Abstract

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. Aday 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1(LGL1; GenBank accession no. AF109674) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmedLGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

  • genes in lung development
  • mesenchymal-epithelial signaling

morphogenesis and differentiation of fetal organs is dependent on precise signaling between mesenchymal cells and epithelial cells (derived from foregut endoderm). This signaling regulates cell proliferation, fate, migration, and differentiation (33). Although mesenchymal-epithelial interactions in the late-gestation lung have long been known to influence both growth and differentiation of the epithelium, the exact nature of these interactions remains incompletely understood (8, 17, 18, 26, 27). Organ-specific humoral and extracellular matrix (ECM) factors determine branching morphogenesis and epithelial differentiation of the fetal lung, intestine, and kidney (7, 33). In the lung, glucocorticoid hastens the onset of maturation of type II epithelial cells, an effect mediated in part by the adjacent mesenchyme. Molecules elaborated by embryonic mesenchymal cells (fibroblasts) in response to glucocorticoid stimulation augment epithelial cell differentiation as reflected by the production of pulmonary surfactant (21, 22, 27). Fibroblasts adjacent to the epithelium (adjacent fibroblasts) produce greater amounts of differentiation factors in response to glucocorticoid stimulation than do fibroblasts located more distant from the epithelial cells, with maximal production of differentiation factors on gestationalday 20 in the rat (term = 21.5 days) (27).

A series of events prepares fetal lungs for the onset of gas exchange at birth. Critical among these events is the surge in surfactant production by distal airway epithelial cells that occurs in the latter third of gestation in all mammalian species (31). Surfactant, a complex of phospholipids (90%) and proteins (10%), is an essential modulator of surface tension at the alveolar air-liquid interface (1) that has been widely used as a marker of epithelial cell differentiation.

To investigate the molecular mechanisms by which fibroblasts mediate glucocorticoid action on epithelial cells in the late-gestation lung, we searched for glucocorticoid-inducible genes in the developing lung in a fetal rat model. We applied the techniques of differential display (DD)-PCR, library screening, and a novel approach for the specific amplification of cDNA ends [similar to that of Li and Nicholas (13)] to the identification of novel genes expressed in the late-gestation lung. We screened 25% of the expressed genes in fetal rat lung cell cultures and isolated a panel of cDNAs probes representing mRNAs in which the pattern of developmental and hormonally modulated expression is coordinate with the onset of surfactant synthesis. We then probed a glucocorticoid-induced late-gestation lung mesenchymal cDNA library with randomly selected novel sequence probes. We report here the isolation of a novel gene,late-gestation lung 1(LGL1), that is differentially expressed in fetal lung mesenchymal cells during development but is not detectable in lung epithelium. We investigated the tissue-specific expression, hormonal modulation, and cellular localization ofLGL1 in the fetal rat and showed thatLGL1 is conserved in mammals.

Comparison of LGL1 to sequences in the genome database (BLAST search) showed that the deducedLGL1 gene product (lgl1 polypeptide) shared 68% identity (81% homology) to P25TI. P25TI is a polypeptide with trypsin inhibitor activity that was recently identified in human glioblastoma and neuroblastoma cells (but not in normal tissues), with no homology to other proteinase inhibitors. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich, androgen-regulated secreted proteins. The role of regulatory proteins in developmental processes is well recognized. We postulate a role for lgl1 in the tight control of ECM degradation that is critical to normal cell and organ development.

EXPERIMENTAL PROCEDURES

Materials. Drugs and chemicals were obtained from the following sources: chromatographically purified collagenase (type CLSPA) from Worthington (Freehold, NJ); culture medium [minimal essential medium (MEM)], nylon (Hybond-N) membranes, and Sequenase from Amersham; penicillin, streptomycin, urea, agarose, TRIzol Reagent, ethidium bromide, random hexanucleotide primers, Taq polymerase, and restriction endonucleases from GIBCO BRL (Life Technologies, Burlington, ON); PCR primers from Sheldon Biotechnology (Montreal, PQ); deoxynucleotides and RNAguard RNase inhibitor from Pharmacia Biotech (Baie d’Urfé, PQ); and [α-32P]dCTP from Dupont Canada (Mississauga, ON).

