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During fetal development, the lung is a secretory organ (1–3). The secreted liquid contributes to the volume of amniotic liquid and serves as a liquid template for airway and distal lung development. Fetal lung liquid secretion is osmotically coupled to Cl− transport (1, 4, 5). The cellular pathways for Cl−-coupled liquid secretion across the fetal pulmonary epithelium include CFTR and Ca2+-activated Cl− channels (4–7); however, other pathways may exist. Similarly, the local and systemic factors stimulating lung liquid secretion in vivo are poorly understood.

Zhou et al. (8) recently showed that KGF is a potent stimulant of CFTR-independent liquid secretion in the fetal mouse lung in vitro. KGF (FGF-7) is a member of the FGF superfamily of growth factors and is a secreted product of fibroblasts adjacent to epithelia (9, 10). Recent evidence suggests that another member of the FGF family, FGF-10, may also have effects on liquid secretion in the fetal lung (11, 12). KGF exerts its effects by binding to the KGFr, a splice variant of the FGF receptor 2, which is expressed on epithelia (13). FGF-10 is also able to bind and signal via the KGFr (14). The KGF promoter contains consensus sequences for regulation by proinflammatory cytokines (IL-1, IL-6) and cAMP (15, 16). The temporal and spatial distribution of KGF, FGF-10, and KGFr in the fetal lung suggests that these FGF are important signaling molecules during development (17, 18). Gene-targeting studies showed that disruption of KGF signaling in the lung by overexpression of a dominant negative receptor (19) or expression of KGF using a lung-specific promoter (20) dramatically altered lung development. FGF-10 null mice have tracheas, but all further lung development is arrested (21, 22). These studies suggest important developmental roles for both KGF and FGF-10 in the fetal lung.

In the present study, we analyzed the effects of KGF and FGF-10 on liquid secretion in human fetal lung tissue in an in vitro explant model. We also determined the ontogeny of the KGF and KGFr mRNA in human lung and asked whether their expression was influenced by IL-1 and cAMP.

Methods

Drugs and chemicals.

Cell culture media, FCS, and antibiotics were obtained from the Cell Culture Facility at the University of Iowa. Calf skin collagen solution (Vitrogen 100) was obtained from Collagen Corporation (Palo Alto, CA). Recombinant human KGF (FGF-7) was a gift from Amgen Corporation (Thousand Oaks, CA). FGF-10, FGF-7–blocking antibodies (MAb251, mouse monoclonal), and IL-6 were purchased from R & D Systems (Minneapolis, MN). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Tissue samples.

Human fetal lung tissue specimens were obtained from therapeutic terminations of pregnancies. No terminations were performed for suspected fetal anomalies . Postnatal lung specimens were obtained from autopsy tissues. The present study was approved by the Institutional Review Board of the University of Iowa.

Human fetal lung explant culture and morphometric assessment ofFGF-7 and FGF-10 effects.

Six- to 12-wk gestation human fetal lung tissue explants were embedded in a collagen mixture in the bottom of a 35-mm tissue culture dish and cultured in Ham's F-12 with 10% FCS, 10 mM HEPES, and penicillin/streptomycin by using previously reported methods (5). Explants were used for electrophysiology studies and timed drug exposure experiments if they showed evidence of mesenchymal cell migration into the collagen matrix, were well embedded in the collagen matrix, and had visible epithelial layers with liquid-filled lumina.

Individual explants were examined 0, 24, 48, and 85 h after exposure to evaluate the effects of FGF-7 on the luminal liquid volume present. Each explant was photographed serially with phase contrast microscopy, and the photographs were overlaid with a 100-point grid. In each section, the 100 intersections were scored with the reference grid to determine whether they covered lung tissue or lumen. Agents evaluated included the following: KGF (5–50 ng/mL) and bumetanide (500 μM). Data are presented as the percentage of the explant comprised of liquid-filled lumen (% mean luminal area).

Similar methods were used to quantify the effects of FGF-10 on explant luminal volume. In these experiments, a concentration of 100 ng/mL was used, and we quantified the changes in luminal volume 0, 12, and 24 h after addition of the reagent.

Measurement of PD.

