AJP - Lung Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 290: L250-L258, 2006. First published September 2, 2005; doi:10.1152/ajplung.00244.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/L250    most recent
00244.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Filby, C. E.
Right arrow Articles by Wallace, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Filby, C. E.
Right arrow Articles by Wallace, M. J.

VDUP1: a potential mediator of expansion-induced lung growth and epithelial cell differentiation in the ovine fetus

C. E. Filby, S. B. Hooper, F. Sozo, V. A. Zahra, S. J. Flecknoe, and M. J. Wallace

Fetal and Neonatal Research Group, Department of Physiology, Monash University, Victoria, 3800, Australia

Submitted 3 June 2005 ; accepted in final form 31 August 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The degree of fetal lung expansion is a critical determinant of fetal lung growth and alveolar epithelial cell (AEC) differentiation, although the mechanisms involved are unknown. As VDUP1 (vitamin D3-upregulated protein 1) can modulate cell proliferation, can induce cell differentiation, and is highly expressed in the lung, we have investigated the effects of fetal lung expansion on VDUP1 expression and its relationship to expansion-induced fetal lung growth and AEC differentiation in fetal sheep. Alterations in fetal lung expansion caused profound changes in VDUP1 mRNA levels in lung tissue. Increased fetal lung expansion significantly reduced VDUP1 mRNA levels from 100 ± 8% in control fetuses to 37 ± 4, 46 ± 4, and 45 ± 9% of control values at 2, 4, and 10 days of increased fetal lung expansion, respectively. Reduced fetal lung expansion increased VDUP1 mRNA levels from 100 ± 16% in control fetuses to 162 ± 16% of control values after 7 days. VDUP1 was localized to airway epithelium in small bronchioles, AECs, and some mesenchymal cells. Its expression was inversely correlated with cell proliferation during normal lung development (R2 = 0.972, P < 0.002) as well as in response to alterations in fetal lung expansion (R2 = 0.956, P < 0.001) and was positively correlated with SP-B expression during normal lung development (R2 = 0.803, P < 0.0001) and following altered lung expansion (R2 = 0.817, P < 0.001). We suggest that VDUP1 may be an important mediator of expansion-induced lung cell proliferation and AEC differentiation in the developing lung.

vitamin D3-upregulated protein 1; fetal lung development; lung expansion; cell proliferation; surfactant protein B

DURING FETAL LIFE, LUNG DEVELOPMENT is largely determined by the degree to which the lungs are distended by liquid. This liquid is secreted across the pulmonary epithelium into the lung lumen and leaves the lungs via the trachea. The fetal glottis and upper airway (18) restrict the efflux of lung liquid causing it to accumulate within the future airways. At rest (during fetal apnea), this maintains an internal distending pressure on the fetal lungs of 1–2 mmHg (43), which is vital for fetal lung growth and development. If this internal distending pressure is abolished by draining the fetal lungs of liquid, lung growth ceases (1, 19). On the other hand, obstruction of the fetal trachea [tracheal obstruction (TO)] prevents the loss of lung liquid, causing it to accumulate within the future airways. As a result, the fetal lungs expand with accumulated liquid, which is a potent stimulus for fetal lung growth and structural maturation (6, 19, 31, 32).

The acceleration in lung growth induced by TO is time dependent, causing a maximum increase in DNA synthesis rates at 2 days of TO, which returns to control levels by 10 days of TO (31–33); the cell types in the distal lung that proliferate include type II alveolar epithelial cells (AEC), fibroblasts, and endothelial cells (33). The basal level of lung expansion is also an important determinant of AEC phenotype, as alterations in fetal lung expansion induce major changes in type I and type II AEC proportions. Increases in lung expansion induce type II to type I AEC trans-differentiation, whereas reductions in lung expansion favor the type II cell phenotype (3, 12, 15). Although it is clear that the basal degree of lung expansion regulates lung cell proliferation and AEC differentiation, the cellular and molecular mechanisms that mediate these effects are unknown.

As lung expansion is an important determinant of normal fetal lung development, much attention has focused on identifying the genes that mediate expansion-induced fetal lung growth, particularly as they are likely to be normal endogenous regulators of lung cell proliferation and differentiation. We have recently identified VDUP1 (vitamin D3-upregulated protein 1) as a gene that is differentially expressed by 36 h of increased fetal lung expansion (40a). VDUP1 is a 46-kDa intracellular protein that was initially isolated in HL-60 cells, and its expression is upregulated by vitamin D3 administration (8). VDUP1 is highly expressed in the lung (23) and has well described roles in cell cycle inhibition and tumor suppression (8, 17, 23, 38) as well as cellular differentiation (7, 8, 10, 41). Thus it is possible that VDUP1 is an important modulator of fetal lung growth and development, but its potential role in lung development has not been investigated previously. Our primary aim was to determine the effect of normal, accelerated, and reduced lung growth, induced by changes in fetal lung expansion, on VDUP1 expression in fetal sheep; we have also correlated these changes with the known changes in fetal lung growth and maturation induced in these models. We hypothesized that VDUP1 mRNA levels would be low during periods of rapid lung cell proliferation and elevated when lung growth is inhibited and type II AEC differentiation is enhanced.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental groups. The lung tissues used in this study were previously collected from fetal and newborn sheep during 1) normal fetal and postnatal development (14) as well as following 2) accelerated (24, 32) and 3) retarded (19) fetal lung growth, induced by alterations in fetal lung expansion. The Monash University Committee for Ethics in Animal Experimentation approved all experimental procedures involving the use of animals. The animals included in the ontogeny analysis were not exposed to any experimental manipulation, and lung tissue was collected at 90, 105, 111*, 128, 138, and 142 days of gestational age (GA; term ~147 days) and at 2 wk postnatal age (PNA; n = 5 for each, except *n = 4) as described previously (14).

Fetal lung growth rates were manipulated in chronically catheterized fetal sheep by increasing and decreasing the degree of fetal lung expansion (19, 24, 33). To implant fetal catheters, anesthesia of the ewe and fetus was induced by sodium thiopentone (1 g iv) and was maintained, after tracheal intubation, by inhalation of 1.5% halothane in O2. Two large-bore, saline-filled silicon rubber catheters were inserted into the midcervical trachea of each fetus; one catheter was directed toward the lungs, and the other was directed toward, but did not enter, the larynx. These catheters were exteriorized from the ewe and joined together to form a continuous tracheal loop that allowed normal flow of tracheal liquid.

