Ontogeny and localization of TGF-beta type I receptor expression during lung development

Yun Zhao1,2, Stephen L. Young1,2, J. Clarke McIntosh3, Mark P. Steele1, and Robert Silbajoris2

1 Department of Medicine and 3 Department of Pediatrics, Duke University Medical Center, and 2 Research Service, Durham Veterans Affairs Medical Center, Durham, North Carolina 27710


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor (TGF)-beta is a family of multifunctional cytokines controlling cell growth, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-beta are mediated by type I (Tbeta R-I) and II (Tbeta R-II) receptors. Our previous studies show that the expression of Tbeta R-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of Tbeta R-I during lung development. The expression level of Tbeta R-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. Tbeta R-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of Tbeta R-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. Tbeta R-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense Tbeta R-I signal was observed in the epithelial lining of the developing bronchi. In gestational day 16 lungs, the expression of Tbeta R-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of Tbeta R-I expression. In postnatal lungs, Tbeta R-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of Tbeta R-I in cultured rat lung cells. Tbeta R-I was expressed by cultured rat lung fibroblasts, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of Tbeta R-I that is different from that of Tbeta R-II during lung development. Tbeta R-I, Tbeta R-II, and TGF-beta isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for Tbeta R-I in mediating TGF-beta signal transduction during lung development.

transforming growth factor-beta ; mesenchyme; epithelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR (TGF)-beta is a family of multifunctional cytokines that is involved in controlling many biological activities including cell growth, differentiation, extracellular matrix deposition, and cell cycle progression in a variety of cell types (14, 19). The biological effects of TGF-beta are known to be exerted on cells through interaction of the ligands with their specific transmembrane receptors. A number of different types of putative receptors for TGF-beta , including three distinct size classes termed type I (Tbeta R-I; 50-60 kDa), type II (Tbeta R-II; 75-85 kDa), and type III (Tbeta R-III; a 280-kDa proteoglycan with a 120-kDa core protein), have been identified by affinity cross-linking experiments (3, 20). Sequence analysis of Tbeta R-III revealed that it is a transmembrane proteoglycan with a short and highly conserved cytoplasmic domain that has no apparent signal motif (17, 34). Tbeta R-I and Tbeta R-II are transmembrane serine/threonine kinases that consist of a short extracellular domain, a single transmembrane region, and a kinase domain (9, 16). Although Tbeta R-I and Tbeta R-II share similar domain structures, Tbeta R-I distinguishes itself from Tbeta R-II by several features. Tbeta R-I has a unique highly conserved GS motif (SGSGSG) in the juxtamembrane region immediately preceding the kinase domain. Ligand-induced phosphorylation of the serines and threonines in the GS domain is required for activation of signaling (31). After the GS sequence, Tbeta R-I has an LP motif. The immunophilin 12-kDa FK506 binding protein binds the LP sequence of Tbeta R-I, inhibiting TGF-beta signaling (5). Tbeta R-I also has a short extracellular domain and essentially no COOH-terminal extension after the kinase domain.

Both Tbeta R-I and Tbeta R-II are indispensable for TGF-beta signaling (2, 9, 16). Tbeta R-II is capable of binding TGF-beta independently, but binding of Tbeta R-I to TGF-beta requires the presence of Tbeta R-II. Once Tbeta R-II binds to TGF-beta , it results in the recruitment of Tbeta R-I to form a heteromeric complex. Activated Tbeta R-II transphosphorylates Tbeta R-I kinase, thereby activating Tbeta R-I, which then propagates the signal to downstream substrates (33, 36). Recent studies (8, 15) revealed that Mothers against dpp (Mad) in Drosophila and its homologs play important roles in the intracellular signal transduction of the serine/threonine kinase receptors. TGF-beta induces heteromeric complexes of Smad2, Smad3, and Smad4 and their concomitant translocation to the nucleus, which is required for TGF-beta signal transduction (21, 25).