In vitro measurements of PD were made using previously described methods (5). Briefly, fetal lung explants (6–12 wk gestation) bathed with HEPES buffered NaCl Ringer's solution in 35-mm tissue culture dishes were mounted on the stage of an inverted microscope and maintained at 37°C with a water-jacketed heater on a vibration-isolation table. A glass microelectrode (resistance ∼10 Mohms) filled with 150 mM KCl was advanced with a hydraulic manipulator under direct observation. The potential-measuring reference Ag-AgCl electrode was connected to the bathing solution with a 1-M KCl-agar bridge. Transepithelial voltage was measured with an electrometer and recorded with a strip chart recorder. In all cases, the most distal dilated ends of branching tubules (ductal or acinar epithelia) were chosen for the impalement sites.

Expression of KGF and KGFr mRNA.

Ribonuclease protection assays (RPA) were used to quantify KGF and KGFr mRNA expression in tissues by using previously described methods (23). Plasmids containing the cDNA for human KGF and KGFr were the generous gift of Dr. Paul Finch, Mount Sinai Medical Center, New York, NY (24). The cDNA encoding KGF and KGFr were cloned into pT3/T7 18U (Pharmacia) as previously reported (24). The KGF cDNA consisted of a 324-bp Eco RI/ Bam H1 fragment (nt 145–469) of the human KGF cDNA. The KGFr cDNA consisted of a 148-bp Eco RI/ Hin dIII fragment (nt 1267–1414) of the human KGFr cDNA. Total RNA was isolated from lung tissue pieces by using the acid guanidinium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (25). [α-32P]dCTP antisense riboprobes for KGF and KGFr were prepared using the MAXIscript in vitro transcription kit using T3 polymerase according to the manufacturer's instructions (Ambion, Austin, TX). Hybridization was performed using 1 ng of each probe and 20 μg of total RNA according to the manufacturer's instructions in the Hybspeed RPA kit (Ambion). RNA-RNA hybrids were digested with RNase to yield protected fragments of 324, 148, and 80 bp for KGF, KGFr, and 18S, respectively. Results were visualized by PAGE and autoradiography. Expression in the lung was quantified by counting 32P emissions from the protected KGF and KGFr fragments and normalizing to the 18S signal. Replicate studies were performed on the same specimens and representative results presented.

Regulation of expression of KGF and KGFr mRNA by cAMP andIL-6.

First trimester human fetal lung explants (6–12 wk gestation) were treated with either 10 μM forskolin with 10 μM IBMX (cAMP agonist), 50 ng/mL IL-6, or left as controls in basal culture media for 12, 24, and 48 h. RPA were used to quantify KGF and KGFr mRNA expression as above. The 32P emission data were log-transformed and then analyzed using a mixed-model ANCOVA (SAS version 6.12 for Windows, Cary, NC) (26). The experiment was performed on three separate tissue preparations.

Effect of KGF-blocking antibody on cAMP-induced liquidsecretion.

First trimester human fetal lung explants (7–9 wk gestation) were treated with either 50 μM CPT-cAMP with 10 μM IBMX or left as controls in basal culture media. In both conditions, explants were studied in the presence or absence of a murine MAb directed against KGF (concentration 10 μg/mL). This dose was 10 times the neutralization dose50 (ND50) reported for this antibody (R & D Systems). Tissues were sequentially photographed 0, 24, and 58 h after treatment to assess changes in the luminal fluid content.

RESULTS

KGF and FGF-10 stimulate liquid secretion in human fetal lungtissue.

Previous work in transgenic mice and cultured mouse fetal mouse lung explants showed that KGF stimulated fetal lung liquid secretion (8, 20). Other work in murine lung suggested that FGF-10 may have similar effects (11, 12). We tested the hypothesis that these factors stimulate liquid secretion in human fetal lung by quantifying the effects of these growth factors on explant luminal volume over time in culture. As shown in Figure 1, A and B, KGF caused a dose-dependent increase in explant luminal area that was apparent within the first 24 h of application. The effect was most pronounced at a dose of 50 ng/mL. Liquid secretion was inhibited by coincubation of KGF with bumetanide, an inhibitor of the electroneutral Na+/K+/Cl− cotransporter, consistent with effects mediated by Cl− secretion. These results were qualitatively similar to those previously reported in cultured fetal mouse lung (8). The effects of FGF-10 on explant luminal area and liquid secretion are shown in Figure 1, C and D. Similar to KGF, a significant increase in explant luminal area was observed within 12 h of FGF-10 treatment.