To accelerate fetal lung growth, the exteriorized tracheal loop was obstructed (TO) for 2, 4, or 10 days (n = 5 for each) during the alveolar stage of lung development (24, 33). This prevents the normal efflux of lung liquid via the trachea, causing the fetal lungs to expand with accumulated liquid, which markedly increases fetal lung growth rates (32) and induces type II to type I cell trans-differentiation (12). In control fetuses (0 day TO, n = 5), lung liquid was allowed to flow through the tracheal loop unimpeded, thereby maintaining a normal level of lung expansion (33). To retard fetal lung growth, fetal lung expansion was reduced by draining the fetal lungs of liquid via the tracheal catheter for 7 days (19) (n = 4). This causes lung growth to cease (1, 19, 31) and favors type I to type II AEC differentiation (15). In age-matched control fetuses (n = 4) lung liquid was again allowed to flow normally through the tracheal loop unimpeded (19). For all experiments in which lung liquid volumes were manipulated, the experimental period ended at ~128 days of gestation.

All animals were humanely killed with an overdose of pentobarbital sodium (130 mg/kg iv). At autopsy, the lungs were drained of liquid, removed, and weighed; the left bronchus was ligated; and the left lung was removed distal to the ligature. Small portions of the left lung were then snap frozen in liquid nitrogen and stored at –70°C for Northern blot analyses. The right lungs were fixed via the trachea at 20 cmH2O with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), and portions were processed for electron and light microscopy.

Ovine VDUP1 cDNA fragment. The ovine VDUP1 cDNA fragment used in these studies (clone R23; accession no. DQ004561) was isolated by subtraction hybridization in a separate experiment, designed to identify genes that are up- and downregulated by 36 h of increased lung expansion (40a). This fragment was one of the most highly downregulated clones isolated, is 406 nt in length, and has 78% nucleotide identity to nucleotides 2328–2701 of the published 3'-untranslated region of human VDUP1 (accession no. NM_006472.1). In total we isolated and cloned 15 other cDNA fragments aligning to four different regions of the human VDUP1 gene. In two clones that contained cDNA fragments corresponding to coding region of the human VDUP1 gene, one had 93% nucleotide identity and 100% amino acid identity (nt 221–282), whereas the other had 90% nucleotide identity and 98% amino acid identity (nt 963–1497); the latter corresponded to the last ~40% of the coding region of the human VDUP1 gene. Clearly, therefore, the sequence of the VDUP1 gene is well conserved over the coding region between sheep and humans but is less well conserved in the untranslated regions.

Northern blot analysis. Fetal lung VDUP1 and surfactant protein (SP)-B mRNA levels were quantified by Northern blot analysis using ovine 32P-labeled cDNA probes. Total RNA was extracted from fetal lung tissue by a modified guanidine-thiocyanate method, and 20 µg of denatured RNA were electrophoresed on a 1% agarose gel containing formaldehyde. The RNA was transferred to a nylon membrane (Duralon-UV membranes, Stratagene) by capillary action in 5x saline sodium citrate buffer and cross-linked to the membrane with UV light (Hoeffer UVC 500, Amrad). The membrane was prehybridized in hybridization buffer (deionized formamide, 7% wt/vol SDS, 5x SSPE, and 0.1 mg/ml salmon sperm) at 42°C for ~4 h before the addition of radiolabeled denatured cDNA probe (2 x 106 cpm/ml). The cDNA probes used were generated from the ovine VDUP1 cDNA fragment described above and a 330-nt ovine SP-B cDNA fragment (27). Hybridization occurred at 42°C overnight before a series of low (1x SSC, 0.1% SDS) and high (0.1x SSC, 0.1% SDS) stringency washes were performed to remove nonspecifically bound probe. Membranes were sealed in plastic and exposed to a phosphor storage screen for 1–3 days before being imaged by a phosphorimager (Storm 860, Molecular Dynamics). Membranes were subsequently reprobed with a radiolabeled ovine cDNA fragment for 18S rRNA to adjust for minor differences in loading. The intensity of each band was determined with the image analysis software Image QuaNT (Molecular Dynamics). The ontogeny analysis was performed on two blots due to the large number of samples. To allow comparisons between the two blots, the 128-day GA samples were run on each blot, and then all values were expressed as a percentage of the mean 128-day GA value. The mRNA levels for VDUP1 and SP-B are expressed relative to the level of 18S rRNA in individual animals and then as a percentage of either the mean control value or the 128-day GA ontogeny value.

VDUP1 immunohistochemical analysis. The fixed right lung was separated into the upper, middle, and lower lobes, and each lobe was accurately cut into 5-mm slices. Every second slice from each lobe was further subdivided into three sections. Six sections were then chosen at random from each lobe, and the tissue was cut into ~1 cm x 1 cm sections (x5 mm thick) and embedded into paraffin blocks. Blocks were selected at random, and tissue sections were cut at 5 µm. The sections were incubated at 60°C for 2 h, deparaffinized in xylene, rehydrated using graded alcohol washes, and washed in PBS. The sections were then boiled in sodium citrate (0.01 M, pH 6.0) for 20 min (in microwave, on high) to enhance antigen retrieval, washed in PBS (2 x 5 min), and then incubated in hydrogen peroxide (3%) for 5 min to block endogenous peroxidase activity. The sections were then rinsed in water, washed again in PBS, and incubated in blocking-permeabilization buffer (3% normal goat serum, 0.1% Triton X-100 in PBS) in a humidity chamber (30 min at room temperature). The sections were incubated with primary antibody (1:1,000, mouse anti-human VDUP1; MBL, Nagoya, Japan) for 90 min at room temperature, washed in PBS (with 0.1% Tween 20) for 5 min (3x), and then incubated with a biotinylated secondary antibody (goat anti-mouse diluted 1:700; Vector Laboratories, Burlingame, CA) in PBS for 1 h at room temperature. The sections were again washed in PBS/0.1% Tween 20 for 5 min (3x), and then the biotinylated secondary antibody was detected using the Vectastain ABC detection kit (Vector Laboratories). The sections were washed, dehydrated, and permanently mounted.