Lung branching morphogenesis and alveolarization are a complex process of reciprocal interactions between epithelial and mesenchymal tissue that require precise regulatory control. A regulatory role for TGF-beta in the developing lung has been implicated because the TGF-beta family is composed of highly conserved cytokines with demonstrated pleiotropic effects on their own biosynthesis, mesenchymal mitogenesis, and epithelial differentiation. Tbeta R-II and Tbeta R-I have been shown to be required for growth regulation and extracellular matrix production by TGF-beta in lung fibroblasts (39, 41). Until the question of cellular localization of its receptors is resolved, the mechanism by which TGF-beta may regulate lung development will remain speculative. A previous study by Zhao and Young (40) showed that expression of the Tbeta R-II is temporally and spatially regulated during rat lung development (40). In an effort to gain further clues about the function and mechanism of TGF-beta signaling during lung development, we examined the temporal expression of Tbeta R-I by Northern blot analysis and the spatial localization of Tbeta R-I mRNA by in situ hybridization of rat embryo and rat lung tissue. Tbeta R-I mRNA shows a distinctive developmental localization and is highly regulated during lung development. These studies provide direct evidence for differential expression and localization of Tbeta R-I compared with Tbeta R-II during rat lung development.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and tissue preparation. Timed-pregnant Sprague-Dawley rats were purchased from Zivic Miller (Allison Park, PA). At least two and as many as five separate litters of donor animals or adults (7-8 wk of age) were used for analysis at each time point. The lungs were removed while the animals were under pentobarbital sodium (50 mg/kg) anesthesia, frozen in liquid nitrogen, and stored at -70°C. Lung tissues were fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated through graded ethanol, and embedded in paraffin. Five-micrometer-thick sections were cut and mounted on 3-aminopropyltriethoxysilane (Sigma, St. Louis, MO)-treated glass slides.

Cell lines and culture techniques. Fetal rat lung fibroblasts were isolated from gestational day 16 fetal rat lung tissue by culture of tissue explants in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal calf serum. After two passages, a confluent, homogeneous monolayer of cells with fibroblast morphology was present. These cells were positive for vimentin and negative for cytokeratin by immunocytochemistry. Adult rat lung fibroblasts were isolated from 9-wk-old rats. These cells were in approximately the 12th to 15th passages and were derived from lung tissue that had been minced and pressed through a 100-mesh screen before culture. After three passages, a confluent, homogeneous population of cells with a fibroblast morphology was obtained. These cells were vimentin positive and cytokeratin negative. Rat lung microvascular endothelial cells isolated from adult rats with the technique of Magee et al. (18) were a generous gift from Dr. Andrew Canada (Duke University Medical Center, Durham, NC). A rat lung alveolar epithelial cell line was derived from rat primary neonatal type II cells by transfection with SV40 large T antigen. These epithelial cells were cytokeratin positive and demonstrated serum-dependent proliferation. The cells were maintained in DMEM with 10% fetal calf serum. All media were supplemented with penicillin and streptomycin. Cultures were grown in humidified 5% CO2 and 95% air at 37°C.

RNA extraction and Northern blot analysis. Total RNA was prepared from lung tissues and cells essentially with the method of Chirgwin et al. (6) by extracting tissue with 4 M guanidinium thiocyanate and pelleting through a cushion of cesium chloride. The cDNAs for rat Tbeta R-I and Tbeta R-II were isolated from rat lung as previously described (40). The cDNAs for rat TGF-beta 1 (28), mouse TGF-beta 2 (23), and mouse TGF-beta 3 (7) were obtained from the American Type Culture Collection (Manassas, VA). Poly(A)+ RNAs were selected with the PolyATtract mRNA isolation kit (Promega, Madison, WI). The integrity and quantity of the RNA were evaluated by ultraviolet spectrophotometry and denaturation of agarose gels stained with ethidium bromide. Two micrograms of mRNA were fractionated on an agarose gel, transferred to Biotrans nylon membrane (ICN, Irvine, CA), and fixed in a Stratalinker UV cross-linker (Stratagene, La Jolla, CA). Filters were hybridized at 42°C in 50% formamide solution containing 5× saline-sodium phosphate-EDTA (SSPE; 1× SSPE is 0.18 M NaCl, 10 mM Na2HPO4, and 1 mM EDTA), 5× Denhardt's solution [5× Denhardt's is 0.1% (wt/vol) each of polyvinylpyrrolidone, bovine serum albumin, and Ficoll], 0.1% SDS, and 0.1 mg/ml of denatured and sonicated salmon sperm DNA with 106 counts/min of 32P-labeled Tbeta R-I, Tbeta R-II, TGF-beta 1, TGF-beta 2, or TGF-beta 3 probe. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was used as a control. Filters were washed twice with 2× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 15 mM trisodium citrate) containing 0.1% SDS for 15 min at room temperature and finally washed with 0.1× SSC containing 0.1% SDS for 20 min at 60°C. The filters were autoradiographed.

