Stretch-stimulated surfactant synthesis is coordinated by the paracrine actions of PTHrP and leptin

J. S. Torday1,2 and V. K. Rehan2

Departments of 2 Pediatrics and 1 Obstetrics and Gynecology, Harbor-University of California Los Angeles Research and Education Institute, Torrance, California 90502


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intrauterine lung development, culminating in physiological pulmonary surfactant production by epithelial type II (TII) cells, is driven by fluid distension through unknown mechanisms. Differentiation of alveolar epithelial and mesenchymal cells is mediated by soluble factors like parathyroid hormone-related protein (PTHrP), a stretch-sensitive TII cell product. PTHrP stimulates pulmonary surfactant production by a paracrine feedback loop mediated by leptin, a soluble product of the mature lipofibroblast (LF). When LFs and TIIs are stretched in coculture, there is a fivefold increase in surfactant phospholipid synthesis that can be "neutralized" by inhibitors of PTHrP or leptin, implicating a paracrine feedback loop in this mechanism. Stretching LFs stimulates PTHrP binding (2.5-fold) and downstream stimulation of triglyceride uptake quantitatively (15-25%) due to upregulation of adipose differentiation-related protein expression. Stretching TII cells increases leptin stimulation of their surfactant phospholipid synthesis threefold, suggesting that retrograde signaling by leptin to TII cells is also stretch sensitive. We conclude that the effect of stretch on alveolar LF and TII differentiation is coordinated by PTHrP, leptin, and their receptors.

lipofibroblast; parathyroid hormone-related protein; leptin; adipose differentiation-related protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTRAUTERINE LUNG DEVELOPMENT is driven by lung fluid secretion, which distends the alveolar acinus, forming a "bubblelike" template for its structure and function (2, 16). Stretch stimulates the growth and differentiation of both the epithelial and mesenchymal cells of the alveolar septal wall in preparation for air breathing at the time of birth. Of particular importance to the transition from intrauterine to extrauterine adaptation is the synthesis and secretion of pulmonary surfactant by alveolar type II cells. The surfactant reduces surface tension of the lung lining layer, preventing atelectasis of the air-filled lung. The surfactant is a complex of both proteins and phospholipids, the latter being responsible for the lowering of surface tension.

It has previously been shown that stretching stimulates the growth of cultured mixed lung cells (15) and the secretion of pulmonary surfactant phospholipid by epithelial type II (TII) cells (38). It has subsequently been shown that stretch increases the expression of surfactant proteins (27). However, there is no information regarding how stretch stimulates surfactant phospholipid synthesis. Developing TII cell surfactant phospholipid synthesis is controlled by a wide variety of growth factors (13), cytokines (41), and eicosanoids (10), which mediate the paracrine regulation of fetal lung development. Among these, prostaglandin E2 (PGE2) and parathyroid hormone-related protein (PTHrP) production by TII cells is stimulated by stretch, and each can stimulate surfactant phospholipid production (25, 36). The mechanism of PTHrP action on surfactant phospholipid synthesis is paracrine in nature (25), being expressed and secreted by TII cells (34) and binding to its cognate receptor on neighboring mesenchymal fibroblasts (unpublished observations), stimulating both cAMP and D-myo-inositol 1,4,5-trisphosphate production (25). The cAMP-dependent protein kinase A (PKA) pathway stimulates the uptake, storage, and release of triglycerides, which are characteristic of the mature lipofibroblast (LF) (20, 24, 36). The triglycerides stored by LFs have been found to act as substrate for TII cell surfactant phospholipid synthesis (31). We have recently demonstrated that uptake, storage, and trafficking of neutral lipids may be mediated by adipose differentiation-related protein (ADrP), a 53-kDa protein associated with the limiting membrane that surrounds these lipids within the cytoplasm (unpublished observations).