Fetal rat lung cell primary culture.Isolation and primary culture of fetal rat lung cells were performed as previously described (29). Wistar rats of known gestational age (day 0 = mating; term =day 21.5) were obtained from Charles River Laboratories (St. Constant, PQ) and killed with diethyl ether. The fetuses were immediately removed from the uterus, and the fetal lungs were dissected out. Epithelial and adjacent fibroblast cells were isolated from the fetal lungs as previously described (29). Briefly, after trypsin dispersion, collagenase digestion, and several steps of differential centrifugation and adherence to plastic of adjacent fibroblasts, the cells were incubated for attachment of the epithelial cells. Nonadherent cells were removed from all cell cultures after an overnight incubation. The cells were grown to confluence over 1–6 days in MEM-10% fetal bovine serum, thoroughly rinsed in MEM (serum and glucocorticoid free), and then incubated in MEM alone. Cells incubated in MEM plus a specified concentration of hormone were in all other respects handled exactly as were the MEM controls. An equal volume of the solvent in which the hormone was dissolved was added to the control medium. All experiments were performed 24–48 h after confluence. Viability and purity of the cultures were comparable to previously published data (16). Epithelial cells express phenotypic features of type II cells and possess antigenic determinants of mature type II cells (16).

Hormonal treatment of fetal lung fibroblasts. Adjacent fibroblasts in culture (days 2021) were exposed to glucocorticoid (10−7 M cortisol, 24 h) or androgen (10−8 M 5α-dihydrotestosterone, 24 h). Various aspects of prenatal lung development are modulated by exposure to hormones at these concentrations. Glucocorticoid enhances epithelial cell differentiation as measured by surfactant phospholipid production (32) and decreases ECM synthesis (6). By contrast, surfactant phospholipid production is delayed by androgen (32). Furthermore, Sweezey and colleagues (29, 30) have previously shown that these doses also modulate levels of glucocorticoid-receptor mRNA and protein in the same model systems, fetal rat lung cells in culture, and lung tissue. mRNA levels were compared by Northern blot analysis in the presence and absence of factors added to the medium.

RNA isolation. Total (nuclear and cytoplasmic) RNA was isolated by lysing the cells in TRIzol Reagent. After extraction with chloroform, RNA was ethanol precipitated, collected by centrifugation, lyophilized, and dissolved in water. RNA integrity was confirmed by fractionation on 1% (wt/vol) agarose-formaldehyde gels and staining the rRNA bands with ethidium bromide.

DD-PCR. To identify glucocorticoid-inducible genes, the expression of which is required for normal lung development, we used the technique of DD-PCR to screen for tissue-specific genes that are conserved across species, expressed in particular cell types during well-defined developmental windows, and responsive to known modulators of lung maturation. The method relies on the ability of an arbitrarily chosen cDNA to be amplified by a pair of short primers with PCR. The key element in the protocol is to use a set of oligonucleotide primers, one anchored to the poly(A) tail of a subset of mRNAs and the other being a short and arbitrary sequence so that it anneals at different positions relative to the first primer. mRNA subpopulations defined by these primer pairs are amplified after reverse transcription and resolved on a DNA-sequencing gel. Unique fragments can be reamplified, cloned, and used to probe Northern blots, cDNA libraries, and zoo blots to identify tissue-specific, abundant, and conserved sequences (28).

Total RNA (1 μg) prepared from tissue or cultured cells (or from other fetal rat tissues for comparison) was reverse transcribed with one of four anchored oligo(dT) primers [T12MN, where M is degenerate (A, C, or G) and N is A, C, G, or T] (14, 15). One microliter of reverse transcription reaction product was then amplified in a volume of 20 μl (2 mM each deoxynucleotide 5′-triphosphate, 2.5 mM T12MN, 0.5 mM arbitrary decamer, and 0.6 μl of [α-32P]dCTP, 3,000 Ci/mmol). PCR products were resolved on an 8% DNA-sequencing gel and subjected to autoradiography. All reactions were run in triplicate to avoid the possibility of losing rarer mRNAs and to minimize PCR errors that lead to spurious bands. For each gestational time point (except where whole lung tissue is used), triplicates of culture types were analyzed. cDNAs from non-lung rat tissues are displayed as controls on every gel.