Reverse transcription-polymerase chain reaction. We isolated lungs from 25 gestational day 14 rat fetuses. Reverse transcription-polymerase chain reaction (RT-PCR) assay was performed as described earlier (38). Briefly, cDNA was synthesized from total RNA primed with oligo(dT)12-18 and reverse transcribed in a final volume of 20 µl with Superscript II reverse transcriptase (GIBCO BRL, Life Technologies, Grand Island, NY). The target sequences were amplified by PCR in a final volume of 50 µl. PCR cycling conditions were 28 cycles consisting of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a 5-min final extension at 72°C. The PCR product was analyzed by agarose gel electrophoresis.

The primers for Tbeta R-I were 5'-TGGTCTTGCCCATCTTCACA-3' (sense) and 5'-ATTGCATAGATGTCAGCACG-3' (antisense), which yielded a PCR product of 279 bp (11). The primers for Tbeta R-II were 5'-TTGTGGGAGGCCCAAGATGC-3' (sense) and 5'-TGGTTGAGCCAGAAGCTGGG-3' (antisense), which yielded a PCR product of 420 bp (40). The primers for beta -actin were 5'-TGACGAGGCCCAGAGCAAGA-3' (sense) and 5'-ATGGGCACAGTGTGGGTGAC-3' (antisense), which yielded a PCR product of 330 bp (1).

In situ hybridization. Sections of paraformaldehyde-fixed and paraffin-embedded rat lungs were subjected to in situ hybridization with 35S-cRNA. Sense and antisense cRNA riboprobes were generated from rat Tbeta R-I cDNA subcloned into pGEM-7Zf (Promega). The plasmid was linearized with restriction enzymes. cRNA was obtained in the sense and antisense orientation by transcribing from T7 and SP6 promoters, respectively, with the riboprobe transcription kit (Stratagene). Riboprobes were labeled with 35S-UTP (Amersham, Arlington, IL). Primary transcripts were reduced to an average fragment length of 200 bp by alkaline hydrolysis. Sections were rehydrated, fixed in 4% paraformaldehyde in PBS, and treated with proteinase K. The sections were treated again with 4% paraformaldehyde in PBS and acetylated with acetic anhydride. After dehydration through ethanol, the sections were prehybridized in hybridization solution containing 50% formamide, 0.3 M NaCl, 20 mM Tris · HCl, pH 8.0, 5 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 500 µg/ml of tRNA, and 100 mM dithiothreitol. Hybridization was performed with 2-5 × 107 counts · min-1 · ml-1 of 35S-labeled cRNA probe in hybridization solution (with 10% dextran sulfate) and incubated overnight at 55°C. The slides were washed with 5× SSC containing 10 mM dithiothreitol at 37°C, followed by washing with 2× SSC at 65°C, and then treated with RNase A. Washing was then continued with 2× SSC at 65°C and 0.1× SSC at 37°C. Sections were next dehydrated, dried, and dipped in Kodak NTB-2 autoradiographic emulsion. After exposure at 4°C for 1-2 wk, the slides were developed and counterstained with hematoxylin and eosin.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Detection of Tbeta R-I mRNA in developing rat lung by Northern blot analysis. Developmental expression of Tbeta R-I was studied in rat lungs. We isolated RNA from embryonic rat lungs of day 14 and day 16 gestational ages and performed RT-PCR analysis. We detected both Tbeta R-I and Tbeta R-II expression in gestational day 14 and day 16 rat lungs, and the level of Tbeta R-I and Tbeta R-II expression in gestational day 14 rat lung was slightly lower than that in day 16 rat lung (Fig. 1). We analyzed Tbeta R-I mRNA levels in rat lungs at different embryonic and postnatal stages by Northern blotting (Fig. 2). A 6.1-kb Tbeta R-I transcript was detected in rat lung tissue. The level of expression of Tbeta R-I mRNA changed significantly during lung development. Tbeta R-I mRNA was detected in rat fetal lung tissues early in development, reached maximal concentration at 16 days of gestation, and then decreased as development proceeded. Tbeta R-II showed a different expression pattern from that of Tbeta R-I over the same developmental times. Tbeta R-II mRNA was increased as development progressed, reached maximal concentration in the early postnatal period, and then decreased.