PTHrP also stimulates LF expression and secretion of leptin, which stimulates surfactant phospholipid synthesis and surfactant protein B expression by developing TII cells (35). We have hypothesized that stretch stimulates surfactant phospholipid synthesis by coordinately upregulating the receptor-mediated mechanisms that mediate both the PTHrP and leptin target cell effects in this developmental, regulatory paracrine loop.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Leptin antibody (rat, polyclonal) was acquired from Linco (St. Charles, MO). PTHrP(7-34) amide was obtained from Bachem (Torrance, CA). [125I]-Tyr34-PTHrP(1-34) was purchased from Peninsula Laboratories, Belmont, CA. [3H]choline chloride and [3H]triolein were purchased from NEN (Boston, MA).

Animals. Time-mated Sprague-Dawley rats (time E0 = day of mating) were obtained from Charles River Breeders (Holister, CA). These experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Cell culture method for isolation of LFs and TII cells from fetal lung. This method has been used extensively in our laboratory (32, 33). Three to five dams were used per preparation on day 19 of gestation (E0 = day of mating). The dams were killed with an overdose of pentobarbital sodium (100 mg/ml ip), and the pups were removed from the uterus by laparotomy and kept on ice. The lungs were removed en bloc under a laminar flow hood using sterile technique and put into ice-cold sterile Hanks' balanced salt solution without calcium or magnesium. The solution was decanted, and 5 volumes of 0.05% trypsin (Worthington) were added to the lung preparation.

The lungs were dissociated in a 37°C water bath using a Teflon stirring bar to physically disrupt the tissue. When the tissue was completely dispersed into a unicellular suspension (~20 min), the cells were spun down at 500 g for 10 min at room temperature in a 50-ml polystyrene centrifuge tube. The supernatant was decanted, and the cell pellet was resuspended in DMEM (GIBCO) containing 10% fetal bovine serum (FBS) to yield a mixed cell suspension of ~3 × 108 cells, as determined by a Coulter Particle Counter (Hayaleah, FL). The cell suspension was added to 75-cm2 culture flasks (Corning Glass Works, Corning, NY) for 30-60 min at 37°C in a CO2 incubator to allow for differential adherence of the fibroblasts (28). Fibroblasts were maintained in DMEM/10% FBS or DMEM/0.1% bovine serum albumin (BSA) until further processing. These cells were LFs based on vimentin staining (>95% positive), and all the cells contained neutral lipid pools by electron microscopy (29).

The nonadherent cells from the above-described cell preparation were transferred to another set of 75-cm2 culture flasks for an additional 60-min period. After this second culture period, the medium and nonadherent cells were removed from the flask and diluted with 1 volume of culture medium. This diluted suspension was cultured overnight in 75-cm2 flasks at 37°C in a CO2 incubator to allow the TII cells to adhere (3). TII cells were identified by their appearance in culture under phase-contrast microscopy, lamellar body content, and microvillar processes or by cytokeratin-positive staining.

Fibroblast/TII cell coculture. Embryonic day 19 (E19) fibroblasts and TII cells were cocultured on Bioflex silicone-Elastomer multiwell plates (Flexcell, Hillsborough, NC) precoated with type I collagen (Collagen, San Francisco, CA) as follows. Fibroblasts and TII cells were harvested from monolayer cultures with 0.25% trypsin/5 mM EDTA. The numbers of cells were determined by a Coulter Particle Counter, and they were recombined in a 1:1 ratio, spun down at 500 g for 10 min, and allowed to incubate as a pellet at 37°C in a CO2 incubator. At the end of the incubation, the cells were resuspended in DMEM to yield a cell suspension equivalent to 2 × 107 fibroblasts and 2 × 107 TII cells; one-twentieth of this suspension (i.e., 1 × 106 fibroblasts and 1 × 106 TII cells) was then seeded per well. The cells were quantitatively adherent and remained attached to the membrane throughout the experimental procedure. The cells were maintained in DMEM at 37°C in 5% CO2-95% air.