All tissue sections were viewed under a light microscope, and at least four sections (from different regions of the lungs) were viewed from each animal; care was taken to avoid sections containing major airways and blood vessels. Multiple fields of view were examined per section (at least six or seven per section), at different magnifications, and cellular localization was ascertained by examining the nucleus and cytoplasm of the cell by varying the focus through the tissue. Digital images were captured and stored electronically.

Cell proliferation. DNA synthesis rates were determined for fetuses exposed to increases and decreases in fetal lung expansion by measuring the incorporation of [3H]thymidine into DNA (disintegrations per min/µg DNA) as previously described (19, 24, 33); values are expressed as a percentage of control values. In fetuses used for the ontogeny analysis, lung cell proliferation rates were determined using an immunohistochemical marker of cell proliferation (Ki67). Random portions of frozen lung tissue from three fetuses each at 90, 110, 128, 138, and 142 days GA were sectioned at 10 µm. The sections were thawed, air-dried, and fixed in 4% paraformaldehyde in PBS for 20 min, followed by a PBS wash (5 min). The sections were then boiled in sodium citrate (0.01M, pH 6.0) for 20 min (in microwave, on high) to enhance antigen retrieval and washed in PBS (2 x 5 min). Endogenous peroxidases were blocked by incubating in 75% methanol/2% H2O2 in PBS for 5 min. Nonspecific binding was reduced by incubating the sections for 30 min in blocking buffer (10% normal goat serum, 0.1% Triton, 0.05 M Tris·Cl, pH 7.2). The sections were then incubated with a 1:150 dilution of primary antibody (mouse anti-human Ki67, Clone MIB-1; Dakocytomation) for 90 min at room temperature and washed (3 x 5 min in PBS/0.1% Tween 20). Sections were then incubated with a biotinylated secondary antibody (polyclonal goat anti-mouse diluted 1:700, Dakocytomation) for 1 h and washed (3 x 5 min in PBS/0.1% Tween 20), and then the secondary antibody was detected using the Vectastain ABC detection kit (Vector Laboratories). The sections were washed, dehydrated, and permanently mounted. A minimum of 500 nuclei were counted per fetus, and the percentage of Ki67-labeled cells (Ki67 labeling index) was measured.

Statistical analysis. All values are expressed as means ± SE. Differences between control fetuses and fetuses exposed to decreased lung expansion were analyzed by an unpaired t-test. Differences between control fetuses and fetuses exposed to increased lung expansion (accelerated lung growth), as well as the ontogenic changes in VDUP1, SP-B mRNA levels, and the Ki67 labeling index, were analyzed by a one-way ANOVA followed by a least significant difference post hoc test. In the ontogeny study, all mRNA values were expressed as a percentage of the 128-day GA values, which were run on each blot, so that comparisons could be made between blots. Similarly, all values from the increased and decreased lung expansion studies were expressed as a percentage of the control values run on the same blot. The relationship 1) between VDUP1 and SP-B mRNA levels and 2) between VDUP1 mRNA levels and the percentage of Ki67-labeled cells (during normal lung development) or the previously described DNA synthesis rates (following alterations in lung expansion) was tested by the most appropriate regression for the data set. All statistical tests were performed using the software package SPSS, and significance was taken at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Changes in VDUP1 mRNA levels during normal and altered lung growth. The relative levels of VDUP1 mRNA in the ovine fetal lung at 90 days (14.0 ± 2.7%), 105 days (39.7 ± 8.9%), and 111 days (28.1 ± 8.1%) of gestation, which corresponds to the canalicular stage of lung development, were low compared with values measured during the alveolar stage of lung development: 128-day (100.0 ± 8.5%) and 138-day GA fetuses (111.7 ± 7.7%) (P < 0.05, Fig. 1). Near term (142 days GA), VDUP1 mRNA levels in fetal lung tissue were significantly increased to 180.0 ± 18.3% relative to the 128-day GA levels (P < 0.05) and remained elevated at 2 wk PNA (156.8 ± 21.2%).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1. A: vitamin D3-upregulated protein 1 (VDUP1) mRNA levels (means ± SE) determined by Northern blot, expressed as a proportion of the 18S rRNA level in each sample and as a percentage of the mean 128 days (d) gestational age (GA) levels. VDUP1 mRNA levels increased over gestation, with notable increases in expression occurring between 111–128 d and 138–142 d GA. Values that do not share a common letter are significantly different from each other (P < 0.05). B: Northern blots for VDUP1 and 18S rRNA in tissue collected from lungs of fetal sheep at 90, 105, 111, 128, 138, and 142 d GA and from lambs at 2 wk postnatal age (PNA); each lane represents mRNA from a different animal. The 18S rRNA bands were used to adjust for minor differences in loading between lanes.

 
In fetuses exposed to increased lung expansion induced by TO, VDUP1 mRNA levels in fetal lung tissue were significantly decreased from 100.0 ± 8.3% in control fetuses to 37.2 ± 3.7% of control levels at 2 days of TO (P < 0.01, Fig. 2). The effect of increased lung expansion on VDUP1 mRNA levels in fetal lung tissue persisted at 4 days (45.9 ± 3.6%) and 10 days (45.0 ± 8.6%) of TO. On the other hand, lung liquid drainage for 7 days significantly increased VDUP1 mRNA levels in fetal lung tissue from 100.0 ± 15.8% in control fetuses to 162.4 ± 15.6% in lung liquid-drained fetuses (P < 0.05, Fig. 3).



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2. VDUP1 (A) and surfactant protein B (SP-B, B) mRNA levels (means ± SE) in control fetuses and fetuses exposed to increased fetal lung expansion, expressed as a proportion of the 18S rRNA level in each sample and as a percentage of the mean value in control fetuses. Both VDUP1 and SP-B mRNA levels decreased significantly at 2 d of tracheal obstruction (TO) and remained low at 4 and 10 d of TO. *Values that are significantly different (P < 0.001) from the control values run on the same Northern blot. C: Northern blots for VDUP1, SP-B, and 18S rRNA in lung tissue from control fetuses and from fetuses exposed to 2, 4, and 10 d of TO; each lane represents mRNA from a different fetus. The 18S rRNA bands were used to adjust for minor differences in loading between lanes.