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Fig. 1.   RT-PCR analysis of transforming growth factor-beta receptor (Tbeta R) in RNA isolated from embryonic rat lung tissues. Total RNA was isolated from gestational day (Gd) 14 and Gd16 rat lungs. PCR was performed with primers specific for Tbeta R type I (Tbeta R-I), Tbeta R type II (Tbeta R-II), and beta -actin, and products were analyzed on a 1.2% agarose gel. Std, standard molecular-weight markers.



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Fig. 2.   Northern blot analysis of Tbeta R-I and Tbeta R-II mRNAs in developing rat lung tissue. Poly(A)+ RNA (2 µg) isolated from lung was subjected to electrophoresis through a formaldehyde-denaturing 0.8% agarose gel and after Northern blotting was hybridized with a radiolabeled Tbeta R-I or Tbeta R-II cDNA probe and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for loading (left). Nos. on right, size of the transcripts. Pd, postnatal day. Band densities were quantified by scanning densitometry (right). Scan values for Tbeta R-I and Tbeta R-II mRNA signals were normalized with scan data for GAPDH. Ratios are expressed in arbitrary units.

The level of TGF-beta 1, TGF-beta 2, and TGF-beta 3 mRNA expression in rat lungs at different embryonic and postnatal stages was also analyzed by Northern blotting (Fig. 3). All three TGF-beta isoforms were expressed during lung development. One transcript (2.6 kb) was detected for TGF-beta 1, whereas five transcripts (5.9, 4.8, 4.1, 3.3, and 2.2 kb) for TGF-beta 2 and two transcripts (3.6 and 2.4 kb) for TGF-beta 3 were seen. There was a distinct temporal difference among TGF-beta 1, TGF-beta 2, and TGF-beta 3 expression. Expression of TGF-beta 1 was low at early gestational days when the same samples showed high expression of TGF-beta 2 and TGF-beta 3. However, TGF-beta 1 mRNA was subsequently increased, reached maximal concentration in the early postnatal period, and then decreased. TGF-beta 2 and TGF-beta 3 were expressed in similar patterns during lung development. Expression of TGF-beta 2 and TGF-beta 3 was high in rat fetal lung tissues early in development, decreased near birth, and then increased as development proceeded. GAPDH was used as a control for equal loading.


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Fig. 3.   Northern blot analysis of TGF-beta isoforms in developing rat lung tissue. Poly(A)+ RNA (2 µg) isolated from lung was subjected to electrophoresis through a formaldehyde-denaturing 0.8% agarose gel and after Northern blotting was hybridized with radiolabeled TGF-beta 1, TGF-beta 2, or TGF-beta 3 cDNA probe and reprobed with a GAPDH cDNA as a control for loading (left). Nos. on right, size of transcripts. Band densities were quantified by scanning densitometry (right). Scan values for TGF-beta 1, TGF-beta 2, and TGF-beta 3 mRNA signals were normalized with scan data for GAPDH. Ratios are expressed in arbitrary units.

Localization of Tbeta R-I in fetal and postnatal rat lung tissues by in situ hybridization. The spatial localization of Tbeta R-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with antisense cRNA probes. Figure 4 shows in situ hybridization of Tbeta R-I mRNA in a gestational day 14 rat embryo. Tbeta R-I mRNA was observed in the mesenchyme and epithelium of gestational day 14 rat lungs. The most prominent labeling signal of Tbeta R-I was noted in the developing epithelium.


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Fig. 4.   In situ hybridization of Tbeta R-I mRNA in Gd14 rat fetal lung tissue. Gd14 rat fetal lung was hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe, and sections were counterstained with hematoxylin and eosin for demonstration of morphology. A: bright-field photomicrograph. B: dark-field photomicrograph of the same area in A. Tbeta R-I mRNA was observed in mesenchymal tissue and epithelial cells. The most prominent labeling signal of Tbeta R-I was noted in the epithelial lining of developing bronchi. A and B are at the same magnification; bar in A, 25 µm.

In day 16 gestational age rat lung tissue, Tbeta R-I mRNA was expressed in the mesenchymal tissue of the future parenchyme and in the epithelial lining of the developing airway (Fig. 5). The hybridization signal of Tbeta R-I mRNA in the mesenchymal tissue was intensified compared with that on gestational day 14, and the expression of Tbeta R-I remained high in the epithelium of the growing airway. Both the undifferentiated airway epithelial cells of the small distal branches (Fig. 5, A and B) and the differentiated airway epithelial cells of the proximal branches (Fig. 5, C and D) had similar levels of Tbeta R-I expression.