Figure 1
figure 1

Effects of KGF and FGF-10 on liquid secretion in human fetal lung tissue. A, Serial photographs of explants treated with KGF (5–50 ng/mL), KGF with 500 μM bumetanide, or basal control media. KGF caused a dose-dependent increase in explant luminal volume that was apparent within the first 24 h of treatment. Representative photos shown from experiments replicated on three different tissue preparations. For each experimental condition, six to eight explants were photographed. B, Measured changes in explant percent luminal volume in response to the treatments shown in (A). Graphical representation of data shown in (A). Bars represent mean ± SE from a representative experiment. Explants from three different tissue preparations were analyzed as described for (A). (★p< 0.05, compared with the control by a paired t test). C, Serial photographs of explants treated with FGF-10 (100 ng/mL) vs basal control media. FGF-10 caused a dose-dependent increase in explant luminal volume that was apparent by 12 h of treatment. Representative photos shown from experiments performed on three different tissue preparations. For each experimental condition, four to six explants were photographed and a representative photo is shown. Panels C-1 and C-2 show control tissues at 0 and 24 h, respectively. Panels C-3 and C-4 show FGF-10 treated tissues at 0 and 24 h, respectively. D, Changes in explant percent luminal volume in response to the treatments shown in (C). Bars represent mean ± SE from a representative experiment as described in (C). (★p< 0.05, compared with the control by a paired t test).

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Effects of KGF on PD.

Liquid secretion in the fetal lung is coupled to the transport of Cl− (1, 5). We hypothesized that if KGF stimulates liquid secretion via the active transport of Cl−, the PD would hyperpolarize acutely. Unexpectedly, KGF had no acute effect on PD across the fetal lung epithelium (Fig. 2A). This included a broad range of KGF doses from 0.1 to 1000 ng/mL. Explant tissues were viable as shown by hyperpolarization of PD in response to the subsequent addition of IBMX and forskolin (5).

Figure 2
figure 2

Effects of KGF on PD. A, Representative PD tracing. KGF had no acute effects on PD across the fetal epithelium in experiments on tissues from three separate specimens. The PD hyperpolarized in response to cAMP agonists [IBMX, forskolin (Fsk)], indicating tissue viability and responsiveness to other agonists. In indicates tissue impaled with microelectrode;out, microelectrode removed. B, Graphical representation of PD recordings from explants treated with KGF for 60 h. Chronic KGF exposure (5–50 ng/mL) caused a dose-dependent decrease in PD. The experiment was performed on three separate tissue preparations. Bars indicate mean ± SE for five to eight cysts/condition. Results were replicated on three different tissue specimens. ★p< 0.05, compared with the control by a paired t test.

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In view of this result, explants were exposed to KGF at doses of 5 or 50 ng/mL for 60 h, conditions that stimulated luminal liquid secretion (Fig. 1), and the PD was recorded. As shown in Figure 2B, chronic KGF exposure caused a significant dose-dependent decrease in PD. There was a statistically significant decrease in PD over time with 5 ng/mL KGF (−1.13 ± 0.13 mV, n= 8, p< 0.01) and 50 ng/mL KGF (−0.79 ± 0.10 mV, n= 10, p< 0.001) compared with controls (−1.88 ± 0.25 mV, n= 6, Fig. 2B). KGF-treated and control tissues responded to IBMX/forskolin with characteristic hyperpolarization of PD (not shown). The PD in bumetanide-treated explants was unobtainable as no appreciable lung cysts were present.

Ontogeny of KGF and KGFr mRNA in human lung.

In murine lung models, the expression of the mRNA for KGF and KGFr is developmentally regulated (17, 18). Similar data in humans are not available. We, therefore, determined the ontogeny of KGF and KGFr mRNA in the lung by using RPA. As shown in Figure 3, A–C, both KGF and KGFr mRNA were expressed in fetal and postnatal lung. Because of the limited number of samples available for analysis, it is difficult to conclude how mRNA abundance changes with development. However, there was a suggestion that the KGFr mRNA abundance declined postnatally (Fig. 3C).

Figure 3
figure 3

Ontogeny of KGF and KGFr mRNA in human lung. A, KGF and KGFr mRNA expression were determined by RPA. Columns 1–6: 8, 12, 18, 22 wk gestation, 5 mo, 12 y. A representative result from three similar experiments is shown. 18S RNA abundance was used for data normalization. B, KGF mRNA expression normalized to 18S RNA abundance. KGF mRNA was expressed in fetal and postnatal lung. C, KGFr mRNA expression normalized to 18S RNA abundance. KGFr mRNA was expressed in the first and second trimester tissues. There was a suggestion of a decline in expression in postnatal tissues.

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Effects of cAMP and IL-6 on KGF and KGFr mRNA expression.