Cell stretching method. Cocultured fibroblasts and TII cells in multiwell plates were mounted on a Flexercell FX-3000 strain unit (Flexcell) on top of flat-headed Delrin cylinders. Application of a vacuum stretched each membrane over the central cylinder post, creating uniform radial and circumferential strain across the membrane surface. Equibiaxial elongation of 0-9% was applied at intervals of 50 cycles/min for 15 min/h.

Choline incorporation assay. The rate of saturated phosphatidylcholine synthesis was determined as previously described (31). Briefly, the mixed cell cultures were incubated with [3H]choline chloride (1 µCi/ml) for 4 h at 37°C in 5% CO2-balance air. Phospholipids were extracted by the method of Bligh and Dyer (4) and dried under a stream of nitrogen at 50°C. The phospholipid extracts were mixed with 0.5 ml of an osmium tetroxide solution (70 mg/100 ml of carbon tetrachloride) and reacted for 15 min at room temperature (30). The reaction mixture was dried under a stream of nitrogen at 50°C, and the dried extract was chromatographically separated by thin-layer chromatography in chloroform:methanol:water (65:25:4 vol/vol) (30). The phospholipids were subsequently scraped from the chromatography plates and analyzed for radioactive content by liquid scintillation spectrometry.

Triglyceride uptake assay. The method used to quantitate triglyceride uptake by fetal rat lung fibroblasts has previously been described (31). Briefly, culture medium was replaced with DMEM containing 20% adult rat serum mixed with [3H]triolein (5 µCi/ml). The cells were incubated at 37°C in 5% CO2-balance air for 4 h. At the termination of the incubation, the medium was decanted, the cells were rinsed twice with 1 ml of ice-cold DMEM, and the cells were removed from the culture plate after a 5- to 10-min incubation with 2 ml of a 0.05% trypsin solution. An aliquot of the cell suspension was taken to determine the cell number, and the remaining cell suspension was extracted for neutral lipid content.

RNA extraction. Total cellular RNA was isolated using previously described methods (5).

Semiquantitative RT-PCR. The appropriate cDNA fragments were amplified using 400 ng of total RNA from lung tissues or cells, avian myeloblastosis virus reverse transcriptase, random hexamers, and deoxyribonucleotides. The reactions were run at 42°C for 75 min and terminated by heating at 95°C for 5 min. Coamplification with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as an internal standard. PCR was initiated by Taq DNA polymerase and allowed to proceed for 30 cycles with an annealing temperature of 50°C. The following primers were used in the RT-PCR assay: rat ADrP, sense, 5'-ACAGCTCACTTATGGTCGTGCC; antisense, 3'-AAACAGTGATGAAGCCTGCTCG; GAPDH, sense, 5'-CCATGACAACTTTGGTATCGTGG; antisense, 3'-TTGCTGTAGCCAAATTCGTTGTC. The identities of all RT-PCR products were confirmed by Southern blotting. mRNA expression was quantitated by densitometry (Eagle Eye, Stratagene).

PTHrP receptor binding assay. The receptor binding assay was carried out using a method similar to that previously described (23) to determine the number of PTHrP receptors per cell, based on Scatchard plots. The assay mixture, in a total volume of 0.1 ml, contained 50 mM Tris · HCl (pH 7.4), 2 mM dithiothreitol, 10 mM EDTA, 10 µg/ml each of protease inhibitors (leupeptin, pepstatin, antipain, and aprotinin), 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml BSA, 5 mM MgCl2, 10-500 pmol [125I]-Tyr34-PTHrP-(1-34) (sp act 1,064 Ci/mmol), 10-12 µg membrane protein, and 10-10 to 10-6 M PTHrP-(1-34). Triplicate samples were incubated for 30 min at 30°C.