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Fig. 5.   In situ hybridization of Tbeta R-I mRNA in Gd16 rat fetal lung tissue. Gd16 rat fetal lung was hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe, and sections were counterstained with hematoxylin and eosin for demonstration of morphology. A: bright-field photomicrograph of distal epithelium of developing airway (solid arrows) hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe. star , Pleural border. B: dark-field photomicrograph of the same area in A. C: bright-field photomicrograph of proximal epithelium of developing airway (open arrows) hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe. D: dark-field photomicrograph of the same area in C. Hybridization signal of Tbeta R-I mRNA in mesenchymal tissue was intensified, and expression of Tbeta R-I remained high in the epithelium of both the distal bronchioles and the proximal conducting airway of the lung. All photomicrographs are at the same magnification; bar in A, 5 µm.

Figure 6, A and B, shows in situ hybridization of Tbeta R-I mRNA in newborn rat lung tissue. The presence of Tbeta R-I mRNA in alveolar epithelial cells and blood vessels was detected. Tbeta R-I mRNA was observed in the smooth muscle surrounding the endothelium and adventitial layer of blood vessels. Figure 6C shows a higher magnification photomicrograph of a blood vessel hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe. The expression of Tbeta R-I mRNA was detectable in smooth muscle cells and the endothelium. The absence of hybridization in the erythrocytes within blood vessels confirmed the specificity of the hybridization signal of Tbeta R-I. Tbeta R-I was also observed in the interalveolar septa of the lung (Fig. 6D). The localization of Tbeta R-I mRNA in postnatal day 3 rat lung is showed in Fig. 7. Tbeta R-I mRNA was detected in the parenchymal tissues and blood vessels. A pattern similar to the newborn lung expression of Tbeta R-I was observed. Hybridization of rat lung with Tbeta R-I sense probe revealed no signal (Fig. 7D).


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Fig. 6.   In situ hybridization of Tbeta R-I mRNA in newborn rat lung tissue. Sections of newborn rat lung were hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe, and sections were counterstained with hematoxylin and eosin for demonstration of morphology. A: bright-field photomicrograph. B: dark-field photomicrograph of the same area in A. C: higher-magnification photomicrograph of a blood vessel hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe. D: higher-magnification photomicrograph of parenchymal tissues of developing airway hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe. Tbeta R-I was expressed throughout the developing lung parenchyma. Tbeta R-I mRNA was detected in alveolar epithelial cells and blood vessels. Hybridization signal of Tbeta R-I mRNA was also observed in smooth muscle sheath surrounding the endothelium and adventitial layer of small blood vessels. A significant level of Tbeta R-I expression was noted in the cells of the interstitium. Absence of hybridization signal in red blood cells within blood vessels confirmed the specificity of the hybridization of Tbeta R-I. A and B are at same magnification; bar in A, 2.5 µm. C and D are at same magnification; bar in C, 1 µm.



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Fig. 7.   In situ hybridization of Tbeta R-I mRNA in Pd3 rat lung tissue. Pd3 rat lung was hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe, and sections were counterstained with hematoxylin and eosin for demonstration of morphology. A: bright-field photomicrograph. B: dark-field photomicrograph of the same area in A. C: higher-magnification photomicrograph of the interstitium of the lung hybridized with a 35S-UTP-labeled antisense Tbeta R-I cRNA probe. Tbeta R-I is detected throughout the lung parenchyma. Tbeta R-I was seen in interalveolar septa and alveolar epithelial cells. D: no labeling is seen with sense probe. A and B are at same magnification; bar in A, 2.5 µm; bar in C, 1 µm; bar in D, 2.5 µm.

Expression of Tbeta R-I by cultured lung cells. To investigate the cell-type expression of Tbeta R-I in vitro, we examined the expression of Tbeta R-I by four lung-derived stable cell lines (Fig. 8). Tbeta R-I mRNA was detected as a 6.1-kb species expressed in adult rat lung microvascular endothelial cells, alveolar epithelial cells, fetal lung fibroblasts, and adult lung fibroblasts. Two Tbeta R-I mRNA species of ~6.1 and 4.0 kb were observed in lung microvascular endothelial cells and fetal lung fibroblasts.