The promoter region of hKGF contains consensus binding sites for IL-6 and cAMP (15, 16). We hypothesized that KGF mRNA expression would increase in human fetal lung in response to these agents. As shown in Figure 4, A–C, cAMP agonists and IL-6 both increased expression of KGF mRNA in human fetal lung explants. The cAMP effects were most notable at 12 h incubation (p< 0.005), whereas IL-6 effects were greatest at 24 h (p< 0.050). There were no significant changes in KGFr mRNA expression in response to cAMP or IL-6 (not shown).

Figure 4
figure 4

Regulation of KGF and KGFr mRNA expression. Eight to ten explants were cultured for each condition as described in “Methods.” Replicate experiments were performed on three separate tissue specimens. A, RPA for KGF and KGFr mRNA abundance in cAMP- or IL-6–stimulated first trimester human lung explants. 18S RNA abundance was used for data normalization. Lanes 1–4: control tissue 0, 12, 24, and 48 h; l anes 5–7: IL-6–treated tissue 12, 24, and 48 h;lanes 8–10: cAMP-treated tissue 12, 24, and 48 h. B, KGF mRNA expression increased with exposure to cAMP and was statistically significant at 12 h compared with controls (★p< 0.005). C, KGF mRNA expression increased with exposure to IL-6 and was statistically significant at 24 h compared with controls (★p< 0.05).

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KGF-blocking antibody does not inhibit cAMP-induced liquidsecretion.

Explants treated with or without cAMP agonists were examined sequentially for changes in luminal area in the presence or absence of KGF-blocking antibodies. At a concentration of 10 μg/mL, KGF-blocking antibodies had no significant effect on the liquid secretion in control or cAMP-treated tissues (Fig. 5).

Figure 5
figure 5

Effect of KGF-blocking antibodies (Ab) on liquid secretion in control or cAMP-treated explants. Tissues from three different first trimester samples were studied ± KGF-blocking antibodies and photographed sequentially at the indicated time points. Each time point represents data from four to six explants. KGF-blocking antibodies failed to inhibit the cAMP-induced increase in luminal area. cAMP agonists induced a significant increase in luminal area at each time point compared with controls (★p< 0.05).

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DISCUSSION

The present study presents the first evidence that KGF and FGF-10 stimulate liquid secretion in human fetal lung. The observation that the effects of KGF on liquid secretion were bumetanide-sensitive suggests that the underlying mechanism is, at least in part, mediated by Cl− secretion. Interestingly, KGF-stimulated liquid secretion did not acutely change the PD, whereas chronic KGF exposure depolarized the PD. The mRNA for KGF and KGFr were both expressed in human lung, and their abundance was greatest in fetal samples. We also showed that IL-6 and cAMP both increased KGF mRNA expression, effects that may be mediated through cis-acting elements in the promoter region of the KGF mRNA (15). Thus, the expression of KGF and KGFr mRNA in fetal lung and the knowledge of the local effects of KGF and FGF-10 on epithelia suggest that these FGF family members are paracrine factors regulating lung liquid secretion in the human fetus.

The local factors regulating lung liquid secretion during fetal development are poorly understood. Studies in fetal lambs and goats show that lung liquid production is linked to Cl− secretion and that fluid challenges and prolactin both stimulate secretion (1, 27, 28). Several agents, including atrial natriuretic factor (29), vasopressin (30), vasotocin (30), and epidermal growth factor (31), were reported in decreased lung liquid production in the fetal lamb model. In vitro studies in fetal rat (32, 33) and human explant models (5, 6, 34) indicate that β-agonists, prostaglandins, and cAMP agonists stimulate Cl− and liquid secretion and that these effects may be altered by the hormonal conditions of the culture (7, 35). Subsequent studies in primary cultures of distal lung epithelia derived from rat (4) or human (36, 37) fetal lung showed that cAMP agonists and Ca2+ agonists stimulate Cl− secretion. In addition to Cl− secretion, amiloride-sensitive Na+ absorption was detected in some studies (38, 39). Work in murine models first suggested that KGF may be an important regulator of fetal lung liquid production. Simonet et al. (20) showed that transgenic mice expressing KGF driven by the human SP-C promoter developed cystic dilatation of the lung reminiscent of cystic adenomatoid malformation in humans. Subsequent in vitro studies confirmed that fetal mouse lung liquid secretion was potently stimulated by KGF and that liquid secretion was CFTR-independent (8, 20). In addition to stimulating liquid secretion, KGF may play other important roles in the fetal lung through its effects on cell proliferation, differentiation, and morphogenesis (40–45).