Reactions were stopped by the addition of 0.1 ml of homogenization buffer containing 20 mg/ml of BSA and placed in ice-cold water for 30 min, followed by centrifugation at 15,000 g for 1 min. The supernatant was aspirated, and the pellet was counted for radioactive content with a gamma counter (model 1470; Wallace, Gaithersburg, MD). Nonspecific binding was determined in the presence of 1 µM nonradioactive PTHrP-(1-34). Specific binding of [125I]- Tyr34-PTHrP-(1-34) was calculated as total binding minus nonspecific binding and expressed as femtomoles per milligrams of protein. Specificity of binding was determined in the presence of 1 µM PTHrP-(7-34), a selective PTHrP receptor antagonist, showing that it inhibited the binding of radioactive ligand. In preliminary studies, the binding was found to be linear with time for up to 60 min of incubation.

Statistical analyses. Analysis of variance was used to compare data within experimental groups. The null hypothesis was rejected when P < 0.05 was not obtained.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Initially, we tested the effect of stretch on de novo saturated phosphatidylcholine (Sat PC) synthesis by TII cells cultured alone or cocultured with fibroblasts derived from E19 fetal rat lung (Fig. 1). There was a modest (15%) but significant increase in Sat PC synthesis by TII cells when stretched alone in culture. Coculture of TII cells with fibroblasts had no effect on Sat PC synthesis per se, but when the cocultures were stretched there was a dramatic fivefold increase in Sat PC synthesis. We subsequently repeated this experiment in the presence of either the specific PTHrP receptor antagonist PTHrP-(7-34) amide or a leptin antibody (Fig. 2). Note that the effect of stretch on Sat PC synthesis by TII cell-fibroblast cocultures was completely blocked by the addition of either the PTHrP receptor antagonist or the leptin antibody, reducing the rate of Sat PC synthesis to that of unstretched cocultures.


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Fig. 1.   Effect of stretch on saturated phosphatidylcholine (Sat PC) synthesis. Comparison of Sat PC synthesis by embryonic day 19 (E19) fetal epithelial type II (TII) cells alone; alone, stretched (6-9% radial elongation, 50 cycles · min for 15 min-1 · h × 15 h-1); cocultured with E19 lipofibroblast (LF); and cocultured with E19 LF, stretched (15 h). The cells were incubated with [3H]choline for the last 4 h of the experiment. *P < 0.05, ***P < 0.00001, static vs. stretched cells, by analysis of variance (ANOVA); n = 5.



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Fig. 2.   Effect of stretch on surfactant phospholipid synthesis is due to parathyroid hormone-related protein (PTHrP) and leptin. Sat PC synthesis (a) by stretched E19 TIIs (6-9% radial elongation, 50 cycles · min for 15 min-1 · h × 15 h-1) cocultured with LFs (b) is inhibited by either PTHrP receptor antagonist (PTHrP ANT, 5 × 10-6 M) or by leptin antibody (c; Leptin Ab, 1:100 dilution). The cells were incubated with choline chloride for the last 4 h of the experiment. *P < 0.001, TII/LF/stretch vs. TII/LF/stretch/PTHrP ANT or Leptin Ab by ANOVA; n = 5.

On the basis of these initial observations, we sought to determine the nature of the PTHrP- and leptin-dependent stretch effects on TII cell Sat PC synthesis. Given that stretch stimulates TII cell PTHrP expression (34), we began by examining the effect of stretch on fibroblast PTHrP receptor binding (Fig. 3). Stretching fetal rat lung fibroblasts under the same conditions as those used for the cocultures, we observed a 2.5-fold increase in PTHrP binding to its receptor, indicating that stretch coordinates the increase in PTHrP expression (34) with the increase in PTHrP receptor binding.


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Fig. 3.   Stretch increases the number of LF PTHrP receptors. E19 LFs were stretched (6-9% radial elongation, 50 cycles · min for 15 min-1 · h × 15 h-1), and PTHrP binding was assayed. *P < 0.01 by ANOVA; n = 5.