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 3. VDUP1 (A) and SP-B (B) mRNA levels (means ± SE) in control fetuses and fetuses exposed to reduced lung expansion, expressed as a proportion of 18S rRNA and as a percentage of the mean value in control fetuses. Both VDUP1 and SP-B mRNA levels increased significantly and by a similar magnitude following 7 d of reduced lung expansion induced by lung liquid drainage. *Values that are significantly different (P < 0.05) from the control values run on the same Northern blot. C: Northern blots for VDUP1, SP-B, and 18S rRNA in tissue collected from lungs of control fetuses and from fetuses exposed to 7 d of lung liquid drainage; each lane represents mRNA from a different fetus. The 18S rRNA bands were used to adjust for minor differences in loading between lanes.

 
Immunohistochemistry for VDUP1 in fetal lung tissue. The VDUP1 protein was found to be highly abundant in fetal lung tissue, particularly at 142 days of gestation when expression levels are high compared with earlier in gestation (90–111 days of gestation). VDUP1 was localized to the epithelium of the small bronchioles and was abundant in the distal perialveolar parenchyma (see Fig. 4). In particular, VDUP1 appeared to be present in both type I and type II AECs as well as in some cells of mesenchymal origin (most probably fibroblasts, Fig. 4). In type II AECs, which were classified by their distinctive shape and position within the alveolar walls, the VDUP1 staining was relatively light and appeared to be primarily localized to the cytoplasm. Similarly, in cells that had flattened nuclei and cytoplasmic extensions resembling type I AECs (or possibly vascular endothelial cells), VDUP1 was predominantly localized within the cytoplasm, although a number of heavily stained flattened nuclei were observed (see Fig. 4). It is most likely that these VDUP1-positive cells with flattened nuclei and cytoplasmic extensions are type I AECs and not vascular endothelial cells, as VDUP1 was not localized to cells lining larger blood vessels when cut in cross section (not shown).



View larger version (58K):
[in this window]
[in a new window]
  		Fig. 4. Low power (x40, A) and higher power (x100, B) images of fetal lung tissue demonstrating the cellular localization of VDUP1 (brown staining) in airway epithelium (indicated by arrow, A) and in the alveolar parenchyma (B). Note the positive staining for VDUP1 in type II (TII) alveolar epithelial cells (AECs), in type I (TI) AECs (cytoplasmic extensions are indicated by arrows) and in interstitial cells (IC) of the lung parenchyma.

 
Relationship between VDUP1 and SP-B expression. During normal lung development, the temporal pattern of SP-B expression was very similar to and was significantly correlated with VDUP1 expression. SP-B mRNA levels were low during the canalicular phase of lung development at 90 days (37.5 ± 4.1%), 105 days (45.8 ± 6.6%), and 111 days GA (40.6 ± 7.0%) compared with its expression during the alveolar stage at 128 days (100.0 ± 2.6%, P < 0.05) GA. SP-B mRNA levels continued to increase at 138 days (168.7 ± 22.7%, P < 0.05) and again at 142 days GA (264.2 ± 40.6%, P < 0.05) but then decreased to 170.0 ± 11.6% (P < 0.05) at 2 wk PNA (14). These changes in SP-B expression were closely correlated with changes in VDUP1 expression throughout normal lung development; the relationship was a highly significant, positive linear relationship (y = 16.8 + 0.9x, R2 = 0.803, P < 0.0001; Fig. 5A).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 5. The relationship between VDUP1 and SP-B mRNA levels in ovine fetal lung tissue are shown during normal lung development (A) and in fetuses exposed to increases ({bullet}) or decreases ({circ}) in fetal lung expansion (B). The relationship between VDUP1 mRNA levels and cell proliferation rates are shown during normal lung development (C) and in fetuses exposed to increases ({bullet}) and decreases ({circ}) in fetal lung expansion (D). Cell proliferation rates were determined by the percentage of Ki67-labeled cells during normal lung development and by the incorporation of [3H]thymidine into DNA, in fetuses exposed to increases and decreases in lung expansion. In A and B, each point represents values from a single fetus. In C and D, each point represents a group mean ± SE (n = 3–5 for each), as the cell proliferation rates and VDUP1 expression were not performed in the same fetuses.

 
Similarly, the changes in VDUP1 mRNA levels were positively correlated with the changes in SP-B mRNA levels following alterations in fetal lung expansion (Fig. 5B). SP-B mRNA levels were decreased from 100.0 ± 6.7% in control fetuses to 29.6 ± 2.5% of control values at 2 days TO and remained low at 4 days (33.2 ± 3.9%) and 10 days (18.8 ± 0.4%) of TO (P < 0.001, Fig. 2B). On the other hand, lung liquid drainage significantly increased SP-B mRNA levels from 100.0 ± 8.5% in control fetuses to 142.3 ± 11.7% in lung liquid-drained fetuses (P < 0.05, Fig. 3B). The relationship between VDUP1 mRNA levels and SP-B mRNA levels in both control (128 days GA) fetuses and fetuses exposed to alterations in fetal lung expansion (both increases and decreases) was highly significant (R2 = 0.817, P < 0.0001) and best described by the quadratic function: y = 1.4x – 20.6 – 0.002x2 (Fig. 5B).

Relationship between VDUP1 and lung growth. A significant inverse relationship was also found between VDUP1 mRNA levels and lung cell proliferation rates during normal lung development and following alterations in fetal lung expansion (both increases and decreases). The percentage of lung cells proliferating during normal lung development, determined by a Ki67-labeling index, was highest at 90 days of GA (8.7 ± 1.7%) when VDUP1 mRNA levels were lowest. There was a nonsignificant decrease in cell proliferation at 111 days GA (6.0 ± 0.4%), which corresponded to a small, nonsignificant increase in VDUP1 mRNA levels. However, compared with the 90- and 111-day levels, cell proliferation rates were significantly lower at 128 days GA (2.8 ± 0.5%, P < 0.05), which coincided with a threefold increase in VDUP1 mRNA levels. Cell proliferation rates remained low at 138 days (2.3 ± 0.4%) and 142 days GA (1.7 ± 0.3%) when VDUP1 mRNA levels remained high. A highly significant inverse exponential relationship was found between VDUP1 mRNA levels and the rate of lung cell proliferation (y = 1.6 + 104.4/x, R2 = 0.972, P < 0.002; Fig. 5C), indicating that high VDUP1 mRNA levels are associated with low lung cell proliferation rates during normal lung development.