The temporal and spatial distribution of the KGF and KGFr mRNA in developing lung shows that the cells expressing KGF are situated near the respiratory epithelium expressing its receptor, consistent with a role in the local regulation of liquid secretion (17, 18). In the human lung, the ontogeny of KGF and KGFr expression suggests that KGF may be an important locally acting factor that regulates secretion during development. The abundance of KGF and KGFr mRNA was greatest in the fetal human lung (Fig. 3, A–C). Further studies are needed to correlate changes in KGF and KGFr mRNA abundance with changes in their respective protein products.

The signaling pathway for KGF in fetal pulmonary epithelia is unknown, but KGFr is a membrane-spanning tyrosine kinase and presumably exerts its effects through phosphorylation of proteins (13). Similarly, FGF-10 also signals via the KGFr with high affinity (14). The 5′ flanking region of the human KGF gene contains cis-acting elements responsive to regulation by IL-1, IL-6, and cAMP agonists (15, 16). We observed that both cAMP and IL-6 significantly increased expression of KGF but not KGFr mRNA in vitro, suggesting that specific intracellular messengers and secreted factors may mediate the paracrine effects of KGF in the fetal lung. However, KGF-blocking antibodies failed to inhibit the cAMP-induced swelling of the explants. One interpretation of these data is that the KGF antibodies were not fully blocking under these experimental conditions. Alternatively, the cAMP effects may be mediated through other mechanisms. Although we have not shown that a change in KGF mRNA abundance is paralleled by changes in KGF protein levels, the observation that cAMP agonists induced KGF mRNA expression in human fetal lung explants may, in part, explain our previous observation that cAMP stimulates liquid secretion in this model (5, 6).

The observation that KGF null mice have no obvious pulmonary developmental abnormalities suggested that other FGF family members may signal through the KGF receptor (46). In addition to the effects of KGF noted in this study, we found that FGF-10 stimulated liquid production. Of note, both FGF-1 and FGF-10 are expressed in the developing lung mesenchyme and may act as ligands for KGFr (11, 14, 47). FGF-1 had marked effects on mouse lung-branching morphogenesis in vitro but little impact on liquid production (11). In the same study, FGF-10 stimulated expansion of the lung bud similarly to KGF, consistent with stimulation of liquid secretion (11). FGF-10 null mice die prenatally and show absence of lung development beyond a tracheal rudiment (21, 22). These data, in addition to the present study, suggest that KGF and FGF-10 play important roles in lung development, including the regulation of liquid secretion in human fetal lung.

The cellular pathways for Cl− transport and osmotically coupled liquid secretion after stimulation of human fetal lung by KGF or FGF-10 are largely unknown. However, KGF- or FGF-10–stimulated secretion may be responsible for regulating liquid production by the normal fetal lung and the fetal lung affected by cystic fibrosis (6, 8). Presumably, Cl− secretion in response to these agents involves a basolateral membrane entry step, such as the bumetanide-sensitive Na+/K+/Cl− cotransporter, and an apical membrane exit step. The apical membrane channel or transporter responsible for KGF-induced Cl− secretion is likely to be CFTR-independent on the basis of this study and previous murine studies, but its identity is uncertain (8). Possible candidates include a Ca2+-activated Cl− channel (37), CFTR-independent cAMP-activated pathways (48), a member of the ClC family of Cl− channels (49, 50), or a novel fetal Cl− channel. Neither the acute nor the chronic effects of KGF on PD support the notion of stimulation of electrogenic Cl− secretion. It is also possible that KGF and FGF-10 stimulate Cl− transport via an electroneutral pathway such as an anion exchanger, although the observed depolarization of the PD argues against this. Alternatively, the effects of these growth factors may be indirect. Rather than directly stimulating secretion, they may exert an inhibitory effect on an absorptive pathway such as the epithelial sodium channel or an apical membrane cation channel (51–53). In this case, inhibition of an apical membrane cation conductance would hyperpolarize the apical membrane and depolarize the PD, thereby increasing the driving force for Cl− secretion. Consistent with this model, PD decreased in explants chronically exposed to KGF, whereas luminal size increased. Finally, it is conceivable that KGF or FGF-10 have no direct effects on Cl− secretion and exert their effects on liquid secretion by increasing the epithelial mass (41, 54). However, this seems unlikely due to the time course and magnitude of the changes in luminal area that were observed in the present study. Understanding the cellular basis for KGF- and FGF-10–stimulated liquid secretion may provide further insights into normal lung development and cystic fibrosis pathogenesis and treatment.