To assess the effects of glucocorticoid and androgens on mRNA synthesis, RNA from cells grown in the presence and absence of these reagents was reverse transcribed, amplified, and displayed side by side.

After autoradiography, cDNA bands that were unique or particularly intense in distal airway epithelial cells or adjacent lung fibroblasts from day 20 rat fetuses were isolated by cutting through the film. Gel slices were excised, incubated in 100 μl of distilled H2O for 10 min, eluted by boiling for 15 min, recovered by ethanol precipitation, and redissolved in 10 μl of H2O. Four microliters of eluted cDNA sequence were reamplified in a 40-μl reaction volume with the same primer set (or a pair of arbitrary primers) and conditions as in the display except that the deoxynucleotide 5′-triphosphates are at 20 mM and no isotope was added. Reamplified probes recovered from 1.5% agarose gels were cloned into the PCRII vector with the TA cloning system (Invitrogen), sequenced directly, and compared with sequences in GenBank to determine whether they are novel. All sequences were analyzed for open reading frames and for functionally important protein sequence motifs of putative translated products with PC Gene and Omiga 1.1 software.

Construction of full-length cDNA library. A glucocorticoid-induced day 20 fetal lung adjacent fibroblast cDNA library was prepared from 4 to 5 μg of poly(A)+ RNA with a Stratagene ZAP synthesis kit according to the supplier. The fibroblast cDNA library had an average insert size of 1.8 kb.

Screening of full-length cDNA library.Probes derived from DD-PCR clones were used to screen the cDNA library constructed from glucocorticoid-treated adjacent fibroblasts (Stratagene λZAP cDNA Gigapack II Gold Cloning Kit). The cDNA library was titered and plated with host cells on NZY plates at a density of 5 × 104 plaque-forming units/plate. The plaques were grown at 37°C for 6–8 h until near confluence and then chilled at 4°C. Duplicate filters were made by transferring the plaques to Hybond-N filters, and then each filter was denatured (1.5 M NaCl and 0.5 M NaOH), neutralized (1.5 M NaCl and 0.5 M Tris-Cl, pH 8.0), washed [0.2 M Tris-Cl, pH 7.5, and 2× saline-sodium citrate (SSC) buffer], and baked at 80°C for 1.5 h under vacuum. The filters were prehybridized in 10% (wt/vol) dextran sulfate, 40% (vol/vol) formamide, 4× SSC, 0.2 M Tris-Cl, pH 7.5, 1× Denhardt’s reagent, and 0.1 mg/ml of sonicated herring sperm DNA for 3 h at 42°C. The filters were hybridized overnight at 42°C in the same solution after the addition of 32P end-labeled probe [10 × 106counts ⋅ min−1(cpm) ⋅ 150-mm filter−1 for primary screenings and 5 × 106cpm/100-mm filter for subsequent screenings]. Filters were washed 1–3 times with 2× SSC and 0.1% SDS for 20 min/wash and ≤2 times for 30 min at 50°C in 0.1× SSC and 0.1% SDS. After being air-dried, the filters were exposed overnight to Kodak Biomax MR film. Positive plaques were cored from the plates and incubated in 1 ml of suspension medium buffer and 20 μl of chloroform overnight at 4°C. Each of these phage solutions was diluted 104 times and used for secondary screenings. Tertiary screenings were also performed. The pBluescript plasmids were excised from the phage according to the supplier’s instructions.

System for PCR identification of cDNA ends. A 300-bp probe specific to glucocorticoid-treatedday 20 adjacent fibroblasts isolated by DD-PCR (Fig. 1) and used to screen a glucocorticoid-treated day 20 fetal rat lung cDNA library identified a 2.7-kb clone. Northern analysis (Fig. 2) indicated that this cDNA did not include the 5′ region of LGL1. The following procedure was adapted to isolate the 5′ region ofLGL1.

Fig. 1.