To link the stretch effect on PTHrP expression by TII cells to fibroblast differentiation relevant to surfactant phospholipid synthesis (31), we next examined the effect of stretch on PTHrP stimulation of triglyceride uptake by LFs (Fig. 4). As expected, PTHrP (5 × 10-7 M) alone significantly increased triglyceride uptake. In combination with stretch, PTHrP progressively and significantly increased triglyceride uptake over PTHrP by 15-25% at 3-9% elongation. We next examined the effect of PTHrP on ADrP expression, since ADrP mediates the uptake of triglyceride by fetal rat lung fibroblasts (unpublished observations). As can be seen in Fig. 5, PTHrP treatment enhanced the expression of ADrP mRNA by these cells, thus providing a mechanism for the effect of PTHrP on triglyceride uptake.


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Fig. 4.   Effect of stretch on PTHrP-stimulated triglyceride uptake by fetal rat lung fibroblasts. Primary E19 fetal rat lung fibroblasts were stretched (0-9% radial elongation, 50 cycles · min for 15 min-1 · h × 15 h-1) ± PTHrP (5 × 10-7 M) for 15 h. The cells were incubated with [3H]triolein for the last 4 h of the experiment. At the termination of the incubation, the cells were analyzed for triglyceride content. phi P < 0.05 vs. control; *P < 0.05; **P < 0.01; ***P < 0.001 vs. PTHrP by ANOVA; n = 5.



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Fig. 5.   PTHrP (5 × 10-7 M for 15 h) upregulates adipose differentiation-related protein (ADrP) expression vs. control (CTR). Representative gel, n = 5. Lane loading controlled for with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Because PTHrP stimulates TII cell surfactant phospholipid synthesis by a paracrine effect on fetal rat lung fibroblast leptin expression, we next examined the effect of stretch on leptin stimulation of TII cell Sat PC synthesis (Fig. 6). Note that both leptin and stretch stimulated Sat PC synthesis by three- to fourfold, but that the combination of leptin and stretch increased Sat PC synthesis by eightfold, suggesting a synergistic interaction between stretch and leptin.


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Fig. 6.   Effects of leptin and stretch on [3H]Sat PC synthesis. E19 TII cells were stretched (6-9% radial elongation, 50 cycles · min for 15 min-1 · h × 15 h-1) ± leptin (100 ng/ml) and incubated with [3H]choline chloride for the last 4 h of the experiment to determine the rate of saturated phosphatidylcholine synthesis. *P < 0.05, **P < 0.01 control vs. treated by ANOVA; n = 5.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There is extensive experimental evidence to indicate that lung inflation increases the production of surfactant phospholipids in intact animals (7, 21, 40), isolated organs (7, 18, 19), and by cells in culture (6, 38). For example, Wirtz and Dobbs (38) showed the effect of a single mechanical stretch on TII cell surfactant phospholipid secretion, although surprisingly, the effect was not sustained by subsequent stretches. One possible reason for the failure to progressively increase surfactant secretion with stretch is that this process may be substrate dependent, as suggested by the effects of nutrition (14, 17) and ventilation (19, 21) on surfactant production. In support of such a hypothetical substrate-dependent mechanism, previous studies from our laboratory have demonstrated that 1) triglycerides of fibroblast origin can be mobilized by TII cells for surfactant phospholipid synthesis (31), 2) this process is hormonally regulated (31), and 3) stretch increases PGE2 production by TII cells, which increases the release of triglycerides from lung LFs (36). The trafficking of the triglycerides from fibroblasts to TII cells may be mediated by ADrP (unpublished observations). In the present series of experiments, we have evaluated the potential role of the stretch-sensitive PTHrP-dependent signaling pathway (34) on the effect of stretch on pulmonary surfactant phospholipid synthesis.

