaOH. The amount of [^3H]thymidine incorporated was analyzed by liquid scintillation counting.

Receptor Constructs

A 1762-base pair cDNA containing the full-length coding sequence for TbetaRII was isolated from rat lung as described(20) . A 1542-base pair cDNA containing the full-length coding sequence for TbetaRI was isolated from rat lung by using reverse transcriptase PCR cloning technique with primers (sense: 5`-dACAGTGGCAGCGGGACCAT-3`; antisense: 5`-GAGCAGAGTTCCCACGGTG-3`). TbetaRI cDNA was cloned into plasmid pNoTA (5 Prime 3 Prime, Inc., Boulder, CO) and was characterized by sequencing analysis. Sequencing was carried out in both directions by the dideoxy chain termination method (21) using Sequenase version 2.0 (U. S. Biochemical Corp.) kit and S-dATP (Amersham).

Northern Analysis

Total RNA was from cells prepared by the guanidine thiocyanate/cesium chloride method. Poly(A) RNAs were selected with the PolyATtract® mRNA isolation kit (Promega, Madison, WI). Two µg of mRNA was fractionated on an agarose gel, transferred to Nytran nylon membrane (Schleicher and Schuell) and fixed by a Stratalinker UV cross-linker (Stratagene, La Jolla, CA). Filters were hybridized at 42 °C in 50% formamide solution containing 5 times SSPE (1 times SSPE is 0.18 M NaCl, 10 mM Na(2)HPO(4), and 1 mM EDTA), 1 times Denhart's solution (1 times Denhart's is 0.02% (w/v) each of polyvinylpyrrolidone, bovine serum albumin, and Ficoll), 0.5% SDS, and 0.2 mg/ml of denatured and sonicated fish sperm DNA with 10^6 cpm of P-labeled TbetaRI or TbetaRII cDNA probes. Equivalent RNA loading and transfer were confirmed by subsequent reprobing with a rat glyceraldehyde phosphate dehydrogenase cDNA probe. Filters were washed twice with 2 times SSC (1 times SSC is 0.15 M NaCl, 15 mM trisodium citrate), 0.1% SDS for 15 min at room temperature and finally washed with 0.1 times SSC, 0.1% SDS for 20 min at 60 °C. The filters were autoradiographed. Scanning densitometry (Molecular Dynamics, Sunnyvale, CA) was performed to quantify the relative amounts of mRNA species.

Construction of TbetaRII Dominant-negative Mutant and Transfection

A kinase-defective rat TbetaRIIDeltaK was generated by PCR using primers (sense: 5`-dGCCGGTCTATGACGAGC-3`; antisense: 5`-CCGCTACACCAGCGTGTCCAGCTC-3`). PCR conditions were 1 min at 92 °C, 1 min at 60 °C, and 1 min at 72 °C for 25 cycles. The resulting PCR product was cloned into plasmid pNoTA (5 Prime 3 Prime, Inc.) and was characterized by sequencing analysis. For expression in eukaryotic cells, the truncated TbetaRII cDNA (TbetaRIIDeltaK) or the full-length wild type TbetaRII cDNA was released from pNoTA with restriction enzyme EcoRI and EcoRV, and the fragment of TbetaRII or TbetaRIIDeltaK was subcloned into pcDNA3 (Invitrogen, San Diego, CA). This plasmid contains the cytomegalovirus major intermediate early transcriptional promoter and enhancer. The subcloned TbetaRII or TbetaRIIDeltaK cDNA was verified by restriction enzyme digestion and confirmed by sequence analysis.

Transfection

Cells were plated in DMEM and 5% fetal bovine serum 24 h before transfection. Cells were cultured overnight, and the medium was replaced on the following day by serum-free medium. Transfection of cells with the dominant-negative mutant and wild type construction was performed by using the lipofectamine transfection system (Life Technologies, Inc.). The medium were replaced at 16 h following the start of transfection, and TGF-beta1 was added at the final concentration of 5 ng/ml for fetal lung fibroblasts and 1 ng/ml for adult lung fibroblasts. The control cells were transfected with the cloning plasmid without TbetaRII cDNA. The mammalian reporter vector pCMVbeta (Clontech Laboratories, Inc., Palo Alto, CA), which has the lacZ gene under the transcriptional control of the cytomegalovirus immediate early gene promoter, was used to optimize the transfection conditions.