Differential display-PCR comparing DNA profiles fromday 20 fetal rat cells or tissues. Total RNA (1 μg) prepared from lung or other fetal rat tissue or primarily cultured cells was reversed transcribed with an anchored oligo(dT) primer (T12MN) and amplified with T12MN and an arbitrary decamer. PCR products were resolved on 8% DNA-sequencing gels. Lane 1, day 20 fetal rat lung epithelial cells;lane 2, epithlial cells treated with 5α-dihyrotestosterone (DHT; 10−8 M);lane 3, day 20 fetal rat lung adjacent fibroblasts treated with DHT; lane 4, adjacent fibroblasts;lanes 5 and6, adjacent fibroblasts treated with glucocorticoid; lane 7, fetal spleen;lane 8, fetal liver. Arrow, probe used to screen for late-gestation lung 1gene (LGL1).

Fig. 2.

Expression of LGL1 in fetal rat lung. Equal amounts of total RNA (30 μg) isolated from fetal rat lung cell cultures on day 20 or fetal whole lungs at indicated times were electrophoresed, blotted, and hybridized to a 1.7-kb LGL1 probe as described inexperimental procedures. A 0.8-kb probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to control for loading, quality, and transfer of RNA. AF, adjacent fibroblasts; EPI, epithelial cells; D, fetal day. Nos. on left, no. of kb.

RNA from rat embryonic day 20 lungs was used to create a cDNA library in the λZAPII vector (Stratagene). A portion of this library was mass excised with VCSM13 helper phage to yield a cDNA library in the pBluescript phagemid (Stratagene) with the protocol supplied with the Stratagene ZAP cDNA synthesis kit. One hundred nanograms of this circular library was then used as a template for long-accurate, inverse PCR with Advantage KlenTaq Polymerase Mix (Clontech). This system for PCR identification of cDNA ends (SPICE) utilizes a strategy similar to that published by Li and Nicholas (13). The primers used for the first round of PCR were 5′-TCTGTGTGTAGTGGGTGCACATGGCTCCTGAGC-3′ and 5′-GGGGCACAGTTGCCTGTGAATGTTGGCCTCAAAC-3′. The PCR product consisting of phagemid plus additional cDNA sequences at either end was visualized on an agarose gel as a band migrating at >3 kb (the size of pBluescript). To increase the odds of obtaining a correct clone, a second round of PCR was performed with the nested primers 5′-CACTCGTGGGGGTACGGGTAGGTGTAATCCTTC-3′ and 5′-TCTACCAGAGGACCTGAGCTCAGTTCCCACGAC-3′. The PCR band was then excised from the gel, treated with kinase, and self-ligated. Transformation of Escherichia coliDH5α with this DNA yielded a clone containing the complete phagemid sequence plus the cDNA sequence both upstream and downstream from the PCR primers joined together. This DNA was then autosequenced (Li-Cor model 4000L) from the T7 primer site in pBluescript.

DNA sequencing. LGL1 radiolabeled with α-35S-dATP was sequenced by the dideoxy method according to the supplier with a T7 sequenase version 2 sequencing kit (Amersham Life Sciences). DNA was also autosequenced (Li-Cor) from the T7 primer site in pBluescript and from the T7 and M13 reverse-primer sites in pT-Adv (Clontech).

Northern analysis. Total RNA (20–30 μg) was size fractionated on a 1% agarose gel containing formaldehyde and ethidium bromide with a 1× MOPS buffer. RNA was passively transferred to Hybond-N nylon membranes (Amersham) and ultraviolet cross-linked. A solution of 40% deionized formamide, 10% dextran sulfate, 1× Denhardt’s solution, 0.4% SDS, 4× SSC, 20 mM Tris-Cl, pH 7.4, 0.2 mg/ml of yeast tRNA, and 0.1 mg/ml of salmon sperm DNA was used for prehybridization for a minimum of 6 h. Then hybridization occurred overnight at 42°C with 1 × 106 cpm/ml of a randomly primed (Multiprime DNA kit, Amersham) [α-32P]CTP-labeled cDNA probe in a solution that was of the same composition as for prehybridization except for the omission of SDS. Blots were then washed (highest stringency: 0.1× SSC and 0.1% SDS at 50°C for 30 min), and the transcripts were visualized with standard autoradiography. To control for loading, quality, and transfer of RNA, the membranes were stripped and then reprobed with the 0.8-kbPstI-Xba I fragment of the human glyceraldehyde-3-phosphate dehydrogenase cDNA radiolabeled as above.