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Fig. 8.   Northern blot analysis of Tbeta R-I in RNA isolated from rat lung-derived cell lines. Each lane contained 2 µg of mRNA prepared from adult rat lung microvascular endothelial cells, alveolar epithelial cells, fetal lung fibroblasts, and adult lung fibroblasts. mRNA was subjected to electrophoresis through a formaldehyde-denaturing agarose gel and after Northern blotting was hybridized with radiolabeled Tbeta R-I probe. A loading control was done by hybridization of the same blot with a cDNA to another gene, GAPDH. Lane 1, rat lung microvascular endothelial cell line cultured from adult rats; lane 2, alveolar epithelial cell line derived from rat primary neonatal type II cells by transfection with SV40 large T antigen; lane 3, fetal lung fibroblast cell line isolated from Gd16 fetal rat lung tissue; lane 4, adult lung fibroblast cell line isolated from 9-wk-old rats. Nos. on right, size of transcripts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism of lung development involves epithelial-mesenchymal interactions, which are spatially and temporally controlled. TGF-beta is a family of multifunctional cytokines that are involved in lung development. Overexpression of TGF-beta 1 in the developing respiratory epithelium of transgenic mice inhibits lung sacculation and epithelial cell differentiation, indicating a role for TGF-beta 1 in lung morphogenesis and differentiation (42). TGF-beta 2 knockout mice show postnatal defects in the conducting airways of the lung but have no gross morphological defects in prenatal lungs (30). The TGF-beta 3 knockout, in contrast to the TGF-beta 1 and TGF-beta 2 knockouts, produced a profound inhibition of lung development and was lethal at birth due to respiratory insufficiency (13). Tbeta R-I and Tbeta R-II have been identified as the signaling receptors for TGF-beta . Zhao and Young (40) previously demonstrated that the expression of Tbeta R-II is spatially and temporally regulated during rat lung development. To gain further clues about the function and mechanism of TGF-beta signaling during lung development, we examined the temporal and spatial expression of Tbeta R-I during lung development.

The expression of Tbeta R-I mRNA was found in mesenchymal tissues and epithelium at all stages of lung development, which overlapped the expression of Tbeta R-II (40). The finding that Tbeta R-I is ubiquitously expressed in the lung was anticipated because TGF-beta has been found to act on many different cell types and to regulate a wide variety of cellular activities in the lung (26), and both Tbeta R-I and Tbeta R-II are directly involved in receptor signal transduction (4, 36). Expression of a truncated Tbeta R-II lacking the intracellular kinase domain suppresses cell growth signal by TGF-beta in Mv1Lu mink lung epithelial cells (4, 35) and in lung fibroblasts (41). Both Tbeta R-I and Tbeta R-II have been shown to be required for the regulation of extracellular matrix production in rat lung fibroblasts by TGF-beta (39).

The expression of Tbeta R-I by four lung-derived stable cell lines was examined. Tbeta R-I mRNA was expressed in adult rat lung microvascular endothelial cells, alveolar epithelial cells, fetal lung fibroblasts, and adult lung fibroblasts. Numerous in vitro studies show that TGF-beta elicits a multiplicity of effects in various lung-derived cell types (reviewed in Ref. 26). The expression of TGF-beta receptors by these lung-derived cell types, together with those previously reported in vitro studies of TGF-beta biological effects on lung-derived cell types, suggests that TGF-beta s and TGF-beta receptors might be needed for the ongoing regulation of growth in the normal function of mature lung cells beyond the fetal period.

TGF-beta was initially named by its ability to promote anchorage-independent growth and a transformed phenotype in nontumorigenic mesenchymal cells (24, 29). The localization of Tbeta R-I in mesenchymal cells of the developing lung supports a role for TGF-beta signaling in regulating the growth of embryonic lung connective tissues during development. In addition to mediating signals controlling the growth of lung connective tissue, Tbeta R-I is also involved in TGF-beta signaling that regulates the synthesis of extracellular matrix components in lung fibroblasts (39, 41). It is clear that the mesenchyme controls the airway branching pattern and that processes inherent in the epithelial cells alter cell and tissue shape to produce branch points (32). Cell-extracellular matrix interactions are known as one class of important mechanisms that direct development of structural and functional lungs (10, 22). Regulation of lung fibroblast extracellular matrix molecule biosynthesis through TGF-beta -receptor signal transduction could pass signals from the mesenchyme to the epithelium, thus governing a cascade of reciprocal interactions between the mesenchyme and epithelium and regulating the ensuing morphogenesis of the forming lung.