The reduction in VDUP1 mRNA levels induced by increased lung expansion (Fig. 2A) coincided with a large increase in lung cellular proliferation rates, as determined by the incorporation of [3H]thymidine, at both 2 days (777%) and 4 days (66%) of increased lung expansion, relative to controls (32). On the other hand the increase in VDUP1 mRNA levels induced by lung liquid drainage (Fig. 3A) for 7 days was associated with a substantial decrease in lung cell proliferation to 37.3% of control levels (19). An inverse exponential relationship was found between VDUP1 expression and the rate of lung cell proliferation (y = 1.9*106 e–0.2x, R2 = 0.956, P < 0.001; Fig. 5D), indicating that high VDUP1 mRNA levels following alterations in fetal lung expansion are associated with low lung cell proliferation rates.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first time that VDUP1 has been implicated as a regulator of any developmental process that we are aware of. We have shown that VDUP1 expression is reduced in response to an increase in lung expansion that induces a marked acceleration (eightfold increase) in lung cell proliferation. On the other hand, lung deflation, which causes lung growth to cease, was associated with a large increase in VDUP1 expression. As VDUP1 is involved in cell growth and differentiation in a wide range of tissues, we have related the changes in VDUP1 mRNA levels in the developing lung to changes in indexes of lung growth and to expression of the type II AEC marker, SP-B. VDUP1 mRNA levels were inversely related to cellular proliferation and were positively related to the mRNA levels for the type II AEC marker SP-B following alterations in fetal lung expansion (Fig. 5). Similarly, VDUP1 mRNA levels were found to increase in parallel with increases in SP-B expression and were inversely related to cell proliferation rates, over the last third of gestation. When combined with the known roles of VDUP1, these data support a role for VDUP1 in the control of cell proliferation and type II AEC differentiation in the developing lung.

VDUP1 has well-described roles in growth inhibition and tumor suppression. Its expression is downregulated in tumors of the lung (16, 17), colon (5, 21), mammary glands (5, 17, 46, 50), lymph glands (9), and melanoma cells (40). On the other hand, overexpression of VDUP1 is associated with the suppression of both tumor growth (5, 17, 22, 34, 42) and the formation of metastases (16). VDUP1 has been shown to arrest cell cycle progression at G0/G1 (23, 34), likely via its interaction with thioredoxin (38) and/or by forming part of a zinc-finger transcriptional co-repressor complex (17). Thioredoxin is a cellular redox protein that promotes cell proliferation and alters gene expression via redox-dependent alterations in the activity of growth-promoting proteins (25) and by enhancing the binding of transcription factors, such as NF-{kappa}B, activator protein-1, and p53, to target DNA (25, 48). VDUP1 is a potent negative regulator of thioredoxin as it downregulates thioredoxin mRNA levels and synthesis of the thioredoxin protein as well as inhibiting thioredoxins' reducing activity (35, 48); the latter decreases thioredoxins' potential to activate growth-promoting proteins. VDUP1 overexpression also inhibits cell proliferation by causing downregulation of the cyclin A2 promoter (17), which contains promyelocytic leukemia zinc-finger (PLZF) response elements (47). Cyclin A2 is a regulator of the cell cycle that modulates the function of two different cyclin-dependent kinases and is able to control progression of the cell cycle at S phase during mitosis (45). The effect of VDUP1 on cyclin A2 is likely due to the interaction of VDUP1 with a zinc-finger transcriptional co-repressor complex that consists of PLZF, Fanconi anemia zinc-finger, and histone deacetylase 1 (17). Those studies suggest that VDUP1 is likely to exert its antiproliferative effects by inhibition of cyclin A2 expression by interacting with the PLZF transcriptional co-repressor complex and/or by inhibiting the ability of thioredoxin to regulate proliferation-related proteins and transcription factors.

In view of the well described roles for VDUP1 in growth inhibition, our finding that VDUP1 expression in the fetal lung is differentially regulated by stimuli that accelerate and retard fetal lung growth is consistent with the suggestion that VDUP1 is an important regulatory factor of these processes. We found that VDUP1 was significantly downregulated by increased fetal lung expansion, which is a potent stimulus for fetal lung cell proliferation (6, 19, 31, 32). On the other hand, VDUP1 expression was significantly upregulated by lung deflation, which causes lung cell proliferation to effectively cease (1, 19). Furthermore, when all the data was combined, we showed that VDUP1 expression was inversely correlated with the rate of lung cell proliferation, during both alterations in lung expansion and normal lung development. Consequently, we suggest that VDUP1 may be an important inhibitory modulator of cell proliferation in the developing lung and that lung cell proliferation may depend upon the removal of its inhibitory influence. Although the pathways by which VDUP1 could exert this modulatory effect on cell proliferation are unknown, they likely involve the inhibition of thioredoxin and/or cyclin A2 as described above and may depend upon the cellular location of VDUP1, specifically, nucleus vs. cytoplasm (see below). Future studies involving VDUP1 activation or inhibition will be needed, however, to elucidate a causal role for VDUP1 as an inhibitor of cell proliferation in the developing lung.