In situ hybridization. In situ hybridization was performed essentially as described by Motoyama et al. (19) with an LGL1 digoxigenin-labeled RNA probe. Briefly, tissue sections were rehydrated and washed in PBS. Pretreatment included postfixation in 4% paraformaldehyde (20 min) followed by proteinase K digestion (20 mg/ml, 17 min at 25°C) and acetylation (0.1 M triethanolamine and 0.25% acetic anhydride, 10 min at 25°C). Sections were then dehydrated and air-dried before addition of the hybridization solution. Digoxigenin-labeled probes were then added to freshly prepared hybridization solution (50% deionized formamide, 10% dextran sulfate, 1.5× Denhardt’s reagent, 0.5 mg/ml of yeast tRNA, 0.3 M NaCl, 5 mM EDTA, and 25 mM Tris, pH 7.5) at a concentration of 1 ng/μl. After denaturation at 80°C, the probe was added to the tissue section and incubated overnight at 55°C. After brief washes with 5× SSC and 50% formamide at 55°C, the tissue was treated with RNase A (10 μg/ml) for 30 min at 37°C. The digoxigenin nucleic acid detection kit (Boehringer Mannheim) was used for immunological detection of the hybridized probe. Tissue was then counterstained with methyl green and prepared for viewing.

RESULTS

Identification of LGL1 among glucocorticoid-induced genes in fetal rat lung adjacent fibroblasts. We used DD-PCR to screen 25% of the expressed genes in fetal rat lung (gestational days 1920) cell cultures and isolated 50 cDNA probes. These probes, which ranged in size from 200 to 500 bp, represented mRNAs in which the pattern of developmental and hormonally modulated expression is coordinate with the onset of surfactant synthesis. Among 23 fibroblast-specific sequences, 14 appeared to represent mRNAs in which the expression is increased when adjacent fibroblasts are incubated in the presence of glucocorticoid and decreased when these cells are incubated in the presence of androgen. Figure 1 illustrates a display comparing cDNA profiles from day 20 distal airway epithelial cells and adjacent fibroblasts. TheLGL1 300-bp probe (Fig. 1, arrow) appeared to represent an mRNA that was upregulated by glucocorticoid and downregulated by 5α-dihydrotestosterone in adjacent fibroblasts.

Thirty cDNA probes (18 epithelial and 12 fibroblast) were subcloned and sequenced. A single probe was randomly selected (among novel sequence clones) and used to screen a glucocorticoid-inducedday 20 fetal rat lung cDNA library. A unique clone containing a 2.7-kb insert was isolated, digested withRsa I,Sma I, andKpn I to create overlapping sequences and further subcloned for sequence analysis. Northern blot analysis (see Developmental expression of LGL1) with a 1.7-kb probe (Fig. 2) indicated a transcript close to 4 kb. Because DD-PCR relies on the cloning of probes at the 3′-ends and the average size of inserts in the cDNA library was 1.8 kb, these results suggested that the cDNA did not include the complete 5′-end of the message sequence. A novel technique based on inverse PCR of a circular cDNA library, similar to that of Li and Nicholas (13), was developed to clone the 5′ region of the LGL1 cDNA (seeexperimental procedures). The fetal rat lung LGL1 sequence is shown in Fig. 3. An open reading frame ofLGL1 identified a polypeptide of 188 amino acids (Fig. 3). The coding region ofLGL1 demonstrated 68% identity (81% homology) at the amino acid sequence level to P25TI, a polypeptide with weak trypsin inhibitor activity previously identified in human glioblastoma and neuroblastoma cells but not present in normal human tissues (Fig. 4). Interestingly, virtually no significant homology was observed at the level of DNA sequence, suggesting that although lgl1 and P25TI may share functional properties, they are unlikely to have evolved from a single ancestral gene. P25TI has no homology with other proteinase inhibitors. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich, androgen-regulated secreted proteins (Fig.5). The exact function of these extracellular proteins is not yet known. Two signature protein motifs have been identified and are indicated in Fig. 5. The second signature contains a cysteine residue that is involved in a disulfide bridge in members of the family.

Fig. 3.

Sequence of LGL1. Nucleotide sequence with presumed reading frame (uppercase letters) is shown. cDNA sequence has been deposited with GenBank (accession no. AF109674).