Receptor Affinity Cross-linking

The affinity labeling of the TGF-beta receptor was carried out according to a procedure described previously(22) . The monolayer cells were washed with binding buffer (DMEM, 2 mg/ml bovine serum albumin, 25 mM HEPES, pH 7.4) and then incubated for 3 h at 4 °C with 100 pM of I-TGF-beta1 (6440 kBq/µg, DuPont NEN) or 100 pM of I-TGF-beta1 plus 80 nM unlabeled TGF-beta1 in binding buffer. Cells were washed three times with binding buffer without bovine serum albumin at 4 °C. Bound I-TGF-beta1 was cross-linked to cell membranes by adding 0.25 mM bis-sulfosuccinimidyl suberate (Pierce) and incubating on ice for 20 min. Cells were harvested and were solubilized in 125 mM NaCl, 10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.3 µM aprotinin, and 1 µM pepstatin. Cross-linked proteins were resolved by SDS-polyacrylamide gel electrophoresis on 12% gels under reducing conditions and autoradiographed.


RESULTS

Growth responses to TGF-beta1 by rat lung fibroblasts were examined. Confluent fetal rat lung fibroblasts and adult rat lung fibroblasts were made quiescent and exposed to different concentration of TGF-beta1. The adult rat lung fibroblast cell line was responsive to the growth stimulation of TGF-beta as expected of most fibroblast cells, whereas fetal rat lung fibroblasts displayed an unexpected response, growth inhibition (Fig. 1). The effects of TGF-beta1 on the proliferation of adult lung fibroblasts were dependent on concentration (Fig. 1A). TGF-beta1 stimulated the proliferation of adult lung fibroblasts at concentration less than 5 ng/ml. Maximal effects were observed at a concentration of 1 ng/ml, and TGF-beta1 had no effect at concentrations of over 5 ng/ml. In contrast, TGF-beta1 inhibited the proliferation of fetal lung fibroblasts in a dose-dependent manner up to 10 ng/ml after a 20-h treatment with TGF-beta1 (Fig. 1B). [^3H]Thymidine incorporation peaked at 24-36 h in adult lung fibroblasts treated with TGF-beta1, but inhibition of DNA synthesis of fetal lung fibroblasts by TGF-beta1 persisted for up to 72 h (data not shown).


Figure 1: Effects of TGF-beta1 on growth of adult lung fibroblast cells (A) and fetal lung fibroblasts (B). Lung fibroblasts were plated at a density of 2 times 10^4 cells/well in a 24-well plate and were made quiescent in serum-free DMEM for 24 h. Cells were treated with the indicated concentration of TGF-beta1 for 20 h and then labeled with [^3H]thymidine for 6 h. DNA was precipitated with 10% trichloroacetic acid, and the amount of [^3H]thymidine incorporated was analyzed by liquid scintillation counting. Each value represents the average of three separate cultures measured for each TGF-beta1 concentration used. Error bars are S.E.



In order to elucidate a possible mechanism whereby TGF-beta1 exerts its actions on fibroblast proliferation, the expression of TGF-beta receptors by adult and fetal lung fibroblasts were evaluated. Cross-linking of I-TGF-beta1 to fetal lung fibroblasts or adult lung fibroblasts revealed three species of receptors with apparent molecular masses of 60, 85 and 280 kDa (Fig. 2A). These proteins were equivalent to the TGF-beta type I, II, and III receptors, respectively. A 40-kDa component was seen in fetal lung fibroblasts, but not in adult lung fibroblasts. It is not clear whether it was an isoform of the TGF-beta type I receptor or an uncharacterized TGF-beta binding protein. A lower molecular mass species (35 kDa) was also detected in both adult and fetal lung fibroblasts.


Figure 2: Expression of endogenous TGF-beta receptors in lung fibroblasts. A, receptor cross-linking assays were used to measure cell surface TGF-beta receptor expression. Monolayer cultures of adult lung fibroblast cells (lane 1) or fetal rat lung fibroblasts (lane 2) in six-well plates were incubated with 100 pM of I-TGF-beta1. The receptors were cross-linked with disuccinimidyl suberate. Cell extracts were resolved on a SDS-polyacrylamide gel under reducing conditions. The TGF-beta receptor type III (TbetaRIII), type II (TbetaRII), and type I (TbetaRI) were visualized after autoradiography; B, Northern blotting was used to analyze expression of TbetaRI and TbetaRII mRNAs. Each lane contained 2 µg of mRNA prepared from adult lung fibroblasts (lane 1) or fetal lung fibroblasts (lane 2). mRNA was subjected to electrophoresis through a formaldehyde denaturing agarose gel and, after Northern blotting, hybridized with radiolabeled TbetaRI or TbetaRII cDNA probe and quantified by scanning densitometry. Scan values for TbetaRI and TbetaRII mRNA signals were normalized with the scan data of glyceraldehyde phosphate dehydrogenase. Ratios were expressed in arbitrary units.