The immunohistochemical analysis demonstrated that, in the distal airways, VDUP1 was predominantly localized within airway epithelial cells of the small bronchioles and appeared to be present in both type I and type II AECs (Fig. 4). Because of their close apposition to and similar morphology with capillary endothelial cells, it is difficult to identify type I AECs using light microscopy without specific cell markers. However, we believe that the VDUP1-labeled cells observed within the alveolar walls that had flattened nuclei and cytoplasmic extensions were unlikely to be endothelial cells. This is because VDUP1 staining was not present in endothelial cells of the small blood vessels that could be clearly identified. In a number of these "type I AECs," both nuclear and cytoplasmic staining was observed, with some nuclei being darkly stained for VDUP1. Although it is difficult to clearly demonstrate cytoplasmic vs. nuclear staining in the flat images (see Fig. 4), following a careful in-depth examination, it appeared that type I AECs were the only cell type that had evidence of nuclear staining; VDUP1 staining appeared to be confined to the cytoplasm in type II AECs, with little or no staining of nuclei (see Fig. 4). The possible significance of differential staining patterns between type I and type II AECs is unclear, although the presence of VDUP1 in the nuclei of type I AECs could relate to its inability to divide; previous studies have provided compelling evidence to indicate that type I AECs cannot divide (11). Thus it is possible that the presence of VDUP1 in the nucleus suppresses nuclear specific activities that are critical for cell proliferation such as cyclin A2 expression and/or thioredoxin activity, as indicated above. Previous studies have predominantly localized VDUP1 staining to the cytoplasm in a number of different cell types (7, 17, 23) although nuclear staining has been observed in HL-60 cells plated at high cell densities (17); these conditions would be expected to inhibit cell proliferation. Under the latter conditions, VDUP1 within the nucleus is colocalized with the PLZF transcriptional co-repressor complex that inhibits cyclin A2 expression (17).

Our findings that VDUP1 is differentially regulated by alterations in fetal lung expansion is consistent with the findings of previous studies demonstrating that mechanical strain is an important regulator of VDUP1 expression (44, 49). In those studies, mechanical strain was shown to downregulate VDUP1 expression and VDUP1 protein synthesis in rat cardiac myocytes both in culture (44) and in vivo, in response to a pressure overload (49). Although the mechanism by which VDUP1 expression is altered by mechanical forces is unknown, it may be secondary to alterations in signaling pathways activated by cell stretch. These include activation of extracellular matrix receptors (2, 30), stretch-sensitive ion channels (2, 4), or direct alteration of gene transcription by elements within its promoter such as the shear stress responsive element (37, 39). Indeed, the promoter region of VDUP1 contains two Sp1 sequence elements that have been shown to act as a shear stress responsive element (26).

High levels of VDUP1 have also been associated with cellular differentiation in mammary (10, 20), epidermal (7), T-cell (34), myeloid (8), and gastrointestinal epithelial (41) cells, although the mechanisms involved are unknown. It is possible that thioredoxin or the zinc-finger transcriptional co-repressor complex could affect the expression of genes that determine cell phenotype and may, therefore, mediate the effect of VDUP1 on cellular differentiation. In the developing lung, differentiation of AECs into the two specialized phenotypes is essential for providing the lung with a thin barrier for efficient gas exchange (type I AECs) and for producing pulmonary surfactant (type II AECs). Surfactant reduces the surface tension generated at the air-liquid interface once the lung becomes filled with air at birth. We have previously reported the changes in AEC proportions throughout gestation and have demonstrated that the degree of basal lung expansion is an important determinant of AEC phenotype. Increased fetal lung expansion favors the type I cell phenotype, whereas reduced fetal lung expansion favors the type II AEC phenotype (12, 15). As the changes in type II AEC proportions are closely associated with concomitant alterations in SP-B mRNA levels (12, 13, 14), we chose SP-B as a marker of differentiated type II AECs. We found a highly significant positive correlation between the expression levels of VDUP1 and SP-B throughout normal lung development and following increases and reductions in fetal lung expansion (Fig. 5, A and B). Given the relationship between VDUP1 and differentiation in other cell types, it is possible that VDUP1 may play a role in the regulation of AEC phenotypes. Indeed, as VDUP1 is highly induced by vitamin D3 (8), it may mediate the stimulatory effects of vitamin D3 on type II AEC differentiation and SP-B expression (28, 29, 36). The presence of VDUP1 in type I cells that do not express SP-B may, on the surface, appear to contradict this suggestion; however, the expression of all proteins, including SP-B, is dependent upon a balance between positive and negative regulators. Thus a lack of SP-B expression in type I cells is likely due to the presence of high levels of repressors of SP expression. However, further studies will be required to demonstrate a causal role for VDUP1 in the regulation of alveolar epithelial cell differentiation.

In summary, we have shown that lung VDUP1 expression is regulated by the degree of basal lung expansion during fetal life, is inversely related to the rate of cellular proliferation, and is positively related to type II AEC differentiation during normal, accelerated, and retarded fetal lung growth. These findings suggest that VDUP1 may mediate the effects of alterations in lung expansion on lung cell proliferation and differentiation and that it may be an important endogenous regulator of fetal lung growth and type II AEC differentiation during normal lung development.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The experiments described in this study were funded by the National Health and Medical Research Council of Australia.