Fig. 4.

Sequence alignment of LGL1 gene product (lgl1) and P25TI. The 2 sequences share 68% identity and 81% homology. *, Identical sequence; +, similar sequence; #, cysteine residues.

Fig. 5.

Sequence alignment of lgl1 and 7 members of CRISP family of cysteine-rich, androgen-inhibited secreted proteins. HRTT1,Halocynthia roretzi gene expressed in tip of tail in tadpole larvae; GLIP, glioma pathegenesis-related protein; CRS3, cysteine-rich secretory protein-3 percursor; AEG1, sperm-coating glycoprotein-1 precursor; TPX1, testis-specific protein precursor; SGP28, specific granule protein precursor 28k. *, Identical residues; ‖, well-conserved residues (identical in 5 or more proteins); #, cysteine residues. Rectangles, signature motifs for CRISP proteins.

Developmental expression of LGL1. To determine the developmental profile ofLGL1 expression, we performed Northern analysis with a 1.7-kb probe isolated from the glucocorticoid-induced adjacent fibroblast cDNA library. The probe was hybridized to a 3.9-kb mRNA species in whole fetal rat lung (Fig. 2). mRNA levels were high in fetal lung tissue, whereas weaker expression could be detected in the fetal heart, kidney, and intestine and also in the adult rat lung and heart (Fig. 6). Moreover,LGL1 mRNA was detected in primary cell cultures of fetal lung adjacent fibroblasts but not distal airway epithelial cells (Figs. 2 and 7). The relative abundance of LGL1 mRNA in adjacent fibroblasts increased during the late-canalicular stage of gestation (day 20) and was maximal during the saccular stage (day 21; Fig. 2), with a subsequent decline in expression during the neonatal period.

Fig. 6.

Expression of LGL1 mRNA in adult (A) and fetal (B) rat organs. Equal amounts of total RNA (20 μg) isolated from various fetal and adult rat tissues were electrophoresed, blotted, and hybridized to a 1.7-kbLGL1 probe as described inexperimental procedures. A 0.8-kb probe for human GAPDH was used to control for loading, quality, and transfer of RNA. Nos. on left, no. of kb.

Fig. 7.

Glucocorticoid induction of LGL1expression in day 20 fetal rat lung adjacent fibroblasts. Equal amounts of total RNA (20 μg) isolated from day 20 fetal rat lung cell cultures were electrophoresed, blotted, and hybridized to a 1.7-kbLGL1 probe as described inexperimental procedures. Lung cell cultures were untreated or incubated in presence of cortisol (CORT; 10−7 M) for 24 h. A 0.8-kb probe for human GAPDH was used to control for loading, quality, and transfer of RNA.

Effect of glucocorticoid on LGL1 expression. To examine the effect of glucocorticoid onLGL1 mRNA expression,day 20 fetal lung fibroblasts and epithelial cells in culture were exposed to cortisol (10−7 M) at 24 h. Cortisol increased expression of LGL1 in day 20 fetal rat lung adjacent fibroblasts 5- to 10-fold (Fig. 7). No detectible expression ofLGL1 in day 20 fetal lung epithelial cells was observed in the presence of glucocorticoid.

LGL1 is conserved and developmentally regulated in the human fetal lung. The 1.7-kbLGL1 probe was also used to detectLGL1 expression in adult human and mouse lungs (Fig. 8). PCR amplification of reverse-transcribed RNA isolated from human fetal lung with primers specific to the rat sequence detectedLGL1 mRNA at weeks 12, 16, and18 (data not shown). In marked contrast to the lack of homology ofLGL1 to P25TI at the DNA level, partial DNA sequencing of the coding region of humanLGL1 shows 100% identity with the rat sequence.

Fig. 8.

LGL1 is conserved in humans and rodents. Equal amounts of total RNA (30 μg) isolated from various rat, mouse, and human lungs were electrophoresed, blotted, and hybridized to a 1.7-kb LGL1 probe as described in experimental procedures. Nos. on left, no. of kb.

Cellular localization of LGL1 in the whole lung. In situ hybridization studies of sections ofday 16 fetal rat tissues revealed expression of LGL1 mRNA in nonepithelial, interstitial (mesenchymal) cells throughout the lungs and intestinal tract (and kidney; data not shown). Epithelial cells from these organs were substantially free of hybridization signal (Fig.9).