The expression of TGF-beta type I and II receptor were further examined by Northern blot analysis (Fig. 2B). TbetaRII mRNA was detected as a 5.1-kilobase species expressed in adult lung fibroblasts and in fetal lung fibroblasts. Two TbetaRI mRNA species of approximately 6.1 and 4.0 kilobases were observed in adult lung fibroblasts and in fetal lung fibroblasts. TbetaRII and TbetaRI were differentially expressed in fetal and adult lung fibroblasts.

To examine the significance of type II receptor in mediating the growth-promoting and growth inhibition effects of TGF-beta, we used a dominant-negative inhibitory approach to create a loss-of-function mutation of TbetaRII. We constructed a kinase-deficient cytoplasmic deletion mutant of rat TbetaRII (TbetaRIIDeltaK). The truncated TbetaRII and wild type TbetaRII were cloned into pcDNA3, under the transcriptional control of the cytomegalovirus immediate early gene promoter and enhancer. We transfected rat lung fibroblasts with rat TbetaRIIDeltaK or TbetaRII. The expression level of the truncated receptor was tested by affinity labeling of transfected cells with I-TGF-beta1 (Fig. 3). Fibroblasts transfected with TbetaRIIDeltaK yielded a TGF-beta affinity-labeled product of 45 kDa, the predicted size of the truncated receptor TbetaRII. A high level of TbetaRIIDeltaK were expressed on the cell surface of fetal and adult lung fibroblasts. The truncated receptor was able to bind ligand. I-TGF-beta1 binding to TbetaRIIDeltaK was efficiently competed by unlabeled ligand, the same as the wild type TbetaRII binding.


Figure 3: Expression of the truncated TbetaRII lacking the cytoplasmic kinase domain in lung fibroblasts. Monolayer cultures of fetal rat lung fibroblasts or adult rat lung fibroblasts transiently transfected with rat TbetaRIIDeltaK or empty pCDNA3 vector were incubated with I-TGF-beta1 in the absence(-) or in the presence (+) of 80 nM unlabeled TGF-beta1. Bound I-TGF-beta1 was cross-linked with disuccinimidyl suberate. Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and then were autoradiographed. Lanes 1, 2, 5, and 6, fetal lung fibroblasts; lanes 3, 4, 7, and 8, adult lung fibroblasts.



[^3H]-thymidine incorporation was measured to assess the proliferation rate of lung fibroblasts expressing the truncated TbetaRII. Adult lung fibroblasts were transfected with TbetaRIIDeltaK or empty vector. Transfected fibroblasts were cultured in the absence or presence of TGF-beta1 (1 ng/ml) for 20 h. TbetaRIIDeltaK completely blocked the TGF-beta1-dependent DNA synthesis (Fig. 4A). When adult lung fibroblasts were transfected with 1 µg of TbetaRIIDeltaK and 1 µg of wild type rat TbetaRII, the growth responsiveness of adult lung fibroblasts to TGF-beta1 was restored by adding the wild type TbetaRII cDNA (Fig. 4B). Fetal lung fibroblasts transfected with TbetaRIIDeltaK were unable to convey TGF-beta1-dependent growth inhibition (Fig. 5A). Similarly, the diminished response to TGF-beta1 by fetal fibroblasts was rescued by cotransfection of TbetaRIIDeltaK with the wild type TbetaRII (Fig. 5B). The results show that the kinase-defective deletion of TbetaRII is a dominant-acting inhibitor of signal transduction by TGF-beta receptor complex and that TbetaRII was required for TGF-beta-dependent positive and negative growth response.


Figure 4: Dominant-negative effects of TbetaRII on TGF-beta-induced growth. A, adult lung fibroblasts were transfected with TbetaRIIDeltaK or empty vector. Transfected fibroblasts were cultured in the absence or presence of TGF-beta1 (1 ng/ml) for 20 h. DNA synthesis was assayed by measuring [^3H]thymidine incorporation; B, restoration of TGF-beta1-dependent growth response. Adult lung fibroblasts were transfected with 1 µg of TbetaRIIDeltaK and 1 µg of wild type rat TbetaRII. The cells were treated with TGF-beta1 for 20 h and then assayed for DNA synthesis. The bars represent the means ± S.E. (n = 3).