   ACKNOWLEDGMENTS
 
We are indebted to Alex Satragno for assistance with the surgical preparation of animals, to Gosia Zieba for the immunohistochemical analysis of Ki67, and to Alison Thiel for assistance in the laboratory.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. E. Filby, Dept. of Physiology, Bldg. 13F, Monash Univ., Vic 3800, Australia (e-mail: Caitlin.Filby{at}med.monash.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, and Robinson PM. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123: 649–660, 1977.[Web of Science][Medline]
  2. Banes AJ, Tsuzaki M, Yamamoto J, Fischer T, Brigman B, Brown T, and Miller L. Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses to mechanical signals. Biochem Cell Biol 73: 349–365, 1995.[Web of Science][Medline]
  3. Benachi A, Delezoide AL, Chailley-Heu B, Preece M, Bourbon JR, and Ryder T. Ultrastructural evaluation of lung maturation in a sheep model of diaphragmatic hernia and tracheal occlusion. Am J Respir Cell Mol Biol 20: 805–812, 1999.[Abstract/Free Full Text]
  4. Berridge MJ. Calcium signalling and cell proliferation. Bioessays 17: 491–500, 1995.[CrossRef][Web of Science][Medline]
  5. Butler LM, Zhou X, Xu WS, Scher HI, Rifkind RA, Marks PA, and Richon VM. The histone deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin. Proc Natl Acad Sci USA 99: 11700–11705, 2002.[Abstract/Free Full Text]
  6. Carmel JA, Friedman F, and Adams FH. Fetal tracheal ligation and lung development. Am J Dis Child 109: 452–456, 1965.[Abstract/Free Full Text]
  7. Champliaud MF, Viel A, and Baden HP. The expression of vitamin D-upregulated protein 1 in skin and its interaction with sciellin in cultured keratinocytes. J Invest Dermatol 121: 781–785, 2003.[CrossRef][Medline]
  8. Chen KS and DeLuca HF. Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3. Biochim Biophys Acta 1219: 26–32, 1994.[Medline]
  9. De Vos S, Hofmann WK, Grogan TM, Krug U, Schrage M, Miller TP, Braun JG, Wachsman W, Koeffler HP, and Said JW. Gene expression profile of serial samples of transformed B-cell lymphomas. Lab Invest 83: 271–285, 2003.[Web of Science][Medline]
  10. Escrich E, Moral R, Garcia G, Costa I, Sanchez JA, and Solanas M. Identification of novel differentially expressed genes by the effect of a high-fat n-6 diet in experimental breast cancer. Mol Carcinog 40: 73–78, 2004.[Medline]
  11. Evans MJ, Cabral LJ, Stephens RJ, and Freeman G. Renewal of alveolar epithelium in the rat following exposure to NO2. Am J Pathol 70: 175–198, 1973.[Web of Science][Medline]
  12. Flecknoe S, Harding R, Maritz G, and Hooper SB. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am J Physiol Lung Cell Mol Physiol 278: L1180–L1185, 2000.[Abstract/Free Full Text]
  13. Flecknoe SJ, Boland RE, Wallace MJ, Harding R, and Hooper SB. Regulation of alveolar epithelial cell phenotypes in fetal sheep: roles of cortisol and lung expansion. Am J Physiol Lung Cell Mol Physiol 287: L1207–L1214, 2004.[Abstract/Free Full Text]
  14. Flecknoe SJ, Wallace MJ, Cock ML, Harding R, and Hooper SB. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol 285: L664–L670, 2003.[Abstract/Free Full Text]
  15. Flecknoe SJ, Wallace MJ, Harding R, and Hooper SB. Determination of alveolar epithelial cell phenotypes in fetal sheep: evidence for the involvement of basal lung expansion. J Physiol 542: 245–253, 2002.[Abstract/Free Full Text]
  16. Goldberg SF, Miele ME, Hatta N, Takata M, Paquette-Straub C, Freedman LP, and Welch DR. Melanoma metastasis suppression by chromosome 6: evidence for a pathway regulated by CRSP3 and TXNIP. Cancer Res 63: 432–440, 2003.[Abstract/Free Full Text]
  17. Han SH, Jeon JH, Ju HR, Jung U, Kim KY, Yoo HS, Lee YH, Song KS, Hwang HM, Na YS, Yang Y, Lee KN, and Choi I. VDUP1 upregulated by TGF-beta1 and 1,25-dihydroxyvitamin D3 inhibits tumor cell growth by blocking cell-cycle progression. Oncogene 22: 4035–4046, 2003.[CrossRef][Web of Science][Medline]
  18. Harding R, Bocking AD, and Sigger JN. Influence of upper respiratory tract on liquid flow to and from fetal lungs. J Appl Physiol 61: 68–74, 1986.[Abstract/Free Full Text]
  19. Hooper SB, Han VKM, and Harding R. Changes in lung expansion alter pulmonary DNA synthesis and IGF-II gene expression in fetal sheep. Am J Physiol Lung Cell Mol Physiol 265: L403–L409, 1993.[Abstract/Free Full Text]
  20. Huang L and Pardee AB. Suberoylanilide hydroxamic acid as a potential therapeutic agent for human breast cancer treatment. Mol Med 6: 849–866, 2000.[Web of Science][Medline]
  21. Ikarashi M, Takahashi Y, Ishii Y, Nagata T, Asai S, and Ishikawa K. Vitamin D3 up-regulated protein 1 (VDUP1) expression in gastrointestinal cancer and its relation to stage of disease. Anticancer Res 22: 4045–4048, 2002.[Web of Science][Medline]
  22. Joguchi A, Otsuka I, Minagawa S, Suzuki T, Fujii M, and Ayusawa D. Overexpression of VDUP1 mRNA sensitizes HeLa cells to paraquat. Biochem Biophys Res Commun 293: 293–297, 2002.[CrossRef][Medline]
  23. Junn E, Han SH, Im JY, Yang Y, Cho EW, Um HD, Kim DK, Lee KW, Han PL, Rhee SG, and Choi I. Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function. J Immunol 164: 6287–6295, 2000.[Abstract/Free Full Text]
  24. Keramidaris E, Hooper SB, and Harding R. Effect of gestational age on the increase in fetal lung growth following tracheal obstruction. Exp Lung Res 22: 283–298, 1996.[Web of Science][Medline]
  25. Kwon YW, Masutani H, Nakamura H, Ishii Y, and Yodoi J. Redox regulation of cell growth and cell death. Biol Chem 384: 991–996, 2003.[CrossRef][Web of Science][Medline]
  26. Lin MC, Almus-Jacobs F, Chen HH, Parry GC, Mackman N, Shyy JY, and Chien S. Shear stress induction of the tissue factor gene. J Clin Invest 99: 737–744, 1997.[Web of Science][Medline]
  27. Lines A, Nardo L, Phillips ID, Possmayer F, and Hooper SB. Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep. Am J Physiol Lung Cell Mol Physiol 276: L239–L245, 1999.[Abstract/Free Full Text]
  28. Marin L, Dufour ME, Nguyen TM, Tordet C, and Garabedian M. Maturational changes induced by 1{alpha},25-dihydroxyvitamin D3 in type II cells from fetal rat lung explants. Am J Physiol Lung Cell Mol Physiol 265: L45–L52, 1993.[Abstract/Free Full Text]