Fig. 9.

Cellular localization of LGL1 in fetal rat lung and intestine. Fetal tissue section in situ hybridization was performed with an LGL1 antisense RNA probe. Digoxigenin nucleic acid detection kit was used for immunologic detection of hybridized probe. Tissue was then counterstained with methyl green. A: day 16 fetal rat lung (high power).B: day 16 fetal rat intestine (high power).C andD: day 16 fetal rat lung (low power). Dark blue color,LGL1 staining of mesenchyme (fibroblasts) in both lung and intestine.

DISCUSSION

We sought to identify genes that are highly expressed in fetal rat lung adjacent fibroblasts at the onset of augmented production of pulmonary surfactant in late gestation, that are not expressed in epithelial cells, and the expression of which is vigorously enhanced when cells are pretreated with glucocorticoid. We cloned a novel gene,LGL1, that is expressed in the mesenchyme but not in epithelial cells of late-gestation fetal rat lung, kidney, and intestine. Each of these organs depends on mesenchymal stimuli for normal maturation of adjacent epithelium and hence normal organ function. LGL1 is developmentally regulated, strongly induced by glucocorticoid, and conserved across species (human, mouse, and rat).

Locally expressed molecules in lung mesenchymal and epithelial cells regulate branching and differentiation. These regulatory molecules include transcription factors acting upstream (3, 19, 34) as well as other growth and differentiation factors (4, 20, 23, 25, 34) that are involved in the complex network of reactions essential to terminal lung organogenesis. Until recently, the major approaches to identifying specific genes expressed during these processes were differential screening and subtractive hybridization. These approaches to the cloning and characterization of lung-specific genes have had limited success. We therefore adapted the approach of DD-PCR to the identification of novel genes expressed in the developing lung in our fetal rat model. Among the advantages of this method over previous screening approaches are its high sensitivity, the small amount of tissue required, the ability to rapidly identify transcripts that respond to multiple overlapping developmental or environmental cues, and the ability to process multiple RNA samples in parallel (28). Advantages of this approach for isolation of novel lung-specific genes are that 1) multiple sets of mRNA subpopulations (e.g., expressed in distal airway epithelial cells vs. fibroblasts; glucocorticoid or androgen stimulated vs. unstimulated;day 12 vs. day 19 gestation) can be visualized in a single display, allowing rapid identification of variant gene expression;2) sequences of interest can be quickly and easily amplified, subcloned, sequenced, and compared with sequences in databanks; 3) although very rare mRNAs may not be identified, very abundant expression is not crucial for detection; and 4) a single display can provide an estimate of the proportion of transcripts that respond to specific developmental cues.

The use of a global screening method such as DD-PCR for gene identification requires a strategy for rapid identification of transcripts that are of greatest interest. To screen cDNA libraries, we used probes representing mRNAs that are expressed atday 20 gestation but absent atday 12, are enriched in fibroblasts versus distal airway epithelial cells, and in which the expression is upregulated by glucocorticoid and inhibited by androgen in primary cell cultures.

A single probe was used to screen a glucocorticoid-inducedday 20 fetal rat lung adjacent fibroblast library to isolate a 2.7-kb fragment ofLGL1 cDNA. Because Northern analysis indicated that this sequence did not represent a full-length cDNA, we used a novel inverse PCR protocol, SPICE [similar to that described by Li and Nicholas (13)], to clone the 5′ region of LGL1. This method offers a rapid and efficient approach to cloning of cDNA termini that precludes the necessity of multiple rounds of library screening. Moreover, because the average insert size of even high-quality cDNA libraries is ∼2 kb (our library inserts averaged 1.8 kb), we avoided the time-consuming assembly of multiple vector inserts that can be necessary to the cloning of very long mRNA transcripts. This approach will likely prove useful in concert with DD-PCR screening for novel genes, which produces cDNA probes enriched for the 3′-end of genes.

Although not all cDNA sequences identified by DD-PCR will encode proteins that play either a regulatory or functional role in lung maturation, these cDNA sequences furnish a limited subset of cDNA probes, representing genes in which expression is developmentally and hormonally regulated in fetal lung cells. These cDNA sequences can be used to probe gene function in primary cell cultures highly enriched for particular cell types (e.g., fetal lung epithelial cells or adjacent fibroblasts).