As shown in Fig. 7, we have observed coordinating effects of stretch on TII cell expression of PTHrP (34) and PGE2 (36) (step 1), the LF PTHrP receptor (step 2) (34), its downstream effect on LF ADrP expression (step 3) and triglyceride uptake (34) (step 4), and on the interaction between LF-produced leptin (step 5) (35) and the TII cell leptin receptor (step 6), stimulating de novo surfactant phospholipid synthesis by TII cells (step 7) (35). We have previously reported that stretching TII cells in culture increases the production of PTHrP [1.4-2.3 pmol/h, static vs. stretched cells (34)] and leptin [5-22 ng/h, static vs. stretched cells (35)]. The effects of PTHrP and leptin are mutually exclusive, i.e., independent and unidirectional, since LFs express the PTHrP receptor and leptin, but fetal TII cells do not (25, 34), although it should be noted that adult TII cells do (8); conversely, TII cells express PTHrP and the leptin receptor, but fibroblasts do not (35). Although it is difficult to compare the independent quantitative effects of stretch on PTHrP and leptin with their integrated effects on de novo surfactant phospholipid synthesis, the fold increase in each approximates the combined effect. We have previously demonstrated that PTHrP stimulation of TII cell differentiation is dependent on leptin expression by LFs (35).


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Fig. 7.   Coordinating effects of stretch on TII cell-LF interactions leading to increased surfactant synthesis. PGE2, prostaglandin E2; ADRP, adipose differentiation-related protein; TG, triglyceride.

PTHrP is necessary for the differentiation of the alveolar acinus (26). Its effects on the cytoarchitecture of the mesenchymal and epithelial components of the lung are dependent on paracrine interactions between the epithelium and mesenchyme (25, 34, 35), mediated by a PTHrP receptor-dependent PKA pathway (25) leading from the epithelium to the mesenchyme and back again to the epithelium (35). The parathyroid hormone/PTHrP receptor was initially cloned from rat osteoblastic and opossum renal cell cDNA libraries (1). The identity and conformational homology of the corresponding amino-terminal domains of parathyroid hormone and PTHrP allow both molecules to interact with one common receptor equipotently on lung fibroblasts (25). Usdin et al. (37) subsequently identified a second receptor that selectively recognizes parathyroid hormone, termed the PTH2 receptor. We have recently shown that fluid formation within the alveolus is the driving force for the upregulation of PTHrP expression by TII cells (33), and we have previously shown that stretching increases the sensitivity of target mesenchymal fibroblasts for PTHrP stimulation of triglyceride uptake (33), a PKA-dependent mechanism important in providing substrate for surfactant phospholipid synthesis (30). These observations and the more recent evidence that leptin mediates the PTHrP effect on TII cell surfactant phospholipid synthesis (34) have provided a paracrine loop whose receptor intermediates were candidates for a stretch-mediated mechanism. We have found that cell stretch enhances PTHrP signaling from the epithelium to the mesenchyme by upregulating both PTHrP itself (33) and PTHrP receptor expression coordinately, and cell stretching enhances the signaling from the mesenchyme to the epithelium by increasing leptin stimulation of surfactant phospholipid synthesis by TII cells, thus completing this epithelial-mesenchymal-epithelial paracrine loop.

Our studies have focused on the role of PTHrP in normal lung development. This focus provides evidence for the role of stretch in physiological lung development and may help us to understand the pathophysiology of barotrauma/volutrauma. By identifying the up- and downstream regulators of the PTHrP signaling pathway, we hope to identify the molecular mechanisms responsible for this important mechanism of lung development and pathophysiology.


    FOOTNOTES

Address for reprint requests and other correspondence: J. S. Torday, Dept. of Pediatrics, Center for Developmental Biology, RB-1, 1124 W. Carson St., Torrance, CA 90502 (E-mail: jtorday{at}gcrc.rei.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 22, 2002;10.1152/ajplung.00380.2001

Received 24 September 2001; accepted in final form 15 February 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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