  29. Marin L, Dufour ME, Tordet C, and Nguyen M. 1,25(OH)2D3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants. Biol Neonate 57: 257–260, 1990.[Web of Science][Medline]
  30. Meyer CJ, Alenghat FJ, Rim P, Fong JH, Fabry B, and Ingber DE. Mechanical control of cyclic AMP signalling and gene transcription through integrins. Nat Cell Biol 2: 666–668, 2000.[CrossRef][Web of Science][Medline]
  31. Moessinger AC, Harding R, Adamson TM, Singh M, and Kiu GT. Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86: 1270–1277, 1990.[Web of Science][Medline]
  32. Nardo L, Hooper SB, and Harding R. Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid volume. Pediatr Res 43: 184–190, 1998.[Web of Science][Medline]
  33. Nardo L, Maritz G, Harding R, and Hooper SB. Changes in lung structure and cellular division induced by tracheal obstruction in fetal sheep. Exp Lung Res 26: 105–119, 2000.[CrossRef][Web of Science][Medline]
  34. Nishinaka Y, Nishiyama A, Masutani H, Oka S, Ahsan KM, Nakayama Y, Ishii Y, Nakamura H, Maeda M, and Yodoi J. Loss of thioredoxin-binding protein-2/vitamin D3 up-regulated protein 1 in human T-cell leukemia virus type I-dependent T-cell transformation: implications for adult T-cell leukemia leukemogenesis. Cancer Res 64: 1287–1292, 2004.[Abstract/Free Full Text]
  35. Nishiyama A, Matsui M, Iwata S, Hirota K, Masutani H, Nakamura H, Takagi Y, Sono H, Gon Y, and Yodoi J. Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem 274: 21645–21650, 1999.[Abstract/Free Full Text]
  36. Rehan VK, Torday JS, Peleg S, Gennaro L, Vouros P, Padbury J, Rao DS, and Reddy GS. 1Alpha,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1alpha,25-dihydroxy vitamin D3: production and biological activity studies in pulmonary alveolar type II cells. Mol Genet Metab 76: 46–56, 2002.[CrossRef][Web of Science][Medline]
  37. Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF Jr, and Gimbrone MA Jr. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. Proc Natl Acad Sci USA 90: 4591–4595, 1993.[Abstract/Free Full Text]
  38. Schulze PC, De Keulenaer GW, Yoshioka J, Kassik KA, and Lee RT. Vitamin D3-upregulated protein-1 (VDUP-1) regulates redox-dependent vascular smooth muscle cell proliferation through interaction with thioredoxin. Circ Res 91: 689–695, 2002.[Abstract/Free Full Text]
  39. Shinoki N, Kawasaki T, Minamino N, Okahara K, Ogawa A, Ariyoshi H, Sakon M, Kambayashi J, Kangawa K, and Monden M. Shear stress down-regulates gene transcription and production of adrenomedullin in human aortic endothelial cells. J Cell Biochem 71: 109–115, 1998.[CrossRef][Medline]
  40. Song H, Cho D, Jeon JH, Han SH, Hur DY, Kim YS, and Choi I. Vitamin D(3) up-regulating protein 1 (VDUP1) antisense DNA regulates tumorigenicity and melanogenesis of murine melanoma cells via regulating the expression of fas ligand and reactive oxygen species. Immunol Lett 86: 235–247, 2003.[CrossRef][Web of Science][Medline]
  41. Sozo F, Wallace MJ, Zahra VA, Filby CE, and Hooper SB. Gene expression profiling during increased fetal lung expansion identifies genes likely to regulate development of the distal airways. Physiol Genomics (October 25, 2005), 10.1152/physiolgenomics.00148.2005.
  42. Takahashi Y, Ishii Y, Murata A, Nagata T, and Asai S. Localization of thioredoxin-interacting protein (TXNIP) mRNA in epithelium of human gastrointestinal tract. J Histochem Cytochem 51: 973–976, 2003.[Abstract/Free Full Text]
  43. Takahashi Y, Nagata T, Ishii Y, Ikarashi M, Ishikawa K, and Asai S. Up-regulation of vitamin D3 up-regulated protein 1 gene in response to 5-fluorouracil in colon carcinoma SW620. Oncol Rep 9: 75–79, 2002.[Medline]
  44. Vilos GA and Liggins GC. Intrathoracic pressures in fetal sheep. J Dev Physiol 4: 247–256, 1982.[Medline]
  45. Wang Y, De Keulenaer GW, and Lee RT. Vitamin D(3)-up-regulated protein-1 is a stress-responsive gene that regulates cardiomyocyte viability through interaction with thioredoxin. J Biol Chem 277: 26496–26500, 2002.[Abstract/Free Full Text]
  46. Yam CH, Fung TK, and Poon RY. Cyclin A in cell cycle control and cancer. Cell Mol Life Sci 59: 1317–1326, 2002.[CrossRef][Web of Science][Medline]
  47. Yang X, Young LH, and Voigt JM. Expression of a vitamin D-regulated gene (VDUP-1) in untreated- and MNU-treated rat mammary tissue. Breast Cancer Res Treat 48: 33–44, 1998.[CrossRef][Medline]
  48. Yeyati PL, Shaknovich R, Boterashvili S, Li J, Ball HJ, Waxman S, Nason-Burchenal K, Dmitrovsky E, Zelent A, and Licht JD. Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A. Oncogene 18: 925–934, 1999.[CrossRef][Web of Science][Medline]
  49. Yodoi J, Nakamura H, and Masutani H. Redox regulation of stress signals: possible roles of dendritic stellate TRX producer cells (DST cell types). Biol Chem 383: 585–590, 2002.[CrossRef][Web of Science][Medline]
  50. Yoshioka J, Schulze PC, Cupesi M, Sylvan JD, MacGillivray C, Gannon J, Huang H, and Lee RT. Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity. Circulation 109: 2581–2586, 2004.[Abstract/Free Full Text]
  51. Young LH, Yang X, and Voigt JM. Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea. Mol Carcinog 15: 251–260, 1996.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
T. E. Tipple, S. E. Welty, L. D. Nelin, J. M. Hansen, and L. K. Rogers
Alterations of the Thioredoxin System by Hyperoxia: Implications for Alveolar Development
Am. J. Respir. Cell Mol. Biol., November 1, 2009; 41(5): 612 - 619.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
F. Sozo, S. B. Hooper, and M. J. Wallace
Thrombospondin-1 expression and localization in the developing ovine lung
J. Physiol., October 15, 2007; 584(2): 625 - 635.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/L250    most recent
00244.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Filby, C. E.
Right arrow Articles by Wallace, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Filby, C. E.
Right arrow Articles by Wallace, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.