The observed localization of LGL1expression in time and space and the glucocorticoid responsiveness ofLGL1 are of interest based on the current understanding of late-gestation lung development. During development of the respiratory tract, embryonic cells are instructed to organize along an axis (pattern formation) and differentiate such that the proximal structures (trachea) differ greatly from those of the more distal alveoli (3). LGL1 is maximally expressed in the saccular stage of development, concordant with the onset of rapid surfactant production in the lung.LGL1 appears to be spatially distributed, with elevated expression in the mesenchyme adjacent to the epithelium, the same mesenchyme that is enriched in differentiation factors in late gestation (27, 33).LGL1 expression in theday 20 fetal rat lung is stimulated 5- to 10-fold by glucocorticoid. Taken together, these observations suggest a regulatory or functional role for lgl1 in late-gestation lung development.

The nature of any such potential regulatory or functional role for lgl1 remains unknown. However, the significant homology between lgl1 and a recently described protein, P25TI, suggests an intriguing possibility. P25TI is a 25-kDa trypsin-binding protein secreted by human glioblastoma cells that has weak trypsin-inhibitory activity (12). It was long believed that trypsin (a proteinase) is synthesized exclusively in the pancreas. However, Koshikawa et al. (11) have demonstrated that trypsin is widely expressed in epithelial cells of the skin, esophagus, stomach, small intestine, liver, kidney, and lung. Trypsin is also expressed in neoplasms of the lung (10). Trypsin potently hydrolyzes a wide variety of proteins, including ECM proteins, which are thought to be important in late-gestation lung development (5, 24). In addition, trypsin effectively activates the latent forms of various matrix metalloproteinases that are involved in ECM degradation (11). Early models of branching morphogenesis of the lung focused exclusively on the assembly and degradation of ECM as major determinants of branching patterns (2). More recent evidence (9) has emphasized the critical role of secreted signaling molecules in regulating mesenchymal-epithelial interactions that underlie the branching process. In the tissue microenvironment of developing organs, proteolytic activity is regulated by the balance between the local concentration of activated enzymes and their endogenous inhibitors (10). It has been suggested that P25TI is involved in the regulation of ECM proteolysis that contributes to tumor invasion (12).

We propose a model for a role of lgl1 in the regulation of ECM remodeling based on 1) the temporal and spatial pattern of lgl1 expression in fetal lung mesenchyme,2) the enrichment of lgl1 in cysteine residues, 3) the likelihood that it is an extracellular protein (based on homology with the CRISP family of secreted proteins, maintaining two signature CRISP protein motifs in the deduced polypeptide), and4) the homology of lgl1 to the trypsin inhibitor P25TI. LGL1 is maximally expressed by adjacent fibroblasts during the late-canalicular early-saccular stage of lung development, a time and place of active proteolytic thinning of ECM (24). We postulate that lgl1 is secreted and acts to regulate ECM degradation by modulation of levels of trypsin activity. Trypsin, in turn, may be involved in ECM degradation either directly via hydrolysis of ECM proteins or indirectly by activation of matrix metalloproteinases.

In summary, we report the cloning ofLGL1, a novel gene expressed in the late-gestation lung. The developmental and spatial expression and hormonal responsiveness of LGL1 are consistent with a role for lgl1 in lung development. The homology of lgl1 to CRISP proteins suggests that it is a secreted extracellular protein. The very significant homology to the weak trypsin inhibitor P25TI suggests that LGL1 may have a role in the regulation of trypsin and thereby in the modulation of ECM proteolysis. These homologies invite postulation of potential functional roles for LGL1, suggesting areas for further investigation.

Acknowledgments

This work was supported by a grant from the Medical Research Council of Canada (to F. Kaplan).

Footnotes

  • Address for reprint requests and other correspondence: F. Kaplan, McGill University-Montreal Children’s Hospital Research Institute, 2300 Tupper St, Montreal, Quebec, Canada H3H 1P3 (E-mail:fkaplan{at}www.debelle.mcgill.ca).

  • 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.

REFERENCES

View Abstract