Figure 5: Dominant-negative effects of TbetaRII on TGF-beta-induced antiproliferative response. A, fetal lung fibroblasts were transfected with TbetaRIIDeltaK or empty vector. Transfected fibroblasts were cultured in the absence or presence of TGF-beta1 (5 ng/ml) for 20 h. DNA synthesis was assayed by measuring [^3H]thymidine incorporation; B, restoration of TGF-beta1-dependent growth inhibition. Fetal lung fibroblasts were cotransfected with 1 µg of TbetaRIIDeltaK and 1 µg of wild type rat TbetaRII. The cells were treated with TGF-beta1 for 20 h and then assayed for DNA synthesis. The bars represent the means ± S.E. (n = 3).




DISCUSSION

Proliferation of fibroblasts is an important aspect of lung development and is also a key feature of repair of lung injury. Expression of TGF-beta isoforms has been detected in the lung at critical times during development(2, 23) . The expression pattern and known biological activities of TGF-beta suggest an important role for this factor in lung development. In this study, the effects of TGF-beta on growth of fetal and adult lung fibroblasts were characterized, and the role of TbetaRII in TGF-beta-induced signaling was examined.

Our data show that TGF-beta stimulates proliferation of adult fibroblasts at low concentration but remains inactive at higher concentration, while inhibiting growth of fetal fibroblasts regardless of concentration. An example of the bifunctional nature of TGF-beta has been reported in human smooth muscle cells(24) . TGF-beta is mitogenic only when used at lower concentration, whereas at higher concentration it inhibits DNA synthesis. More recent studies have demonstrated that TGF-beta can act as a negative or positive growth regulator in two sublines of the same epithelial cells, depending on their commitment to differentiation(25) . TGF-beta activates two different signal transduction pathways through activating different Ras proteins and myelin basic protein kinases(26) .

We examined the possible relationship between the bifunctional action of TGF-beta and the expression of its cellular receptors. The two fibroblast cell lines possess all three TGF-beta receptors; however, fetal fibroblasts expressed a much higher level of cell surface type II receptor at the mRNA and the protein level than adult lung fibroblasts. The finding is in agreement with our previous study (20) that the expression of TbetaRII is developmentally regulated in the lung. This raises the possibility that the bifunctional action of TGF-beta on lung fibroblasts may depend on their developmental stage.

We show that the kinase-deleted truncation of TbetaRII acts as a dominant inhibitor of TGF-beta signal transduction. The proliferative action and the growth inhibition of TGF-beta were abolished by overexpression of TbetaRIIDeltaK. TGF-beta-induced signaling events leading to growth stimulation are likely to be different at some level from that of growth inhibition. TGF-beta increases c-fos expression in growth-inhibited cells but not in growth-stimulated cells (27) . An increase in retinoblastoma protein phosphorylation is seen in TGF-beta-induced proliferation(28) . However, our results indicated TbetaRII is essential for both pathways. This finding suggests that binding of TGF-beta to TbetaRII may be a common step required for the TGF-beta signaling cascade. The TGF-beta signal transduction pathways for the growth stimulation and the growth inhibition may diverge after binding of TGF-beta to TbetaRII and then different intracellular signals might be activated. An intriguing question raised by this study is how TGF-beta exerts its multiplicity of effects through interaction with its transmembrane receptors. The signaling specificity could occur through interaction of TbetaRII with different types of type I receptor. The characterization of other members of TGF-beta receptor family, particularly type I receptors, might allow us to determine the mechanism of multiple effects of TGF-beta.

In conclusion, we found that TGF-beta stimulates proliferation of adult lung fibroblasts, while inhibiting growth of fetal lung fibroblasts. We have shown that the truncated form of TbetaRII blocked both growth-stimulating and inhibition effects of TGF-beta. The results suggest that TGF-beta type II receptor is indispensable for the signal transduction pathway and the diverse regulatory effects of TGF-beta.


FOOTNOTES

*
This work was supported by the Department of Veterans Affairs and National Institutes of Health Grant HL32188. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of the Clifford W. Perry Research Award from American Lung Association of North Carolina. To whom correspondence should be addressed: P. O. Box 3177, Dept. of Medicine, Duke University Medical Center, Durham, NC 27710. Fax: 919-286-6824; :zhaoyun{at}acpub.duke.edu.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; TbetaRI, transforming growth factor-beta type I receptor; TbetaRII, transforming growth factor-beta type II receptor; TbetaRIII, transforming growth factor-beta type III receptor; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Rob Silbajoris and Erick Larson for technical support. We also thank Dr. Jo Rae Wright for her critical reading of the manuscript.


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