We found that a high level of Tbeta R-I mRNA was expressed in rat fetal lung tissue early in development, but the expression of Tbeta R-I mRNA decreased as development proceeded. The temporal expression of Tbeta R-I was different from that of Tbeta R-II during rat lung development. The expression of Tbeta R-II mRNA was low in rat fetal lung tissue early in development, but the expression of Tbeta R-II mRNA increased as development continued. TGF-beta receptors and their ligands exhibit distinct but overlapping patterns of expression during lung development. The time course of Tbeta R-I expression was similar to those of TGF-beta 2 and TGF-beta 3 at early developmental stages, and their expression levels were highest in rat fetal lung tissue early in development. However, the expression of Tbeta R-I was different from that of TGF-beta 2 and TGF-beta 3 during the postnatal period. A similar temporal expression was observed for Tbeta R-II and TGF-beta 1, and their expression levels were maximal in postnatal day 1 rat lung. The apparent differential expression of the three TGF-beta isoforms and their receptors at different developmental stages raises interesting possibilities of specific roles for each ligand and for Tbeta R-I and Tbeta R-II during lung development in vivo.

The finding that Tbeta R-I and Tbeta R-II were differentially expressed in the process of lung development was not anticipated. A ligand-induced heterooligomer model was proposed for TGF-beta signal transduction (33, 36, 37), in which the biological effects of TGF-beta are mediated by Tbeta R-II in concert with Tbeta R-I. TGF-beta binds directly to Tbeta R-II, which then recruits Tbeta R-I to form a heteromeric complex. The signaling TGF-beta -receptor complex contains two molecules each of Tbeta R-I and Tbeta R-II bound to one TGF-beta molecule. Our results show that the expression level of Tbeta R-I was distinct from that of Tbeta R-II during rat lung development, which indicates that an excessive amount of either Tbeta R-I or Tbeta R-II may exist. The individual roles of excessive Tbeta R-I and Tbeta R-II remain to be elucidated. The ratio of Tbeta R-I to Tbeta R-II may influence the biological activities of TGF-beta .

The expression of Tbeta R-I and Tbeta R-II is regulated distinctly not only in a temporal manner but also in a spatial manner during lung development. A previous study by Zhao and Young (40) showed an interesting expression pattern of Tbeta R-II in day 16 gestational age rat fetal lung tissue. Tbeta R-II was expressed along a proximal-distal gradient in the developing airway epithelium. A concentrated expression of Tbeta R-II was detected in the distal cuboidal epithelium, whereas the Tbeta R-II transcript was absent or in low concentration in the proximal pseudostratified columnar epithelium. In contrast, we found that the epithelial cells in both the distal and proximal bronchioles had a similar level of Tbeta R-I expression. TGF-beta has been recognized as being important in lung epithelial cell differentiation (12, 27). Based on these observations, we speculate that both Tbeta R-I and Tbeta R-II may participate in the TGF-beta signaling pathway to regulate lung epithelial cell differentiation, but Tbeta R-II may provide positional information during epithelial morphogenesis.

In conclusion, we described the expression and localization of Tbeta R-I mRNA during lung development. The expression of Tbeta R-I was found to be highly regulated during lung development. Compared with Tbeta R-II, Tbeta R-I shows a unique developmental expression and localization pattern in rat lung. The temporal and spatial distribution of Tbeta R-I mRNA in rat lung tissues along with its regulated expression pattern during development supports the notion that TGF-beta plays an important role as a multifunctional autocrine/paracrine regulator during lung development. The distinction of the temporal and spatial expression of Tbeta R-I and Tbeta R-II mRNAs in the rat lung suggests that each receptor may play a different role in lung development. To ascertain their roles in the signal transduction pathway and the diverse regulatory effects of TGF-beta in lung development processes, further experiments remain to be done.


    ACKNOWLEDGEMENTS

This work was supported by a Department of Veterans Affairs grant; National Heart, Lung, and Blood Institute Grant HL-32188; and an American Lung Association grant.


    FOOTNOTES

Y. Zhao is a recipient of the Clifford W. Perry Research Award from the American Lung Association of North Carolina.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Y. Zhao, Medical Research 151, Durham VA Medical Center, Durham, NC 27705 (E-mail: zhaoyun{at}duke.edu).

Received 7 October 1999; accepted in final form 11 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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